Cell suspensions

Biophys J, 2003 May, 84(5), 3087 - 101
An experimental and theoretical analysis of ultrasound-induced permeabilization of cell membranes; Sundaram J et al.; Application of ultrasound transiently permeabilizes cell membranes and offers a nonchemical, nonviral, and noninvasive method for cellular drug delivery . Although the ability of ultrasound to increase transmembrane transport has been well demonstrated, a systematic dependence of transport on ultrasound parameters is not known . This study examined cell viability and cellular uptake of calcein using 3T3 mouse cell suspension as a model system . Cells were exposed to varying acoustic energy doses at four different frequencies in the low frequency regime (20-100 kHz) . At all frequencies, cell viability decreased with increasing acoustic energy dose, while the fraction of cells exhibiting uptake of calcein showed a maximum at an intermediate energy dose . Acoustic spectra under various ultrasound conditions were also collected and assessed for the magnitude of broadband noise and subharmonic peaks . While the cell viability and transport data did not show any correlation with subharmonic (f/2) emission, they correlated with the broadband noise, suggesting a dominant contribution of transient cavitation . A theoretical model was developed to relate reversible and irreversible membrane permeabilization to the number of transient cavitation events . The model showed that nearly every stage of transient cavitation, including bubble expansion, collapse, and subsequent shock waves may contribute to membrane permeabilization . For each mechanism, the volume around the bubble within which bubbles induce reversible and irreversible membrane permeabilization was determined . Predictions of the model are consistent with experimental data.

World J Gastroenterol, 2003 May, 9(5), 941 - 5
Biological characteristics of HCC by ultrasound-guided aspiration biopsy and its clinical application; Lin LW et al.; AIM: To probe the pathological biological characteristics of hepatocellular carcinoma (HCC) by the ultrasound-guided aspiration biopsy and assess the clinical application value of this method . METHODS: The biopsy and DNA analysis by flow cytometry (FCM) were taken in 46 cases with HCC nodules, including 26 cases and 20 cases with nodules < or =3 cm and >3 cm in diameters respectively, and 12 cases with intrahepatic benign hyperplastic nodules . They were taken in 22 cases of 46 cases with HCC before and after the therapy . Fine-needles and automatic histological incised biopsy needles were used . The fresh biopsy tissue was produced into the single cell suspension, which was sent for DNA detection and ratio analysis of cell period . The ratio of each DNA period of cell proliferation of each group was calculated and compared with each other . The DNA aneuploid (AN) and apoptosis cell peak were observed and their percentages were calculated . RESULTS: The ratios of S and G(2)/M periods of DNA, which reflect cell hyperproliferation, in the group with HCC tumors >3 cm in diameter were markedly higher than those of the group with HCC nodules < or =3 cm in diameter and the group with the benign hyperplastic nodules (P<0.01 except A:B of S period, P<0.05) . The ratios of the middle group were also apparently higher than those of the latter group (P<0.01) . The ratio of DNA AN of 46 cases with HCC nodules was 34.8 % (16/46) . None of the cases with the intrahepatic hyperplastic nodules appeared AN . The DNA AN appeared more apparently with the growth of the tumors . The AN ratio of the group with tumors >3 cm in diameter was 55 % (11/20), markedly higher than that of the group with tumors < or =3 cm in diameter which was 19.2 % (5/26) (P<0.01) . The FCM DNA analysis of 22 specimens of hepatic carcinoma tissue before therapy showed that the aneuploid peaks appeared in 5 cases (22.7 %) . The ratio of G(1) period rose after therapy while the S period and G(2)/M ratios fell (P<0.01) . The aneuploid peak disappeared in the 5 cases after the therapy, while the apoptosis peaks in 12 cases (54.5 %) appeared . CONCLUSION: Addition to supply the information of the pathological morphology of the tumor, the ultrasound-guided fine-needle aspiration tissue could be sent for FCM DNA analysis to comprehend its pathological biological characteristics . This can not only provide the clinic the reliable information about the occurrence, development, diagnosis, curative effect and prognosis of tumors but also supply biological information for clinic to choose therapeutic schemes.

Anesthesiology, 2003 May, 98(5), 1139 - 46
Local anesthetics modulate neuronal calcium signaling through multiple sites of action; Xu F et al.; BACKGROUND: Local anesthetics (LAs) are known to inhibit voltage-dependent Na+ channels, as well as K+ and Ca2+ channels, but with lower potency . Since cellular excitability and responsiveness are largely determined by intracellular Ca2+ availability, sites along the Ca2+ signaling pathways may be targets of LAs . This study was aimed to investigate the LA effects on depolarization and receptor-mediated intracellular Ca2+ changes and to examine the role of Na+ and K+ channels in such functional responses . METHODS: Effects of bupivacaine, ropivacaine, mepivacaine, and lidocaine (0.1-2.3 mm) on evoked {Ca2+}(i) transients were investigated in neuronal SH-SY5Y cell suspensions using Fura-2 as the intracellular Ca2+ indicator . Potassium chloride (KCl, 100 mm) and carbachol (1 mm) were individually or sequentially applied to evoke increases in intracellular Ca2+ . Coapplication of LA and Na+/K+ channel blockers was used to evaluate the role of Na+ and K+ channels in the LA effect on the evoked {Ca2+}(i) transients . RESULTS: All four LAs concentration-dependently inhibited both KCl- and carbachol-evoked {Ca2+}(i) transients with the potency order bupivacaine > ropivacaine > lidocaine >/= mepivacaine . The carbachol-evoked {Ca2+}(i) transients were more sensitive to LAs without than with a KCl prestimulation, whereas the LA-effect on the KCl-evoked {Ca2+}(i) transients was not uniformly affected by a carbachol prestimulation . Na+ channel blockade did not alter the evoked {Ca2+}(i) transients with or without a LA . In the absence of LA, K+ channel blockade increased the KCl-, but decreased the carbachol-evoked {Ca2+}(i) transients . A coapplication of LA and K+ channel blocker resulted in larger inhibition of both KCl- and carbachol-evoked {Ca2+}(i) transients than by LA alone . CONCLUSIONS: Different and overlapping sites of action of LAs are involved in inhibiting the KCl- and carbachol-evoked {Ca2+}(i) transients, including voltage-dependent Ca2+ channels, a site associated with the caffeine-sensitive Ca2+ store and a possible site associated with the IP(3)-sensitive Ca2+ store, and a site in the muscarinic pathway . K+ channels, but not Na+ channels, seem to modulate the evoked {Ca2+}(i) transients, as well as the LA-effects on such responses.

Br J Haematol, 2003 May, 121(3), 439 - 47
Aurora2/BTAK/STK15 is involved in cell cycle checkpoint and cell survival of aggressive non-Hodgkin's lymphoma; Hamada M et al.; Non-Hodgkin's lymphoma (NHL) has a wide biological heterogeneity and shows extremely variable responses to therapeutic measures . However, markers that indicate disease activity and determine treatment strategies for this malignancy are little recognized . Using the differential display method, we have identified Aurora2/BTAK/STK15, a centrosome-associated serine/threonine kinase, whose overexpression leads to centrosome amplification, chromosomal instability and transformation of mammalian solid tumours . Northern analysis with mRNA from a single tumour cell suspension of NHL confirmed that Aurora2/BTAK/STK15 was highly expressed in histologically aggressive types . To elucidate the function of Aurora2/BTAK/STK15 in NHL, Aurora2/BTAK/STK15 sense or antisense genes were transfected to B-cell lymphoma cell lines to generate overexpressed or under-regulated tumour cells . Aurora2/BTAK/STK15 antisense transfectant was barely established compared with a sense or vector-only transfectant . Two clones were finally established that exhibited a low proliferation rate and significantly increased G1 arrest compared with vector-only transfectants . Moreover, antisense oligo treatment in vitro showed that restriction of cell growth appeared in proportion to antisense oligo concentration . These results suggest that Aurora2/BTAK/STK15 is an effective candidate to indicate not only disease activity but also tumorigenesis of non-Hodgkin's lymphoma . Retardation of tumour cell growth resulting from the restriction of this gene's functions may be a novel therapeutic approach for non-Hodgkin's lymphoma.

Biol Chem, 2003 Mar, 384(3), 437 - 46
The role of octadecanoids and functional mimics in soybean defense responses; Fliegmann J et al.; Oxylipins of the jasmonate pathway and synthetic functional analogs have been analyzed for their elicitor-like activities in an assay based on the induced accumulation of glyceollins, the phytoalexins of soybean (Glycine max L.), in cell suspension cultures of this plant . Jasmonic acid (JA) and its methyl ester showed weak phytoalexin-inducing activity when compared to an early jasmonate biosynthetic precursor, 12-oxo-phytodienoic acid (OPDA), as well as to the bacterial phytotoxin coronatine and certain 6-substituted indanoyl-L-isoleucine methyl esters, which all were highly active . Interestingly, different octadecanoids and indanoyl conjugates induced the accumulation of transcripts of various defense-related genes to different degrees, indicating distinct induction competencies . Therefore, these signaling compounds and mimics were further analyzed for their effects on signal transduction elements, such as the transient enhancement of the cytosolic Ca2+ concentration and MAP kinase activation, which are known to be initiated by a soybean pathogen-derived beta-glucan elicitor . In contrast to the beta-glucan elicitor, none of the other compounds tested triggered these early signaling elements . Moreover, endogenous levels of OPDA and JA in soybean cells were shown to be unaffected after treatment with beta-glucans . Thus, OPDA and JA, which are functionally mimicked by coronatine and a variety of 6-substituted derivatives of indanoyl-L-isoleucine methyl ester, represent highly efficient signaling compounds of a lipid-based pathway not deployed in the beta-glucan elicitor-initiated signal transduction.

An Acad Bras Cienc, 2003 Mar, 75(1), 55 - 69 Epub 2003 Apr 17.
Karyotypic analysis in species of the genus Dasyprocta (Rodentia: Dasyproctidae) found in Brazilian Amazon; Ramos RS et al.; A total of 30 animals of the genus Dasyprocta were cytogenetically studied . They belong to the following species: D . prymnolopha (N=20), D . leporina (N=6), D . fuliginosa (N=1) and Dasyprocta sp . (N=3) (Dasyproctidae, Hystricognathi) . Cell suspensions were obtained by peripheral blood culture, besides bone marrow and spleen cells, from D . prymnolopha and D . leporina . The diploid number was 64/65 for all samples . The karyotypes showed similarity, and chromosomal polymorphism was not detected by Giemsa conventional staining and G banding . The constitutive heterochromatin distribution at the pericentromeric region of all the chromosomes was similar in all species . D . prymnolopha, D . leporina and Dasyprocta sp . presented variation in the heterochromatical block size at one of the homologues of the A18 pair . D . fuliginosa presented the heterochromatin uniformly distributed in all chromosomes . There was not variation in the NORs pattern in the species studied.

Surg Endosc, 2003 Jul, 17(7), 1098 - 104 Epub 2003 Apr 28.
The influence of adhesion prophylactic substances and taurolidine/heparin on local recurrence and intraperitoneal tumor growth after laparoscopic-assisted bowel resection of colon carcinoma in a rat model; Opitz I et al.; BACKGROUND: The goal of the study was to investigate the influence of adhesion prophylactic substances (Interceed/lntergel) as well as taurolidine/heparin on intraperitoneal tumor growth and the local recurrence rate after laparoscopic cecum resection in a rat tumor model . METHODS: Sixty BDIX rats were randomized in three therapy groups and one control group . A laparoscopic-assisted cecum resection was performed via three-trocar method after intraperitoneal tumor cell application (10,000 cells) of a colon carcinoma cell line (DHD/K1/TRb) in all animals . According to the randomization, the cecum suture and a 1 x 1-cm peritoneal defect were either covered with Intergel/Interceed or 1 ml of 0.5% taurolidine 10 IU heparin . The control group underwent instillation of 1 ml 0.9% NaCl solution . After 4 weeks the animals were euthanized and intraperitoneal tumor growth, local recurrence rate, and the number of intraperitoneal adhesions were determined . RESULTS: The local recurrence rate was not significantly affected by any of the substances . Nevertheless, taurolidine/heparin significantly reduced the total number and weight of intraperitoneal metastases . The formation of adhesions was not significantly influenced by adhesion prophylaxis substances or by taurolidine/heparin . CONCLUSIONS: Taurolidine/heparin led to a significant reduction of intraperitoneal tumor growth after intraperitoneal application, whereas local tumor recurrence was not significantly influenced . This might be due to the number of injected tumor cells in this cell suspension model . Interceed and Intergel did not reduce intraperitoneal tumor growth . Furthermore, adhesion formation was not reduced by any of the substances.

Theor Appl Genet, 2003 Aug, 107(3), 406 - 12 Epub 2003 Apr 24.
Field performance of transgenic tall fescue (Festuca arundinacea Schreb.) plants and their progenies; Wang ZY et al.; Tall fescue (Festuca arundinacea Schreb.) is a hexaploid, outcrossing grass species widely used for forage and turf purposes . Transgenic tall fescue plants were generated by biolistic transformation of embryogenic cell suspension cultures that were derived from single genotypes of widely used cultivar Kentucky-31 . Primary transgenics from two genotypes, their corresponding regenerants from the same genotypes and control seed-derived plants were transferred to the field and evaluated for 2 years . Progenies of these three classes of plants were obtained and evaluated together with seed-derived plants in a second field experiment . The agronomic characteristics evaluated were: heading date, anthesis date, height, growth habit, number of reproductive tillers, seed yield and biomass . The agronomic performance of the primary transgenics and regenerants was generally inferior to that of the seed-derived plants, with primary transgenics having fewer tillers and a lower seed yield . However, no major differences between the progenies of transgenics and the progenies of seed-derived plants were found for the agronomic traits evaluated . Primary transgenics and regenerants from the same genotype were more uniform than plants from seeds . Progenies of transgenics performed similarly to progenies of the regenerants . The addition of a selectable marker gene in the plant genome seems to have had little effect on the agronomic performance of the regenerated plants . No indication of weediness of the transgenic tall fescue plants was observed . Our results indicate that outcrossing grass plants generated through transgenic approaches can be incorporated into forage breeding programs.

J Steroid Biochem Mol Biol, 2003 Feb, 84(2-3), 279 - 89
Expression and localization of estrogen receptor alpha, estrogen receptor beta and progesterone receptor in the bovine oviduct in vivo and in vitro; Ulbrich SE et al.; This study examined the regulation and localization of estrogen receptors alpha and beta (ERalpha, ERbeta) and progesterone receptor (PR) in the bovine oviduct . Oviduct epithelial cells from cycling cows (in vivo) were investigated . In addition, the reactivity of a cell suspension culture stimulated with physiological doses of estradiol-17beta (E2) or progesterone (P4) was tested (in vitro) . The specific steroid receptor expression of oviductal cells was quantified for mRNA using real-time RT-PCR . Furthermore, steroid receptor proteins were analyzed by Western blotting and localized by immunohistochemistry in situ . Obvious cyclic changes of receptor expression in vivo were observed and concurrent expression patterns were detected in vitro . PR and ERalpha mRNA transcripts were elevated in vivo during the follicular phase . The highest PR and ERalpha protein expression was detected subsequently during the early-luteal phase . In vitro, E2-supplementation resulted in an upregulation of PR and ERalpha . Both ERbeta mRNA and protein expression were highest during the luteal phase in vivo and elevated ERbeta expression levels were observed in vitro after P4 treatment . Evidence is provided for a varying expression of ERalpha, ERbeta and PR in bovine oviducts at different cycle stages in vivo, respectively under steroid supplementation in vitro . The region specific and cycle dependent expression differences point towards a functional importance of the three steroid receptors in the bovine oviduct, the site of fertilization and early embryonic development.

Radiat Res, 2003 May, 159(5), 612 - 20
Comparison of the radiotoxicity of two alpha-particle-emitting immunoconjugates, terbium-149 and bismuth-213, directed against a tumor-specific, exon 9 deleted (d9) E-cadherin adhesion protein; Miederer M et al.; We investigated the effects of the alpha-particle emitters (149)Tb and (213)Bi coupled to a tumor-specific antibody targeting the mutated delta 9 E-cadherin (d9 E-Cad) on single cells and cell pellets . The d9 mutation of the adhesion molecule E-cadherin is found in 10% of diffuse-type gastric cancers and is not expressed in normal tissue . Human breast cancer cells (MDA-MB-435S) transfected with d9 E-Cad or the wild-type E-cadherin gene were used to study the effects of anti-d9 E-Cad MAb coupled to (149)Tb and (213)Bi ((149)Tb-d9 MAb and (213)Bi-d9 MAb) . The density of binding sites determined on transfected MDA tumor cells by Scatchard analysis and flow cytometry varied from 4 x 10(4) to 6 x 10(4) antigens per cell . Internalization of radioimmunoconjugates by cells expressing d9 E-Cad was less than 10% of bound antibody within 240 min . The effect of the radioimmunoconjugates on cell suspensions and cell pellets was quantified by {(3)H}thymidine incorporation, and the dose to the cell nuclei was determined using microdosimetric calculations . (149)Tb and (213)Bi immunoconjugates affected cells in suspension similarly . Significant differences in the proliferation capacity of d9 E-cadherin- and wild-type E-cadherin-expressing cells were observed at activity concentrations around 185 kBq/ml, corresponding to antibody concentrations between 200 ng/ml and 1000 ng/ml . Proliferation after incubation with (213)Bi-d9 MAb was 50% greater in pelleted wild-type E-Cad-expressing cells compared to wild-type E-Cad cells in suspension . In contrast, the proliferation of pelleted d9 E-Cad cells was similar to that of d9 E-Cad cells in suspension . For (149)Tb-d9 MAb, no significant difference was found between pelleted cells and cells in suspension for low activity concentrations . However, at high activity concentrations, (149)Tb-d9 MAb had only a small effect on pelleted cells . These in vitro studies demonstrate different effects of (149)Tb and (213)Bi conjugated to a tumor-specific antibody toward single cells and tumor cell pellets . Microdosimetric simulation of single cell survival after alpha-particle irradiation modeled the experimental results with reasonable accuracy.

Int J Dermatol, 2003 Feb, 42(2), 132 - 6
Melanocyte-keratinocyte cell transplantation for stable vitiligo; Mulekar SV; BACKGROUND: Vitiligo is a common disorder with a worldwide prevalence of 1-2% . In India the psychological and social impact of the disease is significant and is detrimental to patients . OBJECTIVE: To evaluate the usefulness of epidermal cell transplantation in the treatment of vitiligo . METHODS: A simpler and modified method based on that of Olsson and Juhlin has been used . It utilizes a shave biopsy skin sample of up to one-tenth the size of the recipient area . The skin sample is incubated, the cells mechanically separated using trypsin EDTA solution, and then centrifuged to prepare a suspension . The cell suspension is then applied to the derm-abraded depigmented skin area and collagen dressing is applied to keep it in place . RESULTS: One hundred and twenty-two patients with generalized vitiligo, 43 with segmental and 19 with focal vitiligo were treated and observed for a period of 1 year . In the generalized vitiligo group 65 (53%) showed excellent pigmentation, 10 (8%) showed good pigmentation, 11 (9%) showed fair pigmentation and 28 (23%) patients showed poor pigmentation . Eight (7%) patients did not follow up . Thirty-six (84%), five (12%) and two (4%) patients showed excellent, good and poor pigmentation, respectively, in the segmental vitiligo group . Thirteen (69%) and five (26%) patients showed excellent and poor results, respectively, in the focal vitiligo group . One (5%) patient did not appear for follow up . Recurrence was observed in 15 patents . CONCLUSION: This surgical treatment gives its best results in segmental and focal vitiligo, even with large affected areas, and in at least 50% of patients with generalized vitiligo, thus improving their appearance.

Cancer Sci, 2003 Jan, 94(1), 125 - 33
Usefulness of combined treatment with mild temperature hyperthermia and/or tirapazamine in the treatment of solid tumors: its independence of p53 status; Masunaga S et al.; Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both hind legs of Balb/cA nude mice . Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors . The mice then received tirapazamine (TPZ) with or without mild temperature hyperthermia (40 degrees C, 60 min) (MTH), gamma-ray irradiation with or without MTH and/or TPZ, cisplatin (CDDP) with or without MTH and/or TPZ, or paclitaxel (TXL) with or without MTH and/or TPZ . After each treatment, the tumors were excised, minced and trypsinized . The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling (i.e., quiescent (Q) cells) was determined by using immunofluorescence staining for BrdU . Meanwhile, 6 h after gamma-ray irradiation or 24 h after other cytotoxic treatments, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells . The MN frequency and apoptosis frequency in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU . On the whole, gamma-ray irradiation and CDDP injection induced a higher frequency of apoptosis and lower frequency of MN in SAS/neo cells than SAS/mp53 cells . There were no apparent differences in the induced frequency of apoptosis and MN between SAS/neo and SAS/mp53 cells after TPZ or TXL treatment . MTH sensitized cells to TPZ-inducing cytotoxicity more markedly in SAS/mp53 and Q cells than in SAS/neo cells and total cells, respectively . In gamma-ray irradiation and CDDP treatment, the enhancement in combination with MTH and/or TPZ was more remarkable in SAS/mp53 cells and Q cells than in SAS/neo and total tumor cells, respectively . Also in the case of TXL treatment, the combination with MTH and/or TPZ induced a slightly greater enhancement effect in SAS/mp53 cells and Q cells . In view of the difficulty in controlling mutated p53 status tumors and intratumor Q cells, combination treatment with MTH and/or TPZ as a cooperative modality in cancer therapy is considered to have potential for controlling solid tumors as a whole.

Cancer, 2003 Apr 25, 99(2), 118 - 27
Fine-needle aspiration biopsy and flow cytometry immunophenotyping of lymphoid and myeloproliferative disorders of the spleen; Zeppa P et al.; BACKGROUND: Flow cytometry (FC) is a useful adjunct to fine-needle aspiration biopsy (FNAB) in the evaluation of lymphoproliferative disorders . The application of FC to FNAB of the spleen (sFNAB) is reported . METHODS: Flow cytometry was performed on 18 sFNAB collected over 3 years . The series comprised 10 cases of non- Hodgkin lymphomas (NHL), 2 cases insufficient for diagnosis, 2 cases of reactive hyperplasia (RH), and 4 cases of myeloid metaplasia (MM) . FNAB was performed under ultrasound guidance using a 22-gauge needle . One or two passes were sufficient to prepare a conventional smear that was immediately evaluated to select the cases studied and to prepare a cell suspension for FC . The following fluoresceinated antibodies were used: CD3, CD19/kappa/lambda, FMC7/CD23/CD19, Bcl-2, and CD13/HLA-DR . In six cases, cytospins were also prepared for immunocytochemistry and were tested for CD20 (L26), CD45Ro, and kappa and lambda light chain expression . RESULTS: Flow cytometry contributed to the diagnosis of all cases of NHL by assessing light chain restriction . The specific subtype was also diagnosed by CD19/CD5 and CD 19/CD10 coexpression in two cases . Flow cytometry quantified the percentage of myeloid cells in MM cases and contributed to the cytologic diagnosis showing a polyclonal light chain expression in RH cases . Immunocytochemistry was effective and concordant in four cases . Patients tolerated the sFNAB well and no complications were reported . Cytologic and FC diagnoses were confirmed by follow-up and by histologic evaluation in cases in which splenectomy was performed for therapeutic purposes . CONCLUSION: Flow cytometry applied to sFNAB corroborates the cytologic diagnosis in lymphoid and myeloproliferative disorders of the spleen and allows therapeutic decisions avoiding splenectomy .

J Allergy Clin Immunol, 2003 Apr, 111(4), 869 - 74
Atopy patch test reactions show a rapid influx of inflammatory dendritic epidermal cells in patients with extrinsic atopic dermatitis and patients with intrinsic atopic dermatitis; Kerschenlohr K et al.; BACKGROUND: Normal human skin harbors a single epidermal dendritic cell (DC) population, the CD1a(+++)CD11b(-) Langerhans cells . In many chronic inflammatory skin diseases, the epidermal DC pool bears a second population, the CD1a(+)CD11b(+++) inflammatory dendritic epidermal cells (IDECs) . Immunophenotypic, ultrastructural, and functional aspects of IDECs have been investigated in chronic untreated skin lesions of intrinsic and extrinsic atopic dermatitis (AD), contact dermatitis (CD), and psoriasis, but little is known about freshly induced early skin lesions . OBJECTIVE: We sought to characterize enumerative and immunophenotypic changes in the epidermal DC pool during the development of eczematous skin lesions . METHODS: The atopy patch test with aeroallergens and food-protein allergens and a conventional patch test with standard-series haptens were performed as models for early skin lesions of extrinsic and intrinsic AD and CD, respectively . After 72 hours, epidermal cell suspensions were prepared, analyzed in a standardized flow cytometric technique, and compared with the results obtained from chronic lesions . RESULTS: The migration of IDECs into the epidermis occurs within 72 hours and is thus an early event . It continues in chronic AD, but not in chronic CD, lesions . The specific upregulation of FcepsilonRI, especially on IDECs, occurs later during formation of extrinsic but not intrinsic AD lesions . LCs were negative for Cd36 in patch test lesions, whereas in chronic skin lesions, LCs expressed Cd36 . CONCLUSION: The DC alteration during skin lesion formation can be subdivided into early and late events, with the influx of IDECs as an early event and the alteration of the DC phenotype as a late event.

Int J Artif Organs, 2003 Mar, 26(3), 235 - 40
Carboxyfluorescein diacetate succinimidyl ester facilitates cell tracing and colocalization studies in bioartificial organ engineering; Mueller-Stahl K et al.; BACKGROUND: We demonstrate a method that includes colocalization studies to analyze cell suspensions after isolation and to characterize 3-dimensional grafts consisting of cells and matrix in vitro and in vivo . MATERIALS AND METHODS: Neonatal rat cardiomyocytes were labelled by CFDA-SE after harvest . Cells in the isolated cell suspension, the embodied cells in the seeded scaffolds were characterized measuring features such as viability and distribution of the cell types . RESULTS: Selective cell count revealed high yields of viable cardiomyocytes . After seeding cells in collagen matrix, viability of the cells decreased gradually in the time process in vitro . Histology of implanted bioartificial myocardial tissue detected viable cardiomyocytes within the graft . CONCLUSION: Using colocalization histology we could label and track cells within the bioartificial myocardial tissue graft in vitro and post implant and assess viability and distribution.

Exp Dermatol, 2003 Apr, 12(2), 172 - 80
Ultraviolet-B irradiation decreases IFN-gamma and increases IL-4 expression in psoriatic lesional skin in situ and in cultured dermal T cells derived from these lesions; Piskin G et al.; Type 1 cytokine producing T cells play an important role in the pathogenesis of psoriasis . Ultraviolet-B (UVB) irradiation is effective in the treatment of this disease . In normal skin, UVB causes a change in dermal microenvironment, leading to a decrease of IFN-gamma expressing type 1 T cells and a concurrent increase of IL-4 expressing type 2 T cells . The aim of this study was to show whether UVB irradiation causes a like-wise shift of type 1 and type 2 responses in psoriatic skin . For this purpose, biopsies were obtained from the lesional skin of psoriatic patients before, 2 days and 14 days after a single exposure to 4 MED UVB . Sections from these biopsies were immunostained (CD3, IFN-gamma and IL-4) or RNA was extracted and analyzed for the expressions of IFN-gamma and IL-4 by PCR . In addition, primary cultures of T cells from dermal cell suspensions were stained intracellularly for IFN-gamma and IL-4 expression and CD4+ and CD8+ T subsets were analyzed by flow cytometry . IFN-gamma was abundantly expressed in situ before irradiation and decreased in all patients after UVB irradiation, whereas IL-4 expression was variably expressed before irradiation and increased in different degrees after irradiation . Cytokine mRNA expressions determined by PCR showed a clear decrease of IFN-gamma and increase of IL-4 following UVB irradiation . Both CD4+ and CD8+ dermal T cells were found to produce less IFN-gamma and more IL-4 following UVB irradiation as determined by flow cytometry . Decrease in IFN-gamma expression and increase in IL-4 expression of dermal T cells in psoriatic lesions after UVB irradiation may lead to decrease in local immunoreactivity . These changes could be part of the therapeutic effects of UVB on psoriasis.

Biotechnol Bioeng, 2003 Jun 30, 82(7), 778 - 83
High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture; Shin YJ et al.; Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures . However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation . To overcome this problem, we sought an expression system in which heterologous gene expression could be induced at high levels . We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation . This induction system was found to give good yield of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system .

Cytometry A, 2003 May, 53(1), 55 - 65
High-throughput flow cytometry: validation in microvolume bioassays; Ramirez S et al.; BACKGROUND: We recently reported an automated sample handling system, designated HyperCyt, by which samples are aspirated from microplate wells and delivered to the flow cytometer for analysis at rates approaching 100 samples per minute . In this approach, an autosampler and peristaltic pump introduce samples into a tubing line that directly connects to the flow cytometer . Air bubbles are inserted between samples to prevent sample dispersion . In the present work, we compare results of HyperCyt with those of conventional manual flow cytometric analysis in representative flow cytometric bioassays and describe a cell suspension method in which HyperCyt exploits the use of microvolume wells . METHODS: Human eosinophils and neutrophils were treated with trypsin to generate a wide (>25-fold) range of membrane P-selectin glycoprotein ligand-1 (PSGL-1) expression and then stained with fluorescent anti-PSGL-1 antibodies . Human peripheral blood mononuclear cells were stained with fluorescein isothiocyanate- and phycoerythrin-conjugated monoclonal antibodies for multiparameter immunophenotype analysis . U937 cells labeled with PKH62GL were used to assess cell settling in microplate wells . RESULTS: Differences in PSGL-1 expression levels were detected by HyperCyt autosampling of leukocytes from 96-well plates at an analysis rate of approximately 1.5 s/well . HyperCyt measurements linearly correlated with parallel manual measurements (r(2) = 0.98) . Lymphocyte subpopulations were accurately distinguished and reproducibly quantified in multiparameter immunophenotyping assays performed over a range of HyperCyt analysis rates (1.4-5.5 s/sample) . When assay volumes were reduced to 10 microl/well in 60-well Terasaki plates, cells could be maintained in uniform suspension for up to 30 min by periodically inverting plates on a rotating carousel before HyperCyt analysis . HyperCyt analysis of five fluorescence-level Cyto-Plex beads sampled from Terasaki plate microwells at 2.5 s/well produced highly reproducible results over a wide range of input bead concentrations (from 7 x 10(5) to 20 x 10(6) beads/ml) that linearly correlated with manual analysis results . CONCLUSIONS: The HyperCyt autosampling system enabled a 10-fold or greater increase in sample throughput compared with conventional manual flow cytometric sample analysis, with comparable analysis results . Assays were performed efficiently in 10-microl volumes to enable significant reagent cost savings, use of quantity-limited reagents at otherwise prohibitive concentrations, and maintenance of uniform suspensions of cells for prolonged periods .

Front Biosci, 2003 May 01, 8, s522 - 32
Strategies for the augmentation of grafted dopamine neuron survival; Sortwell CE; The percentage of grafted embryonic DA neurons that survive transplantation is low, estimated at 5-20% . Significant agreement has emerged from the work of research groups worldwide that specific conditions associated with the transplant procedure and post-transplantation interval render grafted mesencephalic cells susceptible to apoptotic death . Detrimental triggers including hypoxia/ischemia, trophic factor withdrawal, and oxidative stress appear to exert the most impact on grafted DA neuron survival . Treatment strategies that aim to reduce or eliminate the triggers of grafted cell death appear to be more successful than approaches that target the intracellular apoptotic cascade . In particular, treatment of mesencephalic cell suspensions with isolated neurotrophic factors (GDNF, BDNF, NT 4/5) as well as glial-derived factors, antioxidant therapies and augmentation of graft vasculature have demonstrated consistent survival promoting effects . Caspase inhibition, although initially quite promising, has not been demonstrated to reliably increase grafted cell survival . Bcl-2 overexpression similarly has yet to prove beneficial, although this may be due to biologically irrelevant levels of bcl-2 present during the critical immediate post-grafting interval . Future strategies will target a "cocktail" approach in which effective treatment agents are combined to maximize grafted DA neuron survival . Refinements in ex vivo transduction parameters will allow for efficient sustained delivery of survival promoting agents to grafted cells . Once identified, the optimal survival-enhancing treatment of grafted primary embryonic DA neurons should also benefit future transplant therapies utilizing alternatively derived DA neurons.

Mikrobiologiia, 2003 Jan-Feb, 72(1), 14 - 8
{Bacterial degradation of EDTA}; Satrutdinov AD et al.; Degradation of EDTA (ethylenediaminetetraacetic acid) or metal-EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied . The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium . Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg2+, Ca2+, Mn2+, Pb2+, Co2+, Cd2+, Zn2+, Cu2+, or Fe3+ . The metal-EDTA complexes (Me-EDTA) studied could be divided into three groups according to their degradability . EDTA complexes with stability constants K below 10(16) (lg K < 16), such as Mg-EDTA, Ca-EDTA, and Mn-EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5-10 h of incubation . Me-EDTA complexes with lg K above 16 (Zn-EDTA, Co-EDTA, Pb-EDTA, and Cu-EDTA) were not completely degraded during a 24-hour incubation, which was possibly due to the toxic effect of the metal ions released . No degradation of Cd-EDTA or Fe(III)-EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.

J Surg Res, 2003 Mar, 110(1), 255 - 65
Divalent cations modulate human colon cancer cell adhesion; Thamilselvan V et al.; BACKGROUND: Iatrogenic tumor implantation within surgical sites can compromise curative cancer surgery . Cancer cell adhesion to extracellular matrix proteins is mediated by diverse matrix receptors, most notably integrins . Divalent cations may modulate integrin-ligand interactions in some cells . MATERIALS AND METHODS: We studied adhesion of SW620 and Caco-2 human colon cancer cells to collagen I, the dominant collagen of the interstitial matrix, and confirmed our results in primary human colon cancer cells from surgical specimens . Single cell suspensions in either HEPES/NaCl buffer or media supplemented with 0-1 mM Mn2+ or Mg2+, and 0-10 mM Zn2+ or Ca2+ were plated onto collagen-I-precoated dishes for 30 min . RESULTS: Supplementation of the HEPES/NaCl/BSA buffer with 1 mM Mn2+, Mg2+, Zn2+, or Ca2+ affected adhesion differently . Mn2+ (1 mM) markedly promoted SW620 adhesion vs control (21.17 +/- 0.08-fold) . Mg2+ (1 mM) had a similar but lesser effect (14.71 +/- 0.02-fold) . However, 1-10 mM Ca2+ inhibited basal cell adhesion by 22.0 +/- 3.1 to 88.0 +/- 7.3 % inhibition . Ca2+ (2.5-10 mM) also inhibited Mn2+-induced adhesion . Zn2+ stimulated basal adhesion slightly at lower concentrations but inhibited Mn2+-stimulated adhesion similarly to Ca2+ at higher concentrations . Results were duplicated in conventional serum containing culture medium supplemented with these cations . Caco-2 cells and primary cancer cells yielded similar results . All results are significant to P < 0.01 . DISCUSSION: Integrin-mediated colon cancer cell adhesion is affected by extracellular divalent cation concentrations . Washing the surgical site with dilute calcium or zinc solutions might diminish perioperative tumor implantation.

Clin Chim Acta, 2003 May, 331(1-2), 103 - 10
Flow cytometry study of polymorphonuclear neutrophil oxidative burst: a comparison of three fluorescent probes; Walrand S et al.; BACKGROUND: The use of 2',7'-dichlorofluorescein diacetate (DCFH), dihydrorhodamine 123 (DHR) and hydroethidine (HE) has been described for detecting respiratory burst activity by flow cytometry in polymorphonuclear neutrophil (PMN) suspension . However, their specificities for reactive oxygen species are not well defined . We investigated the reactivity of these probes for detecting superoxide anion (O(2)(* -)), hydrogen peroxide (H(2)O(2)) and/or nitric oxide (NO(z.rad;))-dependent mechanisms . METHODS: PMNs (10(6)/ml) were preincubated for 15 min at 37 degrees C with DCFH (5 micro mol/l), DHR (1 micro mol/l) or HE (10 micro mol/l) . Cell suspensions were then split for each probe into five different aliquots containing either no effector or one effector: N-ethylmaleimide (NEM, 150 micro mol/l, NADPH oxidase inhibitor), sodium azide (NaN(3), 50 micro mol/l, peroxidase and catalase inhibitor), N-nitro-L-arginine methyl ester (L-NAME, 1.5 micro mol/l, NO(z.rad;) synthase inhibitor) or H(2)O(2) (30%) . At the same time, PMNs were stimulated with phorbol myristate acetate (PMA, 10 micro mol/l) for 10 min at 37 degrees C . Analyses were carried out on a Beckman-Coulter Epics XL equipped with an argon laser (488 nm) . Green fluorescences from DCFH and DHR were measured in the FL1 channel and HE fluorescence was analyzed in the FL2 channel . RESULTS: NaN(3) decreased the fluorescence of PMNs incubated with DCFH, indicating that it needs a peroxidase activity to react with H(2)O(2) . L-NAME reduced the oxidation of DCFH, showing that it reacts with reactive nitrogen species . DHR was specifically responsive to H(2)O(2) accumulation . HE seemed to be preferentially oxidized by O(2)(* -) . CONCLUSIONS: Hence the choice of the probe to be used depends on the reactive species of interest.

Receptors Channels, 2002, 8(5-6), 319 - 30
Aequorin-based functional assays for G-protein-coupled receptors, ion channels, and tyrosine kinase receptors; Dupriez VJ et al.; Aequorin is a photoprotein originating from jellyfish, whose luminescent activity is dependent on the concentration of calcium ions . Due to the high sensitivity and low background linked to luminescent assays, as well as to its absence of toxicity and its large linear dynamic range, aequorin has been used as an intracellular calcium indicator since its discovery in the early 1960s . The first applications of aequorin involved its microinjection in cells . The cloning of its gene in 1985 opened the way to the stable expression of aequorin in cell lines or even entire organisms . Here we present the validation of aequorin as a functional assay for the screening of G-protein-coupled receptors, ion channels, and tyrosine kinase receptors, as well as for their pharmacological characterization in agonist and antagonist detection assays . We optimized our cell suspension-based assay and determined that the most sensitive assay was performed at room temperature, with mitochondrially expressed aequorin and using coelenterazine derivative h for reconstitution of aequorin . The robustness of the assay and the current availability of luminometers with integrated injectors allow aequorin to fit perfectly with high throughput functional assays requirements.

Naunyn Schmiedebergs Arch Pharmacol, 2003 Apr, 367(4), 353 - 63 Epub 2003 Mar 06.
Store operated Ca2+ influx by selective depletion of ryanodine sensitive Ca2+ pools in primary human skeletal muscle cells; Weigl L et al.; The contraction and relaxation of skeletal muscle is driven by release of Ca2+ from sarcoplasmic reticulum through the ryanodine receptor type 1 and extruding the ion from the cytosol by Ca2+ ATPases . Efficient refilling of the empty Ca2+ stores is essential for repetitive cycles of muscle contraction and relaxation, but not investigated in human skeletal muscle cells . Here we show that under conditions of selective depletion of the ryanodine-sensitive Ca2+ pool Ca2+ influx occurs in differentiated human skeletal muscle cells using the Ca2+ imaging technique . This Ca2+ influx is not due to permeation through the L-type Ca2+ channel and not observed under conditions of inhibited Ca2+ ATPase . The Ca2+ influx was visualised by quenching the intracellular fura2 signal with Mn2+ on single cell level and also using fluorescence photometry of cell suspensions . The Mn2+ influx was inhibited by the Ca2+ channel blockers La(3+) and SKF96356 . The delineation of the signalling cascade leading to Ca2+ influx evoked by selective depletion of ryanodine sensitive Ca2+ stores showed that phospholipase C or protein kinase C were not involved . Interestingly, a Mn2+ influx was triggered by the cell-permeant analogue of diacylglycerol and further augmented by the application of RHC80267, a diacylglycerol lipase inhibitor . This signalling pathway could be attributed to the participation of a protein kinase C activity . However, Mn2+ influx evoked by selective depletion of ryanodine sensitive Ca2+ stores was not altered by RHC80267 or protein kinase C inhibitors . Using RT-PCR, correctly spliced mRNA fragments were detected corresponding to human transient receptor potential (TRPC) Ca2+ channels type 1, 3, 4 and 6 . These data show that in skeletal muscle at least two independent mechanisms of Ca2+ influx exist . For Ca2+ influx triggered by the selective depletion of ryanodine sensitive Ca2+ stores we propose a phospholipase C independent coupling of ryanodine receptors to voltage insensitive Ca2+ channels.

Cryobiology, 2003 Apr, 46(2), 135 - 45
Vitrification of ECV304 cell suspensions using solutions containing propane-1,2-diol and trehalose; Wusteman MC et al.; In this paper, we report on the suitability of solutions containing propane-1,2-diol (propylene glycol, PD), sugars, and salts for the vitrification of the human cell line, ECV304 . Cooling (at 10 degrees C/min) and rewarming (at 80 degrees C/min) were at rates that are practicable for the tissues to be studied later . Under these conditions, 45% PD in phosphate-buffered saline (PBS) sometimes froze during cooling and always devitrified during rewarming but both events were avoided if the PBS salts were replaced by an osmotically equivalent concentration of sucrose or trehalose . The effect of such solutions on cells was evaluated using a cell culture assay in which the number of cells recovered after 3 days of culture was divided by the number cells plated, giving a cell multiplication factor or CMF . In the absence of PD the cells tolerated a low-salt concentration in solutions that were made isotonic with sugars, but they recovered poorly when 45% PD was also present . Trehalose gave significantly better recovery than sucrose . When 39% PD and 15% trehalose were included in a low-salt vehicle solution (LSV) that contained approximately 5% of the total salt concentration of PBS (this solution was designated LSV/39/15), the cells exhibited approximately 40% of untreated control CMF following exposure for 9min . LSV/39/15 vitrifies with a glass transition temperature of -102 degrees C, does not devitrify when warmed at 80 degrees C/min, and has suitable dielectric properties for uniform and rapid dielectric heating . An improved method for adding and removing LSV/39/15 gave a CMF of approximately 55% of untreated controls . Using this method, 1.0ml suspensions of ECV304 cells was cooled to, and stored briefly at, -120 degrees C and then rewarmed by immersion in a 37 degrees C water bath ( approximately 75 degrees C/min) . The CMF of the cooled samples was similar to that of the exposure-only controls, approximately 50% of the untreated control CMF in both cases.

Microvasc Res, 2003 Mar, 65(2), 132 - 6
Prototype of an in vitro model of the microcirculation; Shevkoplyas SS et al.; We have used microfabrication technology to construct a network of microchannels, patterned after the dimensions and architecture of the mammalian microcirculation . The network is cast in transparent silicone elastomer and the channels are coated with silanated mPEG to provide lubrication . Flow of red and white blood cells through the network is readily visualized by the use of high-speed digital image acquisition . The acquired sequences of high-quality images are used to calculate hematocrits and rates of red cell movement in the microchannels . Our prototype system has significant advantages over scaled-up room-size experimental systems in that it permits experimentation with actual human blood cells . Experiments can be carried out under well-controlled conditions in a network of microchannels with precisely known dimensions using cell suspensions of defined composition . Moreover, there is no need to counteract or anticipate the host's adaptive responses that may confound live animal experiments . Notwithstanding its limitations, the current prototype demonstrates certain features characteristic of the microcirculation, such as parachute and bullet shapes of red cells deformed in capillary channels, rouleaux formation, plasma skimming, and the utilization of collateral flow pathways due to flow obstruction caused by a white cell blocking a microchannel . We present this device as a prototype scale-to-scale model of the mammalian microcirculation . Limitations of the system as well as a variety of possible applications are described.

Photochem Photobiol, 2003 Mar, 77(3), 259 - 64
CD11b+ cells are the major source of oxidative stress in UV radiation-irradiated skin: possible role in photoaging and photocarcinogenesis; Mittal A et al.; Exposure of skin to solar UV radiation induces oxidative stress and suppression of cell-mediated immune responses . These effects are associated with the greater risk of several skin disorders including photoaging and photocarcinogenesis . We have shown that UV-induced infiltrating leukocytes contribute in developing oxidative stress in UV-irradiated skin . The peak period of UV-induced infiltrating leukocytes lies between 48 and 72 h after UV exposure of the skin . In this study we demonstrated that UV (90 mJ/cm2)-induced infiltrating CD11b+ cells in C3H/HeN mice skin were the major source of oxidative stress . Hydrogen peroxide (H2O2) was determined as a marker of oxidative stress . Flow cytometric analysis of viable cells revealed that the number of CD11b+H2O2+ cells were significantly higher (31.8%, P < 0.001) in UV-irradiated skin in comparison with non-UV-exposed skin (0.4%) . Intraperitoneal administration of monoclonal antibodies to CD11b (rat IgG2b) to C3H/HeN mice inhibited UVB-induced infiltration of leukocytes, as evidenced by reduction in myeloperoxidase activity (64-80%, P < 0.0005), concomitant with significant reduction in H2O2 production both in epidermis and dermis (66-83%, P < 0.001-0.0005) when compared with the administration of rat IgG2b isotype of anti-CD11b . Furthermore, CD11b+ and CD11b- cell subsets were separated by immunomagnetic cell isolation technique from total epidermal and dermal single cell suspensions obtained 48 h after UV irradiation of the skin and analyzed for H2O2 production . Analytical data revealed that CD11b+ cell population from UV-irradiated skin resulted in significantly higher production of total H2O2 in both epidermis and dermis (87-89%, P < 0.0001) in comparison with CD11b- cell population (11-13% of total H2O2) . These data revealed that infiltrating CD11b+ cells were the major source of oxidative stress in UV-irradiated skin and thus may contribute to photoaging and promotion of skin tumor growth within the UV-irradiated skin . Together, these data suggest that reduction in UV-induced skin infiltration of CD11b+ cells may be an alternative and effective strategy to reduce solar UV light-induced oxidative stress-mediated skin disorders including photoaging and photocarcinogenesis.

Pflugers Arch, 2003 Jul, 446(4), 470 - 4 Epub 2003 Apr 09.
Evidence that nitric oxide does not directly contribute to methacholine-induced amylase secretion in rabbit parotid acinar cells; Tsunoda S et al.; Nitric oxide (NO) is a short-lived free radical and is a widespread intra- and intercellular messenger molecule involved in various physiological functions . We have demonstrated previously that the muscarinic agonist methacholine induces endogenous generation of NO in rabbit parotid acinar cells . Since methacholine also simultaneously evokes amylase secretion, we investigated the effect of NO on the methacholine-induced exocytotic amylase secretion in rabbit parotid acinar cells . Methacholine-evoked amylase secretion was clearly reduced in the absence of extracellular Ca(2+) . The Ca(2+)-mobilizing reagents A23187 and thapsigargin, which stimulate NO generation, also evoked amylase secretion . This response seemed to be caused by NO generated by the activation of endogenous Ca(2+)-regulated NO synthase . However, N(G)-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor, and the NO scavenger haemoglobin had no effect on methacholine-induced amylase secretion . The NO generator sodium nitroprusside (SNP) failed to evoke amylase release . We further studied the effects of L-NAME and SNP on methacholine-induced amylase secretion in crudely dispersed parotid gland cell clusters containing nerve tissue . In this preparation, L-NAME inhibited methacholine-induced amylase secretion and SNP evoked amylase secretion . It is thus unlikely that NO contributes directly to methacholine-induced amylase secretion in rabbit parotid acinar cells . NO appears rather to affect to nerve tissues in the cell suspension.

Planta, 2003 Jun, 217(2), 327 - 39 Epub 2003 Apr 09.
Pre-formed xyloglucans and xylans increase in molecular weight in three distinct compartments of a maize cell-suspension culture; Kerr EM et al.; Cultured cells of maize ( Zea mays L.) were pulse-labelled with l-{1-(3)H}arabinose (Ara) and then monitored for 7 days . The (3)H-hemicelluloses present in three compartments (protoplasm, cell wall and culture medium) were size-fractionated and the fractions assayed for {(3)H}xyloglucans and {(3)H}xylans . Protoplasmic {(3)H}xylans and {(3)H}xyloglucans initially (15 min after {(3)H}Ara-feeding) had weight-average relative molecular masses ( M(w)) approximately 0.5x10(6) and 0.3x10(6), respectively, both rising to 2x10(6) by 30 min . Thus, newly formed hemicellulose molecules were joined to other polymers, or to each other, presumably within Golgi vesicles . New (3)H-hemicelluloses very rapidly bound to the cell wall; however, after 1 day, some {(3)H}xyloglucan and {(3)H}xylan was sloughed from the wall into the medium . The wall-bound {(3)H}xyloglucans were present in the form of extremely large complexes, of M(w)>17x10(6), even as early as 15 min after {(3)H}Ara-feeding . This M(w) is >70-fold greater than that observed by similar methods in cultures of a dicotyledon ( Rosa sp.) . Thus, during wall-binding, newly secreted xyloglucans greatly increased in size, possibly by transglucosylation . Some modest degradation (trimming) of wall-bound {(3)H}xyloglucan occurred later . The earliest wall-bound {(3)H}xylan had M(w) approximately 2x10(6), similar to the protoplasmic {(3)H}xylan; this increased to approximately 4x10(6) by 6 h . For the first 2 days after {(3)H}Ara-feeding, the soluble extracellular (3)H-hemicelluloses present in the culture medium had M(w) approximately 1x10(6)-2x10(6), comparable to the protoplasmic hemicelluloses . However, between 2 and 3 days after {(3)H}Ara-feeding, the M(w) of the soluble extracellular {(3)H}xylans increased abruptly to approximately 10x10(6); the soluble extracellular {(3)H}xyloglucans underwent a similar but more gradual increase in M(w) . Maize (3)H-hemicelluloses thus underwent increases in M(w) in three episodes: (i) intra-protoplasmically, (ii) during wall-binding (especially xyloglucans), and (iii) after sloughing into the medium . Possible mechanisms and roles of these increases are discussed.

Restor Neurol Neurosci, 1998, 13(3-4), 141 - 51
Impaired learning correlates with size of excitotoxic hippocampal CA3 lesions in adult rats, but shows no amelioration by CA3 transplants; Aznar S et al.; Hippocampal CA3 pyramidal cells grafted as cell suspensions to excitotoxic hippocampal lesions in adult rats can exchange several types of short and long range nerve connections with the host brain . We now examined whether such grafts also had functional effects in terms of ameliorating lesion-induced learning and memory deficits . Adult, male rats with bilateral, one week old, ibotenic acid-lesions of the hippocampal CA3 region, were grafted with suspensions of fetal (E18-19) CA3 cells . Seven weeks later the animals were tested for spatial navigation in the Morris Watermaze, together with groups of lesion-only and sham-operated, control rats . The tests were performed over 5 days, with 4 trials per day . At the end of the trials, the size of the lesions and the size and structural incorporation of the transplants in the host brains were evaluated morphometrically for correlations with the behavioural data . We found significant differences in swim pathlength and latency to find the platform in the Morris Watermaze between the lesion-only group and the grafted group versus the sham operated group, but no significant difference between the lesion-only and the grafted group . There was a significant positive correlation between the size of the CA3 lesions and the paucity of performance of the rat in the Watermaze, just as spontaneous recovery accordingly had not occurred over the 8 weeks postlesion . We conclude that the behavioural improvement exerted by the CA3 cell suspension grafts, at a time point when graft-host connections have had time to establish, is at most incomplete by these transplants, pointing to the difficulties there may be in obtaining full functional integration.

J Biol Chem, 2003 Jun 6, 278(23), 21307 - 13 Epub 2003 Apr 01.
G beta 5.RGS7 inhibits G alpha q-mediated signaling via a direct protein-protein interaction; Witherow DS et al.; A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Ggamma-like domain through which they form obligatory dimers with the G protein subunit Gbeta5 in vivo . In Caenorhabditis elegans, orthologs of Gbeta5.RGS dimers are implicated in regulating both Galphai and Galphaq signaling, and in cell-based assays these dimers regulate Galphai/o- and Galphaq/11-mediated pathways . However, initial studies with purified Gbeta5.RGS6 or Gbeta5.RGS7 showed that they only serve as GTPase activating proteins for Galphao . Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Galphao or Galphaq, indicating that the interaction might require additional factors present in vivo . Here, we asked if the RGS7.Gbeta5 complex binds to Galphaq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells . RGS7, Gbeta5, and Galpha subunits were tagged with yellow variants of green fluorescent protein . First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling . Second, we again demonstrate the interaction between RGS7 and Gbeta5 using FRET . Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7.Gbeta5 complex and cyan fluorescence protein-tagged Galphaq, indicating a direct interaction between these molecules.

Hunan Yi Ke Da Xue Xue Bao, 2002 Dec 28, 27(6), 553 - 5
{Separation of adhering cell colonies with a direct digestion method}; Zhao DC et al.; OBJECTIVE: To establish a highly efficient method for the separation of adhering cell colonies . METHODS: Cell suspension was diluted gradiently to four different densities and then seeded on a cell culture plate covered with parted fibrinous membranes . After colonies appeared, they were digested directly with 0.25% trypsin and picked out with a pipette tip . RESULTS: At the density of 5-10.cm-2, colonies could be separated most efficiently in both continuous and normal diploid cell line colonies . The cell number could reach 10(6) after one month of culture for a continuous cell line colony and one and a half month of culture for a normal diploid cell line colony . CONCLUSION: With the direct digestion method, single cell colonies can be effectively separated from the plate covered with parted fibrinous membranes, when the cell was initially seeded at the density of 5-10.cm2.

Curr Eye Res, 2002 Nov, 25(5), 271 - 7
Expression of B7 molecules in the eye during experimental autoimmune anterior uveitis (EAAU); Shao H et al.; PURPOSE: We have reported that CTLA4-Fc, a fusion protein that binds B7, prevents the induction of EAAU and reduces the severity of disease in Lewis rats . Since B7.1 and B7.2 have distinctive roles in other autoimmune diseases, we investigated their roles in the development of EAAU . METHODS: Lewis rats were immunized with melanin associated antigen (MAA) . Eyes were collected at different stages of EAAU and the expression of B7 on iris and ciliary body (ICB) cell suspensions determined by flow cytometry analysis . The incidence of EAAU after treatment with anti B7, and the requirement of B7.1 and B7.2 for proliferation and cytokine production of lymphoid cells to MAA were also studied . RESULTS: B7.2 is up-regulated in resident ICB cells or bone-marrow derived cells which have infiltrated the ICB by day 10 and remains elevated during the acute phase of disease . B7.1 is expressed later during the acute phase . Both B7.1 and B7.2 are down-regulated during remission, with low levels of B7.2 and no detectable B7.1 . The incidence of EAAU was reduced by anti-B7.2 treatment and completely inhibited by a combination of both B7.1 and B7.2 antibodies . Neither anti-B7.1 nor anti-B7.2 alone affected proliferation or cytokine production . However, administration of both anti-B7.1 and B7.2 completely inhibited proliferation as well as IL-2 and TNF-alpha production . CONCLUSIONS: B7.1 and B7.2 are expressed in the eye at different times during EAAU . Both B7 molecules are required for the induction of EAAU, although they probably have different roles.

Cell Mol Biol Lett, 2003, 8(1), 215 - 9
The effect of hypochlorite on human erythrocytes pretreated with X-radiation; Krokosz A; Both hypochlorite and ionizing radiation induce oxidation processes of biomolecules . The effects are dependent to a large degree on the dose of the oxidizing agent . Previously we observed that split doses of gamma radiation caused lower hemolysis than the same but single doses . The critical factors influencing the occurrence of this effect were: the value of the first dose and the time between the doses . In this work we examined the effect of gamma radiation (40-400 Gy) on hemolysis of human erythrocytes induced by hypochlorite . Erythrocytes in PBS, hematocrit 2 %, were irradiated with doses of 40, 200 or 400 Gy . The dose-rate was 23.8 Gy/min . Cell suspensions were stirred during irradiation . After irradiation the erythrocytes were incubated for 1, 3 or 4 hours at room temperature and then hypochlorite was added to a 250 microM concentration . Control samples were erythrocytes treated only with NaOCl . The level of hemolysis was determined after NaOCl addition . Hemolysis of erythrocytes preirradiated with the dose of 400 Gy was lower than hemolysis of erythrocytes treated only with NaOCl . The effect was dependent on the time between the end of irradiation and the addition of NaOCl . In contrast, slightly higher hemolysis was observed for erythrocytes preirradiated with lower (40 or 200 Gy) doses of radiation . The observed effect is similar to that obtained for radiation-induced hemolysis . It suggests that ionizing radiation may induce structural and/or functional changes in erythrocytes, which make the cell more resistant to further oxidative damage.

Naturwissenschaften, 2003 Mar, 90(3), 121 - 6 Epub 2003 Feb 13.
Hydrocarbon synthesis by enzymatically dissociated oenocytes of the abdominal integument of the German Cockroach, Blattella germanica; Fan Y et al.; In insects, hydrocarbons waterproof the cuticle, protect the insect from the external environment, and serve as semiochemicals or their metabolic precursors . In the German cockroach, Blattella germanica, hydrocarbons are synthesized by the abdominal integument, but the precise site of biosynthesis is not known . We developed a method for separation of oenocytes from other cells in the abdominal integument using enzymatic dissociation followed by Percoll gradient centrifugation . Radiolabeled propionate was then used to monitor de novo synthesis of hydrocarbons by dissociated cells . Oenocyte-enriched cell suspensions of abdominal sternites synthesized hydrocarbons, whereas suspensions enriched with epidermal cells did not . Our results show conclusively that hydrocarbons are produced by oenocytes not only in insects whose oenocytes are localized within the hemocoel, but also in those insects whose oenocytes are within the abdominal integument . Furthermore, these data support a hemolymph pathway for transport and delivery of hydrocarbons to both external and internal tissues, including the epicuticle, fat body, and ovaries.

Dev Biol, 2003 Mar 15, 255(2), 373 - 82
Contrasting activities of the aggregative and late PDSA promoters in Dictyostelium development; Weening KE et al.; Expression of the Dictyostelium PdsA gene from the aggregative (PdA) and late (PdL) promoter is essential for aggregation and slug morphogenesis, respectively . We studied the regulation of the PdA and PdL promoters in slugs using labile beta-galactosidase (gal) reporter enzymes . PdL was active in prestalk cells as was also found with stable gal . PdA activity decreased strongly in slugs from all cells, except those at the rear . This is almost opposite to PdA activity traced with stable gal, where slugs showed sustained activity with highest levels at the front . PdA was down-regulated after aggregation irrespective of stimulation with any of the factors known to control gene expression . PdL activity was induced in cell suspension by cAMP and DIF acting in synergy . However, a DIF-less mutant showed normal PdL activity during development, suggesting that DIF does not control PdL in vivo . Dissection of the PdL promoter showed that all sequences essential for correct spatiotemporal control of promoter activity are downstream of the transcription start site in a region between -383 and -19 nucleotides relative to the start codon . Removal of nucleotides to position -364 eliminated responsiveness to DIF and cAMP, but normal PdL activity in prestalk cells in slugs was retained . Further 5' deletions abolished all promoter activity . This result also indicates that the induction by DIF and cAMP as seen in cell suspensions is not essential for PdL activity in normal development.

J Ocul Pharmacol Ther, 2003 Feb, 19(1), 75 - 81
In vitro inhibition of human conjunctival mast-cell degranulation by ketotifen; Schoch C; Ketotifen relieves the symptoms of allergic conjunctivitis through multiple mechanisms of action . One such mechanism may involve stabilization of conjunctival mast cells . Because of inter- and intra-species variation, however, this hypothesis cannot be adequately tested using mast cells from animals or other human tissues . We therefore employed human conjunctival mast cells . The mast cells were prepared using human conjunctival tissues obtained from US eye banks . Cell suspensions were sensitized with human IgE and incubated with ketotifen fumarate or control . After antigenic challenge of sensitized cells with anti-IgE, levels of histamine and tryptase, two mast-cell granule markers, were measured in the supernatant fluid . Cell viability was assessed with a Trypan Blue assay . Ketotifen at concentrations of approximately 10(-11) to 10(-4) M inhibited mast-cell histamine release by 90% or more . Similarly, ketotifen at approximately 10(-10) to 10(-4) M inhibited tryptase release by 90% or more (apart from a single anomalous reading) . At all ketotifen concentrations that stabilized mast cells, cell viability was preserved . Moreover, ketotifen did not impair cell viability unless concentrations were increased above the clinically relevant range, i.e., above the order of magnitude of 10(-4) M . These data demonstrate that ketotifen can stabilize human conjunctival mast cells, without impairing cell viability.

AAPS PharmSci . 2002;4(4):E31.
Quantitative comparison of functional screening by measuring intracellular Ca2+ with radioligand binding at recombinant human dopamine receptors; Kassack MU; The purpose of this study was to test whether screening at dopamine receptors performed with a recently described functional assay for G-protein coupled receptors (GPCRs) provides data that correlate significantly with radioligand binding data in the literature, thus possibly allowing researchers to replace radioligand binding with nonradioactive functional screening . Human dopamine receptors hD1 and hD2L (representing Gs {hD1} or Gi {hD2L} coupled GPCRs) were recombinantly expressed in human embryonic kidney (HEK293) cells . Cells were loaded with Oregon Green 488 BAPTA-1/AM and evenly distributed in 384 well plates . Seventeen test compounds were screened for agonistic activity by injection into the cell suspension and monitoringH of intracellular Ca2+ with a fluorescence microplate reader . Then, standard agonists (100nM SKF38393 for hD1, 30nM quinpirole for hD2L) were injected into wells preincubated with test compounds (screening for antagonism) . Injection of various agonists resulted in a concentration-dependent increase in fluorescence . Further, preincubation of antagonists with dopamine receptor expressing cells inhibits concentration-dependent the agonist-induced increase in fluorescence . Calculated apparent functional Ki values correlate with radioligand binding data in the literature (r2 = 0.7796 for D1, r2 = 0.7743 for D2) . The correlation between apparent functional Ki values and radioligand binding data for the 17 tested compounds suggests that screening of test compounds at dopamine receptors with the functional Ca2+ assay can replace radioligand binding studies . Furthermore, besides apparent Ki values, information about agonistic or antagonistic properties of a test compound can be obtained with the functional Ca2+ assay.

Cryo Letters, 2003 Jan-Feb, 24(1), 57 - 64
Cryopreservation of carrot (Daucus carota l.) cell suspensions and protoplasts by vitrification; Chen Y et al.; Carrot cell suspensions and protoplasts were successfully cryopreserved by vitrification . Cells were precultured in liquid Murashige and Skoog medium containing 0.175 M sucrose for 3 d and then in liquid MS medium containing 0.4 M sorbitol for 1 d . After loading of the precultured carrot cells in 25 % PVS2 at room temperature for 5 min and treatment with 100 % PVS2 at 0 degrees C for 7.5 min, they were quenched in liquid nitrogen . Optimal survival was 83.3 % (based on the triphenyl tetrazolium chloride reduction assay) following warming and unloading . Recovered cells retained the ability to regenerate plantlets in vitro . In the case of vitrification of protoplasts isolated from carrot cell suspensions, the optimal loading and dehydration durations were 5 min in 25% PVS2 and 3 min 100 % PVS2 respectively . Survival of 47 % of the untreated control (based on the FDA-PI (fluorescein diacetate-propidium iodide) staining) was achieved after cryopreservation.

Plant Physiol, 2003 Mar, 131(3), 1104 - 23
Mapping the proteome of barrel medic (Medicago truncatula); Watson BS et al.; A survey of six organ-/tissue-specific proteomes of the model legume barrel medic (Medicago truncatula) was performed . Two-dimensional polyacrylamide gel electrophoresis reference maps of protein extracts from leaves, stems, roots, flowers, seed pods, and cell suspension cultures were obtained . Five hundred fifty-one proteins were excised and 304 proteins identified using peptide mass fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry . Nanoscale high-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry was used to validate marginal matrix-assisted laser desorption ionization time-of-flight mass spectrometry protein identifications . This dataset represents one of the most comprehensive plant proteome projects to date and provides a basis for future proteome comparison of genetic mutants, biotically and abiotically challenged plants, and/or environmentally challenged plants . Technical details concerning peptide mass fingerprinting, database queries, and protein identification success rates in the absence of a sequenced genome are reported and discussed . A summary of the identified proteins and their putative functions are presented . The tissue-specific expression of proteins and the levels of identified proteins are compared with their related transcript abundance as quantified through EST counting . It is estimated that approximately 50% of the proteins appear to be correlated with their corresponding mRNA levels.

FEMS Microbiol Lett, 2003 Mar 14, 220(1), 99 - 104
Electron transfer from Shewanella algae BrY to hydrous ferric oxide is mediated by cell-associated melanin; Turick CE et al.; Shewanella algae BrY uses insoluble mineral oxides as terminal electron acceptors, but the mechanism of electron transfer from cell surface to mineral surface is not well understood . We tested the hypothesis that cell-associated melanin produced by S . algae BrY serves as an electron conduit for bacterial-mineral reduction . Results from Fourier transform infrared spectroscopy and cell surface hydrophobicity assays indicated that extracellular melanin was associated with the cell surface . With H(2) as electron donor, washed cell suspensions of melanin-coated S . algae BrY reduced hydrous ferric oxide (HFO) 10 times faster than cells without melanin . The addition of melanin (20 microg ml(-1)) to these melanin-free cells increased their HFO reduction rate two-fold . These results suggest that cell-associated melanin acts as an electron conduit for iron mineral reduction by S . algae BrY.

Br J Pharmacol, 2003 Mar, 138(5), 959 - 67
A potent tryptase inhibitor nafamostat mesilate dramatically suppressed pulmonary dysfunction induced in rats by a radiographic contrast medium; Sendo T et al.; (1) Intravenous injection of ioxaglate (4 g iodine kg(-1)), an iodinated radiographic contrast medium, caused a marked protein extravasation, pulmonary oedema and a decrease in the arterial partial oxygen pressure in rats . (2) All of these reactions to ioxaglate were reversed by the pretreatment with gabexate mesilate (10 and 50 mg kg(-1), 5 min prior to injection) or nafamostat mesilate (3 and 10 mg kg(-1)), in which the inhibition was complete after injection of nafamostat mesilate (10 mg kg(-1)) . (3) Both gabexate mesilate and nafamostat mesilate inhibited the activity of purified human lung tryptase, although the latter compound was far more potent than the former . (4) Ioxaglate enhanced the nafamostat-sensitive protease activity in the extracellular fluid of rat peritoneal mast cell suspensions . (5) Tryptase enhanced the permeability of protein through the monolayer of cultured human pulmonary arterial endothelial cells . Ioxaglate, when applied in combination with rat peritoneal mast cells, also produced the endothelial barrier dysfunction . These effects of tryptase and ioxaglate were reversed by nafamostat mesilate . (6) Consistent with these findings, immunofluorescence morphological analysis revealed that tryptase or ioxaglate in combination with mast cells increased actin stress fibre formation while decreasing VE-cadherin immunoreactivity . Both of these actions of tryptase and ioxaglate were reversed by nafamostat mesilate . (7) These findings suggest that tryptase liberated from mast cells plays a crucial role in the ioxaglate-induced pulmonary dysfunction . In this respect, nafamostat mesilate may become a useful agent for the cure or prevention of severe adverse reactions to radiographic contrast media.

Tree Physiol, 2003 Apr, 23(6), 419 - 26
Somaclonal variation in Coffea arabica: effects of genotype and embryogenic cell suspension age on frequency and phenotype of variants; Etienne H et al.; We determined how age of embryogenic cell suspensions affects somaclonal variation in five F1 hybrids of Coffea arabica L . Batches of plants were produced either directly from embryogenic callus, or after 3, 6, 9 and 12 months of embryogenic cell suspension culture . Seven phenotypic variants were characterized . Based on vigor and productivity of the regenerated plants, we classified the variants in order of increasing severity of physiological disorders as: Juvenile leaf color, Giant, Dwarf, Thick leaf (Bullata), Variegata, Angustifolia, and Multi-stem . The Dwarf, Angustifolia and Multi-stem variants were the most frequent among the regenerated plants (1.4, 4.8 and 2.9%, respectively) . The frequency (f) of variants increased exponentially with the age (t) of the embryogenic suspension, in accordance with the function f = 0.99e(0.267t) . For all genotypes, somaclonal variation was low (1.3%) in plants produced from embryogenic callus or 3-month-old cell suspensions and increased in frequency with increasing suspension age (6, 10 and 25% in plants produced from cell suspensions aged 6, 9 and 12 months, respectively) . Large differences in somaclonal variation among genotypes were found only in plants produced from 12-month-old cell suspensions . For two genotypes, the oldest suspensions produced a majority of somaclonal variants (80-90%), whereas somaclonal variation ranged between 8 and 18% in the other genotypes . Cell suspension age and genotype also affected the type of variant produced . The severity of somaclonal variations increased with cell suspension age . For all genotypes combined, the Angustifolia variant was the most common . The other somaclonal variations were specific to certain genotypes or distributed randomly among the genotypes.

Int J Androl, 2003 Apr, 26(2), 84 - 90
Evaluation of damage to the testicular cells of golden hamsters caused by experimental cryptorchidism using flow cytometry and confocal microscopy; Vigodner M et al.; Artificial unilateral cryptorchidism was performed in golden hamsters which were then held for different periods of time . The non-operated side was used as a control . At various times from 4 to 15 days, hamsters were killed, testes were removed and weighed, single cell suspensions were prepared for flow cytometry analysis and seminiferous tubules were fixed for confocal microscopy . Using DNA staining by propidium iodide or acridine orange followed by flow cytometry analysis, a marked decrease in the haploid condensed cell fraction was detected at the beginning stages of experimental cryptorchidism . In correlation with flow cytometry results, spermiogenic arrest at stages IX and X of seminiferous epithelium was detected in these animals by confocal microscopy and there were no mature forms of haploid cells in the cryptorchid testis . In the testis with more severe damage, there were almost no haploid cells in the seminiferous tubules of cryptorchid animals . In addition, a significant decrease in tetraploid cell fraction and an increase in S-phase fraction was obtained in severe cases . This may be explained by cell arrest before entrance into meiosis . Destruction of tubule structure and cell arrangement were also observed by confocal microscopy in such cases . In conclusion, flow cytometry, combined with confocal analysis, added useful information about spermatogenesis disturbances in cryptorchid testis and it may be used as diagnostic tools in other cases of spermatogenic disorders.

Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Feb;67(2 Pt 1):021910 . Epub 2003 Feb 24.
Theory of ac electrokinetic behavior of spheroidal cell suspensions with an intrinsic dispersion; Gao L et al.; The dielectric dispersion, dielectrophoretic (DEP), and electrorotational (ER) spectra of spheroidal biological cell suspensions with an intrinsic dispersion in the constituent dielectric constants are investigated . By means of the spectral representation method, we express analytically the characteristic frequencies and dispersion strengths both for the effective dielectric constant and the Clausius-Mossotti factor (CMF) . We identify four and six characteristic frequencies for the effective dielectric spectra and CMF, respectively, all of them being dependent on the depolarization factor (or the cell shape) . The analytical results allow us to examine the effects of the cell shape, the dispersion strength, and the intrinsic frequency on the dielectric dispersion, DEP, and ER spectra . Furthermore, we include the local-field effects due to the mutual interactions between cells in a dense suspension, and study the dependence of co-field or antifield dispersion peaks on the volume fractions.

J Photochem Photobiol B, 2003 Feb, 69(2), 107 - 20
Cellular uptake, localization and photodynamic effects of haematoporphyrin derivative in human glioma and squamous carcinoma cell lines; Gupta S et al.; Uptake, intracellular concentration, localization and photodynamic effects of a haematoporphyrin derivative (HpD, Photosan-3) were compared in human glioma (BMG-1, wild-type p53) and squamous carcinoma (4451, mutated p53) cell lines . Concentration and time dependence of cellular uptake of HpD was assayed from methanol extracts and whole cell suspension spectroscopy, while localization was studied by fluorescence microscopy-based image analysis . Colony-forming ability, apoptosis, cell-cycle progression and cytogenetic damage (micronuclei formation) were investigated as parameters of photodynamic response following irradiation with red light . BMG-1 cells were more sensitive to the photodynamic treatment than 4451 cells, although the 4451 cells accumulated a higher amount of HpD and did not differ significantly from BMG-1 cells with respect to intracellular localization . Photodynamically-induced cytogenetic damage and apoptosis were considerably higher in BMG-1 cells as compared to 4451 cells . The present results strongly suggest that manifestation of the photodynamically-induced lesions in the form of cytogenetic damage and apoptosis are among the important determinants of cellular sensitivity to HpD-PDT besides the photodynamic dose (intracellular concentration of the photosensitizer and the light dose) .

Anal Biochem, 2003 Mar 1, 314(1), 1 - 7
A versatile assay for the accurate, time-resolved determination of cellular viability; Amano T et al.; A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed . The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe . Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer . Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 micro M were employed . To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998 . This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells . Using this cell viability assay, kinetic analyses of cell death could be performed . Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V(max)) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively.

Anal Quant Cytol Histol, 2003 Feb, 25(1), 31 - 8
DNA ploidy in gastrointestinal B-cell lymphomas . An image analysis study of 43 cases; Krugmann J et al.; OBJECTIVE: To analyze the prognostic importance of DNA ploidy pattern on gastrointestinal (GI) B-cell lymphoma using image cytometry (ICM) and to compare the results with previously published flow cytometry (FCM) data . STUDY DESIGN: Forty-three cases of surgically resected primary GI B-cell lymphomas were examined . Thirty-eight tumors were located in the stomach, 2 in the small intestine, 1 in the large bowel and 2 in both the stomach and small intestine . Six cases were at stage E I 1, 15 at stage E I 2, 20 at stage E II 1 and 1 each at stages III and IV . Histologically, the lymphomas were classified as GI low grade marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type (low grade, 12 cases), low grade MALT lymphoma with a high grade component (mixed type, 10 cases) and GI diffuse large B-cell lymphoma (DLBC) (high grade MALT lymphoma, 21 cases) . After gross removal of nonneoplastic tissue, single cell suspensions were prepared from paraffin blocks and stained according to Feulgen . Ploidy analysis was done using a custom-made DNA cytometer and Optimas image analysis software (Optimas Corp., Seattle, Washington, U.S.A.) . RESULTS: Aneuploidy was found in 42% (5/12 cases) of low grade MALT lymphoma, 90% (9/10 cases) of mixed type lymphoma and 100% (21/21 cases) of GI DLBCL . DNA ploidy had no significant impact on overall survival time (P = .73) . CONCLUSION: ICM analysis showed a higher proportion of aneuploidy in GI lymphomas as compared to that in prior studies using FCM for ploidy determination . Whether DNA ploidy is an independent prognostic factor remains to be determined.

Nephron Exp Nephrol . 2003;93(2):e63.
Phenotyping renal leukocyte subsets by four-color flow cytometry: characterization of chemokine receptor expression; Vielhauer V et al.; To investigate mechanisms of cell-mediated injury in renal inflammatory disease it is critical to determine the surface phenotype of infiltrating renal leukocyte subsets . However, the cell-specific expression of many leukocyte receptors is difficult to characterize in vivo . Here, we report a protocol based on flow cytometry that allows simultaneous characterization of surface receptor expression on different subsets of infiltrating renal leukocytes . The described technique combines an adapted method to prepare single cell suspensions from whole kidneys with subsequent four-color flow cytometry . We recently applied this technique to determine the differential expression of murine chemokine receptors CCR2 and CCR5 on infiltrating renal leukocyte subsets . In this article, we summarize our current findings on the validity of the method as compared with immunohistology and in situ hybridization in two murine models of nonimmune (obstructive nephropathy) and immune-mediated (lupus nephritis) inflammatory renal disease . Flow cytometry analysis revealed an accumulation of CCR5-, but not CCR2-positive lymphocytes in inflamed kidneys, compared to the peripheral blood . Particularly renal CD8+ cells expressed CCR5 (79% in obstructed kidneys, 90% in lupus nephritis) . In both models, infiltrating renal macrophages were positive for CCR2 and CCR5 . These data corresponded to immunohistological and in situ hybridization results . They demonstrate that flow cytometric analysis of single cell suspensions prepared from inflamed kidneys is a rapid and reliable technique to characterize and quantify surface receptor expression on infiltrating renal leukocyte subsets .

Fitoterapia, 2003 Feb, 74(1-2), 62 - 7
Production of anthocyanins by Catharanthus roseus; Filippini R et al.; A stable cell suspension line of Catharanthus roseus producing anthocyanin was obtained . In this strain it was found that approximately 30% of cells regularly accumulated these metabolites and that anthocyanin accumulation occurred between the second half of log phase and the stationary phase of the culture growth cycle . The anthocyanins in the suspension cultures were compared with those biosynthetized in the flowers both of regenerated by somatic embryogenesis and field-grown plants . Six anthocyanins were identified in all the examined samples, three 3-O-glucosides and three 3-O-(6-O-p-coumaroyl) glucosides of petunidin, malvidin and hirsutidin . The hirsutidin coumaroyl glucoside has not been reported previously, and was predominat in all samples . The anthocyanin relative content was similar for cell suspensions and flowers from regenerated plants but different from field-grown plant flowers; instead, the total content was almost the same for the two flower types and higher compared to suspension culture content.

Phytochemistry, 2003 Feb, 62(3), 423 - 31
Phytochelatin synthase catalyzes key step in turnover of glutathione conjugates; Beck A et al.; Conjugation of xenobiotic compounds and endogenous metabolites to glutathione is an ubiquitous process in eukaryotes . In animals, the first and rate-limiting step of glutathione-S-conjugate metabolism is characterized by the removal of the aminoterminal glutamic acid residue of glutathione . In plants, however, glutathione-S-conjugates are generally metabolized by removal of the carboxylterminal glycine residue of the tripeptide glutathione to give rise to the S-glutamylcysteinyl-derivative . Purification of the glutathione-conjugate catabolizing activity from cell suspension cultures of the plant Silene cucubalus indicated that phytochelatin synthase catalyzes the first step of the pathway . Heterologously expressed phytochelatin synthase from Arabidopsis efficiently converted S-bima ne-glutathione to S-bimane-glutamylcysteine, the formation of which was unequivocally identified by mass spectrometry . No further products, such as S-derivatives of phytochelatins, were observed . Several different glutathione-S-conjugates served as substrates for the enzyme and were processed to the corresponding glutamylcysteinyl-adducts . Affinity-purified phytochelatin synthase preparations required divalent heavy metal ions such as Cd(2+), Zn(2+) or Cu(2+) for detectable turnover of glutathione-S-conjugates . Characterization of the enzymatic properties of phytochelatin synthase argues for both cellular functions of the gamma-glutamylcysteinyl-dipeptidyltransferase: (1) formation of heavy-metal binding peptides and (2) degradation of glutathione-S-conjugates . Mechanistically, the former role is the result of gamma-glutamylcysteinyl transpeptidation onto glutathione or derivatives thereof, while the catabolic function reflects transpeptidation of S-glutamylcysteinyl-adducts onto the acceptor molecule water . Thus, phytochelatin synthase seems to fulfil a second crucial role in glutathione metabolism.

J Cancer Res Clin Oncol, 2003 Jan, 129(1), 21 - 8 Epub 2003 Jan 11.
Potential of alpha-amino alcohol p-boronophenylalaninol as a boron carrier in boron neutron capture therapy, regarding its enantiomers; Masunaga S et al.; PURPOSE: We evaluated the potential of a newly developed (10)B-containing alpha-amino alcohol of p-boronophenylalanine-(10)B (BPA), p-boronophenylalaninol (BPAol), as a boron carrier in boron neutron capture therapy . METHODS: C57BL mice bearing EL4 tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously via implanted mini-osmotic pumps to label all proliferating (P) cells . After oral administration of L-BPA or D-BPA, or intraperitoneal injection of L-BPAol or D-BPAol, the tumors were irradiated with reactor thermal neutron beams . Some of the tumors were heated at 40 degrees C for 30 min (mild temperature hyperthermia (MTH)) right before neutron exposure, and/or tirapazamine (TPZ) was intraperitoneally injected 30 min before irradiation . The tumors were then excised, minced, and trypsinized . The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling { =quiescent (Q) cells} was determined using immunofluorescence staining for BrdU . Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the apoptosis frequency in Q cells . The apoptosis and MN frequency in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU . RESULTS: Without TPZ or MTH, L- and D-BPAol increased both frequencies markedly, especially for total cells . Although not significantly larger, L-BPA and D-BPAol increased both frequencies slightly more than D-BPA and L-BPAol, respectively . Combination with both MTH and TPZ markedly reduced the sensitivity difference between total and Q cells . CONCLUSION: Both L- and D-BPAol have potential as a (10)B-carrier in neutron capture therapy, especially when combined with both MTH and TPZ.

Biomaterials, 2003 May, 24(11), 1909 - 16
Synergistic effects of H2O2 with components of dental restorative materials on gluconeogenesis in rat kidney tubules; Franz-Xaver R et al.; No data are available about (toxic) effects of dental materials administered in combination with H(2)O(2) from dental bleaching compounds.The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) as well as mercuric chloride (HgCl(2)) and methylmercury chloride (MeHgCl), each in combination with H(2)O(2), was investigated on gluconeogenesis in kidney cells . From rats kidney tubules were prepared . Every 10 min up to 60 min 1-ml samples were drawn from the cell suspension for quantitating the glucose content . Glucose formation in controls was 3.5+/-0.3 nmol/mg.per min (mean+/-SEM, n=21) . Relative rates of glucose formation were obtained by expressing individual rates as percentage of the corresponding control . X-Y concentration curves (effective concentration, EC) of the substances were calculated by fitting a four-parametric sigmoid function to the relative rates of the glucose formation at various test concentrations.At the end of the incubation period cell viability was assessed by trypan blue exclusion . Cell viability decreased within the 60 min interval from 90% to approx . 80% (controls), <25 (HEMA), <20 (TEGDMA), <20 (H(2)O(2)) <10 (MeHgCl), and <10 (HgCl(2)) . Values of 50% effective concentration (EC(50)) were calculated from fitted curves . EC(50) values were (mmol/l; mean+/-SEM; n=4): HEMA, 17.2+/-2.8; TEGDMA, 1.9+/-0.2; H(2)O(2) 0.22+/-0.03, MeHgCl, 0.016+/-0.0005; and HgCl(2), 0.0017+/-0.0005 . No significant decrease of the EC(50) values was found when kidney cells were exposed to HEMA, HgCl(2), or MeHgCl in addition with H(2)O(2) (1-100 microM), compared to those EC(50) values of each compound without H(2)O(2) addition . A significant decrease of the TEGDMA EC(50) values to about 0.25 or 0.04 (mmol/l) was found when cells were exposed to TEGDMA in combination with H(2)O(2) (75 or 100 microM), compared to that TEGDMA EC(50) value without H(2)O(2) addition.The addition of H(2)O(2) (75 and 100 microM) resulted in a synergistic toxic effect of TEGDMA.

J Pineal Res, 2003 Apr, 34(3), 167 - 72
Melatonin modulates the action of near infrared radiation on cell adhesion; Karu TI et al.; The adhesion of human cervical cancer (HeLa) cells to a glass matrix is evaluated following their irradiation in a suspension with a pulsed near-infrared (IR) light-emitting diode (wavelength 820 nm, pulse repetition frequency 10 Hz, irradiation dose 16-120 J/m2) when melatonin (4 x 10(-11) to 4 x 10(-5) m) is added to cell suspension immediately before or after the irradiation . Also, the dependence of visible-to-near-IR radiation (600-840 nm, 52 J/m2) on cell adhesion (action spectrum) is recorded in absence and presence of melatonin (4 x 10(-6) m) . It is found that melatonin in pharmacological concentrations (but not in physiological range) inhibited cell adherence . Irradiation of cells before or after melatonin treatment normalizes cell adhesion to control level . Melatonin in pharmacological concentrations eliminates stimulation of cell attachment induced by irradiation . Pre-treatment (but not post-treatment) with melatonin in the physiological concentration eliminates cell adhesion stimulation induced by irradiation . Melatonin modifies the light action spectrum significantly in near IR region (760-840 nm only) . Thus, the peak at 820-830 nm characteristic for the light action spectrum is fully reduced.

J Appl Physiol, 2003 Jul, 95(1), 57 - 63 Epub 2003 Feb 28.
Exercise-induced changes to in vitro T-lymphocyte mitogen responses using CFSE; Green KJ et al.; Carboxyfluorescein diacetate succinamidyl ester (CFSE) labeling of lymphocyte populations can provide unique insights into cell function at rest and with exercise, due to its ability to quantify cell division on an individual cell basis . This study aimed to characterize the effect of acute, intense exercise on T-lymphocyte function . Well-trained endurance runners completed 60 min of treadmill running at 95% of individual anaerobic threshold . Blood samples were collected before exercise; after 30 and 60 min of exercise; and after 30, 60, and 90 min of recovery . Isolated peripheral blood mononuclear cells were labeled with CFSE and cultured with or without mitogen (phytohemagglutinin) . After culture, cell suspensions were labeled with CD3 (allophycocyanin) and CD8 (phycoerythrin), and expansion rates and cell death rates were calculated for each sample, as well as mitosis rates for each cell generation . Exercise was associated with a 60% decrease in cell expansion in both CD4 and CD8 cell types from before exercise to midexercise (P < 0.05) . The significant decrease in expansion rate in the midexercise samples for both cell types was mirrored by a 65% increase in cell death (P < 0.05) in both cell types at that sample point . Exercise had no effect on the mitosis rate of either CD4 or CD8 cells in any cell generation (generations 0-3) . This study indicates that 1 h of intense exercise affects in vitro T-lymphocyte function . These data suggest, for the first time, that exercise decreases cell expansion rate via an increase in cell death of both CD4 and CD8 T lymphocytes, rather than a decrease in mitosis.

Nippon Ganka Gakkai Zasshi, 2002 Dec, 106(12), 805 - 35; discussion 836
{Transplantation of corneal endothelial cells}; Amano S; Though conventional corneal transplantation has achieved great success, it still has several drawbacks including limited availability of donor corneas, recurrent allograft rejection, and subsequent graft failure in certain cases . Reconstructing clinically usable corneas by applying the technology of regenerative medicine can offer a solution to these problems, as well as making corneal transplantation a non-emergency surgery and enabling the usage of banked corneal cells . In the present study, we focused on corneal endothelium that is critical for corneal transparency and investigated the reconstruction of cornea utilizing cultured human corneal endothelial cells (HCECs) . We succeeded in steadily culturing HCECs by using culture dishes pre-coated with extracellular matrix produced by calf corneal endothelial cells and culture media that contained basic fibroblast growth factor and fetal bovine serum . We performed the following analysis utilizing these cultured HCECs . The older the donor was, the more frequently large senescent cells appeared in the passaged HCECs . The telomeres of HCECs were measured as terminal restriction fragments (TRF) by Southern blotting . HCECs, in vivo from donors in their seventies had a long TRFs of over 12 kilobases . Passaging shortened the TRFs but there was no difference in TRFs among donors of various ages . These results indicated that shortening of telomere length is not related to senescence of HCECs . We investigated the role of advanced glycation end products (AGEs) in the senescence of in vivo HCECs . The results indicated that AGE-protein in the aqueous humor is endocytosed into HCECs via AGE receptors expressed on the surface of HCECs and damages HCECs by producing reactive oxygen species and inducing apoptosis, suggesting that AGEs, at least partly, cause the senescence of HECEs . HCECs were cultured using adult human serum instead of bovine serum to get rid of bovine material that can be infected with prions . Primary and passage culture of HCECs was possible using adult human serum . We reconstructed the cornea using cultured HCECs and human corneal stroma . The corneal stroma, on which the cell suspension of HCECs was poured, was mildly centrifuged to enhance the HCECs attachment to the stroma . The cell density of HCECs on the reconstructed cornea reached 2,500 cells/mm2 . The pump function of the reconstructed cornea was measured with an Ussing chamber . The potential difference in the reconstructed cornea and normal cornea was 0.30 mV and 0.40 mV, respectively; indicating that the pump function of the reconstructed cornea is 75% of that of the normal cornea . The reconstructed cornea was transplanted to a rabbit eye and stayed transparent for 6 months after the operation . Fluorescein labeled cultured HCECs remained on the graft 1 month after the transplantation, indicating that transplanted HCECs contributed to the transparency of the graft . The possibility of using artificial stroma or porcine corneal stroma as a carrier of cultured HCECs was investigated . The artificial stroma made of alkaline-treated collagen could not be sutured but showed good transparency, biocompatibility, and cell-attachability . Porcine corneal stroma, expressing little xeno-sugar antigen alpha-gal epitope, induced no super acute rejection but mild cellular rejection when transplanted in the cornea of animals possessing natural antibody to alpha-gal epitope . The cornea reconstructed with porcine corneal stroma and HCECs had an average cell density of 1721/mm2 and had approximately 60% of the pump function of a normal cornea . As new technologies in corneal transplantation, the application of self immature cells and the direct delivery of cultured HCECs into the anterior chamber were investigated . Part of rat mononuclear cells that were obtained from the bone marrow and injected into the rat anterior chamber transformed into corneal endothelium-like cells, suggesting that self immature cells can transform into corneal endothelial cells . Cultured rabbit corneal endothelial cells that endocytosed iron were injected into the anterior chamber of rabbits whose corneal endothelium was cryo-injured, and were pulled to Descemet's membrane by putting a magnet on the eyelid . In these rabbits, corneal edema decreased more quickly than in the control group and no intraocular pressure rise was observed during 8 weeks after the operation, suggesting that the direct delivery of cultured HCECs into the anterior chamber can be an alternative method of choice . The following obstacles should be addressed to make the transplantation of cultured corneal endothelial cells clinically applicable . 1 . To reconstruct a cornea that is the same as or superior to the normal cornea, more innovation is necessary in the method of culturing and seeding HCECs . We should consider utilizing HCECs obtained from fetuses after clearing ethical issues . Moreover, we need to develop a method to enhance the cell density and the cell functions . 2 . Porcine corneal stroma is promising as a carrier of HCECs instead of human corneal stroma, which is in very limited supply . The usefulness of porcine corneal stroma acellularized to prevent retrovirus infection should be evaluated . 3 . To make the self immature cells applicable to corneal transplantation, we should elucidate the corneal endothelial cell specific markers and the factors that are necessary to induce self immature cells to become corneal endothelial cells . 4 . The direct delivery of cultured HCECs into the anterior chamber can be an alternative method of choice when its long-term safety is confirmed.

J Asian Nat Prod Res, 2003 Mar, 5(1), 5 - 10
Biotransformation of 4(20),11-taxadienes by cell suspension cultures of Platycodon grandiflorum; Dai JG et al.; Platycodon grandiflorum cell suspension cultures were employed to biotransform the taxane diterpenoids 2alpha,5alpha,10beta,14beta-tetraacetoxy-4(20),11-taxadiene (1) and 9alpha-hydroxy-2alpha,5alpha,10beta,14beta-tetraacetoxy-4(20),11-taxadiene (2) . One product, 10beta-hydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (3) was obtained from 1 and two products, 9alpha,10beta-dihydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (4) and 10beta-hydroxy-2alpha,5alpha,9alpha,14beta-tetraacetoxy-4(20),11-taxadiene (5) were obtained from 2 incubated with Platycodon cultured cells respectively, among which 5 is characterized as a new taxoid compound . The effects of the addition stage for 1 and 2 on the biotransformation were investigated and the results revealed that: (1) the optimal addition stage for 1 was in the early logarithmic phase (6th day) of the cell growth period, in which 78% of 1 was converted and the yield for 3 reached 75%; (2) the optimal addition stage for 2 was on the mid-logarithmic phase (12th day) of the cell growth period, in which 25.3% of 2 was converted and the yields for 4 and 5 reached 18.9 and 14.5%, respectively.

Eur J Haematol, 2003 Mar, 70(3), 136 - 42
Effect of recombinant human deoxyribonuclease on the expression of cell adhesion molecules of thawed and processed cord blood hematopoietic progenitors; Beck C et al.; BACKGROUND AND OBJECTIVES: The integrity of granulocytic cells and platelets is compromised within cryopreserved stem cell transplants, and consequent DNA release during the thawing procedure can therefore lead to clotting phenomena or microaggregate formation and that in turn may cause loss of progenitor cells . To circumvent this problem a new processing protocol was introduced using recombinant human deoxyribonuclease (rhDNase) to prevent cell aggregate formation . In addition, the impact of this new processing protocol on CD34+ umbilical cord blood (UCB) cells was assessed . MATERIALS AND METHODS: Fifty samples derived from 7 buffy coat (BC) volume reduced UCB units were cryopreserved, thawed, and processed with washing solutions that were supplemented with rhDNase in various concentrations . Thereafter, clotting and microaggregate formation was scored microscopically . In addition, expression of the adhesion molecules leukocyte function-associated antigen 1 (LFA-1, n = 6), intercellular adhesion molecule-1 (ICAM-1, n = 11), and L-selectin (n = 11) on CD34+ UCB cells was analyzed by flow cytometry after incubating the samples with either dimethyl sulfoxide (DMSO) 5.5%, rhDNase 10 or 50 U/mL, or a combination of DMSO 5.5% and rhDNase 50 U/mL . RESULTS: At a minimal concentration of 10 U rhDNase/mL, clotting or microaggregate formation could be prevented for all tested samples, whereas cell clots could be observed for concentrations up to 8 U/mL . The expression of adhesion molecules on untreated CD34+ UCB cells (L-selectin: 64.6 +/- 18.8%; LFA-1: 62.6 +/- 7.5%; ICAM-1: 14.8 +/- 4.1%) did not show any significant difference compared with cells that were incubated with up to 50 U/mL rhDNase (L-selectin: 62.2 +/- 19.3%; LFA-1: 63.1 +/- 5.9%; ICAM-1: 17.5 +/- 6.7%) . However, after a combined treatment with DMSO 5.5% and rhDNase 50 U/mL, a slight but significant decrease in L-selectin expression could be observed (P < 0.03) . CONCLUSION: The supplementation of rhDNase to a final concentration of 10 U/mL cell suspension proved to be effective in preventing clot formation under the conditions examined and did not lead to decreased expression levels of adhesion molecules . We therefore recommend the use of rhDNase for the prevention of clot formation and cell loss during the processing of thawed UCB transplants.

Immunol Lett, 2003 Mar 3, 86(1), 7 - 14
Technique for obtaining highly enriched, quiescent immature Langerhans cells suitable for ex vivo assays; Tchou I et al.; Epidermis and surface epithelium-dendritic cells comprise of immature cells termed Langerhans cells (LCs), which express characteristically the Birbeck granules, along with surface markers such as CD1a . These cells can capture a pathogen and then migrate and differentiate to a more mature stage . During this maturation process, dentritic cells express surface markers differentially . In physio-pathological models of infection where LCs are involved, it is critically important to ensure that the LCs tested in vitro are still immature and are not artefactually matured-dentritic cells . For experimental purposes, LCs were isolated from skin epidermis obtained from patients undergoing plastic surgery . This work thus aimed at collecting fresh LCs ex vivo and at testing the cells for phenotypic and functional characteristics of the immature stage . After mechanic disruption of the epidermis and proceeding for single cell suspension obtaining, two methods for purification were tested in parallel: (a) a positive immuno-magnetic separation by anti-CD1a-coated beads and (b) a purely mechanic purification system based on a three-step Ficoll floatation process . Both systems were equally efficient in terms of purification and yield . By using flow cytometry phenotyping, we have demonstrated that the use of magnetic beads led to some degree of maturation of CD1a(+) LCs, contrary to the repeated Ficoll floatation . This work calls attention for the use of certain monoclonal antibodies such as anti-CD1a to purify immature dendritic cells as they pre-activate these cells . Pre-activation would render a number of assays on the early events of LC physiology invalid, contrary to the purification of fresh skin epidermis LCs by means of a repeated Ficoll floatation.

Cytometry B Clin Cytom, 2003 Mar, 52(1), 20 - 31
Vortex disaggregation for flow cytometry allows direct histologic correlation: a novel approach for small biopsies and inaspirable bone marrows; Vos JA et al.; BACKGROUND: Many approaches to obtaining single cells from tissue for flow cytometric immunophenotyping are used; however, these methods result in tissue that is too disrupted for subsequent histologic examination . We introduce a new technique for cell dissociation of hematopoietic malignancies that preserves tissue for histology . This is especially important with small specimens for which this type of correlation is critical . METHODS: Fresh tissue from lymph node, gastrointestinal (GI) tract, skin, and other soft tissue biopsies, in addition to cores of inaspirable bone marrows, were briefly vortexed until the RPMI cell culture medium became cloudy . Larger specimens such as lymph nodes were sectioned before disaggregating, whereas smaller ones were vortexed in toto . Resultant flow cytometric analyses were compared with the histology and, in some cases, the immunohistochemistry (IHC) to determine whether the data were concordant . Cell suspensions of 104 specimens-composed of 48 lymph nodes, 19 bone marrow cores (BMCs), 11 GI biopsies, 11 skin/soft tissue biopsies, and 15 miscellaneous specimens-were prepared via vortex disaggregation . RESULTS: Flow cytometric analysis of 96 specimens (92.3%) showed adequacy of material and diagnostic correlation with the histology and IHC . Of the eight cases (7.7%) that were discordant, seven were attributable to significant specimen fibrosis or necrosis . With respect to tissue type, this method produced diagnostic cell suspensions for most lymph nodes (95.8%), GI biopsies (90.9%), and BMCs (89.5%); however, it was less useful for skin/soft tissue samples (81.8%) . CONCLUSIONS: Disaggregation of tissue for flow cytometric analysis by vortexing appears to provide adequate and representative cellular material . This technique is ideal for inaspirable bone marrows and small biopsies where tissue preservation for histology is paramount . Published 2003 Wiley-Liss, Inc.

Cell Tissue Res, 2003 Feb, 311(2), 187 - 98 Epub 2003 Jan 18.
TGFbeta1 limits the expansion of the osteoprogenitor fraction in cultures of human bone marrow stromal cells; Walsh S et al.; Currently, there is considerable interest in the possibility of using cultured human bone marrow stromal cells (BMSCs) for skeletal tissue engineering . However, the factors that regulate their ex vivo expansion and promote their osteogenic maturation remain poorly defined . Using BMSCs obtained from a large cohort of adult donors, the effects of transforming growth factor (TGF)beta1 on these processes have been determined . BMSCs were found to express TGFbeta receptors (TbetaRs) I, II, III (betaglycan) and CD105/endoglin . The expression of TbetaRs I and II, but not TbetaR III or endoglin, was linked to the cells' state of maturation . Treatment with TGFbeta increased the colony-forming efficiency (CFE) of marrow cell suspensions but reduced the median diameter of the colonies that formed and the number of cells harvested at the end of primary culture . Treatment with TGFbeta also resulted in a significant downregulation in the expression of the developmental markers alkaline phosphatase (AP) and STRO-1 . The reduction in AP was due to a decrease in the absolute number of cells expressing this enzyme and in the level (sites/cell) at which it was expressed . Overall, the changes in the expression of STRO-1 and AP are consistent with TGFbeta acting to decrease the size of the osteoprogenitor fraction, and hence the potential clinical utility of the cultured cell population.

Magn Reson Med, 2003 Mar, 49(3), 450 - 8
Effects of equilibrium exchange on diffusion-weighted NMR signals: the diffusigraphic "shutter-speed"; Lee JH et al.; A general picture is presented of the implications for diffusion-weighted NMR signals of the parsimonious two-site-exchange (2SX) paradigm . In particular, it is shown that the diffusigraphic "shutter-speed," tau(-1) identical with |q(2)(D(A) - D(B))|, is a useful concept . The "wave-number" q has its standard definition (given in the text), and D(A) and D(B) are the apparent diffusion coefficients (ADCs) of molecules in the two "sites," A and B, if there is no exchange between them . At low gradient strengths (center of q-space), tau(-1) is less than rate constants for intercompartmental water molecule exchange in most tissue cases . Thus, the exchange reaction appears fast . However, q is increased during the course of most experiments and, as it is, the shutter-speed becomes "faster" and the exchange reaction, the kinetics of which do not change, appears to slow down . This causes a multiexponential behavior in the diffusion-weighting dimension, b, which also has its standard definition . This picture is found to be in substantial agreement with a number of different experimental observations . It is applied here to literature (1)H(2)O data from a yeast cell suspension and from the human and the rat brain . Since the equilibrium transcytolemmal water exchange reaction appears to be in the fast-exchange-limit at small b, the initial slope represents the weighted-average of the ADCs of intra- and extracellular water . Of course, in tissue the former is in the significant majority . Furthermore, a consideration of reasonable values for the other 2SX parameters suggests that, for resting brain tissue, the intracellular water ADC may be larger than the extracellular water ADC . There are some independent inferences of this, which would have ramifications for many applications of diffusion-weighted MRI.

Phytochemistry, 2003 Apr, 62(7), 1093 - 9
Efficient production and capture of 8-prenylnaringenin and leachianone G-biosynthetic intermediates of sophoraflavanone G--by the addition of cork tissue to cell suspension cultures of Sophora flavescens; Zhao P et al.; It has previously been demonstrated that the addition of cork tissue to cell suspension cultures of Sophora flavescens stimulates the production of sophoraflavanone G, most of which has been recovered from the added cork tissue . In the present study, it was found that two precursors of sophoraflavanone G, 8-prenylnaringenin (sophoraflavanone B) and leachianone G, both of which have never been detected either in cultured cells or in the original plants, also accumulated in the added cork tissue . Thirteen minor flavonoids including three prenylated flavonoids, in addition to 8-prenylnaringenin and leachianone G, were isolated from the cork tissue co-incubated with S . flavescens cells . The new compounds flavescenones A, B and C, were determined to be (3R)-5, 7, 2'-trihydroxy-6-gamma, gamma-dimethylallyl-4', 5'-methylenedioxyisoflavanone; 5, 7, 2'-trihydroxy-6-gamma, gamma-dimethylallyl-4', 5'-methylenedioxyisoflavone and 2-{2',4'-dihydroxy-3'-(gamma-hydroxymethyl-gamma-methylallyl)phenyl}-5,6-methylenedioxybenzofuran, respectively, by means of spectroscopic analyses that included 2D-NMR techniques.

Phytochemistry, 2003 Mar, 62(6), 901 - 9
Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures; Ketchum RE et al.; Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids . Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described . These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv . Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol) . The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.

Gut, 2003 Mar, 52(3), 327 - 33
Characterisation of telomerase immortalised normal human oesophageal squamous cells; Morales CP et al.; BACKGROUND AND AIMS: Oesophageal cell lines derived from malignancies have numerous genetic abnormalities and therefore are of limited value for studying the early events in carcinogenesis . Reported attempts to establish normal human oesophageal cell lines either have failed to achieve immortalisation or have achieved it by disrupting important cell functions . We have used telomerase technology to establish normal human oesophageal cell lines . METHODS: Endoscopic biopsy specimens of normal oesophageal squamous epithelium were trypsinised, dispersed into single cell suspensions, and cocultivated with ATCC Swiss 3T3 cells . Oesophageal cells were infected with the catalytic subunit of human telomerase (hTERT) using a defective retroviral vector . The integrity of cell cycle checkpoints was tested by measuring p53 response to UV irradiation, and p16 response to infection with H-RasGV12 . Expression of a differentiation marker was tested by measuring involucrin response to calcium exposure . RESULTS: Cultures of uninfected oesophageal cells had weak telomerase activity at baseline but exhibited loss of telomerase activity and progressive telomere shortening before undergoing senescence between population doublings (PD) 40-45 . In contrast, hTERT infected cells exhibited sustained telomerase activity and stabilisation of telomere length . These cells have reached PD 100 with no diminution in growth rate, while cell cycle checkpoint integrity and involucrin response to calcium exposure have remained intact . CONCLUSIONS: By introducing telomerase into normal human oesophageal squamous cells cocultivated with feeder layers, we have established a cell line that retains normal cell cycle checkpoints and normal differentiation markers . This cell line may be useful for studying the early events in oesophageal carcinogenesis.

Eur Biophys J, 2003 Feb, 31(8), 563 - 74 Epub 2002 Nov 12.
Simulations of NMR-detected diffusion in suspensions of red cells: the "signatures" in q-space plots of various lattice arrangements; Regan DG et al.; Coherence effects from pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance diffusion experiments have been observed and characterized for diffusants in many heterogeneous systems, ranging from porous materials to cell suspensions . The resulting coherence patterns appear in plots of the normalized PGSE signal intensities as a function of the spatial wave vector Q in a so-called q-space plot . The origin of these phenomena and their mathematical and physical underpinnings are now well established . We have conducted a number of studies of diffusion-coherence phenomena in suspensions of red blood cells and have made extensive use of computer simulations of molecular diffusion in virtual lattices of cells to aid in the interpretation and analysis of experimental data . In the current work we extended the canonical model used in these studies to investigate the effect that varying the packing arrangement of cells in the suspension has on the coherence patterns, as seen in q-space plots . We show that changes in the packing arrangement of cells are reflected in the q-space plots and in the results of diffusion tensor analysis and thus we speculate upon the possible clinical importance of these findings.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2003 Feb, 95(2), 222 - 7
Attachment of human periodontal ligament fibroblasts to 3 different root-end filling materials: Scanning electron microscope observation; Balto H et al.; OBJECTIVE: The attachment behavior of the human periodontal ligament (PDL) fibroblasts to root-end filling materials (amalgam and Super-EBA) was compared in vitro to gutta-percha by means of scanning electron microscope . STUDY DESIGN: Amalgam and Super-EBA were placed in a prepared cavity of root slices of freshly extracted human teeth and evaluated freshly prepared . Root slices of teeth with cold-burnished gutta-percha filling with AH26 sealer were used for comparison . The root slices were placed in tissue culture cluster, and 1 mL of cell suspension was added carefully over the root slice . They were incubated at 37 degrees C and 100% humidity for 4, 24, and 72 hours . RESULTS: Results showed that the cold-burnished gutta-percha provides a better substrate than amalgam and Super-EBA for cell growth and attachment . Amalgam was the most toxic material, showing early manifestation of cell injury . CONCLUSION: It was concluded that the composition and surface texture of the substrate have an influence on the morphology and the attachment of the PDL fibroblasts . It is suggested that cell attachment and morphology might reflect the biocompatibility of the substratum.

Adv Exp Med Biol, 2003, 510, 213 - 8
Functional oxygen-17 magnetic resonance imaging and localized spectroscopy; Mateescu GD; Functional oxygen-17 magnetic resonance (MR) imaging and localized spectroscopy is defined as the ensemble of MR measurements aiming at in vivo, noninvasive characterization of oxygen transport and utilization . After a brief description of the present status of in vivo 17O-MR, preliminary results are reported on oxygen delivery and consumption in cell suspensions of Saccharomyces cerevisiae . It is shown that parallel 31P-MR at high magnetic fields has an important corroborative value.

Zhong Yao Cai, 2000 May, 23(5), 249 - 51
{Study on the callus and cell culture of Hypericum perforatum}; Li J et al.; Callus were induced from the leaves and stems of Hypericum perforatum L . The cell suspension culture and plate culture were also investigated in this paper . The results showed that the medium of MS which contained 2,4-D 1 mg/L and NAA 1 mg/L was the best one for inducing callus . The plant growth regulator of 2,4-D was assential for callus and suspension culture . Factors such as the time of cell suspension culture, the ways of plating and cell density, which affects the plating efficency were studied . The results showed that plating at density of 5 x 10(3) cells/ml and with monocells seperated from the cells cultured 14-21 days, high plating efficiency could be obtained either by conditional plate culture or by nursing plate culture.

J Biotechnol, 2003 Mar 6, 101(2), 157 - 63
Inhibition of paclitaxel and baccatin III accumulation by mevinolin and fosmidomycin in suspension cultures of Taxus baccata; Palazon J et al.; To achieve a better understanding of the metabolism and accumulation of paclitaxel and baccatin III in cell cultures of Taxus, inhibitors of the early steps in the terpenoid pathway were applied to a cell suspension culture of Taxus baccata: fosmidomycin as an inhibitor of the non-mevalonate branch of the pathway, and mevinolin as an inhibitor of the mevalonate branch . Synthesis of both taxanes in the cell suspension was first increased when cultured in the product formation medium supplemented with methyljasmonate (100 microM) . The product formation medium was selected after assaying 24 different culture media . When fosmidomycin (200 microM) was added to the product formation medium together with the elicitor, the accumulation of paclitaxel and baccatin III was reduced by up to 3.0 and 1.5 times, respectively, whereas the inhibitory effect of mevinolin (1 microM) was only clearly exerted in the case of paclitaxel . Under the conditions of our experiment, we conclude that in the synthesis of both taxanes, the non-mevalonate pathway is the main source of the universal terpenoid precursor isopentenyl diphosphate (IPP) .

Biotechnol Bioeng, 2003 Mar 30, 81(7), 870 - 5
Expression and secretion of the heterodimeric protein interleukin-12 in plant cell suspension culture; Kwon TH et al.; It has been suggested that plant cell culture is the most suitable system for producing small-to-medium quantities of specialized, expensive, and high-purity proteins . Here, we report that a heterodimeric protein, human interleukin-12 (hIL-12), was expressed and secreted into culture medium in a biologically active form . A transgenic plant expressing hIL-12 was constructed by sexual crossing of plants that expressed each subunit of the protein . From a piece of transgenic plant, callus was induced and cell suspension culture was established . The biological activity and amount of hIL-12 secreted into culture medium were analyzed using bioassays and ELISA . Analysis of cellular localization demonstrated that the protein was secreted into the culture medium together with its intrinsic signal peptide .

Int J Radiat Biol, 2002 Dec, 78(12), 1149 - 57
The radiation hypersensitivity of cells at mitosis; Stobbe CC et al.; PURPOSE: Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes . To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated . MATERIALS AND METHODS: Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method . Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays . Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2) . The indirect effect of OH radicals was investigated with the radical scavenger, DMSO . DNA strand breakage was measured by the comet assay . RESULTS: Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1) . The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells . More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells . The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells . CONCLUSIONS: The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells . Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal . In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells . How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known . The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.

J Exp Bot, 2003 Feb, 54(383), 623 - 32
Phosphorylation of a member of the MBF1 transcriptional co-activator family, StMBF1, is stimulated in potato cell suspensions upon fungal elicitor challenge; Zanetti ME et al.; StMBF1 (Solanum tuberosum multiprotein bridging factor 1) is a plant member of the MBF1 family of transcriptional co-activators . Previously, it has been described as being up-regulated at the transcriptional level by fungal and abiotic stress . To understand whether StMBF1 is also regulated at the post-translational level, in vitro as well as in vivo phosphorylation assays were performed . StMBF1 is phosphorylated under both experimental conditions and {(32)P} incorporation into StMBF1 increases after treatment of potato cells with hyphal cell wall components (HWC) derived from Phytophthora infestans . The StMBF1-phosphorylating activity is strongly inhibited by the calcium-chelator EGTA and partially inhibited by calmodulin antagonists . Using bacterial purified StMBF1 as a substrate, a 57 kDa calcium-dependent protein kinase (p57) that is able to phosphorylate StMBF1 was detected . The StMBF1 kinase activity of p57 was higher in elicited than in non-treated cells . The role of the elicitor-dependent phosphorylation of StMBF1 is discussed.

Ultrason Sonochem, 2003 Mar, 10(2), 81 - 4
Dose-dependent inhibition of ultrasound-induced cell killing and free radical production by carbon dioxide; Feril LB Jr et al.; Previous studies have shown that if a solution for cell suspension is saturated with CO(2), ultrasound-induced in vitro cell killing and free-radical production are inhibited . However, the dose dependency of this observation has not been explored . Here, we used NaHCO(3) and HCl to produce a predictable concentration of CO(2) within the culture medium . Using 1 MHz continuous wave 4 W/cm(2) ultrasound, we sonicated U937 cells suspension for 1 min at 37 degrees C with CO(2) at different concentrations . At 2 mM, reduced cell killing was observed that further decreased with increasing CO(2) concentration until 100% protection was attained at 20 mM . Ultrasound-induced free-radical production was significantly decreased at 1 mM and became undetectable at 2 mM CO(2) . This finding shows that CO(2)-mediated inhibition is concentration dependent and that the threshold for free-radical production is one order of magnitude higher than the threshold for cell killing induced by ultrasound . In addition, it also cautions researchers when adding acids and acid-based agents to a culture medium, which almost always contains NaHCO(3), in experiments related to the bioeffects of ultrasound.

Shi Yan Sheng Wu Xue Bao, 2000 Sep, 33(3), 283 - 4
{The establishment of two new evaluation criteria for cell state of plant cell suspension cultures}; Chen BT et al.; An experiment on suspending rice cell cultures in sucrose solution and on optical density (OD) value in culture solution at 576 nm wave width was carried out, the result indicates that suspension rate (%) of suspensions, while suspending in 0.76 mol/L sucrose solution for 15 minutes at room temperature, can be employed as an judgment criterion of cell state . Suspending cell suspension in 0.76 mol/L sucrose solution is also a good approach on selecting cells from suspension cell mass, as a result, it is beneficial to establishing cell suspension . The browning of cell suspension is related to the necrosis degree of suspension cells . OD value of cell suspension liquid at 576 nm can be taken as a criterion with which the necrosis degree of cell suspension can be evaluated.

Shi Yan Sheng Wu Xue Bao, 1999 Dec, 32(4), 401 - 8
Regeneration of Astragalus adsurgens via somatic embryogenesis from cell suspension protoplasts; Luo JP et al.; Protoplasts from 4-day-old embryogenic cell suspension cultures of Astragalus adsurgens, when cultured in KM8P medium which ammonium concentration was reduced to 2.5 mmol/L and supplemented with 0.5 mg/L NAA, 1.0 mg/L 2, 4-D, 0.7 mg/L BA and 0.4 mol/L glucose, underwent cell sustained divisions and formed cell colonies at a frequency of 16%-20% . Preplasmolysis or low temperature treatment of suspension cells prior to enzyme incubation enhanced colony formation . Following proliferation on MS medium containing 1.0 mg/L 2, 4-D and 0.5 mg/L BA, cell colonies were cultured on MS medium containing 0.1 mg/L NAA and 1.0 mg/L BA, where approximately 40% of colonies produced somatic embryos ranging in number from 20 to 40 per colony . No significant decrease was found in the potential of somatic embryogenesis when protoplast colonies were obtained from long-term cell suspensions . On hormone-free 1/2 MS medium, somatic embryos developed into intact plants, which showed normal morphology and stable chromosome number.

Shi Yan Sheng Wu Xue Bao, 1999 Jun, 32(2), 168 - 74
{Regulation of fungal elicitors on the cell growth and biosynthesis of saponin of Panax ginseng cell suspension culture}; Liu CJ et al.; Adding fungal elicitors to the Panax ginseng cell suspension cultures, the biosynthesis of saponin was obviously induced, the total productivity of saponin in cultures could increase more than 30% of the control . During elicitation, the accumulation patterns of saponin in suspension cultured cells were changed, the culture time for maximum biosynthesis of saponin was shortened 2-4 days comparing with that of the control, and about 80% of biosynthetic saponin in elicited cells was secreted into medium, meanwhile the uptake for sucrose in medium of cells was enhanced, and the disturbing of pH in medium was observed, which predicated that an ion exchange occurred between elicited cells and medium.

Hua Xi Yi Ke Da Xue Xue Bao, 2000 Sep, 31(3), 330 - 3
{An evaluation of adenosine triphosphate bioluminescence assay for tumor in vitro chemosensitivity testing}; Liu S et al.; This study was aimed at the feasibility of using ATP-bioluminescence assay for tumor in vitro chemosensitivity testing . With the use of this assay, the authors determined dose-response curve in mouse fibroma cell line L929 treated with chemotherapeutic agents, and investigated the different in vitro responses of 6 ovarian carcinomas (5 from fresh tumor tissues, 1 from ascites) treated with etopside, cis-plating, 5-fluorouracil and adriamycin . The results showed that the coefficients of variation for triplicate assays ranged from 1.2% to 15.8% which means high reproducibility of the assay . The single cell suspension (including < 30 cells clusters) could be separated from tissue fragments by means of enzyme cocktail (collagenase, Dnase, pronase) . The viable cells were over 90% . This study demonstrates that ATP-bioluminescence assay is a sensitive, reliable and efficient method for tumor chemosensitivity testing . In this connection, the correlation between in vitro drug sensitivity and in vivo patient response is worth further studying.

J Urol, 2003 Feb, 169(2), 718 - 20
Successful diagnosis of orthotopic rat superficial bladder tumor model by ultrathin cystoscopy; Asanuma H et al.; PURPOSE: In the orthotopic animal bladder tumor model noninvasive evaluation of the tumor establishment and the therapeutic effect has been difficult . To our knowledge we present the first diagnosis of orthotopic rat superficial bladder tumor model by ultrathin cystoscopy . MATERIALS AND METHODS: The established AY-27 rat bladder carcinoma cell line was transplanted orthotopically into 22 female Fischer F344 rats . A cell suspension containing 4 x 10 AY-27 cells was instilled into the bladder, which had been conditioned with mild acid washing . To evaluate tumor growth serial cystoscopy was performed via the urethra with an ultrathin endoscope (diameter 0.75 mm.) 5 to 14 days after tumor cell inoculation . At intervals after cystoscopic tumor detection the rats were sacrificed for autopsy and histological examination . RESULTS: In all 22 rats the orthotopic bladder tumor was established 7 to 10 days after tumor cell implantation and in most it was superficial . Cystoscopy permitted inspection of the urethra and whole bladder surface . We detected all tumors as broad based papillary mass (minimal lesion 1 mm . or less) and inspected its detailed appearance and accurate location . CONCLUSIONS: The orthotopic rat superficial bladder tumor model and the diagnostic procedure by ultrathin cystoscopy would be ideal system for preclinical evaluation of new potential intravesical therapies.

J Urol, 2003 Feb, 169(2), 701 - 5
Isolation of circulating cancer cells from whole blood by immunomagnetic cell enrichment and unenriched immunocytochemistry in vitro; Zigeuner RE et al.; PURPOSE: We improved tumor cell detection compared with currently available immunocytochemical methods by immunomagnetic cell enrichment . MATERIALS AND METHODS: Two methods of immunomagnetic enrichment using antibody coated magnetic beads were tested and compared with unenriched immunocytochemistry, including positive selection of epithelial cells with the antiepithelial antibody BER-EP4 and depletion of mononuclear cells with the anti-leukocyte antibody CD45 . Various numbers of tumor cells from the 4 tissue culture cell lines DU 145, RT-4, KTCTL-2 and KTCTL-30 obtained from urological tumors were added to whole blood and mononuclear cells were isolated by density centrifugation . After incubation of the cell suspensions with beads cell separation was done in a magnetic field . After centrifugation on glass slides immunocytochemical staining for cytokeratin was performed . A total of 96 experiments were completed and negative controls were obtained . RESULTS: The number of tumor cells detected by positive selection and depletion was significantly higher compared with immunocytochemistry (Wilcoxon test p <0.01) . Mean enrichment factor and tumor cell recovery rates were 12.9% and 43.5% for positive selection, and 9.4% and 32.6% for depletion, respectively (p <0.05) . With 1 tumor cell suspended in up to 30 ml . full blood unenriched immunocytochemistry failed to detect cancer cells, whereas positive selection revealed epithelial cells in 12 of 14 cases (85.5%) and depletion in all 14 (p <0.05) . No false-positive results were observed . CONCLUSIONS: Compared with unenriched immunocytochemistry immunomagnetic enrichment significantly improves the detection of epithelial cells added to blood . A significant advantage was observed for positive selection . Immunomagnetic enrichment may be important for clinical practice in the future.

Eur J Biochem, 2003 Feb, 270(3), 533 - 8
Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis; Hefner T et al.; The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1beta family of glycosyltransferases . This enzyme was used to prove, by site-directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation . Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity . Exchange of amino acids at the NRD site leads to a decrease of enzymatic activity, e.g . substitution of Glu368 by Asp . Glu368, which is a conserved amino acid in glycosyltransferases located at position 2 and is important for enzyme activity, does not serve as the nucleophile in the catalytic centre as proposed . When it is replaced by Ala, the resulting mutant enzyme E368A exhibits comparable activity as found for E368D in respect to vanillin . Enzyme activities of wild-type and E368A towards several substrates were not affected at the same level . His360 at position 1 of NRD1beta glycosyltransferases occupies a more crucial role as expected . When it is exchanged against other basic amino acids such as Lys or Arg the enzyme activity decreases approximately 1000-fold . Replacement of His360 by Glu leads to a mutant enzyme (H360E) with an approximately 4000-fold lower activity compared with the wild-type . This mutein still produces a beta-glucoside, not an alpha-glucoside and therefore indicates that generation of the typical E-E motif of NRD1alpha glycosyltransferases does not convert a NRD1beta enzyme into a NRD1alpha enzyme . The presented data do not support several suggestions made in the literature about catalytic amino acids involved in the glycosyltransfer reaction.

Anticancer Res, 2002 Sep-Oct, 22(5), 2869 - 75
Induction of syngeneic intradermal and orthotopic epidermal carcinomas in hairless Skh-1 mice as a reproducible model for experiments; Christophe M et al.; Epidermoid carcinomas, clinically and histologically similar to human squamous cell carcinomas (SCC), were obtained in hairless Skh-1 mice . Tumor cells originated from chemically-induced skin cancers . We developed three models of orthotopic skin tumors: (1) intradermal injection of a tumor cell suspension, (2) superficial abrasion of the skin, cell grafting and application of a hydrocolloid dressing, (3) skin incision, seeding and application of a hydrocolloid dressing . Intradermal injection was 100% successful . Skin incision, displaying histological evidence of rapid invasive tumor growth, was 75% successful . Though skin tumor growth after abrasion was only 20% successful, the tumor histogenesis exactly imitated human SCC development . These carcinomas provide research models for further experiments such as photodynamic therapy or antiangiogenesis therapy.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Feb 25, 785(1), 21 - 37
Alkylation of DNA by melphalan: investigation of capillary liquid chromatography-electrospray ionization tandem mass spectrometry in the study of the adducts at the nucleoside level; Van den Driessche B et al.; Nitrogen mustards are among the oldest cancer chemotherapeutic agents and remain the drugs of choice for treatment of many human cancers . A serious complication of treatment with nitrogen mustards is the increased risk of a secondary leukaemia in long-term survivors because not all alkylating agent interactions with DNA result in cell death . In an earlier study 2'-deoxy-5'-mononucleotide/melphalan adducts have been analysed by us by LC-ES MSMS . In this work we want to present the first results of the analysis of the corresponding 2'-deoxynucleoside/melphalan adducts from DNA hydrolysates by column switching/capillary LC-ES tandem mass spectrometry . Nucleosides, compared to nucleotides, give better chromatographic results and show a good sensitivity under electrospray (+) {ES(+)} ionisation . Several adducts were identified under ES(+) conditions . Mono-alkylated nucleoside adducts alkylated at the base moiety were identified for dGuo, dCyd and dAdo . Structures were identified by recording the low-energy CAD product ion scans . Also a mono-alkylated nucleotide pdA with alkylation position at the phosphate moiety could be detected . This proves that in the case of phosphate alkylation the enzymatic dephosphorylation reaction was inhibited . A Jurkat cell suspension was treated with melphalan (1 mM) and incubated at 37 degrees C (5% CO(2)) . After 6 and 48 h, the DNA was isolated and enzymatically hydrolysed . The corresponding nucleoside pool was evaluated with the developed LC-MS method . In the 48-h experiment, one adduct could be identified as a N-7 alkylated dGuo . In the 6-h experiment, no adducts could be found . Additional experiments were done wherein Jurkat-DNA, isolated from a non-treated cell culture, was treated with melphalan . These results were analogous with the data found in melphalan-treated calf thymus DNA . Additionally, we tried to determine the exact alkylation position by interpreting high-resolution fragmentation spectra.

J Histochem Cytochem, 2003 Feb, 51(2), 253 - 7
A photoreactive fluorescent marker for identifying eosinophils and their cytoplasmic granules in tissues; Eversole RR et al.; Here we describe a simple histochemical technique that provides an improved approach to identifying eosinophil components in tissues through the formation of photoreactive complexes that produce stable fluorescent emissions . This method worked readily with histological tissue sections 6-60 microm thick, which were fixed in neutral buffered formalin (NBF), and with cell suspensions similarly fixed and unfixed . Deep red (>605 nm) fluorescent emissions were produced by eosinophil-specific granules when exposed to broadband excitation spectra from a 100-W mercury lamp source (510-590 nm), as well as single-wavelength excitations from both an argon laser (488 nm) and a UV-visible laser (514 nm) . The fluorophore-granule complex emissions increased in intensity during the first minute of continuous photoexcitation, then remained stable (>10 min) . All nonspecific autofluorescence phenomena associated with these tissues were photobleached in the first minute, including areas of background Biebrich scarlet binding where photoreactive complexes were not formed (i.e., collagen), indicating environmental influences on the fluorophore . This technique allows the visualization of eosinophil granules over a greater period of time than is usually permissible with standard fluorescent markers . Therefore, techniques such as confocal microscopy can be utilized to their fullest extent, providing much more detailed information on the location and distribution of the cytoplasmic contents of eosinophils.

Arch Facial Plast Surg, 2003 Jan-Feb, 5(1), 103 - 8
Defect repair in the rat mandible with bone morphogenic proteins and marrow cells; Arosarena OA et al.; OBJECTIVE: To investigate the ability of a bone growth factor mixture and bone marrow cells to repair a critical size defect of the rat mandibular body . DESIGN: Prospective, randomized controlled trial . SUBJECTS: Thirty-seven male Fischer rats . INTERVENTIONS: Critical size defects 4 mm in diameter were created in the left mandibular bodies of the rats . The defects were filled with a bone marrow cell suspension (group 1), a synthetic bone matrix consisting of bovine collagen and calcium hydroxyapatite cement (group 2), the matrix and marrow cells (group 3), the matrix with 100 micro g of bone growth factor mixture (group 4), or the matrix with bone growth factor mixture and marrow cells (group 5) . Animals were killed after 8 weeks, and the nondemineralized specimens were processed histologically . Specimens from group 1 were not processed because there was no grossly appreciable bone regeneration . Stereologic techniques were used to determine and compare the volume fractions and volume estimates of mature bone, new bone, osteoid, marrow, remaining cement, and fibrous tissue in each defect . RESULTS: Volumes of mature bone, new bone, and remaining cement did not differ significantly among the groups (P =.30 for mature bone, P =.17 for new bone, and P =.34 for cement) . However, group 4 and 5 specimens contained significantly more osteoid and larger marrow spaces than did the group 2 and 3 specimens (P<.001 for both) . The specimens in groups 2 and 3 contained significantly more fibrous tissue ingrowth than did those in groups 4 and 5 (P<.001) . CONCLUSION: The synthetic bone substitute containing bone growth factor mixture was effective in stimulating new bone and osteoid development in the rat mandibular model.

J Biomed Mater Res A, 2003 Feb 1, 64(2), 235 - 41
Flow perfusion culture of marrow stromal osteoblasts in titanium fiber mesh; van den Dolder J et al.; The objective of this study was to evaluate the effect of two cell culture techniques, static and flow perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days . A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cells/300 microL) was seeded into the mesh material . Thereafter, the constructs were cultured under static conditions or in a flow perfusion system for 4, 8, and 16 days . To evaluate cellular proliferation and differentiation, constructs were examined for DNA, calcium content, and alkaline phosphatase activity . Samples were also examined with scanning electron microscopy (SEM) and plastic-embedded histological sections . Results showed an increase in DNA from day 4 to day 8 for the flow perfusion system . At day 8, a significant enhancement in DNA content was observed for flow perfusion culture compared with static culture conditions, but similar cell numbers were found for each culture system at 16 days . Calcium measurements showed a large increase in calcium content of the meshes subjected to flow perfusion at day 16 . The SEM examination revealed that the 16-day samples subjected to flow perfusion culture were completely covered with layers of cells and mineralized matrix . In addition, this matrix extended deep into the scaffolds . In contrast, meshes cultured under static conditions had only a thin sheet of matrix present on the upper surface of the meshes . Evaluation of the light microscopy sections confirmed the SEM observations . On the basis of our results, we conclude that a flow perfusion system can enhance the early proliferation, differentiation, and mineralized matrix production of bone marrow stromal osteoblasts seeded in titanium fiber mesh .

Planta, 2003 Jan, 216(3), 432 - 6 Epub 2002 Sep 18.
Formation of benzoquinol moiety in cornoside by salidroside mono-oxygenase, a cytochrome P450 enzyme, from Abeliophyllum distichumcell suspension cultures; Yamamoto H et al.; A microsomal fraction prepared from Abeliophyllum distichumNakai (Oleaceae) cell suspension cultures oxidized salidroside, a glucoside of 4-hydroxyphenylethyl alcohol, to cornoside possessing a unique benzoquinol ring . The enzyme named salidroside mono-oxygenase required NADPH as the only cofactor, and molecular oxygen . The reaction was strongly inhibited by CO as well as several cytochrome P450 inhibitors, such as cytochrome c and miconazole, indicating the involvement of a cytochrome P450 enzyme . Salidroside mono-oxygenase accepted salidroside as the only substrate, but did not oxidize 4-hydroxyphenylethyl alcohol, the salidroside aglucone, and 4-hydroxybenzoic acid . The optimum pH of the reaction was 7.5, and apparent K(m) values for salidroside and NADPH were 44 micro M and 33 micro M, respectively . The benzoquinol ring formation mechanism is discussed in comparison to the mechanism for ipso substitution of 4-hydroxybenzoate by active oxygen species followed by elimination leading to hydroquinone.

Cell Transplant, 2002, 11(7), 653 - 62
Effects of antioxidant pretreatment on the survival of embryonic dopaminergic neurons in vitro and following grafting in an animal model of Parkinson's disease; Love RM et al.; The effect of pretreating cell suspensions of embryonic rat ventral mesencephala (VM) with antioxidant combinations on the survival of dopaminergic (DA) neurons was studied in vitro and following transplantation into the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease . The in vitro experiments examined the effects of two thiol antioxidants, N-acetyl-L-cysteine (NAC) and reduced glutathione (GSH), and a member of the lazaroid family of 21-aminosteroids, U-83836E, singly and in combination, on survival of DA neurons derived from dissociated E14 rat VM tissue . For in vivo studies, cell suspensions were pretreated with combinations of NAC, GSH, and U-83836E prior to transplanting into 6-OHDA-lesioned rats to investigate whether DA neuron survival could be further improved . NAC, GSH, and U-83836E individually increased DA neuron survival in vitro and a combination of all three resulted in the greatest survival . In vivo, pretreatment with U-83836E alone resulted in a significantly greater reduction in amphetamine-induced rotation 6 weeks postgrafting compared with a control group receiving nontreated graft tissue . This functional effect correlated with a significant improvement in DA neuron survival 6 weeks postgrafting . The thiol combination pretreatment of NAC and GSH, and the triple combination of NAC, GSH, and U-83836E, however, failed to improve both functional recovery and DA neuron survival when compared with the nontreated control grafts.

Cell Transplant, 2002, 11(7), 637 - 52
The release of excitatory amino acids, dopamine, and potassium following transplantation of embryonic mesencephalic dopaminergic grafts to the rat striatum, and their effects on dopaminergic neuronal survival in vitro; Zietlow R et al.; A major limitation to the effectiveness of grafts of fetal ventral mesencephalic tissue for parkinsonism is that about 90-95% of grafted dopaminergic neurones die . In rats, many of the cells are dead within 1 day and most cell death is complete within 1 week . Our previous results suggest that a major cause of this cell death is the release of toxins from the injured CNS tissue surrounding the graft, and that many of these toxins have dissipated within 1 h of inserting the grafting cannula . In the present experiments we measured the change over time in the concentration of several potential toxins around an acutely implanted grafting cannula . We also measured the additional effect of injecting suspensions of embryonic mesencephalon, latex microspheres, or vehicle on these concentrations . Measurements of glutamate, aspartate, and dopamine by microdialysis showed elevated levels during the first 20-60 min, which then declined to baseline . In the first 20 min glutamate levels were 10.7 times, aspartate levels 5 times, and dopamine levels 24.3 times baseline . Potassium levels increased to a peak of 33 +/- 10.6 mM 4-5 min after cannula insertion, returning to baseline of <5 mM by 30 min . Injection of cell suspension, latex microspheres, or vehicle had no significant effect on these levels . We then assayed the effect of high concentrations of glutamate, aspartate, dopamine, and potassium on dopaminergic neuronal survival in E14 ventral mesencephalic cultures . In monolayer cultures only dopamine at 200 microM showed toxicity . In three-dimensional cultures only the combination of raised potassium, dopamine, glutamate, and aspartate together decreased dopaminergic neuronal survival . We conclude that toxins other than the ones measured are the main cause of dopaminergic cell death after transplantation, or the effects of the toxins measured are enhanced by anoxia and metabolic challenges affecting newly inserted grafts.

Biotechnol Bioeng, 2003 Mar 5, 81(5), 625 - 8
Cell-type-specific adhesion onto polymer surfaces from mixed cell populations; Quirk RA et al.; The targeted adhesion of a specific cell type from a mixed cell suspension via the surface presentation of a cell-specific ligand is demonstrated . This generic strategy is illustrated by the covalent attachment of a galactose derivative to a polylysine backbone via the amine functionality . Following adsorption of the resultant material to a polymer surface, hepatocyte adhesion is increased via the interaction between galactose and asialoglycoprotein receptors in a concentration-dependent manner . The selective nature of the material is demonstrated by the approximate doubling in the adhesion of hepatocytes relative to a nontargeted cell type (hepatic stellate cells), and an inability of the modified polymer surface to attract additional numbers of the nontargeted cells . This strategy provides a mechanism for controlling the ratios of cell types adhering to scaffold supports, thus enabling the rapid creation of defined coculture systems from heterogeneous cell suspensions .

Plant Cell Physiol, 2002 Dec, 43(12), 1510 - 7
Utilization and transport of glucose in Olea Europaea cell suspensions; Oliveira J et al.; Cell suspensions of Olea europaea var . Galega Vulgar grown in batch culture with 0.5% (w/v) glucose were able to transport D-{(14)C}glucose according to Michaelis-Menten kinetics associated with a first-order kinetics . The monosaccharide carrier exhibited high affinity (K(m) approximately 50 micro M) and was able to transport D-glucose, D-fructose, D-galactose, D-xylose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not D-arabinose, D-mannitol or L-glucose . D-{(14)C}glucose uptake was associated with proton uptake, which also followed Michaelis-Menten kinetics . The transport of 3-O-methyl-D-glucose was accumulative (40-fold, at pH 5.0) and the protonophore carbonyl cyanide m-chlorophenylhydrazone strongly inhibited sugar accumulation . The results were consistent with the involvement of a monosaccharide: proton symporter with a stoichiometry of 1 : 1 . When cells were grown with 3% (w/v) glucose, the uptake of D-{(14)C}glucose followed first-order kinetics and monosaccharide:proton symporter activity was not detected . The value obtained for the permeability coefficient of hexoses in O . europaea cells supported the hypothesis that the first-order kinetics observed in 0.5% and 3% sugar-grown cells was produced exclusively by passive diffusion of the sugar . The results indicate that in O . europaea cells sugar levels have a regulatory effect on sugar transport, because the activity for monosaccharide transport was repressed by high sugar concentrations.

Plant Cell Physiol, 2002 Dec, 43(12), 1502 - 9
Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa; Flores-Sanchez IJ et al.; Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected . Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase . The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells . Elicited cells also transformed more rapidly a higher percentage of {5-(3)H}mevalonic acid into triterpene acids . Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited . These results suggest that in U . tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.

Thorax, 2003 Jan, 58(1), 23 - 9
Sputum T lymphocytes in asthma, COPD and healthy subjects have the phenotype of activated intraepithelial T cells (CD69+ CD103+); Leckie MJ et al.; BACKGROUND: T cells of intraepithelial phenotype have previously been detected in bronchoalveolar lavage (BAL) fluid in a range of lung diseases; these cells express the adhesion molecule alpha(E)beta(7) integrin, CD103, the ligand for epithelial cell E-cadherin . In subjects with asthma CD4+ lymphocytes are the predominant T cell subtype found in bronchial biopsy specimens and in BAL fluid, whereas CD8+ lymphocytes have been shown to predominate in subjects with chronic obstructive pulmonary disease (COPD) . The aim of this study was to analyse the expression of CD103, activation markers (CD25 and CD69), and chemokine receptors (CXCR3, CCR5 and CCR3) on CD4+ and CD8+ lymphocytes from sputum and peripheral blood of subjects with asthma, COPD, and healthy controls . METHODS: T cell surface markers were assessed by immunofluorescence labelling and flow cytometry of gated lymphocytes among CD45+ leucocytes in sputum cell suspensions . RESULTS: Sputum lymphocytes expressed higher levels of CD103 and CD69 than blood lymphocytes in all subject groups, with CD103 expressed at higher levels on CD8+ than on CD4+ cells . There were no detectable differences in numbers of CD4+ and CD8+ T cells between subjects with asthma, COPD and controls . The percentage of sputum lymphocytes expressing CXCR3 was lower in subjects with asthma or COPD than in healthy controls; CCR3 was not detectable on sputum or blood lymphocytes . CONCLUSIONS: Sputum T lymphocytes are predominantly of activated intraepithelial phenotype (CD103+ CD69+), and normal numbers of CD4+ and CD8+ T cell populations are found in the sputum of patients with asthma and COPD.

Histol Histopathol, 2003 Jan, 18(1), 103 - 10
A comparison between double and triple therapies of octreotide, galanin and serotonin on a rat colon carcinoma; Sitohy B et al.; Sixty female nude mice (C578L/6jBom-nu) were injected with 100 microl cell suspension containing 2 x 10(6) viable cells of an N-methyl-N-nitroguanidine-induced rat colonic adenocarcinoma . After seven days the animals were divided into five groups . The first group received only saline and served as a control group . The second group received a triple therapy of octreotide, galanin and serotonin (20 microg/kg) . The last three groups received double therapies of octreotide/galanin, octreotide/serotonin or galanin/serotonin (20 microg/kg) . They were treated twice a day for five days . Tumour volume and weight, relative volume density of tumour-feeding blood vessels and of tumour necrotic tissue, as well as apoptotic and proliferation indices were determined . Animal weight, food consumption, faeces weight and its water content were recorded before and after treatment . Tumour volume was significantly reduced only in the group that received the triple therapy . The volume density of the tumour-feeding blood vessels was significantly reduced in the treated groups with the exception of the group that received octreotide and serotonin . Increased relative volume density of tumour necrotic tissue occurred only in the group treated with triple therapy . Apoptotic indices were significantly increased in all treated groups . No statistical difference was found between treated animals and controls regarding proliferation indices, food consumption, faeces weight and water content or animal weight . In conclusion, double therapy using two of the gastrointestinal bioactive substances, octreotide, galanin and serotonin, has certain effects on colon cancer cells . To cause a considerable tumour necrosis, triple therapy seems to be required . Both double and triple therapy seem to lack obvious side-effects.

Cytometry A, 2003 Jan, 51(1), 46 - 51
A new method for improving metaphase chromosome spreading; Deng W et al.; BACKGROUND: The success of complex molecular cytogenetic studies depends on having properly spread chromosomes . However, inconsistency of optimum chromosome spreading remains a major problem in cytogenetic studies . METHODS: The metaphase spreading process was carefully timed to identify the most critical phase of chromosome spreading . The effects of dropping height of cell suspension, slide condition, drying time, fixative ratio, and relative humidity on the quality of metaphase spreads were studied by quantitative examination of metaphase chromosome spreads . Normal and immortalized human epithelial ovarian cells, neuroblastoma cells, and normal lymphocytes were tested . RESULTS: Humidity over the slide was the most important variable affecting the quality of chromosome spreads . Consistent improvement in chromosome spreading (larger metaphase area, less chromosome overlaps, or lower frequencies of broken metaphases) was obtained for all cell types if dynamic cell rehydration, occurring as fixative absorbs moisture from air, was made to coincide with the prompt fixation of spread chromosomes to the slide . This was achieved by dropping cells on dry glass slides placed in a shallow metal tray and then quickly lowering the tray into a covered 50 degrees C water bath for slide drying . CONCLUSIONS: A new and simple method for improving metaphase chromosome spreading was developed based on our study on the characteristics of chromosome spreading .

Jpn J Cancer Res, 2002 Dec, 93(12), 1366 - 77
Impact of the p53 status of the tumor cells on the effect of reactor neutron beam irradiation, with emphasis on the response of intratumor quiescent cells; Masunaga S et al.; Human head and neck squamous cell carcinoma cells transfected with mutant p53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both the hind legs of Balb/cA nude mice . Tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors . After administration of sodium borocaptate-10B (BSH) or p-boronophenylalanine-10B (BPA), the tumors were irradiated with neutron beams . The tumors not treated with 10B-compound were irradiated with neutron beams or gamma-rays . The tumors were then excised, minced and trypsinized . The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU . Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells . The MN and apoptosis frequencies in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU . Without 10B-carriers, in both tumors, the relative biological effectiveness of neutrons was greater in Q cells than in total cells, and larger for low than high cadmium ratio neutrons . With 10B-carriers, the sensitivity was increased for each cell population, especially for total cells . BPA increased both frequencies for total cells more than BSH . Nevertheless, the sensitivity of Q cells treated with BPA was lower than that of BSH-treated Q cells . These sensitization patterns in combination with 10B-carriers were clearer in SAS/neo than in SAS/mp53 tumors . The p53 status of the tumor cells had the potential to affect the response to reactor neutron beam irradiation following 10B-carrier administration.

In Vivo, 2002 Nov-Dec, 16(6), 479 - 92
Vascular stroma-derived endothelial colony forming progenitor cells adapt to tumour growth; Lincoln DT et al.; Under appropriate nutrient agar culture conditions, primary or xenografted human and animal tumour biopsy-derived cell suspensions will form two types of colony . The first type, consisting of tight colonies of round cells which form tumours when introduced into nude mice, is of neoplastic origin . The second type of colony, the cells of which fail to form tumours on injection into nude mice, consists of loose colonies of larger, inter-connecting elongated bi- or tripolar cells and is thought to originate from vascular stroma-derived endothelial colony forming progenitor cells (V-ECPC) . The likely importance of V-ECPC to tumour growth is emphasised by a positive correlation between the VECPC-derived endothelial cell colonies and both tumour vascularity and growth rate . A high cloning efficiency obtained from tumours of particularly intense vascular nature indicates that this cell is of importance in vascular adaptation and therefore tumour growth . In contrast, avascular, fibrotic tumour tissue yielded very low numbers of stromal vascular endothelial cell colonies . The results suggest that stromal vascular endothelial cell colonies do not arise from the mature fibroblastic elements of the tumour stroma, but rather from cells within actively growing regions . Tritiated thymidine uptake studies show that the vascular stroma-derived endothelial colony forming progenitor cells cell are cycling . Cell separation studies have characterized the as yet morphologically unidentified V-ECPC as having a sedimentation rate of 4.7 mm./hr and a mean density of 1.068 g/cm3 and hence a calculated volume of 450 microns 3.

Planta Med, 2002 Dec, 68(12), 1113 - 7
Biotransformation of 2alpha,5alpha,10beta,14beta-tetraacetoxy-4(20),11-taxadiene by cell suspension cultures of Catharanthus roseus; Dai J et al.; Catharanthus roseus cell suspension cultures were employed for the biotransformation of 2alpha,5alpha,10beta,14beta-tetraacetoxy-4(20),11-taxadiene ( 1), and four metabolites were obtained . Based on their physical and chemical data, the structures of the four metabolites were respectively elucidated as 10beta-hydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (2), 5alpha-hydroxy-2alpha,10beta, 14beta-triacetoxy-4(20),11-taxadiene (3), 6alpha,10beta-dihydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (4), and 6alpha,9alpha,10beta-trihydroxy-2alpha,5alpha,14beta-triacetoxy-4(20),11-taxadiene (5), among which 3 and 5 were characterized as new taxoids . The effects of the stages of substrate addition on the biotransformation were also investigated . The results revealed that the biotransformation rate for 1 reached 85.3 % and the yield of 2 70 % when 1 was administered during the mid-linear phase (9 - 12 th day) of the cell growth cycle . On the other hand, the yield for 4 reached the highest level of 11.8 % when 1 was added in the early linear phase (6 th day).

Plant J, 2002 Dec, 32(6), 1033 - 48
A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula; Suzuki H et al.; The saponins of the model legume Medicago truncatula are glycosides of at least five different triterpene aglycones: soyasapogenol B, soyasapogenol E, medicagenic acid, hederagenin and bayogenin . These aglycones are most likely derived from beta-amyrin, a product of the cyclization of 2,3-oxidosqualene . Mining M . truncatula EST data sets led to the identification of sequences putatively encoding three early enzymes of triterpene aglycone formation: squalene synthase (SS), squalene epoxidase (SE), and beta-amyrin synthase (beta-AS) . SS was functionally characterized by expression in Escherichia coli, two forms of SE by complementation of the yeast erg1 mutant, and beta-AS by expression in yeast . Beta-amyrin was the sole product of the cyclization of squalene epoxide by the recombinant M . truncatulabeta-AS, as judged by GC-MS and NMR . Transcripts encoding beta-AS, SS and one form of SE were strongly and co-ordinately induced, associated with accumulation of triterpenes, upon exposure of M . truncatula cell suspension cultures to methyl jasmonate . Sterol composition remained unaffected by jasmonate treatment . Molecular verification of induction of the triterpene pathway in a cell culture system provides a new tool for saponin pathway gene discovery by DNA array-based approaches.

Eur J Biochem, 2003 Jan, 270(1), 66 - 75
Bioenergetics of the formyl-methanofuran dehydrogenase and heterodisulfide reductase reactions in Methanothermobacter thermautotrophicus; de Poorter LM et al.; The synthesis of formyl-methanofuran and the reduction of the heterodisulfide (CoM-S-S-CoB) of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) are two crucial, H2-dependent reactions in the energy metabolism of methanogenic archaea . The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures ( pH2) . Formyl-methanofuran synthesis is an endergonic reaction (DeltaG degrees ' = +16 kJ.mol-1) . By analyzing the concentration ratios between formyl-methanofuran and methanofuran in the cells, free energy changes under experimental conditions (DeltaG') were found to range between +10 and +35 kJ.mol-1 depending on the pH2 applied . The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions . Heterodisulfide reduction (DeltaG degrees ' = -40 kJ.mol-1) was associated with free energy changes as high as -55 to -80 kJ.mol-1 . The values were determined by analyzing the concentrations of CoM-S-S-CoB, HS-CoM and HS-CoB in methane-forming cells operating under a variety of hydrogen partial pressures . Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction . Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at pH2<0.12 bar, whilst the number was four for cells grown above that concentration . The shift occurred within a narrow pH2 span around 0.12 bar . The findings suggest that the methanogens regulate the bioenergetic machinery involved in CoM-S-S-CoB reduction and proton pumping in response to the environmental hydrogen concentrations.

Rinsho Byori, 2002 Nov, 50(11), 1079 - 84
{DNA typing for determination of zygosity on multiple pregnancies using fragment analysis for microsatellite}; Kubota N et al.; Zygosity on multiple pregnancies is important factor of prognosis in fetus and new bones . In the past, determination of zygosity has been made mainly on the basis of histological and microscopic observations . Such a morphological distinction, however is not sufficient . Fragment analysis based on capillary electrophoresis has been developed as a rapid and accurate technique for DNA typing . To elucidate the clinical availability of fragment analysis, we examined the zygosity of nine multiple pregnancies (three twins and six triplets) by fragment analysis of microsatellite DNAs and other common DNA tests (PCR-SSCP assay and VNTR analysis using agarose gel electrophoresis) . Fragment analysis and PCR-SSCP identified the heterozygosity in two of three twins and all of six triplets including one presumable dizygotic triplet, while VNTR analysis failed to recognize one of two dizygotic twins and three of five trizygotic triplets . Although detection efficiency of PCR-SSCP seems to be similar to that of fragment analysis, PCR-SSCP could not separate the several microsatellite DNAs that fragment analysis showed to be heterozygous . Moreover, fragment analysis could detect a minor population possessing the different DNA types (> 5%) in the same cell suspension . The present results suggest that fragment analysis of microsatellite DNAs may be an excellent and beneficial method for clinical application of zygosity determination in multiple pregnancies.

Biotechnol Bioeng, 2003 Feb 20, 81(4), 474 - 81
Maximization of hydrogen production ability in high-density suspension of Rhodovulum sulfidophilum cells using intracellular poly(3-hydroxybutyrate) as sole substrate; Maeda I et al.; Growth of and hydrogen production by wild-type (WT) Rhodovulum sulfidophilum were compared with those by one of its mutants lacking the poly(3-hydroxybutyrate) (PHB) biosynthesis ability (PNM2) . During phototrophic growth under aerobic conditions with fixed illumination, changes in the extinction coefficient and PHB content of WT and PNM2 cells revealed interference of light penetration by PHB . WT cells synthesized PHB at an early stage of the cultivation . PHB degradation after exhaustion of acetate during the cultivation of WT resulted in a decrease of the extinction coefficient . The hydrogen production rate under anaerobic conditions with fixed illumination was examined in WT and PNM2 cell suspensions at different densities . The hydrogen production rate was determined not by the light penetration but by the kinds of hydrogen donors and the density of suspension . The highest value of the rate of hydrogen production from PHB, 33.0 ml/l/h, was improved compared with 26.6 ml/l/h, which was the highest value in hydrogen production from succinate . Under the same illumination, conversion to hydrogen from PHB is more efficient than that from succinate, which is one of the best substrates for hydrogen production . These results suggest that the hydrogen production rate can be maximized in the hydrogen production system based on PHB degradation, which is achieved in high-density suspension under external-substrate-depleted conditions after aerobic cultivation in the presence of an excess amount of acetate .

J Control Release, 2003 Jan 9, 86(1), 25 - 32
Stimulation of spleen cells in vitro by nanospheric particles containing antigen; Eyles JE et al.; Activation of cells, in primary culture, by nanospheres containing antigen has been investigated . Single cell suspensions of spleen cells from primed and nai;ve animals were cocultured with escalating quantities of soluble tetanus toxoid (TT) or TT encapsulated within nanospheres fabricated from poly(lactide-co-glycolide) (PLGA) . Concomitantly, spleen cells were also cultured in the presence of 'empty' PLGA nanospheres that contained no TT . Nanospheres loaded with antigen were found to elicit increased proliferation of splenocytes from preimmunised mice in comparison to free antigen during coculture at equivalent doses of immunogen (at low and intermediate doses) . Interestingly, cellular proliferation was abolished if B-cells were removed from the splenocyte cultures . Production of IFN-gamma and IL-6 was increased, for formulated as compared to free antigen, in microcultures from both nai;ve and pre-immunised animals . Secretion of IFN-gamma or IL-6 was not observed when primed or nai;ve spleen cells were stimulated with 'empty' polymeric spheres . Some unspecific cytotoxicity was detected if cells were cocultured with high concentrations of PLGA particles, although toxic effects were not seen at concentrations where maximum levels of cytokine secretion and cellular proliferation were recorded . These cell culture data indicate that, at least in this in vitro model, nanoparticulate TT is able to elicit cytokine production that is probably consistent with increased stimulation . This mechanism is likely to be distinct from non-specific effects caused by components of the delivery vehicle itself.

Mech Dev, 2003 Jan, 120(1), 89 - 98
Embryonic liver cells and permanent lines as models for hepatocyte and bile duct cell differentiation; Strick-Marchand H et al.; Analysis of liver cells during development is facilitated by the possibility of complementing in vivo analysis with experiments on cultured cells . In this review, we discuss results from several laboratories concerning bipotential hepatic stem cells from mouse (HBC-3, H-CFU-C, MMH and BMEL), rat (rhe14321) and primate (IPFLS) embryos . Several groups have used fluorescence-activated cell sorting to identify clonogenic bipotential cells; others have derived bipotential cell lines by plating liver cell suspensions and cloning . The bipotential cells, which probably originate from hepatoblasts, can differentiate as hepatocytes or bile duct cells, and undergo morphogenesis in culture . Disparities in differentiation can be explained by distinct medium compositions, extracellular matrix coated culture surfaces, and gene expression detection methods . Potential applications of these cell lines are discussed.

Zhonghua Wai Ke Za Zhi, 2002 Oct, 40(10), 783 - 5
{Long-term culture and differentiation of neural stem cells of embryonic mice}; Yang L et al.; OBJECTIVES: To assess the culture and differentiation of neural stem cells in embryonic mice and set up a basis for further research in to neural stem cells . METHODS: Embryonic cortices of mice were dissociated and single cell suspensions were achieved by mechanical methods in sterile conditions, and cells were seeded in uncoated plate in N2 medium . The cells were passaged by mechanical methods, frozen and thawed by general procedure . They were identified by immunocytochemical techniques . RESULTS: Neural stem cells from embryonic mice were successfully cultured forming typical neurospheres in suspension . Neurons, astrocytes and oligodendrocytes were differentiated from neural stem cells, with a ratio of 7%, 85% - 90% and 2% - 4% respectively . CONCLUSIONS: Neural stem cells, which can be cultured and passaged steadily in vitro and they are the ideal cell sources for cell transplantation and gene therapy.

Zhonghua Zhong Liu Za Zhi, 2002 Sep, 24(5), 451 - 4
{Anti-tumor effect through human endostatin gene transfer in mice bearing B16 melanoma}; Zhang B et al.; OBJECTIVE: To investigate the effects of direct intratumor injection of the packaged cells with retroviral vector carrying human endostatin (hEN) on the growth inhibition of B16 melanoma in C57/BL6 mice . METHODS: Retroviral vector, pLNC-hEN, was constructed with modified and identified hEN gene . The cell line, PA317, was used to establish ecotropic virus producing cells by transfecting and packing with pLNC-hEN . Then the cells were injected directly into the tumor in C57/BL6 mice bearing B16 melanoma, established by intra-cutaneous injection of B16 cell suspension . The tumor size was measured at different intervals to observe the antitumor effect . Micro-vessel density (MVD) in the tumor tissue was evaluated by immunohistological examination to count the apoptotic cells by TUMEL staining . RESULTS: Tumor with diameter of 2 - 3 mm was observed in all mice after 7 - 9 days . The average tumor volume on D3, D5, D7 and D9 after gene transfection was 4.67 +/- 1.1, 22.25 +/- 13.06, 84.17 +/- 43.5 and 155.08 +/- 81.1 mm(3) in the gene therapy group but 136.17 +/- 30.61, 390.17 +/- 220.47, 1 021.67 +/- 537.4 and 2 920.2 +/- 220.01 mm(3) in the control group, the difference of which was statistically significant . The average MVD in the gene therapy and control groups were 8 +/- 2.28 and 28.17 +/- 5.31 while the average apoptotic cell number in the two groups were 23.33 +/- 3.83 and 2.33 +/- 1.21, both of which were statistically significant . CONCLUSION: The direct injection of packaged cells carrying hEN gene is able to inhibit the growth of micro-blood vessels and promote tumor cell apoptosis, which ultimately inhibits the growth of B16 melanoma.

Phytochemistry, 2003 Jan, 62(2), 127 - 37
A flavonol O-methyltransferase from Catharanthus roseus performing two sequential methylations; Cacace S et al.; Protein extracts from dark-grown cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) contained several O-methyltransferase (OMT) activities, including the 16-hydroxytabersonine O-methyltransferase (16HT-OMT) in indole alkaloid biosynthesis . This enzyme was enriched through several purification steps, including affinity chromatography on adenosine agarose . SDS-PAGE of the purified protein preparation revealed a protein band at the size expected for plant OMTs (38-43 kDa) . Mass spectrometry indicated two dominant protein species of similar mass in this band, and sequences of tryptic peptides showed similarities to known OMTs . Homology-based RT-PCR identified cDNAs for four new OMTs . Two of these cDNAs (CrOMT2 and CrOMT4) encoded the proteins dominant in the preparation enriched for 16HT-OMT . The proteins were closely related (73% identity), but both shared only 48-53% identity with the closest relatives found in the public databases . The enzyme functions were investigated with purified recombinant proteins after cDNA expression in Escherichia coli . Unexpectedly, both proteins had no detectable 16HT-OMT activity, and CrOMT4 was inactive with all substrates investigated . CrOMT2 was identified as a flavonoid OMT that was expressed in dark-grown cell cultures and copurified with 16HT-OMT . It represented a new type of OMT that performs two sequential methylations at the 3'- and 5'-positions of the B-ring in myricetin (flavonol) and dihydromyricetin (dihydroflavonol) . The resulting methylation pattern is characteristic for C . roseus flavonol glycosides and anthocyanins, and it is proposed that CrOMT2 is involved in their biosynthesis.

Immunity, 2002 Dec, 17(6), 737 - 47
Increasing tumor antigen expression overcomes "ignorance" to solid tumors via crosspresentation by bone marrow-derived stromal cells; Spiotto MT et al.; To explain why solid cancers grow or are rejected, we examined how the tumor stroma affected the level of antigen expression necessary to induce an immune response . We applied a tamoxifen-regulated Cre-loxP system to induce a model SIYRYYGL antigen recognized by the 2C T cell receptor . Solid tumors expressing the antigen at lower levels grew, whereas solid tumors expressing antigen induced to 26-fold higher levels were rejected . In contrast, mice rejected cell suspensions expressing higher or lower levels of the antigen . The antigen was likely crosspresented because draining lymph node responses required bone marrow-derived cells in the tumor stroma . Thus, tumor antigens expressed at levels sufficient for crosspresentation by bone marrow-derived stromal cells may overcome immunological "ignorance" to solid tumors.

Ai Zheng, 2002 Aug, 21(8), 833 - 7
{Circadian rhythms of DNA synthesis and apoptosis correlated gene expression in bone marrow cells of nude mice bearing human nasopharyngeal carcinoma}; Sun J et al.; BACKGROUND & OBJECTIVE: No report was found on research of circadian rhythms of nasopharyngeal carcinoma(NPC) . This study was designed to investigate the circadian rhythms of DNA synthesis and apoptosis correlated gene expression in bone marrow cells of nude mice bearing NPC and to collect necessary data for making clinical chronochemotherapy schedule of NPC . METHODS: Sixty-nine BALB/C nude mice were synchronized with an alternative lighting regimen with 12 hours in light and 12 hours in dark (LD 12:12) for at least 3 weeks . Human nasopharyngeal poorly differentiated squamous cell carcinoma (CNE-2) cells were implanted into each mouse . Ten days after transplantation, the mice were sacrificed, bone marrow cells were collected in the 3, 7, 11, 15, 19, and 23 hours after light onset (HALO) . Single cell suspension was obtained and stained with propidium iodide . The cellular DNA content was measured by flow cytometry . Data were analyzed with analysis of variance(ANOVA) and Cosinor analysis . Proteins were extracted from bone marrow cells and determined; Bcl-2, p53, and p21 expression were tested using Western blot analysis . RESULTS: The proportion of bone marrow cells in phase G1, S, and G2/M varied according to circadian sampling time with statistical significance (ANOVA) . Moreover, such variation in G1 and G2/M was statistically valid by Cosinor analysis with an acrophase located at 10.8 HALO and 1.8 HALO, respectively . The distribution curves of phase G1 and G2/M fit to Cosinor changes but not for S phase cells . The expression of p53 and Bcl-2 protein level in bone marrow cells varied in 24 h time scale . No p21 protein expression was found in this experiment . CONCLUSION: DNA synthesis of bone marrow cells in nude mice bearing human nasopharyngeal poorly differentiated squamous cell carcinoma (CNE-2) cells varies according to circadian rhythm . The expression of p53 and Bcl-2 protein level in bone marrow cells varies in 24 h time scale.

Gynecol Oncol, 2002 Nov, 87(2), 193 - 9
Development of an in vitro chemo-radiation response assay for cervical carcinoma; Monk BJ et al.; PURPOSE: To determine if synergistic effects of radiation (RT) and chemotherapy (chemo) on human cervical carcinoma cell lines and fresh tumor explants could be determined using an in vitro assay . EXPERIMENTAL DESIGN: In vitro radiation response was determined for 4 cell lines and 26 fresh tumor explants in an agar-based assay . Cells were exposed to increasing doses of RT with or without cisplatin (CDDP), carmustine (BCNU), buthionine sulfoximine (BSO), or paclitaxel (Tax) . Cell suspensions were cultured for 5 days, with {(3)H}thymidine added on day 3 and proliferation was measured . Results were reported as the fraction of proliferation compared to control (FC) . For each combination of irradiation and drug, synergy was tested using the Chou analysis, where a combination index (CI) <1 indicated synergistic interaction . In simple correlation analysis, an R value of >0.7 indicated cross-resistance . RESULTS: RT dose-dependent proliferation inhibition was observed for 2 of the 4 cell lines, and for all but 1 of the fresh specimens . Significant heterogeneity of tumor response to RT was seen . Four specimens that were 1 standard deviation below the median FC response after exposure to 300 cGy were classified as extremely radiation resistant . Twenty-one tumors were evaluated for synergistic response using the combination of chemo and RT with a median FC of 0.27 (+/-0.27) for 6.0 Gy of RT alone, 0.22 (+/-0.21) for CDDP alone, and 0.05 (+/-0.08) for the combination . A CI of 0.35 and an R value of 0.09 demonstrated synergy between chemo and RT without cross-resistance . Similar synergy without cross-resistance was found for RT in combination with BCNU, BSO, and TAX . CONCLUSIONS: Heterogeneous RT dose-response relationships in the in vitro assay were demonstrated . Explants were more sensitive to RT than cell lines . Unlike cell lines, fresh tumor cells consistently displayed synergy with RT and chemo . The synergy between RT and BSO suggests that glutathione depletion may enhance the effect of RT . The assay was feasible for examining fresh tumors and may be an important tool for studying RT or drug resistance . Clinical trials to evaluate this assay are indicated.

Scand J Immunol, 2002 Dec, 56(6), 652 - 64
Interferon-gamma-secreting T cells localize to the epithelium in coeliac disease; Olaussen RW et al.; Increased levels of interferon-gamma (IFN-gamma) transcripts have previously been found in duodenal biopsy specimens from patients with untreated coeliac disease (CD) . Such samples and duodenal control mucosa were therefore studied to locate and phenotype cells spontaneously secreting IFN-gamma . Specimens were collected from consecutively recruited patients with untreated (seven), treated (four) or refractory (three) CD and from five histologically normal controls . Morphological and immunohistochemical examinations were performed, and epithelial and lamina propria cell suspensions were prepared from parallel samples . Unstimulated viable cells secreting IFN-gamma were identified and phenotyped with a new fluorescence-activated cell sorter-based assay, and IFN-gamma messenger RNA (mRNA) was analysed in snap-frozen aliquots of the same suspensions . Untreated CD cases had the highest fraction of IFN-gamma+ cells in the epithelial compartment (median 2.6%, range 1.6-6.2%) and, less strikingly, in the lamina propria compartment (1.6%, range 0.3-3.6%), followed by refractory (1.4%, 1.0-1.9%; and 0.3%, 0.0-1.2%) and treated (0.8%, 0.5-0.9%; and 0.7%, 0.2-1.1%) disease and finally the controls (0.5%, 0.3-0.9%; and 0.2%, 0.1-0.7%) . IFN-gamma mRNA data supported these findings . IFN-gamma+ intraepithelial lymphocytes were mostly CD3+ and CD8+, whereas many positive lamina propria cells were CD8- . We conclude that isolated T cells spontaneously secreting IFN-gamma localize preferentially in the epithelium of patients with classical and refractory CD.

Biotechnol Prog, 2002 Nov-Dec, 18(6), 1377 - 86
A lab-built respirometer for plant and animal cell culture; Lamboursain L et al.; A very simple off-line respirometer was developed to measure oxygen consumption rates of low respiring and shear-sensitive cell suspensions . The respirometer is composed of a 10 mL glass syringe in which the plunger was substituted with a polarographic dissolved oxygen probe . Mechanical agitation is provided by means of a magnetic stirring bar inside the measuring chamber and a stir plate placed below the respirometer . Abiotic oxygen fluxes occurring in the measurement chamber such as oxygen diffusion and probe oxygen consumption were investigated . The apparent oxygen uptake rate was then corrected for abiotic oxygen fluxes, leading to accurate measurements of respiration rates ranging from 0.5 to 25.0 mM x h(-1) . Additionally, the effect of the stirring bar shape and of the test length on the integrity of plant (Eschschzoltzia californica) and animal (NS0) cells was evaluated . Animal cells showed a higher resistance to mechanical stirring inside the respirometer compared to plant cells (0% of broken cells and 78.1% respectively for a polygonal stirring bar and a 15 min test) . For plant cells, cell damage inside the measurement chamber was reduced by optimizing the stirring bar shape and reducing the test length to 5 min or less . This very simple design was shown to provide reliable and low-cost quantification of the oxygen uptake rate of plant and animal cells and can be use even for more demanding measurements such as oxygen affinity studies.

Neoplasma, 2002, 49(5), 300 - 6
Detection of 13q abnormalities in multiple myeloma using immunomagnetically selected plasma cells; Fiserova A et al.; Accurate prognostic evaluation of patients with multiple myeloma (MM) is required for their stratification for more adequate therapy . Chromosomal G-banding and interphase fluorescence in situ hybridization (FISH) on cell-nonspecific samples and on myeloma cells selected by magnetic-activated cell separation (MACS) were used to study 13 samples from 12 multiple myeloma (MM) patients . Bone marrow (BM) samples were analysed using three approaches . Standard mitotic samples were prepared and analysed after G-banding . Interphase FISH was performed to detect the 13q14 deletion in unselected BM cells . In parallel, myeloma cells were selected from the BM using the CD138-specific antibody . The high-purity myeloma cell suspension was then analysed by interphase FISH for the 13q14 deletion . Magnetic separation yielded enriched myeloma cell suspensions with the mean viability of 98.0% (range: 97.0%-99.0%), and the purity of 97.6% (range: 87.2%-99.2%) as detected morphologically, and 85.2% (range: 44.8%-98.4%) as detected by immunophenotyping for CD138+ cells . Interphase FISH revealed the 13q14.3 deletion in 5 of 13 (38.5%) of cell-nonspecific samples and in 9 of 13 (69.2%) of enriched myeloma cell suspensions . In conclusion, interphase FISH on immunomagnetically selected MM cells increases the detection of the 13q14 deletion in BM samples from the patients with MM.

Gastroenterology, 2002 Dec, 123(6), 1941 - 8
Identification and isolation of candidate human colonic clonogenic cells based on cell surface integrin expression; Fujimoto K et al.; BACKGROUND & AIMS: The surface epithelium of the colon is being replaced constantly with cells derived from the stem cells of the crypt . Although the location of the stem cells is known, there are no markers for these cells . This study tested the hypothesis that colonic stem cells might be isolated and cultured on the basis of specific integrin expression patterns in normal human colonic epithelium . METHODS: Integrin expression in normal human colonic mucosa was determined by using indirect immunofluorescence . Crypt cells were then isolated as single cells from normal colon tissues and the expression pattern of integrins was analyzed by flow cytometry . Based on the specific expression of integrin beta1 in colonic crypts, the cells were sorted by using a flow cytometer, and colony assays in soft agar were performed to evaluate the clonogenicity of the sorted cells . RESULTS: By immunofluorescence, the cells located in the lower one third of crypts expressed higher levels of beta1-integrin than the cells in the remainder of the crypt . When isolated crypt cells were stained with the beta1-integrin antibody and examined in a flow cytometer, there were 2 peaks of fluorescence . Sorting of crypt cells based on staining with anti-beta1 integrin antibody produced a cell population with a significantly enhanced ability to form colonies . CONCLUSIONS: beta1-integrin is a candidate surface marker for the proliferative zone of the human colonic crypt . Our in vitro culture system for the clonal growth of a single colonic crypt cell suspension could facilitate the identification of other candidate stem cell markers.

Eur J Neurosci, 2002 Nov, 16(10), 1839 - 49
5,7-Dihydroxytryptamine lesions enhance and serotonergic grafts normalize the evoked overflow of acetylcholine in rat hippocampal slices; Birthelmer A et al.; Adult rats were subjected to intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7-DHT; 150 micro g) and, 15 days later, to intrahippocampal grafts of fetal raphe cell suspensions . About 11 months later, we assessed baseline and electrically evoked release of tritium ({3H}) in hippocampal slices, preloaded with tritiated ({3H})choline or {3H}serotonin (5-HT), in the presence or absence of the 5-HT1B receptor agonist CP-93,129 and the 5-HT receptor antagonist methiothepine . HPLC determinations of monoamine concentrations were also performed . The lesions reduced the concentration of 5-HT (-90%) and the accumulation (-80%) as well as the evoked release (-90%) of {3H}5-HT . They also decreased the inhibitory effects of CP-93,129 on the evoked release of {3H}5-HT . Most interestingly, they facilitated the evoked release of {3H}acetylcholine (+20%) . In slices from rats subjected to lesions and grafts, the responsiveness of the serotonergic autoreceptors (presumably located on the terminals of the grafted neurons) and the release of acetylcholine were close to normal . These results confirm that grafts rich in serotonergic neurons may partially compensate for the dramatic effects of 5,7-DHT lesions on serotonergic hippocampal functions . The lesion-induced reduction of the 5-HT1B autoreceptor-mediated inhibition of evoked 5-HT release may be an adaptation enhancing serotonergic transmission in the (few) remaining terminals . The facilitated release of acetylcholine is probably caused by a reduced serotonergic tone on the inhibitory 5-HT1B heteroreceptors of the cholinergic terminals . When related to data in the literature, this facilitation may be of particular interest in terms of transmitter-based strategies developed to tackle cognitive symptoms related to neurodegenerative diseases.

Appl Environ Microbiol, 2002 Dec, 68(12), 6435 - 8
Incorporation of DNA and protein precursors into macromolecules by bacteria at -15 degrees C; Christner BC; DNA and protein precursors were incorporated into trichloroacetic acid-precipitated material by bacterial cell suspensions during incubation for 50 to 100 days at -15 degrees C . Incorporation did not occur at -70 degrees C and was inhibited by antibiotics . The results demonstrate that bacteria can perform macromolecular synthesis under conditions that mimic entrapment in glacial ice.

Appl Environ Microbiol, 2002 Dec, 68(12), 6246 - 55
Aerobic metabolism of 4-hydroxybenzoic acid in Archaea via an unusual pathway involving an intramolecular migration (NIH shift); Fairley DJ et al.; A novel haloarchaeal strain, Haloarcula sp . strain D1, grew aerobically on 4-hydroxybenzoic acid (4HBA) as a sole carbon and energy source and is the first member of the domain Archaea reported to do so . Unusually, D1 metabolized 4HBA via gentisic acid rather than via protocatechuic acid, hydroquinone, or catechol . Gentisate was detected in 4HBA-grown cultures, and gentisate 1,2-dioxygenase activity was induced in 4HBA-grown cells . Stoichiometric accumulation of gentisate from 4HBA was demonstrated in 4HBA-grown cell suspensions containing 2,2'-dipyridyl (which strongly inhibits gentisate 1,2-dioxygenase) . To establish whether initial 1-hydroxylation of 4HBA with concomitant 1,2-carboxyl group migration to yield gentisate occurred, 2,6-dideutero-4HBA was synthesized and used as a substrate . Deuterated gentisate was recovered from cell suspensions and identified as 3-deutero-gentisate, using gas chromatography-mass spectrometry and proton nuclear magnetic resonance spectroscopy . This structural isomer would be expected only if a 1,2-carboxyl group migration had taken place, and it provides compelling evidence that the 4HBA pathway in Haloarcula sp . strain D1 involves a hydroxylation-induced intramolecular migration . To our knowledge, this is the first report of a pathway which involves such a transformation (called an NIH shift) in the domain ARCHAEA:

Ann Biomed Eng, 2002 Sep, 30(8), 987 - 1001
Hydrodynamic shear and tethering through E-selectin signals phosphorylation of p38 MAP kinase and adhesion of human neutrophils; Hentzen E et al.; Recently, we reported that tethering and rolling of neutrophils in shear flow over a substrate of E-selectin signals activation of beta 2-integrins and firm adhesion via an intracellular signaling pathway involving phosphorylation of p38 MAP kinase . In the current study the objective was to examine the molecular mechanisms and shear dependence underlying activation and adhesion of beta 2-integrin during shear-induced collisions between human neutrophils and murine B cells (300.19) transfected to express either E-selectin or L-selectin . Three separate parameters of cell activation were assessed over the time course of application of a defined shear field to heterotypic cell suspensions in a cone-plate viscometer . These were the two-body collision doublet lifetime and capture efficiency, surface upregulation of CD11b/CD18, and tyrosine phosphorylation of p38 MAP kinase . The data indicate that neutrophil adhesion to E-selectin expressing 300.19 cells occurs with a fourfold higher efficiency of firm adhesion than do collisions with L-selectin or parent control cells . Visual analysis of aggregation in a transparent cone-plate rheoscope revealed that the lifetime and efficiency of doublet formation increased fourfold as the applied shear stress increased . Neutrophil tethering via E-selectin was associated with rapid activation as indicated by upregulation of surface CD11b/CD18 and phosphorylation of p38 MAP kinase within seconds of application of shear . Activation greatly exceeded that observed for neutrophils sheared alone or with B cells expressing L-selectin . A distinct dependence of activation on the magnitude of the shear rate suggests a coupling between the fluid mechanical effects of shear and signaling of neutrophil adhesion.

Planta, 2002 Dec, 216(2), 265 - 72 Epub 2002 Aug 10.
Monitoring flux through the oxidative pentose phosphate pathway using {1-14C}gluconate; Garlick AP et al.; The aim of this work was to examine the metabolism of exogenous gluconate by a 4-day-old cell suspension culture of Arabidopsis thaliana (L.) Heynh . Release of (14)CO(2) from {1-(14)C}gluconate was dependent on the concentration in the medium and could be resolved into a substrate-saturable component (apparent K(m) of approximately 0.4 mM) and an unsaturable component . At an external concentration of 0.3 mM, the rate of decarboxylation of applied gluconate was 0.2% of the rate of oxygen consumption by the cells . There was no effect of 0.3 mM gluconate on the rate of oxygen consumption, or on the rate of (14)CO(2) release from either {1-(14)C}glucose or {6-(14)C}glucose by the culture . The following observations argue that gluconate taken up by the cells is metabolised by direct phosphorylation to 6-phosphogluconate and subsequent decarboxylation through 6-phosphogluconate dehydrogenase . First, more than 95% of the label released from {1-(14)C}gluconate during metabolism by the cell culture was recovered as (14)CO(2) . Secondly, inhibition of the oxidative pentose phosphate pathway (OPPP) by treatment with 6-aminonicotinamide preferentially inhibited release of (14)CO(2) from {1-(14)C}gluconate relative to that from {1-(14)C}glucose . Thirdly, perturbation of glucose metabolism by glucosamine did not affect (14)CO(2) from {1-(14)C}gluconate . Fourth, stimulation of the OPPP by phenazine methosulphate stimulated release of (14)CO(2) from {1-(14)C}gluconate to a far greater extent than that from {1-(14)C}glucose . It is proposed that measurement of (14)CO(2) from {1-(14)C}gluconate provides a simple and sensitive technique for monitoring flux through the OPPP pathway in plants.

Ann N Y Acad Sci, 2002 Oct, 974, 518 - 40
Flow field measurements in the cell culture unit; Walker S et al.; The cell culture unit (CCU) is being designed to support cell growth for long-duration life science experiments on the International Space Station (ISS) . The CCU is a perfused loop system that provides a fluid environment for controlled cell growth experiments within cell specimen chambers (CSCs), and is intended to accommodate diverse cell specimen types . Many of the functional requirements depend on the fluid flow field within the CSC (e.g., feeding and gas management) . A design goal of the CCU is to match, within experimental limits, all environmental conditions, other than the effects of gravity on the cells, whether the hardware is in microgravity ( micro g), normal Earth gravity, or up to 2g on the ISS centrifuge . In order to achieve this goal, two steps are being taken . The first step is to characterize the environmental conditions of current 1g cell biology experiments being performed in laboratories using ground-based hardware . The second step is to ensure that the design of the CCU allows the fluid flow conditions found in 1g to be replicated from microgravity up to 2g . The techniques that are being used to take these steps include flow visualization, particle image velocimetry (PIV), and computational fluid dynamics (CFD) . Flow visualization using the injection of dye has been used to gain a global perspective of the characteristics of the CSC flow field . To characterize laboratory cell culture conditions, PIV is being used to determine the flow field parameters of cell suspension cultures grown in Erlenmeyer flasks on orbital shakers . These measured parameters will be compared to PIV measurements in the CSCs to ensure that the flow field that cells encounter in CSCs is within the bounds determined for typical laboratory experiments . Using CFD, a detailed simulation is being developed to predict the flow field within the CSC for a wide variety of flow conditions, including microgravity environments . Results from all these measurements and analyses of the CSC flow environment are presented and discussed . The final configuration of the CSC employs magnetic stir bars with angled paddles to achieve the necessary flow requirements within the CSC.

J Virol Methods, 2003 Jan, 107(1), 107 - 13
Development of an automated extraction procedure for detection of human papillomavirus DNA in liquid based cytology samples; Cuschieri KS et al.; Liquid based cytology samples are being used increasingly to improve cervical screening and have the advantage that residual cell suspension is available for other tests such as human papillomavirus (HPV) detection . However, as the transport medium is optimised for downstream cytology, problems can be experienced during extraction of nucleic acid . This study aimed to develop a robust protocol for automated extraction of HPV DNA from cervical, liquid based cytology samples using a high throughput robotic system . Considerable modification of existing clinical extraction protocols for swab specimens, together with optimisation of required sample input volume was required to reduce sample blockage during the extraction to acceptable levels . The blockage rate and optimal processing volume was assessed by extracting a fixed volume (1/4) of re-suspended material from the centrifuged pellets of 10, 5 and 1 ml aliquots of 200 specimens . Analysis revealed 17.5% blockage with specimens originating from 10 ml aliquots; 3% with 5 ml and no blockage with 1 ml aliquots of the same samples . A 3% blockage level is acceptable for an automatic well clearance procedure to be followed . HPV testing of the extracts by real-time PCR showed a 1.5% loss of sensitivity in extracts originating from 1 ml aliquots as compared with 5 ml aliquots with a consequent loss of detectable HPV genotypes after reverse hybridisation . In short, 5 ml of liquid based cytology specimen is recommended for nucleic acid extraction, to allow optimal detection of HPV types in clinical samples while retaining maximum efficiency of the robotic extraction procedure .

J Pharmacol Exp Ther, 2002 Dec, 303(3), 1121 - 9
Primaquine-induced hemolytic anemia: formation of free radicals in rat erythrocytes exposed to 6-methoxy-8-hydroxylaminoquinoline; Bolchoz LJ et al.; Primaquine is an important antimalarial drug that is often dose-limited in therapy by the onset of hemolytic anemia . We have shown recently that an N-hydroxy metabolite of primaquine, 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH), is a direct-acting hemolytic agent in rat red cells and that the hemolytic activity of this metabolite is associated with GSH oxidation and oxidative damage to both membrane lipids and skeletal proteins . To determine whether the formation of free radicals may be involved in this process, rat red cells (40% suspensions) were incubated with hemolytic concentrations of MAQ-NOH (150-750 microM) and examined by EPR spectroscopy using 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) as a spin trap . Addition of MAQ-NOH to red cell suspensions containing 10 mM EMPO gave rise to an EPR spectrum with hyperfine constants consistent with those of an EMPO-hydroxyl radical adduct standard . Of interest, formation of EMPO-OH was constant for up to 20 min and dependent on the presence of erythrocytic GSH . Although no other radical adduct signals were detected in the cells by EPR, spectrophotometric analysis revealed the presence of ferrylhemoglobin, which indicates that hydrogen peroxide is generated under these experimental conditions . The data support the hypothesis that oxygen-derived and possibly other free radicals are involved in the mechanism underlying MAQ-NOH-induced hemolytic anemia.

J Neurol Neurosurg Psychiatry, 2002 Dec, 73(6), 678 - 85
Unilateral transplantation of human primary fetal tissue in four patients with Huntington's disease: NEST-UK safety report ISRCTN no 36485475; Rosser AE et al.; OBJECTIVES: Huntington's disease (HD) is an inherited autosomal dominant condition in which there is a CAG repeat expansion in the huntingtin gene of 36 or more . Patients display progressive motor, cognitive, and behavioural deterioration associated with progressive cell loss and atrophy in the striatum . Currently there are no disease modifying treatments and current symptomatic treatments are only partially effective in the early to moderate stages . Neural transplantation is effective in animal models of HD and offers a potential strategy for brain repair in patients . The authors report a safety study of unilateral transplantation of human fetal striatal tissue into the striatum of four patients with HD . Subjects and methods: Stereotaxic placements of cell suspensions of human fetal ganglionic eminence were made unilaterally into the striatum of four patients with early to moderate HD . All patients received immunotherapy with cyclosporin A, azathioprine, and prednisolone for at least six months postoperatively . Patients were assessed for safety of the procedure using magnetic resonance imaging (MRI), regular recording of serum biochemistry and haematology to monitor immunotherapy, and clinical assessment according to the Core Assessment Protocol For Intrastriatal Transplantation in HD (CAPIT-HD) . RESULTS: During the six month post-transplantation period, the only adverse events related to the procedure were associated with the immunotherapy . MRI demonstrated tissue at the site of implantation, but there was no sign of tissue overgrowth . Furthermore, there was no evidence that the procedure accelerated the course of the disease . CONCLUSIONS: Unilateral transplantation of human fetal striatal tissue in patients with HD is safe and feasible . Assessment of efficacy will require longer follow up in a larger number of patients.

Br J Haematol, 2002 Dec, 119(3), 792 - 802
Long-term ex vivo expansion of human fetal liver primitive haematopoietic progenitor cells in stroma-free cultures; Peters R et al.; Successful expansion of haematopoietic cells in ex vivo cultures will have important applications in transplantation, gene therapy, immunotherapy and potentially also in the production of non-haematopoietic cell types . Haematopoietic stem cells (HSC), with their capacity to both self-renew and differentiate into all blood lineages, represent the ideal target for expansion protocols . However, human HSC are rare, poorly characterized phenotypically and genotypically, and difficult to test functionally . Defining optimal culture parameters for ex vivo expansion has been a major challenge . We devised a simple and reproducible stroma-free liquid culture system enabling long-term expansion of putative haematopoietic progenitors contained within frozen human fetal liver (FL) crude cell suspensions . Starting from a small number of total nucleated cells, a massive haematopoietic cell expansion, reaching > 1013-fold the input cell number after approximately 300 d of culture, was consistently achieved . Cells with a primitive phenotype were present throughout the culture and also underwent a continuous expansion . Moreover, the capacity for multilineage lymphomyeloid differentiation, as well as the recloning capacity of primitive myeloid progenitors, was maintained in culture . With its better proliferative potential as compared with adult sources, FL represents a promising alternative source of HSC and the culture system described here should be useful for clinical applications.

Mol Plant Microbe Interact, 2002 Oct, 15(10), 1040 - 9
Attenuation of Cf-mediated defense responses at elevated temperatures correlates with a decrease in elicitor-binding sites; de Jong CF et al.; The interaction between the fungal pathogen Cladosporium fulvum and its only host, tomato, is a well-described gene-for-gene system and several resistance (Cf) genes of tomato and matching fungal avirulence (Avr) genes have been characterized . Transgenic tobacco suspension cells expressing Cf genes respond to matching elicitors with typical defense responses, such as medium alkalization and an oxidative burst . We found that this response is attenuated at elevated ambient temperatures . Tomato seedlings expressing both a Cf and the matching Avr gene rapidly die as a result of systemic necrosis at normal temperatures, but are rescued at 33 degrees C . We demonstrate that, at 33 degrees C, the Cf/Avr-mediated induction of defense-related genes is reversibly suppressed . Furthermore, in cell suspensions, the AVR-induced medium alkalization response is slowly suppressed upon incubation at 33 degrees C, but is quickly restored after transfer to lower temperatures . A high-affinity binding site (HABS) for AVR9 is present on plasma membranes isolated from solanaceous plants and has been suggested to act as a co-receptor for AVR9 . The amount of AVR9-HABS is 80% reduced in tobacco cell suspensions incubated at 33 degrees C, as compared with cell suspensions incubated at 20 degrees C . Our data suggest that the temperature sensitivity of Cf-mediated defense responses resides at the level of perception of the fungal avirulence factors.

Neuroscience, 2002, 115(3), 815 - 27
Basal forebrain cholinergic cell attachment and neurite outgrowth on organotypic slice cultures of hippocampal formation; Tsai ES et al.; Distributions of somata and neurites of cholinergic neurons were studied after seeding dissociated cells onto organotypic slice cultures . Slice cultures were made from hippocampal formation and adjacent cortical regions from rats or mice . Dissociated cell suspensions of basal forebrain tissue from rat or mouse fetuses were seeded onto the slice cultures . Combined cultures were maintained for 1-21 days in vitro . Cultures processed for acetylcholinesterase (AChE) histochemistry demonstrated non-random patterns of cholinergic cells and their neurites . Labeled cells appeared most frequently in the molecular layer of the dentate gyrus, and in the deeper layers of cortical regions adjacent to the hippocampus . Neurites extending from these labeled cells appeared to target the dentate molecular layer and the cortical subplate layer . By 4 days in vitro, AChE-positive basal forebrain cells display several short and thick neurites that appear to be dendrites, and one long process that appears to be an axon . By 5 days in vitro, dendrites are well developed; by 7 days the presumed axon has extended widely over the cortical target zone . These neurites are maintained through 3 weeks in culture . Distributions of cells varied with the age of the slice . AChE-labeled cells were not seen overlying hippocampal tissue when dissociated cells were seeded on slice cultures made from day 0 rats, but a few labeled cells were seen when seeded on slices from day 2 rats . Clear non-random patterns of labeled cells and neurite outgrowth were seen on slice cultures from day 5 or older pups . The non-random distribution seen with AChE-positive neurons was not seen using other techniques that labeled all cells (non-selective fluorescent labels) or all neurons; these techniques resulted in labeled cells scattered apparently homogenously across the slice culture.These studies demonstrate a non-random pattern of attachment or differentiation of basal forebrain cholinergic neurons when these cells are seeded onto cultured cortical slices; this pattern mimics the normal patterns of basal forebrain cholinergic projections to these cortical regions . These data suggest that the factors that normally guide basal forebrain-derived cholinergic axons to their target cells in vivo are present and detectable in this model system.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Nov, 22(11), 996 - 9
{Improvement upon construction of tissue-engineered allogeneic cartilage molded with polyglycolic acid as the scaffold}; Sun AK et al.; OBJECTIVE: To improve the method for constructing allogeneic molded cartilage by means of tissue engineering techniques . METHODS: The chondrocytes from the rib and articular cartilage of infant rabbits were harvested by type II collagenase digestion, followed by in vitro cell culture for 3 to 4 passages . The chondrocytes were then prepared into cell suspension and seeded onto C -and O -shaped pre-molded polyglycolic acid (PGA) scaffolds form chondrocyte-PGA composites, which were subsequently cultured in vitro for 7 to 10 d before implanted subcutaneously into adult rabbits . Improvement was made upon conventional shaping and implantation procedures . Morphological observation and cartilage regeneration assessment were conducted at different time points following the implantation, in comparison with the observation by conventional shaping and implantation methods . RESULTS: During in vitro cell culture, the rate of viable chondrocytes in the final cell suspension was (92+/-2)% after well-controlled prolongation of digestion trypsin, similar to the viable cell rate (93+/-2) % by traditional procedures (P>0.05) . Gross observation found milk-white, newly generated cartilage which had good flexibility 4 weeks after implantation, and after 8 weeks and later, the cartilage took on the color of porcelain-white . Histological examination showed a few inflammatory cells around the newly generated immature cartilage 4 weeks after implantation, and the inflammation abated when the newly generated cartilage acquired similar histological properties to that of the original cartilage 8 weeks postoperatively and later . After 16 weeks, no blood vessel or capillaries were visible within the new cartilage . CONCLUSION: The chondrocyte viability is not affected when the cells are treated with well-controlled prolonged digestion with trypsin during in vitro cell culture . Improved PGA scaffolds shaping and the implantation procedure facilitate the regeneration of the cartilage after the implantation of the composites.

Brain Res Bull, 2002 Nov 30, 59(3), 245 - 50
A procedure for direct lumbar puncture in rats; De la Calle JL et al.; We have set out to establish a fast, simple and innocuous method for repeatedly obtaining cerebrospinal fluid (CSF) samples from rats that are undergoing different experimental procedures or suffering pathological conditions . Here, we report a method that has been optimized to repeatedly collect 30-50 microl of CSF in rats by direct lumbar puncture using a procedure that generally takes 15-20 min to perform and presents very little hazard to the animal . The rats are anaesthetized with isofluorane and placed on a board in such a way that the spine is curved at the level of the L3-L5 vertebrae . After performing a small incision in the skin of the back, a neonatal lumbar puncture needle is introduced into the intrathecal space and CSF is passively collected in the needle cup (facilitating maneuvers are described herein) . Moreover, we have further adapted this method to permit the intrathecal delivery of pharmacological agents and cell suspensions . In such experiments, behavioral tests can be conducted 10-15 min after the intrathecal injection and the activity of the implanted cells can be assessed by sampling lumbar CSF at later times .

Exp Neurol, 2002 Oct, 177(2), 376 - 84
Combined inhibition of apoptosis and complement improves neural graft survival of embryonic rat and porcine mesencephalon in the rat brain; Cicchetti F et al.; To define potential mechanisms of cell death during neural cell transplantation, we investigated the role of intracellular caspase activation in combination with the activation of serum complement . We demonstrated that ventral mesencephalic (VM) cells are susceptible to complement-mediated cell lysis that can be blocked with an anti-C5 complement inhibitor (18A10) . We also determined that incubating freshly isolated allogenic VM cells with the caspase inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF), followed by immediate striatal implantation, led to a 2.5-fold increase in tyrosine hydroxylase (TH) cell survival 12 weeks postimplantation (P < 0.05) . In contrast, overnight incubation with BAF followed by striatal implantation led to a 2-fold reduction in TH cell survival at 12 weeks (P < 0.05) . Using the optimal BAF treatment and complement inhibition, we tested the hypothesis that these treatments would lead to increased cell survival in both allogeneic and xenogeneic transplantation models . We transplanted cell suspensions of (a) rat E14 VM or VM treated with (b) BAF alone, (c) anti-C5, or (d) a combination of BAF and anti-C5 . There was a significant increase in the relative number of TH-positive cells in the BAF/anti-C5 group versus control at 12 weeks posttransplantation . Similar results were achieved in a pig to rat xenotransplant paradigm . A neuronal xenograft marker (70-kDa neurofilament) also demonstrated relative increases in graft volume in the BAF/anti-C5 treatment group . These studies indicate that more than one mechanism can mediate cell death during neural cell transplantation and that a combined treatment using caspase and complement inhibition can significantly improve cell survival.

Cell Transplant, 2002, 11(6), 583 - 92
Vascular adventitia is a suitable compartment to transplant transduced vascular smooth muscle cells for ex vivo gene expression; Beltrao-Braga PC et al.; Vascular smooth muscle cells (VSMC) are ideal for systemic gene therapy because of their proximity to blood vessels and they have demonstrated long-term exogenous gene expression in vivo . However, the procedure generally followed to seed the transduced VSMC onto arteries denuded of endothelial cells usually induces stenosis and thrombosis, with a consequent high risk for use in humans . We demonstrate here that the vascular adventitia is a suitable place to introduce transduced VSMC and to secrete therapeutic proteins into the blood stream by a simple procedure, avoiding postoperative vascular complications . Transduced VSMC, with the retroviral vectors carrying the human growth hormone gene (hGH), were seeded into the adventitia of the rat abdominal aorta by single injection of a cell suspension . Based on the hGH and anti-hGH production in serum and on histological analysis of the removed aortas, we demonstrated hGH production over the 2-month experimental period . None of the animals used in the experiment showed stenosis, thrombosis, or other vascular or visible physiological abnormalities.

Bull Exp Biol Med, 2002 Feb, 133(2), 199 - 201
A method for estimation of urease activity in gastric mucosa biopsy specimens and Helicobacter pylori cell suspensions; Vartanova NO et al.; A method for measuring urease activity in biopsy specimens and Helicobacter pylori cultures from these specimens is proposed . The method is based on measurement (with a portable pH-meter) of the rate of pH changes in a reaction mixture consisting of buffer, substrate (urea), and biopsy specimen or bacterial cells . This method revealed that urease activity of biopsy specimens correlated with that of H . pylori suspension in the same experiment . High urease activity was found in biopsy specimens containing the greatest number of Helicobacter cells; only one of 14 specimens free of H . pylori cells showed no urease activity . Introduction of this method into clinical practice will help to evaluate the contribution of H . pylori to the pathological process.

J Am Coll Cardiol, 2002 Nov 6, 40(9), 1579 - 88
Multicentric inflammation in epicardial coronary arteries of patients dying of acute myocardial infarction; Spagnoli LG et al.; OBJECTIVES: We sought to test the hypothesis of whether inflammatory cell infiltration in patients dying of an acute myocardial infarction (MI) is a multifocal event involving multiple coronary branches . BACKGROUND: Coronary instability is thought to reflect local disruption of a single vulnerable plaque . However, previous postmortem studies have not addressed the question of whether activation of inflammatory cells, particularly T lymphocytes, is limited to the culprit lesion only or rather diffuse in the coronary circulation . METHODS: We performed a systematic flow cytometric study in three groups of autopsied patients (group 1 = acute MI; group 2 = old MI; group 3 = no ischemic heart disease) . Cell suspensions of enzymatically digested coronary arteries were stained for flow cytometry with CD3, CD68, alpha-smooth muscle actin, and human leukocyte antigen (HLA)-DR antibodies . RESULTS: The coronary plaques showed: 1) a higher proportion of inflammatory cells in groups 1 and 2 than in group 3; 2) a higher percentage of T lymphocytes in group 1 than in group 2 (11.67 +/- 0.70% vs . 5.67 +/- 0.74%, p = 0.001) and in group 2 than in group 3 (p = 0.008); and 3) diffuse cell activation in the whole coronary tree of group 1, but not of group 2 subjects . CONCLUSIONS: Our study suggests that lymphocytes may play a key role in coronary instability by determining activation of various cellular types throughout the coronary circulation . Activated T lymphocytes and their products may well represent a new target in both the treatment and prevention of acute coronary syndromes.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2001 Dec, 145(2), 57 - 60
Cell suspensions, cell cultures, and tissue slices--important metabolic in vitro systems; Cervenkova K et al.; In vitro subcellular and cellular systems have important and irreplaceable roles in the metabolic investigations that precede the development of new potential drugs . Of these model systems, tissue slices are probably the nearest to in vivo conditions . From the experimental and complexity points of view, perfused organs lie midway between tissue slices and whole organism . Preparation and working with liver slices is quick and easy, and, excess material can be cryopreserved and stored untill the next experiment . Slices can be prepared from a wide variety of organs and it is possible to co-incubate them . Another important feature is the possibility of interspecies comparison of slices . Different experiments can be run both in the short-term as well as long-term incubations . Each in vitro system has an important place for example, in the development of new medicaments . It is therefore important to compare and supplement experimental results from different in vitro systems when extrapolating to in vivo situations is done.

J Biotechnol, 2003 Jan 23, 100(2), 169 - 76
Primary cultures from the marine sponge Xestospongia muta (Petrosiidae, Haplosclerida); Richelle-Maurer E et al.; In the context of the investigations on the origin and in vitro production of bioactive compounds, primary cultures were developed from ectosomal and choanosomal cell suspensions from the sponge Xestospongia muta . Dissociated cells aggregated and reorganized into a striking reticulated network of cells, typical for X . muta . Moreover, in some cultures an isotropic reticulation of small spicules, very similar to that found in the ectosome of adult sponges, was observed . Phytohaemagglutinin promoted aggregation and the reorganization of the cells . HPLC analyses revealed that straight-chain acetylenic compounds were recovered from short-term cultures and that they were synthesized during culture . Heterotrophic bacteria were assumed to be involved in the process . Together our results established that X . muta would be an excellent experimental model to study, in laboratory conditions, the differentiation of the skeleton and the in vitro biosynthesis of straight-chain acetylenic compounds.

Phytochemistry, 2002 Nov, 61(6), 669 - 73
Oxidative cleavage of the C-C bond of 3,6-dialkylcyclohexane-1,2-diones by cell suspension cultures of Marchantia polymorpha; Matsuura Y et al.; Biotransformation of 3,6-dialkylcyclohexane-1,2-diones by cell suspension cultures of Marchantia polymorpha involves regioselective oxidative cleavage of the C-C bond to give the corresponding oxocarboxylic acids shortened by one carbon unit . In the case of cyclohexane-1,2-dione, adipic acid was obtained.

Phytochemistry, 2002 Nov, 61(6), 621 - 30
alpha-Galactosidase from cultured rice (Oryza sativa L . var . Nipponbare) cells; Kim WD et al.; The alpha-galactosidase from rice cell suspension cultures was purified to homogeneity by different techniques including affinity chromatography using N-epsilon-aminocaproyl-alpha-D-galactopyranosylamine as the ligand . From 11 l of culture filtrate, 28.7 mg of purified enzyme was obtained with an overall yield of 51.9% . The cDNA coding for the alpha-galactosidase was cloned and sequenced . The enzyme was found to contain 417 amino acid residues composed of a 55 amino acid signal sequence and 362 amino acid mature alpha-galactosidase; the molecular weight of the mature enzyme was thus calculated to be 39,950 . Seven cysteine residues were also found but no putative N-glycosylation sites were present . The observed homology between the deduced amino acid sequences of the mature enzyme and alpha-galactosidases from coffee (Coffea arabica), guar (Cyamopsis tetragonolooba), and Mortierella vinacea alpha-galactosidase II were over 73, 72, and 45%, respectively . The enzyme displayed maximum activity at 45 degrees C when p-nitrophenyl-alpha-D-galactopyranoside was used as substrate . The rice alpha-galactosidase and Mortierella vinacea alpha-galactosidase II acted on both the terminal alpha-galactosyl residue and the side-chain alpha-galactosyl residue of the galactomanno-oligosaccharides.

Toxicol In Vitro, 2002 Dec, 16(6), 675 - 82
Oxidative stress response of rat testis to model prooxidants in vitro and its modulation; Rajesh Kumar T et al.; Understanding the effects of prooxidants on mammalian testis either in vitro or in vivo is important, since recent evidence shows that oxidative stress can play a vital role in the etiology of male infertility . In this investigation, we have examined the oxidative stress response of adult rat testis in vitro as induced by model prooxidants (tert-butyl hydroperoxide (t-bHP) and cumene hydroperoxide (cHP) deploying two models-testicular cell suspensions (TCS) and testicular explants (TE) . Significant induction of oxidative stress was observed in both models as evidenced by increased thiobarbituric acid reactive substances (TBARS) levels on incubation with hydroperoxides . The response was both concentration and time dependent . At the highest concentration (200 microM), both hydroperoxides induced a 100% increase in the TE model, compared with a dramatic (380-560%) increase in the TCS model during a 30-min incubation . Further evidence of oxidative stress such as reduction in the GSH levels and alterations in the activity of antioxidant enzymes (catalase and glutathione peroxidase) were also obtained in the TE model . In the TE model, radical scavengers, namely thiourea, urea and mannitol, as well as antioxidants such as glutathione and catalase inhibited the t-bHP-induced lipid peroxidation response to varying degree . A similar degree of protection was also evident with known antioxidants such as ascorbic acid, butylated hydroxyanisole and butylated hydroxytoluene in the TE model . Further co-incubation of TE either with mercaptosuccinate (a potent glutathione peroxidase inhibitor) or 3-aminotriazole (an irreversible catalase inhibitor) resulted in a marked increase in t-bHP-induced lipid peroxidation, clearly suggesting the importance of both of these enzymic antioxidants in rat testis in vitro . These data suggest that the TE model may be further utilized to screen antioxidants in vitro and also investigate the prooxidant potency of xenobiotics in testicular cells.

Neuroscience, 2002, 115(2), 495 - 504
Rat nigral xenografts survive in the brain of MHC class II-, but not class I-deficient mice; Duan WM et al.; We have examined the role of the indirect pathway of antigen recognition and T cells in neural xenografts rejection by using major histocompatibility complex (MHC) class II-deficient mice as xenograft recipients . Dissociated embryonic ventral mesencephalic tissue from Sprague-Dawley rats was stereotaxically injected as a cell suspension into the striatum of MHC class II-deficient adult mice as well as MHC class I-deficient and wild-type mice as controls . All of the MHC class II-deficient mice had surviving grafts in the striatum 4 weeks post-grafting . In contrast, only a few of the MHC class I-deficient mice exhibited very small grafts and none of the wild-type mice had any surviving grafts . The mean number of surviving transplanted dopamine neurons in the MHC class II-deficient group was significantly larger than that observed in the other two groups . Moderate levels of MHC class I antigen expression were seen in the transplantation sites of some animals in the MHC class II-deficient group . No helper or cytotoxic T cells were observed infiltrating into the graft sites of this group . However, there were markedly increased levels of expression of MHC class I and class II antigens, and a number of T cells infiltrating in the graft sites in both the MHC class I-deficient and wild-type groups . These results show that rat embryonic nigral tissue can survive transplantation in the brain of the MHC class II-deficient mice for at least 4 weeks without any overt signs of rejection, suggesting that the indirect pathway of foreign antigen recognition mediated by host MHC class II molecules and helper T cells plays an important role in the rejection responses to intracerebral xenografts.

Brain Res, 2002 Nov 15, 955(1-2), 268 - 80
Comparison between survival of lazaroid-treated embryonic nigral neurons in cell suspensions, cultures and transplants; Karlsson J et al.; Death of transplanted dopaminergic neurons is induced both during preparation of donor tissue and after intrastriatal grafting . Oxidative stress is thought to be partly responsible for this cell death . In the present study we compared the effects of three lipid peroxidation inhibitors, the lazaroids Tirilazad mesylate, U-83836E and U-101033, on survival of embryonic mesencephalic neurons in different paradigms . The lazaroids were equally potent in preventing serum deprivation-induced death of cultured dopaminergic neurons . In a second set of experiments, mesencephalic suspensions were pretreated with lazaroids and cell survival was analyzed immediately after dissociation, after 2 or 24 h in culture or after intrastriatal transplantation . Lazaroid pretreatment failed to protect mesencephalic neurons in the in vitro paradigms and U-101033E did not protect grafted dopaminergic neurons in contrast to the neuroprotective effects previously reported for U-83836E and Tirilazad . Pretreatment with the iron chelator deferoxamine mesylate did not protect cultured or grafted dopaminergic neurons, nor did it improve neuronal survival in the serum deprivation model . U-83836E and U-101033E, but not Tirilazad, prevented cell death induced by the pro-oxidant tert-butyl hydroperoxide in suspensions . In a final experiment, we found that systemic treatment of the graft recipient rat with Tirilazad mesylate (before and during the first 3 days after grafting) improved survival of transplanted dopaminergic neurons to 180% of control values . Our results show that systemic treatment with a lipid peroxidation inhibitor for 3 days can promote graft survival, but also highlights the poor correlation between neuroprotective effect of pharmacological compounds in vitro and in grafts.

FEBS Lett, 2002 Nov 6, 531(2), 179 - 83
A heat-activated MAP kinase in tomato: a possible regulator of the heat stress response; Link V et al.; Adaptation to elevated temperatures is of major importance for the survival of plants . The role of kinases in heat stress response was studied in tomato by in gel and in solution kinase assays using myelin basic protein as substrate . The application of heat stress in a naturally occurring temperature range resulted in a fast and transient activation of a 50 kDa mitogen-activated protein (MAP) kinase both in a photoautotrophic cell suspension culture and in leaves of mature plants . The heat activation of the MAP kinase was shown to be calcium-dependent . The specific phosphorylation of tomato heat stress transcription factor HsfA3 by a partially purified preparation of the heat-activated MAP kinase supports a physiological role of the identified kinase activity in transducing the heat stress signal.

Biochem J, 2003 Feb 15, 370(Pt 1), 57 - 67
Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization; Hamilton JM et al.; A C-terminal portion of Ara12 subtilisin-like protease (residues 542-757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein . Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12 . The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue . The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves . Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue . Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography . Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases . Optimal proteolytic activity was displayed under acidic conditions (pH 5.0) . Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions . Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates . At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1' position, Asp, Leu and Ala were most favoured . Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis.

Br J Dermatol, 2002 Nov, 147(5), 893 - 904
Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension; Olsson MJ et al.; BACKGROUND: In vitiligo and piebaldism the lack of melanin in the epidermis is due to the fact that melanocytes are missing . The patients suffer psychologically and the white areas have lost the part of the skin barrier protection normally provided by the melanocytes . Medical treatments are ineffective in many of the patients, and surgical methods have therefore been developed . OBJECTIVES: It is important to investigate the long-term results and factors that might influence the outcome of melanocyte transplantations in order to form a basis for guidance in the selection of patients who will benefit most from the treatments . METHODS: A follow-up of 132 patients who had been treated by transplantation on 176 occasions in total, 1-7 years previously, was carried out by questionnaires and clinical examinations . We investigated the responses in five types of leucoderma to three different transplantation methods: autologous cultured melanocytes, ultrathin epidermal sheets and basal layer cell suspension . RESULTS: Stable types of leucoderma, i.e . segmental vitiligo and piebaldism, responded in most cases with 100% repigmentation, regardless of the surgical method used . For these types of leucoderma surgery seems to be the method of choice . The largest group, vitiligo vulgaris, was thoroughly scrutinized and three statistical models were used to analyse the data . The ultrathin epidermal sheet method gave somewhat better overall results, but was the method that gave the worst outcome in knee and elbow areas, emphasizing the importance of the right choice of method depending on the anatomical location to be treated . Irrespective of the method, fingers and elbows were the most difficult areas to repigment . The trunk and the arms and legs (not including elbows and knees) responded best . Patients with increasing and/or extensive vitiligo vulgaris more often showed incomplete repigmentation . They also had a lower chance of retaining their repigmentation compared with those with less extensive vitiligo . Patients in whom untreated white lesions had increased in recent years tended to respond less well to transplantation compared with patients with unchanged or decreased lesions . Within the vitiligo vulgaris group, patients with short disease duration or with small total vitiligo area responded best to transplantation . The subgroup of vitiligo vulgaris patients with hypothyroidism tend to respond less well to the transplantation and they were generally older at vitiligo onset . This information is of great importance for the selection of patients and when informing about the chances of improvement after transplantation . Slight hyperpigmentation was common, especially when ultrathin epidermal sheets had been used . No scars or indurations were seen in treated areas . CONCLUSIONS: Transplantations are the methods of choice in stable types of leucoderma . Progressive, widespread vitiligo vulgaris should never be selected for transplantation.

Cancer Lett, 2002 Dec 15, 188(1-2), 213 - 9
Inhibition of lung metastasis of osteosarcoma cell line POS-1 transplanted into mice by thigh ligation; Kamijo A et al.; Using a model with external ligation of the thigh, the effect of ischemia-reperfusion injury on tumor growth and the activity of lung metastasis was investigated in mice inoculated a spontaneous murine osteosarcoma cell line (POS-1) in vivo . POS-1 cell suspension was inoculated into the right hind footpad of 70 mice . Four weeks after inoculation, the ipsilateral thigh was ligated for 3 h in 15 mice and the contralateral thigh in 15 mice . Another ten mice were inoculated with POS-1 without ligating the thigh . The number of metastatic foci on the lung surface 6 weeks after inoculation was 2.29+/-0.98 (mean+/-SE) foci/lungs in mice with ipsilateral ligation and 6.25+/-2.41 in mice with contralateral ligation, which were significantly lower than control (13.40+/-1.42 in mice no ligation) (P<0.01) . The number of metastatic foci on the lung surface in mice with intraperitoneal injection of superoxide dismutase (SOD) and catalase was 3.25+/-0.65 (mean+/-SE) foci/lungs in mice with ligation which was significantly greater than that in mice without SOD and catalase injection 1.29+/-0.97 (P=0.04).Cell viability was 9.12+/-4.07% with 100 microM H(2)O(2) in 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide assay . It revealed that at concentrations of 100 microM H(2)O(2) or higher was cytotoxic to POS-1 . In cell invasion assay, the number of invading cells with 10 microM H(2)O(2) was 2.80+/-0.53 cells/field, which was significantly lower than control (5.93+/-0.18) (mean+/-SE), indicating that low-dose H(2)O(2) suppressed invasion of POS-1 . These results suggested that reperfusion injury had selective cytotoxicity to POS-1 through producing reactive oxygen species . Activated oxygen was considered to inhibit the regional growth and the ability of lung metastasis of POS-1 cells.

Biotechnol Bioeng, 2002 Dec 30, 80(7), 812 - 22
Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form; Smith ML et al.; The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly . The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity . This antigen was expressed in soybean (Glycine max L . Merr . cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized . The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated . Similar to yeast, the plant-expressed HBsAg was retained intracellularly . The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower . For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network . Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles . The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays . Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay . Possible strategies to increase HBsAg production and improve post-translational processing are discussed .

Appl Biochem Biotechnol, 2002 Jul-Dec, 102-103(1-6), 381 - 93
Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum; Chattopadhyay S et al.; The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20 degrees C . The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone . The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture . It was found that glucose was a better carbon source than sucrose and that NH4+:NO3- ratio (total nitrogen concentration of 60 mM) and PO4(3-) did not have much effect on the growth and product formation . The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH4+:NO3- ratio, and PO4(3-)) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design . This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin . To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated . The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.

Blood, 2003 Feb 15, 101(4), 1487 - 93 Epub 2002 Sep 26.
Functional expression of the eotaxin receptor CCR3 in CD30+ cutaneous T-cell lymphoma; Kleinhans M et al.; Little is known about mechanisms involved in skin-specific homing of cutaneous T-cell lymphoma (CTCL) . Chemokine/chemokine receptor interactions have been implicated in the homing of lymphoma cells to various tissue sites . We investigated tissue samples and tumor cell suspensions of patients with CD30(+) CTCL (n = 8) and CD30(-) CTCL (mycosis fungoides, n = 6; Sezary syndrome, n = 6) for expression of the chemokine receptors CCR3, CCR4, and CCR8 and the CCR3 ligands eotaxin/CCL11, monocyte chemoattractant protein 3 (MCP-3)/CCL7, and RANTES (regulated on activation, normal T expressed and secreted)/CCL5 . Of 8 CD30(+) CTCLs, 7 expressed CCR3, 4 CCR4, and none CCR8 . CCR3 expression was not found in skin tissue samples from 12 CD30(-) CTCLs . Coexpression of CCR3 and CD30 was demonstrated by flow cytometry in tumor cell suspensions . Internalization experiments demonstrated functionality of CCR3 expressed by freshly isolated tumor cells . Actin polymerization as well as migration in response to eotaxin was demonstrated in a CD30(+) cutaneous lymphoma cell line . CCR3 ligand eotaxin/CCL11 was detected in lesional skin of CD30(+) CTCL by immunohistochemistry, preferentially in tumor cells . Eotaxin/CCL11 expression in tumor cells was confirmed by intracellular immunofluorescence . Analysis of cytokine expression pattern of CCR3-bearing infiltrating cells showed a predominance of interleukin-4 (IL-4) but not interferon-gamma (IFN-gamma) protein expression,1 consistent with a T-helper 2 (Th-2) profile . These results suggest that expression of CCR3 and its ligand eotaxin/CCL11 plays a role in the recruitment and retention of CD30(+) malignant T cells to the skin.

J Appl Microbiol, 2002, 93(5), 751 - 7
Extra- and intra-cellular lytic effects of Cytophaga sp . LR2 on the red microalgae Rhodella reticulata; Toncheva-Panova T et al.; AIMS: To evaluate the lytic activities of crude enzymes from Cytophaga sp . LR2 on Rhodella reticulata cells and isolated algal polysaccharide . METHODS AND RESULTS: The Cytophaga compartment was separated after centrifugation in a cell suspension for 30 min at 18,000 g . The extracellular enzyme was obtained from the supernatant and the intracellular from the pelleted cells after sonication and removal of debris . Algal cells were incubated with extra- or intracellular preparations and sowed onto agar medium . The suppressive effect of the extracellular enzyme on colony-forming units was found to be almost twice as high . The result was still more pronounced when treated cells had been shocked osmotically before seeding . Saccharolytic activity was evaluated by changes in the reducing sugars in the media . Concerning isolated algal polysaccharide, the reducing power of the two bacterial preparates was relatively low . A combined fraction showed the highest lytic activity . Using native and SDS electrophoresis some relation between the prevalence of the extra and intracellular protein patterns was registered . Two of the common components' molecular weight masses of 50 and 21 kDa were found to be reproducible in native- and SDS-containing gel . CONCLUSIONS: Cytophaga sp . LR2 produce extra- and intracellular enzymes active in destroying Rhodella cultures . The agents excreted in the medium are more effective.We suppose that two or three different classes of enzymes are involved in the lysis process . The comparative electrophoresis in this case shows the protein components with predictable functions . SIGNIFICANCE AND IMPACT OF THE STUDY: Combining different simple and reproducible approaches to identify the lytic capability of Cytophaga sp . LR2 on R . reticulata.

Planta Med, 2002 Oct, 68(10), 906 - 11
A newly-detected reductase from Rauvolfia closes a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline; Gao S et al.; A new enzyme, 1,2-dihydrovomilenine reductase (E.C . 1.3.1), has been detected in Rauvolfia cell suspension cultures . The enzyme specifically converts 2beta( R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia . A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2',5'-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered an approximately 200-fold enriched enzyme in a yield of approximately 6% . SDS-PAGE showed an M r for the enzyme of approximately 48 kDa . Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 degrees C . The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.

Cryo Letters, 2002 Jul-Aug, 23(4), 229 - 36
A modified differential scanning calorimetry for determination of cell volumetric change during the freezing process; Luo D et al.; A modified analytical and experimental method using differential scanning calorimeter (DSC) was developed to determine the cell volume change during the freezing process . Two cell types were used in the study: human platelets and erythrocytes (red blood cells) . Isotonic cell suspensions with different cytocrits were prepared and used in the DSC experiments . Low cooling rates were used to avoid intracellular ice formation . Cell suspensions were cooled from room temperature to -40 degrees C . Latent heat release from the freezing of cell suspensions was shown to be a linear function of cytocrit . From slope and intercept of the linear function, cell volume change was determined based on a developed theoretical model . From experimental data and theoretical analyses, it was revealed that (a) the final volume of a human platelet at -40 degrees C was 33.7% of its isotonic volume, and 15.2% of the original (at isotonic condition) intracellular water remained unfrozen inside platelets, and (b) the final volume of human erythrocyte at -40 degrees C was 50.0% of its isotonic volume, and 30.3% of the original intracellular water was kept inside cells as residual unfrozen water.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Feb, 22(2), 134 - 6
Effect of nitric oxide on the invasive ability of human osteosarcoma through a filter membrane: an in vitro study; Zhang XY et al.; OBJECTIVE: To study the effect of nitric oxide on the in vitro invasiveness of human osteosarcoma cell line (OS-732) . METHODS: In vitro cultured OS-732 cell suspensions containing different concentrations of sodium nitroprusside (SNP) or PBS (control group) were used to assess the in vitro invasiveness of the tumor cells utilizing Boyden's chamber assay . The tumor cells growing across the filter membrane were counted by means of HE staining and compared between the groups . RESULTS: With the increase of SNP concentration in the cell suspension, the number of tumor cells growing across the membrane tended to decrease . With the exception of the cell suspension containing 500 micromol/L SNP that had a cell number across the membrane significantly different from those of any other groups, significant difference was not observed between any adjacent groups . Between the groups with SNP concentrations that did not come adjacent, however, significant differences were noted . CONCLUSION: Nitric oxide can depress the invasiveness of OS-732 cells across the membrane in vitro.

Helicobacter, 2002 Oct, 7(5), 271 - 80
The effect of Helicobacter pylori infection on levels of DNA damage in gastric epithelial cells; Everett SM et al.; BACKGROUND: Helicobacter pylori infection leads to an increased risk of developing gastric cancer . The mechanism through which this occurs is not known . We aimed to determine the effect of H . pylori and gastritis on levels of DNA damage in gastric epithelial cells . METHODS: Epithelial cells were isolated from antral biopsies from 111 patients . DNA damage was determined using single cell gel electrophoresis and the proportion of cells with damage calculated before and 6 weeks after eradication of H . pylori . Cell suspensions generated by sequential digestions of the same biopsies were assayed to determine the effect of cell position within the gastric pit on DNA damage . RESULTS: DNA damage was significantly higher in normal gastric mucosa than in H . pylori gastritis {median (interquartile range) 65% (58.5-75.8), n = 18 and 21% (11.9-29.8), n = 65, respectively, p <.001} . Intermediate levels were found in reactive gastritis {55.5% (41.3-71.7), n = 13} and H . pylori negative chronic gastritis {50.5% (36.3-60.0), n = 15} . DNA damage rose 6 weeks after successful eradication of H . pylori{to 39.5% (26.3-51.0), p =.007} but was still lower than in normal mucosa . Chronic inflammation was the most important histological factor that determined DNA damage . DNA damage fell with increasing digestion times (r = -.92 and -.88 for normal mucosa and H . pylori gastritis, respectively) . CONCLUSIONS: Lower levels of DNA damage in cells isolated from H . pylori infected gastric biopsies may be a reflection of increased cell turnover in H . pylori gastritis . The investigation of mature gastric epithelial cells for DNA damage is unlikely to elucidate the mechanisms underlying gastric carcinogenesis.

J Exp Clin Cancer Res, 2002 Sep, 21(3), 415 - 20
Development of Dunn osteosarcoma inoculated into subcutaneous air pouch in mice; Hori T et al.; Dunn osteosarcoma cell growth was observed in C3H/Hej mouse subcutaneous air pouch . Inoculation with Dunn osteosarcoma cell suspension was performed by injection into air pouches of 70 mice on Day 7 after the initial injection of air . The developmental pattern of the tumor cell colonies was classified histologically into five stages . In Stage 0 (stage of no tumor cells), there were no tumor cells . In Stage 1 (focal stage), the colonies were limited to the lining-cell layers . In Stage 2, (segmental stage) the colonies protruded into the cavity of the pouch . In Stage 3 (annular stage), the total surface of the inner wall of the pouch was occupied by tumor cells . In Stage 4 (occlusive stage), the cavity of the pouch was fully occupied by tumor cells . The tumor was observed to develop to stage 1 in all mice 7 days after inoculation . The colonies were all found to be settled initially in the deeper layer of lining cells . Thus, the mouse air-pouch model is useful for direct observation of osteosarcoma cell growth.

Cell Transplant, 2002, 11(5), 489 - 94
Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds; Ushida T et al.; Tissue engineering approaches have been clinically tried to repair damaged articular cartilages . It is an essential step to uniformly seed chondrocytes into 3D scaffolds in order to reconstruct tissue-engineered cartilages in vitro, but the tissue engineering could not have been provided with efficient cell seeding methods . Type I collagen is clinically used and known as a cytocompatible material, having recognition sites for integrins . Collagen gel encapsulating chondrocytes has been tried for making regenerated cartilages, but it is found difficult to have the gel keep its original shape after long-term culture, because of shrinking . On the other hand, 3D scaffolds, either of a nonwoven structure or a sponge-like structure, involve difficulty in that chondrocytes could not be uniformly seeded, although they have adequate initial mechanical properties . In this study, by combining collagen gelation with a nonwoven PLLA scaffold, we achieved uniform cell seeding into the 3D scaffold . Bovine articular chondrocytes were mixed with type I collagen solution, and the solution was poured into the nonwoven PLLA scaffold (1.5 mm thick, diameter 15 mm) . The collagen-chondrocyte mixture was made into gel at 37 degrees C for 1 h . The 0.39% collagen mixture was viscous enough to prevent cells from precipitating during gelation . Almost all chondrocytes were able to be incorporated into the PLLA scaffolds by mixing with collagen solution and subsequently making into gel, while 30-40% of the chondrocytes seeded as a cell suspension were not trapped into the PLLA scaffolds . The method presented, where chondrocytes were mixed with collagen solution, and the mixture was incorporated into a 3D scaffold, then made into gel in the scaffold, could serve as an alternative for in vitro cartilage regeneration, also simultaneously having the advantages of both materials.

J Agric Food Chem, 2002 Oct 23, 50(22), 6287 - 94
Simultaneous determination of (45)calcium and (65)zinc uptake by caco-2 cells; Etcheverry P et al.; A simple method for simultaneously determining cell-associated Ca and Zn in Caco-2 cells is described . Calcium and zinc uptake was measured via radioisotopes (45)Ca and (65)Zn . Preliminary studies revealed that (65)Zn, a positron (beta(+)) and gamma emitter, contributed to (45)Ca counts in a liquid scintillation counter (LSC) . However, (45)Ca, being a true beta emitter, did not contribute to the counts in a gamma counter (gammaC) . To differentiate the counts of (45)Ca from those of (65)Zn, first a (65)Zn-labeled cell suspension was read in a gammaC and an LSC, thus obtaining the relationship between the radioactive counts obtained from the gammaC and LSC . This information defined the linear relationship between gammaC (65)Zn counts per minute (CPM) and LSC (65)Zn CPM . Because the (45)Ca and (65)Zn counts obtained in the LSC are additive, giving total LSC CPM, the value of LSC (45)Ca CPM was obtained by subtracting LSC (65)Zn CPM from total LSC CPM for the dual-labeled cell sample, obtaining then LSC (45)Ca CPM . To determine the absolute activity or disintegrations per minute (DPM) of each isotope in the dual-labeled sample, the linear relationship between DPM and CPM was determined for each isotope . The method is simple and straightforward for the determination of (45)Ca counts from a sample also containing (65)Zn, using gamma and liquid scintillation counters.

Cell Motil Cytoskeleton, 2002 Dec, 53(4), 289 - 92
Double-decker chemotaxis: no evidence for photonic stimulation of directed locomotion by human blood polymorphonuclear leukocytes; Kaplow LS et al.; The study was carried out under direct videomicroscopic control to ascertain whether electromagnetic forces (photons) can initiate directed cell motility of human polymorphonuclear neutrophils (PMN) . Cell suspensions containing a mixture of randomly motile white blood cells and erythrocytes (red cells) were placed in a double-decked preparation created by a glass slide and two cover slips and sealed by paraffin . Erythrocytes in the upper or lower chamber were destroyed by a single burst from a narrow ruby laser beam . Directed locomotion of PMN toward the erythrocyte debris occurred exclusively in the chamber in which the erythrocytes had been destroyed . Only random PMN locomotion was observed in the adjacent chamber . The results indicate that in this experimental model, electromagnetic forces do not initiate directed locomotion .

Plant Physiol, 2002 Oct, 130(2), 1022 - 31
Chemical inactivation of the cinnamate 4-hydroxylase allows for the accumulation of salicylic acid in elicited cells; Schoch GA et al.; The cinnamate (CA) 4-hydroxylase (C4H) is a cytochrome P450 that catalyzes the second step of the main phenylpropanoid pathway, leading to the synthesis of lignin, pigments, and many defense molecules . Salicylic acid (SA) is an essential trigger of plant disease resistance . Some plant species can synthesize SA from CA by a mechanism not yet understood . A set of specific inhibitors of the C4H, including competitive, tight-binding, mechanism-based irreversible, and quasi-irreversible inhibitors have been developed with the main objective to redirect cinnamic acid to the synthesis of SA . Competitive inhibitors such as 2-hydroxy-1-naphthoic acid and the heme-coordinating compound 3-(4-pyridyl)-acrylic acid allowed strong inhibition of C4H activity in a tobacco (Nicotiana tabacum cv Bright Yellow {BY}) cell suspension culture . This inhibition was however rapidly relieved either because of substrate accumulation or because of inhibitor metabolism . Substrate analogs bearing a methylenedioxo function such as piperonylic acid (PIP) or a terminal acetylene such as 4-propynyloxybenzoic acid (4PB), 3-propynyloxybenzoic acid, and 4-propynyloxymethylbenzoic acid are potent mechanism-based inactivators of the C4H . PIP and 4PB, the best inactivators in vitro, were also efficient inhibitors of the enzyme in BY cells . Inhibition was not reversed 46 h after cell treatment . Cotreatment of BY cells with the fungal elicitor beta-megaspermin and PIP or 4PB led to a dramatic increase in SA accumulation . PIP and 4PB do not trigger SA accumulation in nonelicited cells in which the SA biosynthetic pathway is not activated . Mechanism-based C4H inactivators, thus, are promising tools for the elucidation of the CA-derived SA biosynthetic pathway and for the potentiation of plant defense.

Appl Immunohistochem Mol Morphol, 2002 Sep, 10(3), 258 - 62
Immunohistochemical detection of immunoglobulin light chain expression in B-cell non-Hodgkin lymphomas using formalin-fixed, paraffin-embedded tissues and a heat-induced epitope retrieval technique; Marshall-Taylor CE et al.; Definitive diagnosis of B-cell non-Hodgkin lymphomas often requires demonstration of B-cell monoclonality . Immunohistochemical detection of monotypic immunoglobulin light chain expression, and thereby B-cell monoclonality, may be accomplished readily using fresh cell suspensions or frozen tissue sections . However, immunohistochemical detection of immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues is more difficult; with few exceptions, techniques suitable for formalin-fixed, paraffin-embedded tissues are not widely available . This report describes and validates a method for detecting immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues using a heat-induced epitope retrieval technique . This method was evaluated in a series of 113 cases of B-cell non-Hodgkin lymphoma, including 73 cases with correlative flow cytometric immunophenotyping data . Monotypic light chain expression was demonstrated in 91 (81%) of 113 cases, including several small core biopsy specimens with extremely limited tissue . Compared with the reference method (flow cytometric immunophenotyping), the specificity of the assay was 100% . Interobserver reproducibility was excellent, with 87% concordance between two independent observers categorizing cases as indeterminate, suggestive or diagnostic of kappa or lambda light chain restriction (Cohen kappa statistic: 0.81) . In summary, the described method permits demonstration of immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues in approximately 80% of cases of B-cell non-Hodgkin lymphoma with a high degree of specificity and excellent interobserver reproducibility . The assay is sufficiently robust for diagnostic use in small biopsies in which fresh tissue is unavailable.

Plant Cell, 2002 Oct, 14(10), 2627 - 41
Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells; Lecourieux D et al.; Cell suspensions obtained from Nicotiana plumbaginifolia plants stably expressing the apoaequorin gene were used to analyze changes in cytosolic free calcium concentrations ({Ca(2+)}(cyt)) in response to elicitors of plant defenses, particularly cryptogein and oligogalacturonides . The calcium signatures differ in lag time, peak time, intensity, and duration . The intensities of both signatures depend on elicitor concentration and extracellular calcium concentration . Cryptogein signature is characterized by a long-sustained {Ca(2+)}(cyt) increase that should be responsible for sustained mitogen-activated protein kinase activation, microtubule depolymerization, defense gene activation, and cell death . The {Ca(2+)}(cyt) increase in elicitor-treated cells first results from a calcium influx, which in turns leads to calcium release from internal stores and additional Ca(2+) influx . H(2)O(2) resulting from the calcium-dependent activation of the NADPH oxidase also participates in {Ca(2+)}(cyt) increase and may activate calcium channels from the plasma membrane . Competition assays with different elicitins demonstrate that {Ca(2+)}(cyt) increase is mediated by cryptogein-receptor interaction.

Brain Res, 2002 Oct 11, 952(1), 78 - 85
IL-1 beta is released from the host brain following transplantation but does not compromise embryonic dopaminergic neuron survival; Clarke DJ et al.; Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease . We have examined the temporal release profile of an inflammatory cytokine, interleukin-1 beta (IL-1 beta) following transplantation of fetal mesencephalic tissue into the rat striatum . The amounts of IL-1 beta released in vivo when added to cultures of embryonic DA neurons, did not significantly reduce the survival of DA neurons in vitro, and inclusion of the naturally-occurring IL-1 receptor antagonist, IL-1ra, did not appear to affect the numbers of surviving DA neurons present after 5 days in vitro . Neither did inclusion of IL-1ra in cell suspensions during transplantation increase the survival of transplanted fetal DA neurons . Thus, although IL-1 beta is released following implantation of a neural transplant, we suggest that this pro-inflammatory cytokine does not play an active role in reducing survival of transplanted DA neurons, unlike other cytokines such as tumor necrosis factor alpha . Modulation of IL-1 beta activity, therefore, will not offer significant improvements to neural transplantation as a treatment for PD.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Nov 5, 779(2), 347 - 52
Measurement of histamine in individual rat peritoneal mast cells by capillary zone electrophoresis with electrochemical detection; Weng Q et al.; Capillary zone electrophoresis was employed for the analysis of histamine in single rat peritoneal mast cells using an amperometric detector with a carbon fiber microdisk bundle electrode . In this method, individual mast cells and then 0.02 mol/l NaOH as a lysing solution are injected into the front end of the separation capillary by electromigration with an aid of a inverted microscope . A cell injector was constructed . Using it, the cell suspension was static, when a voltage for injecting single cells was applied . Histamine in single rat peritoneal mast cells have been identified . Quantitation has been accomplished through the use of calibration curves . The mean amount of histamine for nine cells is 95.8 fmol, which is consistent with the literature value.

Plant Cell, 1989 Oct, 1(10), 1003 - 1009
Receptor-Mediated Endocytosis in Plant Cells; Horn MA et al.; We have employed fluorescein and 125l-labeled elicitors of the defense response in soybeans to monitor the cellular distribution and movement of elicitors following their addition to a soybean cell suspension culture . Our results indicate that the macromolecular elicitors first bind to the cell surface and then internalize in a temperature- and energy-dependent endocytotic process . Within a few hours, virtually all of the elicitor is concentrated in the major vacuole or tonoplast of the cell . Nonspecific (control) proteins neither bound to the cell surface nor internalized in parallel assays.

J Comp Pathol, 2002 Aug-Oct, 127(2-3), 142 - 9
Cultivation of an ovine strain of Ehrlichia phagocytophila in tick cell cultures; Woldehiwet Z et al.; Ehrlichia phagocytophila (previously known as Cytoecetes phagocytophila) which causes tick-borne fever (TBF) in sheep and pasture fever in cattle in the UK and mainland Europe is transmitted by the temperate hard tick Ixodes ricinus . The disease in sheep is characterized by fever, leucopenia and immunosuppression . Studies on the pathogenesis and other aspects of the disease have been hampered because the organism has not been cultivated in continuous or primary cell culture systems . This paper describes the first successful cultivation of a European isolate of E . phagocytophila in two continuous cell lines, IDE8 and ISE6, derived from the temperate hard tick Ixodes scapularis . Once adapted to tick cell cultures the organism was serially sub-cultured in new cells by transferring small portions of infected cell suspension every 2 to 3 weeks . The identity of the organism was confirmed by polymerase chain reaction (PCR), with primers specific to the granulocytic ehrlichiae . Sequence analysis of the PCR products amplified from infected tick cells were shown to be identical with those amplified from the blood of sheep infected with the same strain of E . phagocytophila . A susceptible sheep inoculated with a third passage of the tick cell-adapted E . phagocytophila reacted with fever and rickettsiaemia 5 days later, thus satisfying Koch's postulates.

Mol Biotechnol, 2002 Sep, 22(1), 1 - 8
Vindoline biosynthesis is transcriptionally blocked in Catharanthus roseus cell suspension cultures; Vazquez-Flota F et al.; Catharanthus roseus cell cultures were exposed to different conditions in order to induce alkaloid metabolism . The exposure to jasmonate and fungal elicitors resulted in the transcriptional activation of tryptophan decarboxylase and in the accumulation of the monoterpenoid indole alkaloids ajmalicine and catharanthine, but not of vindoline . The inability of the cell cultures to produce vindoline was related to a lack of expression of the desacetoxyvindoline 4-hydroxylase (D4h) gene . Southern blot analysis revealed that D4h gene was not lost in the cell cultures.

Magn Reson Med, 2002 Oct, 48(4), 649 - 57
Human erythrocyte ghosts: exploring the origins of multiexponential water diffusion in a model biological tissue with magnetic resonance; Thelwall PE et al.; A tissue model composed of erythrocyte ghosts was developed to study the effects of compartmentation on the MR signal acquired from biological tissues . This simple and flexible model offers control over the biophysical parameters that contribute to multicomponent signals arising from cellular systems . Cell density, size, intra- and extracellular composition, and membrane permeability can be independently altered . The effects of cell density and cell size on water diffusion properties were assessed . The data demonstrate non-monoexponential water diffusion in ghost cell suspensions of 17-67% cell density . Data were analysed with the widely employed two-compartment (biexponential) model, and with a two-compartment model that accounted for exchange between compartments . Water exchange between the intra- and extracellular compartments appeared to be significant over the range of diffusion times studied (7-35 ms) . The biexponential fit to the ghost data appeared to be underparameterised as the ADCs and relative fractions of the fast and slow components were dependent on the experimental acquisition parameters, specifically the diffusion time . However, both analysis methods proved effective at tracking changes in the ghost model when it was perturbed . This was demonstrated with cell density variation, cell swelling and shrinkage experiments, and reduction of membrane water permeability using a water channel blocker (pCMBS) . We anticipate that this model system could be used to investigate compartmental diffusion effects to simulate a range of pathologies, especially ischemic stroke .

Prikl Biokhim Mikrobiol, 2002 Jul-Aug, 38(4), 381 - 8
{Kinetic characteristics of enzymatic hydrolysis of complex protein substrates for preparing culture media}; Nekliudov AD et al.; Hydrolysis of a protein mixture from muscle and bone tissues with the enzymatic system from procine pancreatic cell suspension was studied . Kinetic constants and the values of activation energy were determined for individual processes of the release of 15 amino acids . The kinetic characteristics of the overall enzymatic hydrolysis calculated from analysis of the changes in concentrations of terminal amino groups were compared with the characteristics obtained while studying the accumulation patterns of individual amino acids.

J Biotechnol, 2002 Oct 23, 99(2), 133 - 48
Determination of dissolved CO(2) concentration and CO(2) production rate of mammalian cell suspension culture based on off-gas measurement; Frahm B et al.; The determination of dissolved CO(2) and HCO(3)(-) concentrations as well as the carbon dioxide production rate in mammalian cell suspension culture is attracting more and more attention since the effects on major cell properties, such as cell growth rate, product quality/production rate, intracellular pH and apoptosis, have been revealed . But the determination of these parameters by gas analysis is complicated by the solution/dissolution of carbon dioxide in the culture medium . This means that the carbon dioxide transfer rate (CTR; which can easily be calculated from off-gas measurement) is not necessarily equal to carbon dioxide production rate (CPR) . In this paper, a mathematical method to utilize off-gas measurement and culture pH for cell suspension culture is presented . The method takes pH changes, buffer and medium characteristics that effect CO(2) mass transfer into account . These calculations, based on a profound set of equations, allow the determination of the respiratory activity of the cells, as well as the determination of dissolved CO(2), HCO(3)(-) and total dissolved carbonate . The method is illustrated by application to experimental data . The calculated dissolved CO(2) concentrations are compared with measurements from an electrochemical CO(2) probe .

ANZ J Surg, 2002 Sep, 72(9), 655 - 9
New technique to assess the axilla for breast cancer metastases using cell separation technology; Edwards M et al.; INTRODUCTION: Accurate staging of the axilla for metastatic disease is critical in deciding on the optimal management of patients with breast cancer . Lymph node status is the most powerful prognostic factor . Current standard surgical management of breast cancer involves axillary dissection for staging . Pathological staging by routine histology, however, is known to understage the disease extent because only one or two sections are taken from each node, a sampling of less than 1% of most nodes . Sentinel node biopsy is currently under trial to determine if thorough pathological staging of the most likely involved node is more accurate than standard pathological assessment of all nodes . The present pilot study was undertaken to investigate an alternative method of assessing all axillary nodes for cancer cells . METHODS: After routine material was taken from lymph nodes for standard pathological assessment, discarded parts of nodes were used for the study technique . These node parts were mechanically disaggregated, and the cell suspension centrifuged on a density gradient to separate any tumour cells (into the pellet) from lymphocytes (at the top of the gradient) . The pellet was then assessed by haematoxylin and eosin and immunohistochemistry . RESULTS: The results of the present study proved highly significant . The technique detected metastatic cells in three nodes which were negative on routine pathology, in one case changing the status of the patient from node-negative to node-positive . DISCUSSION: It is concluded that the technique examined in the present paper has the potential to reduce sampling error, may offer far more accurate axillary staging than routine histopathology, and should be further evaluated in a controlled trial.

Exp Cell Res, 2002 Oct 1, 279(2), 330 - 43
Purification of fetal mouse hepatoblasts by magnetic beads coated with monoclonal anti-e-cadherin antibodies and their in vitro culture; Nitou M et al.; A simple, rapid, and reproducible method of fetal hepatoblast purification was established to investigate mechanisms controlling interactions between hepatoblasts and nonparenchymal cells during liver development . Because E-cadherin is exclusively expressed on the cell membrane of hepatoblasts, magnetic beads coated with monoclonal antibodies to an extracellular epitope of its molecule were used to purify hepatoblasts from a cell suspension prepared from 12.5-day fetal mouse livers . The purity and yield in the hepatoblast fraction prepared in our protocol were more than 90% and approximately 30%, respectively . The nonparenchymal fraction rarely contained hepatoblasts; the rate of hepatoblast contamination in this fraction was less than 1% . Separate cultures of these two fractions were compared with cocultures of both fractions . In culture of the hepatoblast fraction, hepatoblasts formed aggregates similar to a bunch of grapes via their loose adhesion, floating in the medium after 24 h, and dissociated into single cells from the aggregates after 120 h of culture . By contrast, in the mixed culture, the majority of hepatoblasts formed multicellular spheroids after 24 h, and these spheroids changed into monolayer cell sheets after 120 h of culture . The cells comprising these monolayer sheets abundantly expressed albumin and carbamoylphosphate synthase I . In the mixed culture, fibroblastic cells also proliferated extensively with spreading on glass slides and surrounded the hepatoblast or hepatocyte colonies . On the other hand, fibroblastic cells spreading on glass slides decreased gradually in cultures of the nonparenchymal cell fraction alone . These findings indicated that the coexistence of hepatoblasts and nonparenchymal cells may be essential for their mutual survival, proliferation, differentiation, and morphogenesis . The conditioned medium of fetal liver cell cultures could partially replace the effects of the nonparenchymal cells on hepatoblasts in vitro . Our isolation protocol for fetal mouse hepatoblasts using immunobeads can greatly facilitate studies on mechanisms of cell-cell interactions during liver development.

Chemosphere, 2002 Oct, 49(1), 51 - 9
Metabolism of the environmental estrogen bisphenol A by plant cell suspension cultures; Schmidt B et al.; The metabolism of the environmental estrogen bisphenol A (BPA) was studied in heterotrophic plant cell suspension cultures of soybean (Glycine max), wheat (Triticum aestivum), foxglove (Digitalis purpurea), and thorn apple (Datura stramonium), which were regarded as metabolic model systems for intact plants . Three main metabolic routes of BPA were observed in the tissues . Most of the radioactivity found in the cell extracts consisted of carbohydrate conjugates of BPA amounting to about 85% (foxglove), 80% (wheat), 7% (soybean) and 15% (thorn apple) of applied 14C . The second main route was formation of non-extractable residues . Portions detected were low in foxglove (3.9% of applied 14C), moderate in wheat (13.5%), high in thorn apple (27.4%) and soybean (49.4%) . With thorn apple, BPA derived bound residues were preponderantly resistant towards acid treatment; only traces of BPA were released . The third route was the formation of a highly polar, presumably polymeric material detected in media of soybean and thorn apple (29.3% and 36.0% of applied 14C, respectively) . The mechanism of its formation remained unknown . In thorn apple, this highly polar material was formed extremely rapidly, and was considerably stable . Only traces of BPA were liberated by hydrolytic treatment with cellulase or acid . During hydrolysis experiments with glycoside fractions, non-extractable residues and highly polar materials, low amounts of presumably primary metabolites of BPA (up to 6% of applied 14C) were detected besides the parent compound; their chemical structures remained unclear.

Anal Biochem, 2002 Sep 1, 308(1), 168 - 77
Quantitative high-performance liquid chromatography/electrospray ionization tandem mass spectrometric analysis of 2- and 3-series prostaglandins in cultured tumor cells; Yang P et al.; This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE(2), PGE(3), and other closely related prostaglandins from cultured cells using liquid chromatography/electrospray ionization tandem mass spectrometry . This method permits quantification of selected individual prostaglandins derived either from arachidonic acid (AA) or eicosapentaenoic acid (EPA) from cell extracts without tedious derivatization, lengthy sample preparation, and separation required by GC-MS- or HPLC-UV-based methods . The validation assessment showed that the quantitative determination is linear (r(2)>0.999) for both PGE(2) and PGE(3) in the range tested (1-500 ng/ml, 0.0028-1.4 microM) and a coefficient of variation lower than 10% was obtained for samples analyzed on 3 separate days . The detection limit was 2.5 pg for both PGE(2) and PGE(3) . Extraction efficiency of PGE(2) and PGE(3) from cell suspensions ranged from 89.4 to 98.2% . As an application of the method, prostaglandins formed by EPA in human lung cancer A549 cells were determined . A 62% reduction of PGE(2) formation was noted when A549 cells were treated with 10 microM of EPA . Concomitantly, EPA increased formation of PGE(3) by 10-fold in A549 cells . This is the first report that unequivocally demonstrates that EPA can be converted to PGE(3) by cyclooxygenase in human cancer cells.

J Appl Microbiol, 2002, 93(4), 647 - 55
Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato; Bentsink L et al.; AIMS: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt . The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death . METHODS AND RESULTS: It was demonstrated by RNase treatment of extracted nucleic acids from R . solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA . The ability of NASBA to assess viability was demonstrated in two sets of experiments . In the first experiment, viable and chlorine-killed cells of R . solanacearum were added to a potato tuber extract and tested in NASBA and PCR . In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals . In the second experiment, the survival of R . solanacearum on metal strips was studied using NASBA, PCR-amplification and dilution plating on the semiselective medium SMSA . A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR-amplification resulted in positive reactions also long after cells were dead . The detection level of NASBA for R . solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction . With purified RNA a detection level of 104 rRNA molecules was found . This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA . Preliminary experiments demonstrated the potential of NASBA to detect R . solanacearum in naturally infected potato tuber extracts . CONCLUSIONS: NASBA specifically amplifies RNA from viable cells of R . solanacearum even present in complex substrates at a level of 100 cfu per reaction . SIGNIFICANCE AND IMPACT OF THE STUDY: The novel NASBA assay will be particularly valuable for detection of R . solanacearum in ecological studies in which specifically viable cells should be determined.

Plant Physiol, 1994 Dec, 106(4), 1413 - 1419
Identification of Posttranslationally Modified 18-Kilodalton Protein from Rice as Eukaryotic Translation Initiation Factor 5A; Mehta AM et al.; Using anther-derived rice (Oryza sativa L.) cell-suspension cultures, we have identified an 18-kD protein that is posttranslationally modified by spermidine and is influenced by endogenous polyamine levels . The posttranslationally modified residue has been identified as the unusual amino acid hypusine {N{epsilon}-(4-amino-2-hydroxybutyl)lysine} by reverse-phase high-performance liquid chromatography and gas chromatography-mass-spectrometry analyses . Differential labeling of the protein with labeled amines provided evidence that the butylamine moiety of spermidine is the immediate precursor of the hypusine residue in the protein . The eukaryotic translation initiation factor 5A (eIF-5A) is the only known mammalian protein that undergoes a similar posttranslational modification with hypusine . The purified 18-kD protein co-electrophoreses with human translational initiation factor eIF-5A in both isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels . The purified protein from rice stimulated methionyl-puromycin synthesis in vitro, indicating its functional similarity to mammalian eIF-5A . The results presented provide evidence that the posttranslationally modified 18-kD protein from rice containing hypusine is eIF-5A and suggest the conservation of hypusine-containing translation initiation factor eIF-5A in eukaryotes.

Plant Physiol, 1994 Nov, 106(3), 941 - 947
Light Stress and Oxidative Cell Damage in Photoautotrophic Cell Suspension of Euphorbia characias L; Bladier C et al.; A photoautotrophic cell-suspension culture of Euphorbia characias L . grown at 70 {mu}mol photons m-2 s-1 was very sensitive to light stress: the gross photosynthesis measured by using a mass spectrometric 16O2/18O2 isotope technique showed a fast decrease at a rather low light intensity of 100 {mu}mol photons m-2 s-1, far below the photosynthetic saturation level . The contribution of activated oxygen species on photosystem II photoinhibition was examined for a given light intensity . A protective effect on gross photosynthesis was observed with 1% oxygen . When light stress was applied to a methyl viologen-adapted cell suspension, photoinhibition was reduced . When 50 {mu}mol L-1 methyl viologen was added, photoinhibition was slightly enhanced . These responses suggested an involvement of superoxide radicals in the photoinhibition process of E . characias photoautotrophic cells . The long-term (16 h) effects of photoinhibition were then studied . Aldehyde (malondialdehyde and 4-hydroxyalcenals) production resulting from lipid peroxidation was stimulated in long-term stressed cells . When 50 {mu}mol L-1 methyl viologen were added, increased aldehyde production was measured . Under 1% oxygen, the aldehyde production was comparable to that of nonstressed cells . The relationship among lipid peroxidation, light intensity, and net photosynthesis suggests that aldehyde production may result from cell death provoked by a prolonged energy deficit due to the inhibition of photosynthesis.

Plant Physiol, 1994 Oct, 106(2), 723 - 730
Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study); Hagendoorn M et al.; In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2{prime},7{prime}-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (acetoxymethyl ester) and bis- {3-propyl-5-oxoisoxazol-4-yl}pentamethine oxonol, and the production of secondary metabolites for several plant cell-suspension systems . Anthraquinone production in Morinda citrifolia suspensions is negligible in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), whereas with naphthalene acetic acid (NAA) a significant accumulation is realized . NAA-grown cells showed a lower cytoplasmic pH than did 2,4-D-grown cells . Addition of 2,4-D or parachlorophenoxy acetic acid to NAA-grown cells resulted in an inhibition of anthraquinone production and an increase of the cytoplasmic pH, whereas addition of parachlorophenyl acetic acid had no effect on either parameter . Lignin production in Petunia hybrida cells could be induced by subculturing them in a medium without iron . These cells showed a lower cytoplasmic pH than control cells . Addition of Fe3+ led to a decreased lignin content and an increased cytoplasmic pH . Two cell lines of Linum flavum showed a different level of coniferin and lignin concentration in their cells . Cells that accumulated coniferin and lignin had a lower cytoplasmic pH than cells that did not accumulate these secondary metabolites . Apparently, in different species and after different kinds of treatment there is a correlation between acidification of the cytoplasm and the production of different secondary metabolites . The possible role of this acidification in secondary metabolite production is discussed.

Plant Physiol, 1994 Oct, 106(2), 703 - 712
Comparative Analysis of Short- and Long-Term Changes in Gene Expression Caused by Low Water Potential in Potato (Solanum tuberosum) Cell-Suspension Cultures; Leone A et al.; To dissect the cellular response to water stress and compare changes induced as a generalized response with those involved in tolerance/acclimation mechanisms, we analyzed changes in two-dimensional electrophoretic patterns of in vivo {35S}methionine-labeled polypeptides of cultured potato (Solanum tuberosum) cells after gradual and long exposure to polyethylene glycol (PEG)- mediated low water potential versus those induced in cells abruptly exposed to the same stress intensity . Protein synthesis was not inhibited by gradual stress imposition, and the expression of 17 proteins was induced in adapted cells . Some polypeptides were inducible under mild stress conditions (5% PEG) and accumulated further when cells were exposed to a higher stress intensity (10 and 20% PEG) . The synthesis of another set of polypeptides was up-regulated only when more severe water-stress conditions were applied, suggesting that plant cells were able to monitor different levels of stress intensity and modulate gene expression accordingly . In contrast, in potato cells abruptly exposed to 20% PEG, protein synthesis was strongly inhibited . Nevertheless, a large set of polypeptides was identified whose expression was increased . Most of these polypeptides were not induced in adapted cells, but many of them were common to those observed in abscisic acid (ABA)-treated cells . These data, along with the finding that cellular ABA content increased in PEG-shocked cells but not in PEG-adapted cells, suggested that this hormone is mainly involved in the rapid response to stress rather than long-term adaptation . A further group of proteins included those induced after long exposure to both water stress and shock . Western blot analysis revealed that osmotin was one protein belonging to this common group . This class may represent induced proteins that accumulate specifically in response to low water potential and that are putatively involved in the maintenance of cellular homeostasis under prolonged stress.

Plant Physiol, 1994 Oct, 106(2), 513 - 520
Ammonium Assimilation and the Role of {gamma}-Aminobutyric Acid in pH Homeostasis in Carrot Cell Suspensions; Carroll AD et al.; In vivo 15N NMR spectroscopy was used to monitor the assimilation of ammonium by cell-suspension cultures of carrot (Daucus carota L . cv Chantenay) . The cell suspensions were supplied with oxygen in the form of either pure oxygen ("oxygenated cells") or air ("aerated cells") . In contrast to oxygenated cells, in which ammonium assimilation had no effect on cytoplasmic pH, ammonium assimilation by aerated cells caused a decrease in cytoplasmic pH of almost 0.2 pH unit . This led to a change in nitrogen metabolism resulting in the accumulation of {gamma}-aminobutyric acid . The metabolic effect of the reduced oxygen supply under aerated conditions could be mimicked by artificially decreasing the cytoplasmic pH of oxygenated cells and was abolished by increasing the cytoplasmic pH of aerated cells . The activity of glutamate decarboxylase increased as the cytoplasmic pH declined and decreased as the pH recovered . These findings are consistent with a role for the decarboxylation of glutamate, a proton-consuming reaction, in the short-term regulation of cytoplasmic pH, and they demonstrate that cytoplasmic pH influences the pathways of intermediary nitrogen metabolism.

Plant Physiol, 1994 Jul, 105(3), 823 - 830
Effects of Abscisic Acid Metabolites and Analogs on Freezing Tolerance and Gene Expression in Bromegrass (Bromus inermis Leyss) Cell Cultures; Robertson AJ et al.; Optical isomers and racemic mixtures of abscisic acid (ABA) and the ABA metabolites abscisyl alcohol (ABA alc), abscisyl aldehyde (ABA ald), phaseic acid (PA), and 7{prime}hydroxyABA (7{prime}OHABA) were studied to determine their effects on freezing tolerance and gene expression in bromegrass (Bromus inermis Leyss) cell-suspension cultures . A dihydroABA analog (DHABA) series that cannot be converted to PA was also investigated . Racemic ABA, (+)-ABA, ({plus or minus})-DHABA, and (+)-DHABA were the most active in inducing freezing tolerance, (-)-ABA, ({plus or minus})-7{prime}OHBA, (-)-DHABA, ({plus or minus})-ABA ald, and ({plus or minus})-ABA alc had a moderate effect, and PA was inactive . If the relative cellular water content decreased below 82%, dehydrin gene expression increased . Except for (-)-ABA, increased expression of dehydrin genes and increased accumulation of responsive to ABA (RAB) proteins were linked to increased levels of frost tolerance . PA had no effect on the induction of RAB proteins; however, ({plus or minus})- and (+)-DHABA were both active, which suggests that PA is not involved in freezing tolerance . Both (+)-ABA and (-)-ABA induced dehydrin genes and the accumulation of RAB proteins to similar levels, but (-)-ABA was less effective than (+)-ABA at increasing freezing tolerance . The (-)-DHABA analog was inactive, implying that the ring double bond is necessary in the (-) isomers for activating an ABA response.

Plant Physiol, 1994 Jun, 105(2), 593 - 600
Purification and Characterization of Chloroplastic NADP-Isocitrate Dehydrogenase from Mixotrophic Tobacco Cells (Comparison with the Cytosolic Isoenzyme); Galvez S et al.; Green, mixotrophic tobacco (Nicotiana tabacum) cell cultures in the exponential growth phase were found to have two clearly distinguishable NADP-isocitrate dehydrogenase (ICDH; EC 1.1.1.42) isoenzymes . Their elution behavior during anion-exchange column chromatography was similar to that described previously for the cytosolic (ICDH1) and chloroplastic (ICDH2) enzymes from pea (Pisum sativum) leaves . ICDH2 was absent in etiolated tobacco cell suspensions and appeared during the greening process . Both isoforms were purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation and anion-exchange and affinity chromatography . The isoenzymes were separated on a DEAE-Sephacel column, but the most effective step was a Matrex Red-A column, which enabled an overall purification of 833- and 1328-fold for ICDH1 and ICDH2, respectively . Polyclonal antibodies were raised against each isoform . The ICDH2-specific antibody was used to localize tobacco leaf ICDH2 in situ by an immunogold labeling technique . The enzyme was found largely, if not exclusively, in the chloroplasts of green leaves . ICDH1 and ICDH2 were shown to have apparent native molecular weights of 117,000 and 136,000, respectively, and to consist of identical, 48.5-kD subunits . Similar apparent Km values for NADP, D(+)isocitrate, and Mg2+ were found for the two enzymes when assayed with Mg2+ as the metal cofactor.

Plant Physiol, 1994 Apr, 104(4), 1301 - 1309
Detoxification of Formaldehyde by the Spider Plant (Chlorophytum comosum L.) and by Soybean (Glycine max L.) Cell-Suspension Cultures; Giese M et al.; The phytotoxicity of formaldehyde for spider plants (Chlorophytum comosum L.), tobacco plants (Nicotiana tabacum L . cv Bel B and Bel W3), and soybean (Glycine max L.) cell-suspension cultures was found to be low enough to allow metabolic studies . Spider plant shoots were exposed to 7.1 {mu}L L-1 (8.5 mg m-3) gaseous {14C}-formaldehyde over 24 h . Approximately 88% of the recovered radioactivity was plant associated and was found to be incorporated into organic acids, amino acids, free sugars, and lipids as well as cell-wall components . Similar results were obtained upon feeding {14C}formaldehyde from aqueous solution to aseptic soybean cell-suspension cultures . Serine and phosphatidylcholine were identified as major metabolic products . Spider plant enzyme extracts contained two NAS+-dependent formaldehyde dehydrogenase activities with molecular mass values of about 129 and 79 kD . Only the latter enzyme activity required glutathione as an obligatory second cofactor . It had an apparent Km value of 30 {mu}M for formaldehyde and an isoelectric point at pH 5.4 . Total cell-free dehydrogenase activity corresponded to 13 {mu}g formaldehyde oxidized h-1 g-1 leaf fresh weight . Glutathione-dependent formaldehyde dehydrogenases were also isolated from shoots and leaves of Equisetum telmateia and from cell-suspension cultures of wheat (Triticum aestivum L.) and maize (Zea mays L.) . The results obtained are consistent with the concept of indoor air decontamination with common room plants such as the spider plant . Formaldehyde appears to be efficiently detoxified by oxidation and subsequent C1 metabolism.

Plant Physiol, 1994 Feb, 104(2), 725 - 735
Characterization and Quantification of Intrinsic Ice Nucleators in Winter Rye (Secale cereale) Leaves; Brush RA et al.; Extracellular ice formation in frost-tolerant organisms is often initiated at specific sites by ice nucleators . In this study, we examined ice nucleation activity (INA) in the frost-tolerant plant winter rye (Secale cereale) . Plants were grown at 20{deg}C, at 5{deg}C with a long day, and at 5{deg}C with a short day (5{deg}C-SD) . The threshold temperature for INA was -5 to -12{deg}C in winter rye leaves from all three growth treatments . Epiphytic ice nucleation-active bacteria could not account for INA observed in the leaves . Therefore, the INA must have been produced endogenously . Intrinsic rye ice nucleators were quantified and characterized using single mesophyll cell suspensions obtained by pectolytic degradation of the leaves . The most active ice nucleators in mesophyll cell suspensions exhibited a threshold ice nucleation temperature of -7{deg}C and occurred infrequently at the rate of one nucleator per 105 cells . Rye cells were treated with chemicals and enzymes to characterize the ice nucleators, which proved to be complexes of proteins, carbohydrates, and phospholipids, in which both disulfide bonds and free sulfhydryl groups were important for activity . Carbohydrates and phospholipids were important components of ice nucleators derived from 20{deg}C leaves, whereas the protein component was more important in 5{deg}C-SD leaves . This difference in composition or structure of the ice nucleators, combined with a tendency for more frequent INA, suggests that more ice nucleators are produced in 5{deg}C-SD leaves . These additional ice nucleators may be a component of the mechanism for freezing tolerance observed in winter rye.

Plant Physiol, 1993 Nov, 103(3), 987 - 992
Xyloglucan Endotransglycosylase Activity in Carrot Cell Suspensions during cell Elongation and Somatic Embryogenesis; Hetherington PR et al.; Xyloglucan endotransglycosylase (XET) has been proposed to contribute to cell elongation through wall loosening . To explore this relationship further, we assayed this enzyme activity in suspensions of carrot (Daucus carota L.) cells exhibiting various rates of cell elongation . In one cell line, elongation was induced by dilution into dichlorophenoxyacetic acid (2,4-D)-free medium . During this elongation, 93% of the XET activity was found in the culture medium; in nonelongating controls, by contrast, 68% was found in the cell extracts even though the specific activity of these extracts was lower than in the elongating cells . By far the highest rates of XET secretion per cell were in the elongating cells . A second cell line was induced to undergo somatic embryogenesis by dilution into 2,4-D-free medium . During the first 6 d, numerous globular embryoids composed of small, isodiametric cells were formed in the absence of cell elongation; extracellular XET activity was almost undetectable, and intracellular specific activity markedly declined . After 6 d, heart, torpedo, and cotyledonary embryoids began to appear (i.e . cell elongation resumed); the intracellular specific activity of XET rose rapidly and >80% of the XET activity accumulated in the medium . Thus, nonexpanding cell suspensions (whether or not they were rapidly dividing) produced and secreted less XET activity than did expanding cells . We propose that a XET molecule has an ephemeral wall-loosening role while it passes through the load-bearing layer of the wall on its way from the protoplast into the culture medium.

Plant Physiol, 1993 Nov, 103(3), 763 - 769
Effects of Boron on Proton Transport and Membrane Properties of Sunflower (Helianthus annuus L.) Cell Microsomes; Ferrol N et al.; Boron deficiency and toxicity inhibit ATP-dependent H+ pumping and vanadate-sensitive ATPase activity in sunflower roots and cell suspensions . The effects of boron on H+ pumping and on passive H+ conductance, as well as on fluorescence anisotropy in KI-washed microsomes isolated from sunflower (Helianthus annuus L . cv Enano) cell suspensions, have been investigated . Boron deficiency reduced the total and vanadate-sensitive ATPase activities as well as the vanadate-sensitive ATP-dependent H+ pumping without affecting the amount of antigenic ATPase protein as measured by immunoblotting with an Arabidopsis thaliana plasma membrane anti-H+-ATPase polyclonal antibody . Kinetic studies revealed that boron deficiency reduced Vmax of vanadate-sensitive ATPase activity with little change in the apparent Km for Mg2+-ATP . Proton leakage was greater in microsomal vesicles isolated from cells grown without boron and incubated in reaction medium without added boron, and this effect was reversed by addition of boron to the reaction medium . Fluorescence anisotropy indicated that diphenyl hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probes were immobilized to a greater extent in microsomes from cells grown without boron than in those from cells grown with 100 {mu}M H3BO3 . The apparent decrease of membrane fluidity in microsomes from cells grown without boron was reversed by the addition of boron to the reaction medium . Taken together these data suggest that inhibition of H+ gradient formation in microsomes from sunflower cells grown in the absence of boron could be due to the combined effects of reduced H+-ATPase activity and increased passive conductance across the membrane, possibly resulting from increased membrane rigidity.

Plant Physiol, 1993 Nov, 103(3), 719 - 726
Expression and Localization of Plant Protein Disulfide Isomerase; Shorrosh BS et al.; A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein . The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems . Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment . A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination . PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined . Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco . Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules.

Plant Physiol, 1993 Oct, 103(2), 315 - 321
Pathway of Salicylic Acid Biosynthesis in Healthy and Virus-Inoculated Tobacco; Yalpani N et al.; Salicylic acid (SA) is a likely endogenous regulator of localized and systemic disease resistance in plants . During the hypersensitive response of Nicotiana tabacum L . cv Xanthi-nc to tobacco mosaic virus (TMV), SA levels rise dramatically . We studied SA biosynthesis in healthy and TMV-inoculated tobacco by monitoring the levels of SA and its likely precursors in extracts of leaves and cell suspensions . In TMV-inoculated leaves, stimulation of SA accumulation is accompanied by a corresponding increase in the levels of benzoic acid . 14C-Tracer studies with cell suspensions and mock-or TMV-inoculated leaves indicate that the label moves from trans-cinnamic acid to SA via benzoic acid . In healthy and TMV-inoculated tobacco leaves, benzoic acid induced SA accumulation . o-Coumaric acid, which was previously reported as a possible precursor of SA in other species, did not increase SA levels in tobacco . In healthy tobacco tissue, the specific activity of newly formed SA was equal to that of the supplied {14C}benzoic acid, whereas in TMV-inoculated leaves some isotope dilution was observed, presumably because of the increase in the pool of endogenous benzoic acid . We observed accumulation of pathogen-esis-related-1 proteins and increased resistance to TMV in benzoic acid- but not in o-coumaric acid-treated tobacco leaves . This is consistent with benzoic acid being the immediate precursor of SA . We conclude that in healthy and virus-inoculated tobacco, SA is formed from cinnamic acid via benzoic acid.

Plant Physiol, 1993 Aug, 102(4), 1227 - 1235
Cold-Induced Changes in Freezing Tolerance, Protein Phosphorylation, and Gene Expression (Evidence for a Role of Calcium); Monroy AF et al.; The role of Ca2+ in cold-induced changes in protein phosphorylation, gene expression, and development of freezing tolerance has been studied in cell-suspension cultures of a freezing-tolerant cultivar of alfalfa (Medicago sativa spp . falcata cv Anik) . Chemical treatments to block Ca2+ channels, antagonize calmodulin action, or inhibit protein kinases markedly inhibited the cellular capacity to develop cold-induced freezing tolerance but had little effect on cell viability . An analysis of phosphoprotein profile by two-dimensional polyacrylamide gel electrophoresis revealed that at low temperature the relative level of phosphorylation of several proteins increased, whereas that of several others decreased . When cold acclimation was carried out in the presence of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride, an antagonist of calmodulin and Ca2+-dependent protein kinases, or the Ca2+ channel blocker La3+, the cold-induced changes in protein phosphorylation were strongly inhibited, cells lost their capacity to develop freezing tolerance, and accumulation of transcripts of cold acclimation-specific genes was substantially reduced . An inhibitor of protein kinases, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, had less pronounced effects on the cold-induced protein phosphorylation and caused only a partial inhibition of the cold-induced development of freezing tolerance and accumulation of the transcripts . The level of phosphorylation of one protein, of about 15 kD, increased more than 10-fold at low temperature and showed a strong positive correlation with cold-induced freezing tolerance and gene expression even when the latter were altered with various chemical treatments . These results suggest that Ca2+ and protein phosphorylation, or perhaps a coupling of the two, play an important role during the acquisition of freezing tolerance during cold acclimation.

Plant Physiol, 1993 Jul, 102(3), 851 - 857
Nitrate Fluxes and Nitrate Reductase Activity of Suspension-Cultured Tobacco Cells (Effects of Internal and External Nitrate Concentrations); Zhang N et al.; Cell suspensions of tobacco (Nicotiana tabacum L., cv KY14) were used to determine the responses of NO3- uptake and NO3- reductase activity (NRA) to exogenous NO3- levels in the absence of long-distance NO3- transport . Tobacco cells grown with complete Murashige and Skoog medium for 7 d were subcultured for 3 d with NH4+-free media containing 0, 5, 10, 20, 30, and 40 mM NO3- . Cell NO3-, in vitro NRA, NO3- influx, and efflux of cell NO3- were determined . The NRA increased as cell NO3- increased . Cell NO3- efflux values increased as cell NO3- level increased . Cells with low intracellular NO3- had greater NO3- influx than cells with high intracellular NO3- . Woolf-Augustinsson-Hofstee transformations of the NO3- influx kinetic data revealed patterns characteristic of a high- and low-affinity two-component NO3- uptake system . Apparent Vmax values generally decreased and Km values increased as cell NO3- concentration increased . The NRA of cells supplied with 10 and 20 mM NO3- after 3-d growth in N- free medium increased about 5-fold within 2 h and then remained constant for the next 2 h, whereas NRA of cells supplied with 5 mM NO3- increased only 2-fold during the 4-h period . Intracellular NO3- and other N metabolites associated with cell NO3- levels exerted differential effects on the NO3- influx activity and NRA of tobacco cells cultured in suspension . Expression of high NRA was correlated with both high external and intracellular NO3-, whereas maximum NO3- influx activity required a low (depleted) level of cell NO3-.

Plant Physiol, 1993 Jun, 102(2), 565 - 571
Developmental Profile of Diacylglycerol Acyltransferase in Maturing Seeds of Oilseed Rape and Safflower and Microspore-Derived Cultures of Oilseed Rape; Weselake RJ et al.; Diacylglycerol acyltransferase (EC 2.3.1.20) activity was assayed during the maturation of seeds of oilseed rape (Brassica napus L.) and safflower (Carthamus tinctorius L.) . Developmental studies were also conducted with microspore-derived embryos of oilseed rape (B . napus L . cv Topas) and an embryogenic microspore-derived cell-suspension culture of winter oilseed rape (B . napus L . cv Jet Neuf) . In the maturing seeds, diacylglycerol acyltransferase activity increased to a maximum during rapid accumulation of lipid and declined, thereafter, with seed maturity . In microspore-derived embryos of oilseed rape (cv Topas), high levels of diacylglycerol acyltransferase activity were found throughout the early torpedo to late cotyledonary developmental stages with maximum enzyme specific activity associated with the mid-cotyledonary developmental stage . The cell-suspension culture of winter oilseed rape (cv Jet Neuf) contained 3 to 4% triacylglycerol on a dry weight basis and represented about half of the total lipid . The fatty acid profile of total lipid and triacylglycerol in the cell-suspension culture was similar in samples taken during a 1-year period . The Jet Neuf culture contained diacylglycerol acyltransferase with specific activity similar to that of Topas microspore-derived embryos . Jet Neuf diacylglycerol acyltransferase also displayed an enhanced specificity for erucoyl-CoA over oleoyl-CoA when assayed with 14 {mu}M acyl-coenzyme A in the reaction mixture . The specific activity of diacylglycerol acyltransferase in homogenates prepared from the Jet Neuf culture ranged from 5 to 15 pmol of triacylglycerol min-1 mg-1 of protein when assayed at intervals during a period of 1 year . Thus, the cell-suspension culture may represent an attractive tissue source for purification and characterization of triacyl-glycerol biosynthetic enzymes.

Plant Physiol, 1993 Jun, 102(2), 513 - 520
Further Characterization of Expression of Auxin-Induced Genes in Tobacco (Nicotiana tabacum) Cell-Suspension Cultures; Boot K et al.; We have described the modulation of four auxin-regulated genes during the growth cycle of suspension-cultured tobacco (Nicotiana tabacum {L.} var White Burley) cells . The genes were transiently expressed 2 to 8 h after transfer of stationary phase cells to fresh medium, during the transition from the quiescent phase of cells leaving the mitotic cycle to the synthesis phase of the cell cycle . After this transient induction, the cells showed a decreased sensitivity to auxin . Although the expression pattern suggests that induction of these genes might be important for cell division, over-production of antisense mRNA for one of these genes (pCNT103) did not influence cell division in transgenic tobacco cells . Furthermore, stimuli such as salicylic acid were capable of inducing gene expression but were unable to restore cell division . Although these data do not conclusively exclude a role for these genes in cell division, their significance in this process is discussed in view of their homology with other auxin-induced genes and in view of the specificity of hormone-induced early responses.

Plant Physiol, 1993 Mar, 101(3), 1081 - 1088
Sensitivity to an Ethylene Biosynthesis-Inducing Endoxylanase in Nicotiana tabacum L . cv Xanthi Is Controlled by a Single Dominant Gene; Bailey BA et al.; The ethylene biosynthesis-inducing xylanase (EIX) is known to be a potent elicitor of ethylene biosynthesis and other responses when applied to leaf tissue of Nicotiana tabacum L . cv Xanthi . In contrast, leaf tissue of the tobacco cultivar Hicks was insensitive to EIX at concentrations 100-fold higher than was needed to elicit responses from Xanthi . Cell-suspension cultures of Xanthi and Hicks showed similar differences in sensitivity to EIX . Equivalent levels of ethylene production were elicited in leaf discs of both cultivars after treatment with CuSO4 . The F1 and Xanthi backcross progeny of Hicks and Xanthi crosses were all sensitive to EIX, whereas the F2 and Hicks backcross progeny segregated for sensitivity to EIX . Individual plants from the F2 and Hicks backcross that were insensitive to EIX produced only insensitive progeny when they were self-pollinated . Progeny from sensitive plants either segregated for sensitivity to EIX or produced all sensitive progeny (an F2 plant) . Sensitivity to EIX is controlled by a single dominant gene, based on chi-square analysis of segregation ratios.

Plant Physiol, 1993 Mar, 101(3), 847 - 856
Stress Responses in Alfalfa (Medicago sativa L.) (XIV . Changes in the Levels of Phenylpropanoid Pathway Intermediates in Relation to Regulation of L-Phenylalanine Ammonia-Lyase in Elicitor-Treated Cell-Suspension Cultures); Orr JD et al.; We have used high-resolution gas chromatography to determine the levels of trans-cinnamic acid (CA) and trans-4-coumaric acid (4CA) in alfalfa (Medicago sativa L.) cell-suspension cultures to address the role of these phenylpropanoid pathway intermediates as potential negative regulators of phenylalanine ammonia-lyase (PAL) in vivo . Exogenous addition of CA to elicitor-treated cultures resulted in rapid increases in endogenous CA, 4CA, and CA-conjugate levels associated with inhibition of the appearance of PAL transcripts . Treatment of elicited cultures with {alpha}-aminooxy-{beta}-phenylpropionic acid (AOPP), a potent and specific inhibitor of PAL activity in vivo, resulted in reductions of CA and 4CA, with concomitant increases in PAL transcripts and extractable enzyme activity . In contrast, treatment with tetcyclacis, an inhibitor of CA 4-hydroxylase, resulted in increased CA and CA-conjugate levels, decreased 4CA levels, and decreased PAL transcript levels and enzyme activity . In tetcyclasis-treated cells, the inhibition of PAL transcript appearance preceded the increase in the levels of free CA and its conjugates . In elicited cells in which the phenylpropanoid pathway was not perturbed by metabolic inhibitors, PAL transcripts accumulated rapidly and transiently, beginning to decline by 2 h postelicitation . Changes in levels of total free or conjugated CA or 4CA did not consistently correlate with these changes in transcript levels . We propose that regulation of PAL transcript levels by endogenous phenylpropanoid pathway intermediates could involve compartmentalized pools that may exist because of the microsomal localization of cinnamic acid 4-hydroxylase.

Plant Physiol, 1993 Jan, 101(1), 81 - 88
Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures; Van Duijn B et al.; The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures . The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps . In all protoplasts, a voltage- and time-dependent outward rectifying current was present . The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course . The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration . The outward current did not show inactivation . In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well . The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course . The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.

Mol Ther, 2002 Sep, 6(3), 407 - 14
In vivo generation of dendritic cells by intramuscular codelivery of FLT3 ligand and GM-CSF plasmids; Peretz Y et al.; Dendritic cells (DCs) are the major cells responsible for the uptake and the transport of antigens to regional lymphoid tissues and for the presentation of antigenic peptides to T cells . They are highly effective in immunotherapy . However, in lymphoid and other tissues, DCs constitute only a small population and are difficult to isolate in large numbers . Our objective was to devise a method with which to rapidly expand splenic DCs in vivo . We accomplished this by intramuscular injection of plasmids encoding mouse granulocyte-macrophage colony stimulating factor (GM-CSF) and fms-like tyrosine kinase 3-ligand (FLT3-L) . Gene transfer was amplified by electroporation . Both cytokine vectors significantly increased DC numbers, but they were more effective in combination . When either control plasmid (Blank), or FLT3-L or GM-CSF expression plasmids were injected individually, the mean numbers of CD11c(+)/MHC II(+) DCs in spleen cell suspensions were, respectively, 6, 11, and 23 million . When FLT3-L and GM-CSF plasmids were codelivered, this increased to 36 million . Peak levels occurred 7 days postinjection of DNA . To further characterize these DCs, we stained them with myeloid (CD11b, F4/80)- and lymphoid (CD8alpha)-related markers . FLT3-L cDNA favored lymphoid DC expansion and GM-CSF cDNA favored myeloid DC expansion, whereas combined treatment expanded both types with a myeloid predominance . We confirm the ability of these DCs to present antigen to CD4(+) T cells and to stimulate in mixed lymphocyte cultures . We demonstrate that DCs can be rapidly expanded by this simple gene transfer method, which has numerous potential applications.

Water Res, 2002 Aug, 36(14), 3429 - 38
Elimination of free-living amoebae in fresh water with pulsed electric fields; Vernhes MC et al.; This study investigates the effects of pulsed electric fields on the inactivation of trophozoite form of Naegleria lovaniensis Ar9M-1 in batch and flow processes, systematically examining the lethal effect of field strength, pulse duration, number of pulses, and pulse frequency . Our results show that amoebae eradication is modulated by pulse parameters, composition of the pulsing medium, and physiological state of the cells . Cell survival is not related to the energy delivered to the cell suspension during the electrical treatment . For a given energy a strong field applied for a short cumulative pulse duration affects viability more than a weak field with a long cumulative pulsation . We also determine the optimal electrical conditions to obtain an inactivation rate higher than 95% while using the least energy . Flow processes allow to treat large-scale volumes . Our results show that the most efficient flow process for amoeba eradication requires a field parallel to the flow . Pulsed electric fields are a new and attractive method for inactivating amoebae in large volumes of fresh water.

Am J Chin Med, 2002, 30(2-3), 315 - 25
Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adducts in human promyelocytic leukemia cells; Lin JG et al.; Two components of garlic, diallyl sulfide (DAS) and diallyl disulfide (DADS), inhibited arylamine N-acetyltransferase (NAT) activity and 2-aminofluorene-DNA adduct in human promyelocytic leukemia cells (HL-60) . The NAT activity was measured by high performance liquid chromatography assaying for amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF) . Cellular cytosols and intact cell suspensions were assayed . The inhibition of NAT activity and 2-AF-DNA adduct formation in human leukemia cells by DAS and DADS were dose-dependent and were directly proportional . The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax from human leukemia cells in both assays . This is the first report of garlic components affecting human leukemia cell NAT activity and 2-AF-DNA adduct formation.

Plant Physiol, 1995 Sep, 109(1), 141 - 152
Cell-Free Synthesis of Pectin (Identification and Partial Characterization of Polygalacturonate 4-{alpha}-Galacturonosyltransferase and Its Products from Membrane Preparations of Tobacco Cell-Suspension Cultures); Doong RL et al.; Polygalacturonate 4-{alpha}-galacturonosyltransferase (EC 2.4.1.43) activity has been identified in microsomal membranes isolated from tobacco (Nicotiana tabacum L . cv Samsun) cell-suspension cultures . Incubation of UDP-{14C}galacturonic acid with tobacco membranes results in a time-dependent incorporation of {14C}galacturonic acid into a chloroform-methanol-precipitable and 65% ethanol-insoluble product . The optimal synthesis of product occurs at a pH of 7.8, 25 to 30{deg}C, an apparent Km for UDP-D-galacturonic acid of approximately 8.9 {mu}M, and a Vmax of approximately 150 pmol min-1 mg-1 protein . The product was characterized by scintillation counting, thin-layer chromatography, high-performance anion-exchange chromatography, and gel-filtration chromatography in combination with enzymatic and chemical treatments . The intact product has a molecular mass of approximately 105,000 D based on dextran molecular standards . The product was treated with base to hydrolyze ester linkages (e.g . methyl esters), digested with a homogeneous endopolygalacturonase (EPGase), or base and EPGase treated . Base and EPGase treatment results in cleavage of 34 to 89% of 14C-labeled product into components that co-chromatograph with mono-, di-, and trigalacturonic acid, indicating that a large portion of product contains contiguous 1,4-linked {alpha}-D-galactosyluronic acid residues . Optimal EPGase fragmentation of the product requires base treatment prior to enzymatic digestion, suggesting that 45 to 67% of the galacturonic acid residues in the synthesized homogalacturonan are esterified . At least 40% of the base-sensitive linkages were shown to be methyl esters by comparing the sensitivity of base-treated and pectin methylesterase-treated products to fragmentation by EPGase.

Plant Physiol, 1995 Sep, 109(1), 131 - 139
A Cytochrome P-450 Monooxygenase Catalyzes the First Step in the Conversion of Tabersonine to Vindoline in Catharanthus roseus; St-Pierre B et al.; Hydroxylation at the C-16 position of the indole alkaloid tabersonine has been suggested as the first step toward vindoline biosynthesis in Catharanthus roseus . Tabersonine 16-hydroxylase (16-OH) activity was detected in total protein extracts from young leaves of C . roseus using a novel coupled assay system . Enzyme activity was dependent on NADPH and molecular oxygen and was inhibited by CO, clotrimazole, miconazole, and cytochrome c . 16-OH was localized to the endoplasmic reticulum by linear sucrose density gradient centrifugation . These data suggest that 16-OH is a cytochrome P-450-dependent monooxygenase . The activity of 16-OH reached a maximum in seedlings 9 d postimbibition and was induced by light . The leaf-specific distribution of 16-OH in the mature plant is consistent with the localization of other enzymes in the tabersonine to vindoline pathway . However, in contrast to enzymes that catalyze the last four steps of vindoline biosynthesis, enzymes responsible for the first two steps from tabersonine (16-OH and 16-O-methyltransfersase) were detected in C . roseus cell-suspension cultures . These data complement the complex model of vindoline biosynthesis that has evolved with respect to enzyme compartmentalization, metabolic transport, and control mechanisms.

Plant Physiol, 1995 Aug, 108(4), 1553 - 1560
Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of Hyoscyamus muticus; Medina-Bolivar F et al.; Hairy root cultures of Hyoscyamus muticus have been shown to produce stable levels of tropane alkaloids comparable to those found in whole plants . In contrast, cell cultures of this and other solanaceous species produce only trace amounts of alkaloids but can be used for selection of metabolic variants . We have taken advantage of both systems and the ability to convert between them in vitro in an effort to select for increased production of the tropane alkaloid hyoscyamine . Hairy roots were converted into cell suspensions by addition of 1 mg/L 2,4-dichlorophenoxyacetic acid to Murashige-Skoog medium (T . Murashige and F . Skoog {1962} Physiol Plant 15: 473-497) and screened for resistance to the amino acid analog p-fluorophenylalanine (PFP) . Cells that could grow in media containing 400 {mu}M PFP were selected and cloned from single cells . The resistant cells accumulated high levels of cinnamoyl putrescines, which share the same biosynthetic precursors as hyoscyamine . Hairy root cultures were regenerated from both PFP-sensitive and PFP-resistant cells by removing 2,4-dichlorophenoxyacetic acid from the medium . Resistance to PFP continued to be expressed in regenerated roots . Higher levels of hyoscyamine were found in hairy roots regenerated from PFP-resistant cells than were found in controls . We suggest that the precursors overproduced by the PFP-resistant cells can be diverted into the hyoscyamine pathway upon the regeneration of root cultures.

Plant Physiol, 1995 Jul, 108(3), 1171 - 1178
Pretreatment of Parsley Suspension Cultures with Salicylic Acid Enhances Spontaneous and Elicited Production of H2O2; Kauss H et al.; Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to study the regulation of extracellular H2O2 . After resuspension, the washed cells regulated the H2O2 concentration spontaneously to a constant level that was greatly increased when the cultures were pretreated for 1 d with salicylic acid (SA) . The H2O2 level was further increased on addition of a fungal elicitor preparation, macromolecular chitosan, the sterol-binding polyene macrolide amphotericin B, the G protein-activating peptide mastoparan, or La3+ . In all cases, this induced H2O2 burst was also greatly enhanced in cell suspensions pretreated with SA . Both the spontaneous and the induced H2O2 production were decreased by the protein kinase inhibitor K-252a . It is suggested that production of extracellular H2O2 occurs by an endogenously controlled plasma membrane enzyme complex that requires continuous phosphorylation for function and whose activity is increased by pretreatment of the cells with SA . This system can also receive various external stimuli, including those resulting from binding of fungal elicitor . SA can induce acquired resistance against pathogens . The conditioning of the parsley suspension culture by SA represents, therefore, a model for the long-term regulation of apoplastic H2O2 concentration by this signal substance, as suggested previously for the wound hormone methyl jasmonate.

Plant Physiol, 1995 May, 108(1), 277 - 283
Measurements of in Vivo Ubiquinone Reduction Levels in Plant Cells; Wagner AM et al.; A method is described for the determination of in vivo ubiquinone (UQ) reduction levels in nongreen tissues by extraction and subsequent detection of ubiquinone-10 and ubiquinol-10 with high-performance liquid chromatography . In Petunia hybrida cell suspensions UQ reduction remained at a stable level of about 60%, despite the changing conditions during the batch culture (from excess sugar to starvation) and the concomitant variations in respiration . Also, in the presence of uncoupler, which causes a large increase in respiration via both the cytochrome pathway and the alternative pathway, UQ reduction levels stayed at 60% . In mitochondria isolated from these cells, activity of the alternative pathway was only observed at UQ reduction levels higher than 80% . It is proposed that in vivo the relationship between UQ reduction and the activity of the alternative oxidase is modulated by mechanisms such as thiol modifications and accumulation of organic acids . Accordingly, pyruvate concentration in P . hybrida cells increased in the presence of uncoupler.

Plant Physiol, 1995 Apr, 107(4), 1241 - 1247
Plasma Membrane Redox Enzyme Is Involved in the Synthesis of O2- and H2O2 by Phytophthora Elicitor-Stimulated Rose Cells; Auh CK et al.; An elicitor prepared from the autoclaved cell walls of Phytophthora sp . induced O2- generation and H2O2 accumulation by cultured cells of Rosa damascena Mill . cv Gloire de Guilan . N,N-Diethyldithiocarbamate, a superoxide dismutase inhibitior, blocked H2O2 accumulation and caused a dramatic accumulation of O2- by elicitor-treated rose cells . In the absence of N,N-diethyldithiocarbamate no detectable O2- was accumulated . Diphenyleneiodonium, quinacrine, pyridine, and imidazole, inhibitors of the mammalian neutrophil NADPH oxidase responsible for the generation of O2- during phagocytosis, inhibited O2- generation by elicitor-treated rose cells . Diphenyleneiodonium also inhibited NADH-dependent O2- production by plasma membranes isolated from rose cells . None of the four compounds inhibited the peroxidase activity in the cell-suspension medium . These results demonstrate that elicitor-stimulated accumulation of H2O2 comes only from superoxide dismutase-catalyzed dismutation of O2- . The data are inconsistent with the hypothesis that the synthesis of O2- is catalyzed by extracellular peroxidase and suggest that the enzyme responsible for the synthesis of O2- by elicitor-treated rose cells might be similar to the mammalian neutrophil NADPH oxidase.

Plant Physiol, 1995 Feb, 107(2), 545 - 552
Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum; Hohlfeld H et al.; A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase {THT}, EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L . cv Datura) . The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine) . The apparent native Mr was found to be approximately 49,000 . Highest activity was at pH 6.8 in K-phosphate . The isoelectric point of the enzyme was approximately pH5.2 . The apparent energy of activation was calculated to be 96 kJ mol-1 . The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+ . The apparent Km values were 36 {mu}M for feruloyl-CoA and 85 and 140 {mu}M for cinnamoyl- and 4-coumaroyl-CoA, respectively . The Km value for tyramine in the presence of feruloyl-CoA was 22 {mu}M . In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 {mu}M . The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively . Thus, the reaction occurred in a ternary complex of the enzyme and substrates . The equilibrium constant of the reaction was determined to be 1.3 x 104 . This gave a {delta}G{deg}{prime} eq value of -23.5 kJ mol-1.

Plant Physiol, 2002 Sep, 130(1), 334 - 46
Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase; Wentzinger LF et al.; To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively . These two enzymes catalyze the first two steps involved in sterol biosynthesis . In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases . At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and {(14)C}radioactivity from acetate incorporated into sterols . A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels . Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis . In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways . We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required . Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols . At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.

Plant Physiol, 1996 Nov, 112(3), 1005 - 1014
Interactions between Photosynthesis and Respiration in the Green Alga Chlamydomonas reinhardtii (Characterization of Light-Enhanced Dark Respiration); Xue X et al.; The rate of respiratory O2 consumption by Chlamydomonas reinhardtii cell suspensions was greater after a period of photosynthesis than in the preceding dark period . This "light-enhanced dark respiration" (LEDR) was a function of both the duration of illumination and the photon fluence rate . Mass spectrometric measurements of gas exchange indicated that the rate of gross respiratory O2 consumption increased during photosynthesis, whereas gross respiratory CO2 production decreased in a photon fluence rate-dependent manner . The rate of postillumination O2 consumption provided a good measure of the O2 consumption rate in the light . LEDR was substantially decreased by the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or glycolaldehyde, suggesting that LEDR was photosynthesis-dependent . The onset of photosynthesis resulted in an increase in the cellular levels of phosphoglycerate, malate, and phosphoenolpyruvate, and a decrease in whole-cell ATP and citrate levels; all of these changes were rapidly reversed upon darkening . These results are consistent with a decrease in the rate of respiratory carbon flow during photosynthesis, whereas the increase in respiratory O2 consumption during photosynthesis may be mediated by the export of photogenerated reductant from the chloroplast . We suggest that photosynthesis interacts with respiration at more than one level, simultaneously decreasing the rate of respiratory carbon flow while increasing the rate of respiratory O2 consumption.

Plant Physiol, 1996 Oct, 112(2), 717 - 726
Stress Responses in Alfalfa (XXI . Activation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase Genes Does Not Contribute to Changes in Metabolite Accumulation in Elicitor-Treated Cell-Suspension Cultures); Ni W et al.; Transcription of genes encoding L-phenylalanine ammonia-lyase (PAL), the first enzyme of the phenylpropanoid pathway, and caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT), enzymes involved in the synthesis of lignin and wall-esterified phenolic compounds, was strongly activated in elicitor-treated cell-suspension cultures of alfalfa (Medicago sativa L.) . However, consequent changes in the extractable activities of COMT and CCOMT were small to nonexistent compared with a 15- to 16-fold increase in PAL activity . Only low levels of COMT and CCOMT transcripts were reflected in the total and polysomal RNA fractions compared with PAL transcripts . Elicited cell cultures did not accumulate lignin or the products of COMT and CCOMT in the soluble and wall-esterified phenolic fractions . In one alfalfa cell line in which elicitation resulted in very high PAL activity and increased deposition of methoxyl groups in the insoluble wall fraction, there was still no change in COMT and CCOMT activities . Overall, these results indicate that the initial gene transcription events in elicited cells may be less selective than the subsequent metabolic changes, highlighting the importance of posttranscriptional events in the control of phenylpropanoid biosynthesis.

Plant Physiol, 1996 Sep, 112(1), 311 - 318
Characterization of Novel Sesquiterpenoid Biosynthesis in Tobacco Expressing a Fungal Sesquiterpene Synthase; Zook M et al.; The gene encoding trichodiene synthase (Tri5), a sesquiterpene synthase from the fungus Fusarium sporotrichioides, was used to transform tobacco (Nicotiana tabacum) . Trichodiene was the sole sesquiterpene synthase product in enzyme reaction mixtures derived from unelicited transformant cell-suspension cultures, and both trichodiene and 5-epi-aristolochene were observed as reaction products following elicitor treatment . Immunoblot analysis of protein extracts revealed the presence of trichodiene synthase only in transformant cell lines producing trichodiene . In vivo labeling with {3H}mevalonate revealed the presence of a novel trichodiene metabolite, 15-hydroxytrichodiene, that accumulated in the transformant cell-suspension cultures . In a trichodiene-producing transformant, the level of 15-hydroxytrichodiene accumulation increased after elicitor treatment . In vivo labeling with {14C}acetate showed that the biosynthetic rate of trichodiene and 15-hydroxytrichodiene also increased after elicitor treatment . Incorporation of radioactivity from {14C}acetate into capsidiol was reduced following elicitor treatment of a trichodiene-producing transformant as compared with wild type . These results demonstrate that sesquiterpenoid accumulation resulting from the constitutive expression of a foreign sesquiterpene synthase is responsive to elicitation and that the farnesyl pyrophosphate present in elicited cells can be utilized by a foreign sesquiterpene synthase to produce high levels of novel sesquiterpenoids.

Plant Physiol, 1996 Jul, 111(3), 885 - 891
Relationship between Active Oxygen Species, Lipid Peroxidation, Necrosis, and Phytoalexin Production Induced by Elicitins in Nicotiana; Rusterucci C et al.; Excised leaves of Nicotiana tabacum var Xanthi and Nicotiana rustica were treated with cryptogein and capsicein, basic and acidic elicitins, respectively . Both compounds induced leaf necrosis, the intensity of which depended on concentration and duration of treatment . N . tabacum var Xanthi was the most sensitive species and cryptogein was the most active elicitin . Lipid peroxidation in elicitin-treated Nicotiana leaves was closely correlated with the appearance of necrosis . Elicitin treatments induced a rapid and transient burst of active oxygen species (AOS) in cell cultures of both Nicotiana species, with the production by Xanthi cells being 6-fold greater than that by N . rustica . Similar maximum AOS production levels were observed with both elicitins, but capsicein required 10-fold higher concentrations than those of cryptogein . Phytoalexin production was lower in response to both elicitins in N . tabacum var Xanthi cells than in N . rustica cells, and capsicein was the most efficient elicitor of this response . In cryptogein-treated cell suspensions, phytoalexin synthesis was unaffected by diphenyleneiodonium, which inhibited AOS generation, nor was it affected by tiron or catalase, which suppressed AOS accumulation in the extracellular medium . These results suggest that AOS production, lipid peroxidation, and necrosis are directly related, whereas phytoalexin production depends on neither the presence nor the intensity of these responses.

Plant Physiol, 1996 Mar, 110(3), 791 - 799
Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits); Borejsza-Wysocki W et al.; p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W . Borejsza-Wysocki and G . Hrazdina {1994} Phytochemistry 35: 623-628) . Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases . We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity . This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits . Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures . The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 {mu}M . We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present . The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

J Biol Chem, 2002 Nov 15, 277(46), 43948 - 60 Epub 2002 Sep 10.
The metabolic architecture of plant cells . Stability of central metabolism and flexibility of anabolic pathways during the growth cycle of tomato cells; Rontein D et al.; The changes in the intermediary metabolism of plant cells were quantified according to growth conditions at three different stages of the growth cycle of tomato cell suspension . Eighteen fluxes of central metabolism were calculated from (13)C enrichments after near steady-state labeling by a metabolic model similar to that described in Dieuaide-Noubhani et al . (Dieuaide-Noubhani, M., Raffard, G., Canioni, P., Pradet, A., and Raymond, P . (1995) J . Biol . Chem . 270, 13147-13159), and 10 net fluxes were obtained directly from end-product accumulation rates . The absolute flux values of central metabolic pathways gradually slowed down with the decrease of glucose influx into the cells . However, the relative fluxes of glycolysis, the pentose-P pathway, and the tricarboxylic acid cycle remained unchanged during the culture cycle at 70, 28, and 40% of glucose influx, respectively, and the futile cycle of sucrose remained high at about 6-fold the glucose influx, independently from carbon nutritional conditions . This natural resistance to flux alterations is referred to as metabolic stability . The numerous anabolic pathways, including starch synthesis, hexose accumulation, biosynthesis of wall polysaccharides, and amino and organic acid biosynthesis were comparatively low and variable . The phosphoenolpyruvate carboxylase flux decreased 5-fold in absolute terms and 2-fold in relation to the glucose influx rate during the culture cycle . We conclude that anabolic fluxes constitute the flexible part of plant cell metabolism that can fluctuate in relation to cell demands for growth.

Plant Physiol, 1997 Oct, 115(2), 783 - 791
The Effects of Salicylic Acid and Tobacco Mosaic Virus Infection on the Alternative Oxidase of Tobacco; Lennon AM et al.; Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices . The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco . Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment . Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d . Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA . Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxidase protein, suggesting that steady-state alternative oxidase accumulates in an SA-independent manner . Infection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants . Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.

Plant Physiol, 1997 Oct, 115(2), 683 - 692
A Secreted Factor Inducs Cell Expansion and Formation of Metaxylem-Like Tracheary Elements in Xylogenic Suspension Cultures of Zinnia; Roberts AW et al.; Conditioned medium from mesophyll cell-suspension cultures of Zinnia elegans L . has striking effects on cell expansion and tracheary element differentiation when applied to cultures of freshly isolated mesophyll cells . These effects include (a) induction of early cell expansion, (b) delay in differentiation by 48 h or more, (c) reduction in the synchrony of differentiation, and (d) early formation of very large, metaxylem-like tracheary elements . Like reduced osmotic potential and buffering at pH 5.5, conditioned medium appears to have its primary effect on cell expansion . Partial characterization of the expansion-inducing factor indicates that it is heat stable, of low molecular mass, and is resistant to protease . It also binds reversibly to concanavalin A but is not adsorbed by charcoal . We suggest that the secreted factor may be an oligosaccharide involved in the coordination of cell expansion and differentiation and the regulation of the protoxylem-like to metaxylem-like transition in xylogenic suspension cultures.

Plant Physiol, 1997 Sep, 115(1), 87 - 92
Formation of Di-Isodityrosine and Loss of Isodityrosine in the Cell Walls of Tomato Cell-Suspension Cultures Treated with Fungal Elicitors or H2O2; Brady JD et al.; About 84% of the hydroxyproline residues in a cell culture of tomato (Lycopersicon esculentum x Lycopersicon peruvianum) were present in phenol-inextractable (i.e . covalently wall-bound) material . Treatment of the cells with any of three fungal elicitors (wall fragments from Phytophthora megasperma and Pythium aphanidermatum and xylanase from Aureobasidium pullulans) or with 1 mM H2O2 had little effect on the quantity of phenolinextractable hydroxyproline per milligram of freeze-dried cells . However, each treatment induced a decrease in the content of phenol-inextractable isodityrosine (Idt) residues . Each treatment, except with the P . megasperma fragments, also induced an increase in phenol-inextractable di- (Di-Idt) . The increase in Di-Idt partly accounted for the loss of Idt . We conclude that the elicitors and H2O2 acted to reinforce the existing cross-linking of cell wall (glyco)proteins by evoking oxidative coupling reactions to convert Idt to Di-Idt plus unidentified products . The promotion of cross-linking by elicitor treatment is proposed to be a defensive response that restricts the penetration of pathogens.

Plant Physiol, 1997 Aug, 114(4), 1433 - 1442
Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism; Travert S et al.; A cell-suspension culture obtained from the hybrid Eucalyptus gunnii/Eucalyptus globulus was hardened by exposure to lower temperatures, whereas in the same conditions cells from a hybrid with a more frost-sensitive genotype, Eucalyptus cypellocarpa/Eucalyptus globulus, were not able to acclimate . During the cold exposure the resistant cells accumulated soluble sugars, in particular fructose and sucrose, with a limited increase in cell osmolality . In contrast, the cell suspension that was unable to acclimate did not accumulate soluble sugars in response to the same cold treatment . To an extent similar to that induced after a cold acclimation, frost-hardiness of the cells increased after a 14-h incubation with specific soluble sugars such as sucrose, raffinose, fructose, and mannitol . Such hardening was also observed for long-term cultures in mannitol-enriched medium . This cryoprotective effect of sugars without exposure to lower temperatures was observed in both the resistant and the sensitive genotypes . Mannitol was one of the most efficient carbohydrates for the cryoprotection of eucalyptus . The best hardiness (a 2.7-fold increase in relative freezing tolerance) was obtained for the resistant cells by the cumulative effect of cold-induced acclimation and mannitol treatment . This positive effect of certain sugars on eucalyptus freezing tolerance was not colligative, since it was independent of osmolality and total sugar content.

Plant Physiol, 1997 May, 114(1), 193 - 201
Differential Accumulation of Salicylic Acid and Salicylic Acid-Sensitive Catalase in Different Rice Tissues; Chen Z et al.; We previously proposed that salicylic acid (SA)-sensitive catalases serve as biological targets of SA in plant defense responses . To further examine the role of SA-sensitive catalases, we have analyzed the relationship between SA levels and SA sensitivity of catalases in different rice (Oryza sativa) tissues . We show here that, whereas rice shoots contain extremely high levels of free SA, as previously reported (I . Raskin, H . Skubatz, W . Tang, B.J.D . Meeuse {1990} Ann Bot 66: 369-373; P . Silverman, M . Seskar, D . Kanter, P . Schweizer, J.-P . Metraux, I . Raskin {1995} Plant Physiol 108: 633-639), rice roots and cell-suspension cultures have very low SA levels . Catalases from different rice tissues also exhibit differences in sensitivity to SA . Catalase from rice shoots is insensitive to SA, but roots and cell-suspension cultures contain SA-sensitive catalase . The difference in SA sensitivity of catalases from these different tissues correlates with the tissue-specific expression of two catalase genes, CatA and CatB, which encode highly distinctive catalase proteins . CatA, which encodes a catalase with relatively low sequence homology to the tobacco SA-sensitive catalases, is expressed at high levels exclusively in the shoots . On the other hand, in roots and cell-suspension cultures, with northern analysis we detected expression of only the CatB gene, which encodes a catalase with higher sequence homology to tobacco catalases . The role of catalases in mediating some of the SA-induced responses is discussed in light of these results and the recently defined mechanisms of catalase inhibition by SA.

Plant Physiol, 1997 Feb, 113(2), 621 - 629
Characterization of a Diffusible Signal Capable of Inducing Defense Gene Expression in Tobacco; Chappell J et al.; Treatment of tobacco (Nicotiana tabacum) cell-suspension cultures with cryptogein, an elicitin protein from Phytophthora cryptogea, resulted in the release of a factor(s) that diffused through a 1000-D cutoff dialysis membrane and was capable of inducing sesquiterpene cyclase enzyme activity (a key phytoalexin biosynthetic enzyme in solanaceous plants) when added to fresh cell-suspension cultures . The diffusible factor(s) was released from cells over a 20-h period and induced a more rapid induction of cyclase enzyme activity than did direct treatment of the cultures with pure elicitin protein . The diffusible factor also induced a more rapid accumulation of transcripts encoding for sesquiterpene cyclase, acidic and basic chitinase, and hsr203 (a putative hypersensitive response gene) than did elicitin treatment . The diffusible factor(s) was resistant to protease, pectinase, Dnase, and RNase treatments, was not extractable into organic solvents, and was not immunoprecipitable when challenged with polyclonal antibodies prepared against elicitin protein . The diffusible factor(s) could not induce the release of more factor, suggesting that it was a terminal signal . These results are consistent with the notion that cells directly challenged or stimulated by pathogen-derived elicitors release diffusible secondary signal molecules that orchestrate the induction of complementary defense responses in neighboring cells.

Plant Physiol, 1997 Feb, 113(2), 387 - 395
Polyamines and Pectins (I . Ion Exchange and Selectivity); Messiaen J et al.; The ion-binding and -exchange properties of putrescine, spermidine, and spermine on purified walls of carrot (Daucus carota L.) cell suspensions were investigated by producing ion-exchange isotherms and comparing them with the behavior of Na+, Mg2+, and Ca2+ . The cation exchange capacity of the carrot cell walls was 0.8 equivalent kg-1 dry matter, and the ionic selectivity sequence of the walls for polyamines followed the sequence spermine4+ > spermidine3+ {almost equal to} Ca2+ > putrescine2+ . The polyamines were subjected to only electroselectivity and probably did not induce any favorable supramolecular conformation of pectin like the one induced by Ca2+ . Triangular ion exchanges were also performed with three diamines: ethanediamine, butanediamine, and octanediamine . The shorter the diamine, the higher the total adsorption and selectivity of the exchange . The lower selectivity of the cell wall for putrescine was partly attributed to its inability to access and displace Ca2+ from higher affinity sites within dimerized pectic sequences . The polyamine adsorption and exchange on pectic sequences could result in pectic signal modulation in pathogenesis and in differentiation.

J Microbiol Methods, 2002 Nov, 51(3), 401 - 6
A simple method for the measurement of bacterial particle conductivities; Yunus Z et al.; A method was developed for the measurement of the bacterial particle conductivity, based on the measurement of the conductivity of a bacterial cell suspension sigma(s) and the suspending medium sigma(m) . A line plotted through sigma(s) - sigma(m) versus sigma(m) crosses the x-axis at sigma(m) = sigma(p), independent of the bacterial cell concentration . The method does not require anything more complex than a centrifuge and a conductivity meter . Knowledge of the bacterial particle conductivity is of importance in, for example, the dielectrophoretic separation, manipulation and trapping of bacterial cells, as well as the study of their physiological state .

J Nutr, 2002 Sep, 132(9), 2748 - 56
The capacity of noninflammatory (steady-state) dendritic cells to present antigen in the primary response is preserved in acutely protein- or energy-deficient weanling mice; Zhang X et al.; The objective of this investigation was to determine the influence of wasting protein and/or energy deficits on the capacity of dendritic cells to initiate primary responses . Weanling male and female C57BL/6J mice were permitted free access to a complete purified diet, free access to an isocaloric low protein purified diet (combined deficiencies of protein and energy) or restricted intake of the complete diet (energy deficiency) for up to 14 d; a 19-d-old zero-time control group was also included . Malnourished mice lost 1.5-2% of initial body weight daily . Antigen presentation by dendritic cells from spleen and lymph nodes was assessed in vitro by the primary one-way allogeneic mixed lymphocyte reaction using CBA/J mononuclear or CD4(+) T cells as responders . This function was sustained despite advanced weight loss and, remarkably, was increased in cell suspensions from 14-d energy-deficient mice . Antigen presentation by dendritic cells in mononuclear suspensions was examined in vivo using the host-vs.-graft response in CBA/J recipients, and an ontogeny-related increase was sustained in both malnourished groups through 14 d of weight loss . Neither wasting protocol influenced the proportion of mononuclear cells (1-2%) exhibiting dendritic cell phenotype (CD11c(+)F4/80(-/low)) in the cellular suspensions used to study antigen-presenting activity . Consequently, these functional studies are interpretable on a per dendritic cell basis . In the absence of infectious or inflammatory pressure, the dendritic cell retains antigen-presenting capacity despite acute (wasting) deficiencies of protein and/or energy . The results are relevant to presentation of both foreign (adjuvant role) and self (tolerizing role) antigens by the dendritic cell.

FEBS Lett, 2002 Sep 11, 527(1-3), 43 - 50
Different protein kinase families are activated by osmotic stresses in Arabidopsis thaliana cell suspensions . Involvement of the MAP kinases AtMPK3 and AtMPK6; Droillard M et al.; Five Ca(2+)-independent protein kinases were rapidly activated by hypoosmotic stress, moderate or high hyperosmolarity induced by several osmolytes, sucrose, mannitol or NaCl . Three of these kinases, transiently activated by hypoosmolarity, recognised by anti-phosphorylated mitogen-activated protein (MAP) kinase antibodies, sensitive to a MAP kinase inhibitor and inactivated by the action of a tyrosine phosphatase, corresponded to MAP kinases . Using specific antibodies, two of the MAP kinases were identified as AtMPK6 and AtMPK3 . The two other protein kinases, durably activated by high hyperosmolarity, did not belong to the MAP kinase family . Activation of AtMPK6 and AtMPK3 by hypoosmolarity depended on upstream protein kinases sensitive to staurosporine and on calcium influx . In contrast, these two transduction steps were not involved in the activation of the two protein kinases activated by high hyperosmolarity.

J Chromatogr A, 2002 Aug 16, 967(1), 85 - 113
High-performance liquid chromatographic, capillary electrophoretic and capillary electrophoretic-electrospray ionisation mass spectrometric analysis of selected alkaloid groups; Stockigt J et al.; Systems for efficient separation of selected alkaloid groups by high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE-ESI-MS) are described . The optimized HPLC system was applied for the separation of 23 standard indole alkaloids as well as for qualitative and quantitative analyses of crude alkaloid extracts of Rauvolfia serpentina X Rhazya stricta hybrid cell cultures . The developed conditions for CE analysis proved to be efficient for separation of mixtures of standard indole and beta-carboline alkaloids . The described buffer system is also applicable in the combination of CE with electrospray ionisation mass spectrometry . This analytical technique allowed the separation and identification of components of standard indole alkaloid mixture as well as crude extracts of R . serpentina roots, R . serpentina cell suspension cultures and cortex of Aspidosperma quebracho-blanco . The influence of buffer composition and analyte structures on separation is discussed.

Biochim Biophys Acta, 2002 Sep 5, 1584(1), 1 - 8
Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents; Chiarpotto E et al.; Among the oxidative breakdown products of omega-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes . Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability . As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect . Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation . The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor beta1 (TGFbeta1) production by J774-A1 cells . Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFbeta1 protein synthesis . The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFbeta1 increase . Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde . Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.

Pigment Cell Res, 2002 Oct, 15(5), 348 - 56
Changes in the proliferative activity of epidermal melanocytes in serum-free primary culture during the development of ultraviolet radiation B-induced pigmented spots in hairless mice; Furuya R et al.; Long-term exposure to ultraviolet radiation B (UVB) induced pigmented spots in the dorsal skin of hairless mice of strain (HR-1 X HR/De)F1 . To clarify the cellular mechanism for the development of these UVB-induced pigmented spots, we investigated changes in the proliferative activity of epidermal melanoblasts and melanocytes in the dorsal skin at various weeks after UVB irradiation . Epidermal cell suspensions from the dorsal skin of hairless mice were cultured in a serum-free medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF) . The suspensions were prepared from dorsal skins of mice exposed to UVB for 4 weeks (the stage of hyperpigmentation) . Suspensions were also prepared from mice at 3 (the stage of depigmentation), 8 (the stage of appearance of pigmented spots), 20 (the stage of development of small-sized pigmented spots) and 37 (the stage of development of medium-sized pigmented spots) weeks after the cessation of 8-week UVB exposure . At the stage of hyperpigmentation the proliferative activity of melanoblasts and melanocytes was suppressed . With the development of pigmented spots, the proliferative activity of undifferentiated melanoblasts gradually increased, and then followed the increase in the proliferative activity of differentiated melanocytes . These results suggest that the proliferative activity of epidermal melanoblasts and melanocytes in UVB-irradiated skin increases with the development of pigmented spots.

Electrophoresis, 2002 Jul, 23(13), 1984 - 94
Electrophoresis of cells and the biological relevance of surface charge; Mehrishi JN et al.; Recent developments in electrophoresis of cells are reviewed . Problems and progress in automation and miniaturization of analytical electrophoresis instruments as well as in the interpretation of experimentally determined electrophoretic mobility (EPM) data are summarized: In recent times, the EPM determination techniques not only became more reliable and faster, but also more knowledge could be gained about the cell surface electrical properties, the structure of the glycocalyx as well as its influence on the cell peripheral regions and microenvironment by applying cell electrophoresis . In addition, ways are shown to solve discrepancies between physical requirements of a preparative cell electrophoresis procedure and the quantities of ions, which have to be dissolved in cell suspension media . As the modern machines allow the purification of untagged cells suspended in more cell friendly and physiological media, they are likely to be valuable tools in several useful practical applications in clinical transplantation, gene therapy and treatment of disease states.

J Biomed Mater Res, 2002 Dec 5, 62(3), 350 - 8
Bone formation by rat bone marrow cells cultured on titanium fiber mesh: effect of in vitro culture time; van den Dolder J et al.; The objective of this study was to examine the effect of cell culture time on bone formation by rat bone marrow cells seeded in titanium fiber mesh . As a seeding technique, a high cell suspension was used (3 x 10(6) cells/mL) . Therefore, 30 meshes were repeatedly rotated in a 10 mL tube (containing 30 x 10(6) cells) on a rotation plate (2 rpm) for 3 h . Osteogenic cells were cultured for 1, 4, and 8 days on titanium fiber mesh and finally implanted subcutaneously in rats . Meshes without cells were also implanted subcutaneously in rats . DNA and scanning electron microscopy (SEM) analyses and calcium measurements determined cellular proliferation and differentiation during the in vitro incubation period of the mesh implants . Four weeks after implant insertion, the animals were sacrificed . The implants, with their surrounding tissue, were retrieved and prepared for histologic evaluation and calcium measurements . DNA analysis of the in vitro experiment showed a lag phase from day 1 through day 4, but a 42% increase in DNA between days 4 and 8 . SEM and calcium measurements indicated an increase in calcium from day 1 to day 4, yet only a small but significant increase from days 4 to 8 . Histologic analysis demonstrated that bone was formed in all day 1 and day 4 implants, and that the bone-like tissue was present uniformly through the meshes . The bony tissue was morphologically characterized by osteocytes embedded in a mineralized matrix, with a layer of osteoid and osteoblasts at the surface . The day 8 implants showed only calcium phosphate deposition in the titanium fiber mesh . Calcium measurements of the implants revealed that calcification in day 1 implants was significantly higher (p < 0.05) compared to day 4 and day 8 implants . No significant difference in calcium content existed between day 4 and day 8 implants . On the basis of our results, we conclude that 1) bone formation was generated more effectively in osteogenic cells by a short culture time after seeding in titanium fiber mesh; 2) dynamic cell seeding is probably more effective than static cell seeding; and 3) selection of the right cells from the heterogenous bone marrow population remains a problem .

J Neurosci Res, 2002 Sep 15, 69(6), 934 - 9
Optimal conditions for in vivo induction of dopaminergic neurons from embryonic stem cells through stromal cell-derived inducing activity; Morizane A et al.; A method of inducing dopamine (DA) neurons from mouse embryonic stem (ES) cells by stromal cell-derived inducing activity (SDIA) was previously reported . When transplanted, SDIA-induced DA neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase (TH) expression . In the present study, to optimize the transplantation efficiency, we treated mouse ES cells with SDIA for various numbers of days (8-14 days) . SDIA-treated ES cell colonies were isolated by papain treatment and then grafted into the 6-hydroxydopamine (6-OHDA)-lesioned mouse striatum . The ratio of the number of surviving TH-positive cells to the total number of grafted cells was highest when ES cells were treated with SDIA for 12 days before transplantation . This ratio revealed that grafting cell colonies was more efficient for obtaining TH-positive cells in vivo than grafting cell suspensions . When we grafted a cell suspension of 2 x 10(5), 2 x 10(4), or 2 x 10(3) cells into the 6-OHDA-lesioned mouse striatum, we observed only a few surviving TH-positive cells . In conclusion, inducing DA neurons from mouse ES cells by SDIA for 12 days and grafting cell colonies into mouse striatum was the most effective method for the survival of TH-positive neurons in vivo .

Biochem J, 2002 Dec 15, 368(Pt 3), 865 - 74
Enzyme activity after resealing within ghost erythrocyte cells, and protection by alpha-crystallin against fructose-induced inactivation; Derham BK et al.; The role of alpha-crystallin as a molecular chaperone has been shown in many in vitro studies . In the present paper, we report on the chaperone function of alpha-crystallin within resealed erythrocyte ghosts . Eight enzymes were individually resealed within erythrocyte ghosts and assayed at zero time and at 24 h . The ghost cell suspension was separated into soluble and membrane fractions . Five of the enzymes had significantly greater enzyme activity after 24 h than the control within the soluble fractions . Fructation caused a decrease in enzyme activity (relative to the control) . Resealing of alpha-crystallin within the ghost cell alongside the enzymes protected against inactivation by fructose within the soluble fraction.

Anal Quant Cytol Histol, 2002 Aug, 24(4), 212 - 20
Value of image cytometry in the subclassification of rhabdomyosarcoma; Pohar-Marinsek Z et al.; OBJECTIVE: To test the discriminatory capability of nuclear features in the subclassification of rhabdomyosarcoma (RMS) and especially to differentiate embryonal from alveolar RMS . STUDY DESIGN: The study included 42 patients with RMS . We performed the analysis on Feulgen-stained filtrates of cell suspensions prepared from deparaffinized tissue sections . Image analysis was performed by an automated, high-resolution image cytometer on at least 200 nuclear images . Photometric, morphometric and nuclear texture features were analyzed . Probability density distributions were calculated for each nuclear feature of individual RMS subgroups and compared in order to detect possible differences . RESULTS: There were significant differences between embryonal and alveolar RMS in five nuclear features: DNA index, sphericity, elongation, low_DNA_area and fractall_area . We were able to differentiate between the two main RMS subgroups in 82% of cases on the basis of either sphericity or elongation alone, while the power of differentiation for texture features was 72-79% . CONCLUSION: Differentiation between embryonal and alveolar RMS using one nuclear feature is not an important adjunct to light microscopy . However, the possibility of using a combination of nuclear features would probably increase the discriminatory ability.

Hepatology, 2002 Sep, 36(3), 582 - 91
Cell adhesion regulates platelet-derived growth factor-induced MAP kinase and PI-3 kinase activation in stellate cells; Carloni V et al.; The biologic effects of growth factors are dependent on cell adhesion, and a cross talk occurs between growth factors and adhesion complexes . The aim of the present study was to evaluate the influence of cell adhesion on the major intracellular signaling pathways elicited by platelet-derived growth factor (PDGF) in hepatic stellate cells (HSC) . PDGF signaling was investigated in an experimental condition characterized by lack of cell adhesion for different intervals of time . Basal and PDGF-induced focal adhesion kinase (FAK) tyrosine phosphorylation was maintained in a condition of cell suspension for 2, 4, and 6 hours, whereas it was completely lost after 12 and 24 hours . We examined MAP kinase activity at 2 and 24 hours, corresponding to the higher and lower levels of FAK phosphorylation . In these experiments, MAP kinase activity correlated with FAK phosphorylation . Stimulation with PDGF was able to cause Ras-GTP loading only in adherent cells . The ability of PDGF to induce phosphatidylinositol 3-kinase (PI 3-K) activity was abrogated in cells maintained in suspension . The Ser473 phosphorylation of Akt was only marginally affected by the lack of cell adhesion . We then evaluated the association of FAK with c-Src . This association was found to be cell adhesion dependent, and it did not appear to be dependent from phosphorylated FAK . These changes in PDGF-induced intracellular signaling were associated with a remarkable reduction of PDGF-proliferative potential in nonadherent cells, although no marked differences in the apoptotic rate were observed . In conclusion, these results suggest that cell adhesion differentially regulates major signaling pathways activated by PDGF in HSC.

Ann Bot (Lond), 2002 Aug, 90(2), 269 - 78
Structural and ultrastructural analysis of Solanum lycopersicoides protoplasts during diploid plant regeneration; Tylicki A et al.; Light, fluorescence and electron microscopy were used to analyse the structural properties of protoplasts obtained from established suspension culture of Solanum lycopersicoides Dun, composed of meristematic cell aggregates . Four types of protoplasts were distinguished immediately after isolation: (1) mononuclear; (2) polynuclear, (3) anuclear and (4) homogeneous protoplasts . Only mononuclear protoplasts were capable of complete cell wall regeneration and mitotic division . Other types of protoplasts were eliminated during culture . Three phases were distinguished in the developing protoplast culture: (1) the elimination phase during which protoplasts damaged during isolation underwent complete degradation; (2) a phase of intense division during which both mitotic cell division and amitotic nuclear division took place; and (3) a stabilization phase leading to the formation of suspension culture . The cell suspension culture obtained from protoplasts was capable of regenerating diploid plants.

Chem Pharm Bull (Tokyo), 2002 Aug, 50(8), 1086 - 90
Regulation of lithospermic acid B and shikonin production in Lithospermum erythrorhizon cell suspension cultures; Yamamoto H et al.; Cell suspension cultures of Lithospermum erythrorhizon produced a large amount of lithospermic acid B, a caffeic acid tetramer, as well as shikonin derivatives (each ca . 10% of dry wt.) when cultured in shikonin production medium M-9 . Various culture factors for increasing the production of lithospermic acid B were investigated . Lithospermic acid B production was inhibited by 2, 4-D or NH4+, whereas it was stimulated by Cu2+ . These regulatory patterns were similar to those for the production of shikonin derivatives in these cell cultures, suggestive of close relations and similar metabolic regulation between the production of these compounds . Cultivation under light illumination, however, showed that these metabolisms were independently regulated . In particular, blue light showed a stimulatory effect on lithospermic acid B production, while shikonin production was strongly inhibited, indicative of an effective condition for lithospermic acid B production.

Proc Natl Acad Sci U S A, 2002 Sep 3, 99(18), 11706 - 11 Epub 2002 Aug 15.
Male germ-line stem cell potential is predicted by morphology of cells in neonatal rat testes; Orwig KE et al.; Gonocytes are a transient population of male germ-line stem cells that are derived from primordial germ cells in the embryo and give rise to spermatogonial stem cells, which establish and maintain spermatogenesis in the postnatal testis . In contrast to spermatogonial stem cells, gonocytes can be identified easily in neonatal rat testis cell suspensions based on their large size and distinct morphology . Furthermore, histological analysis of testes from neonatal transgenic rats demonstrated that gonocytes are the only cells that express a lacZ reporter transgene . Two gonocyte subpopulations, designated pseudopod and round, were identified and isolated from neonatal (0-4 days postpartum) rat testis cell suspensions . Male germ-line stem cells, identified by their ability to produce and maintain colonies of spermatogenesis upon transplantation into infertile recipient testes, were present almost exclusively in the pseudopod gonocyte subpopulation . In contrast, annexin V staining indicated that the majority of round gonocytes undergo apoptosis . These results indicate that a nearly pure population of male germ-line stem cells can be prospectively identified in neonatal rat testis cell suspensions by morphological criteria . Together, the pseudopod and round gonocyte populations will provide powerful tools for the study of cellular mechanisms that control cell fates and the establishment of spermatogenesis in the postnatal testis.

Br J Cancer, 2002 Jul 29, 87(3), 344 - 7
Flow cytometric quantification of tumour endothelial cells; an objective alternative for microvessel density assessment; Baeten CI et al.; Assessment of microvessel density by immunohistochemical staining is subject to a considerable inter-observer variation, and this has led to variability in correlation between microvessel density and clinical outcome in different studies . In order to improve the method of microvessel density measurement in tumour biopsies, we have developed a rapid, objective and quantitative method using flow cytometry on frozen tissues . Frozen tissue sections of archival tumour material were enzymatically digested . The single-cell suspension was stained for CD31 and CD34 for flow cytometry . The number of endothelial cells was quantified using light scatter- and fluorescence-characteristics . Tumour endothelial cells were detectable in a single cell suspension, and the percentage of endothelial cells detected in 32 colon carcinomas correlated highly (r=0.84, P<0.001) with the immunohistochemical assessment of microvessel density . Flow cytometric endothelial cells quantification was found to be more sensitive especially at lower levels of immunohistochemical microvessel density measurement . The current method was found to be applicable for various tumour types and has the major advantage that it provides a retrospective and quantitative approach to the angiogenic potential of tumours .

J Exp Biol, 2002 Sep, 205(Pt 18), 2943 - 54
Mechanisms of acid secretion in pseudobranch cells of rainbow trout (Oncorhynchus mykiss); Kern G et al.; Cell suspensions of rainbow trout Oncorhynchus mykiss pseudobranch, prepared by Ca(2+) depletion and mechanical maceration, contained a distinct population of cells that always kept their relatively cuboidal shape and did not round up in suspension or proliferate after adhering to the surface of cell culture dishes . Phasecontrast microscopy revealed an extensive system of basal membrane invaginations, and Na(+)-K(+)-ATPase- and anionexchanger-like immunoreactivity could be localized in cell membranes . The cells were characterized by a high mitochondrial density . Using specific antibodies, V-ATPase subunit B was localized in the plasma membrane . Using a cytosensor microphysiometer, the rate of acid secretion of these cells was measured and compared with the activity of a gill cell preparation . Incubation of pseudobranch cells with bafilomycin A1 (10(-6) mol l(-1)), a specific inhibitor of V-ATPase, reduced the rate of acid secretion by about 10% under control conditions, while no effect of bafilomycin on the rate of acid secretion of gill cells was observed . Application of amiloride (5 x 10(-5) mol l(-1)) reduced the rate of acid secretion in cells of both organs, pseudobranch and gills . Incubation of pseudobranch cells with DIDS (10(-3) mol l(-1)) resulted in a minor increase in the rate of proton secretion, but in cells prepared from the gills of rainbow trout acid secretion was reduced by about 30-40% . It is concluded that pseudobranch cells are equipped with various pathways to secrete protons, and that the anion exchange activity especially of pseudobranch cells appears to be different from that in gills.

J Exp Bot, 2002 Sep, 53(376), 1989 - 90
Expression analysis in plant and cell suspensions of CrCKR1, a cDNA encoding a histidine kinase receptor homologue in Catharanthus roseus (L.) G . Don; Papon N et al.; A full length cDNA (CrCKR1) encoding a hybrid histidine kinase was isolated from a Catharanthus roseus cDNA library . The kinase belongs to the subfamily of cytokinin receptors represented by CRE1/AHK4/WOL in Arabidopsis thaliana . In cell suspensions, the expression of CrCKR1 is not affected by various stress and hormonal treatments but is stimulated in cells continuously exposed to cytokinin . In plants, CrCKR1 is strongly expressed only in the petals of mature flowers . These data suggest that CrCKR1 could take part in the mechanisms leading to the production of secondary metabolites in C . roseus.

J Biomed Opt, 2002 Jul, 7(3), 425 - 34
Photothermal images of live cells in presence of drug; Lapotko D et al.; A new method for the study of drug-cell interaction is suggested . It is based on optical monitoring of cell response to drug impact . To detect this response, a high sensitive photothermal imaging technique has been developed . This technique is based on irradiation of the cells with a focused short pulse double-frequency pump laser (532 nm, 8 ns) and monitoring the laser-induced local thermal effects . This is realized with a second collinear dye laser pulse (630 nm, 6 ns) that probes the heated cell absorbing zones . The temperature-dependent variations of the refractive index in the absorbing zones in cross section of probe beam are recorded with phase-contrast technique . The photothermal (PT) image represents a two-dimensional depth-integrated temperature distribution in the irradiated volume and correlates with the conventional optical absorption images . It was experimentally shown that the introduction of NaN(3) or anti-tumor drug in the cell suspension led to a change in the structure of PT images of human lymphocytes and human lympholeukemia cells, respectively . It was hypothesized that the observed effects could be associated with the change of redox state of respiratory chain component.

Eur J Nucl Med Mol Imaging, 2002 Aug, 29(8), 1006 - 11 Epub 2002 May 17.
Hypoxia-induced alteration of tracer accumulation in cultured cancer cells and xenografts in mice: implications for pre-therapeutic prediction of treatment outcomes with (99m)Tc-sestamibi, (201)Tl chloride and (99m)Tc-HL91; Kinuya S et al.; Weak visualization of tumours in pre-therapeutic scintigrams with technetium-99m sestamibi (MIBI) is likely a predictive sign of unfavourable tumour response to radiotherapy and chemotherapy . However, factors relating to this scintigraphic finding are not well understood . The presence of hypoxic tumour cells is one of the major reasons for therapeutic failure; consequently, we attempted to determine whether oxygenation status affects (99m)Tc-MIBI accumulation in tumour cells . LS180 human colon cancer and T24 human bladder cancer cells were incubated in air or N(2) gas at 37 degrees C . Cellular uptake of (99m)Tc-MIBI was subsequently determined at 15, 60 and 120 min . Uptake of thallium-201 chloride was also assessed . Uptake of (99m)Tc-HL91 was assessed as a hypoxic marker . Accumulation of the tracers in LS180 xenografts was observed in mice treated with 5 mg/kg hydralazine and compared with that in untreated mice . pO(2) in the medium and tumours was measured with O(2) microelectrodes . N(2) gas flow gradually reduced pO(2) in the cell suspension to 1-2 mmHg in 60 min . Cellular uptake of (99m)Tc-MIBI in LS180 cells decreased by approximately 30% in N(2) gas in comparison to that in air throughout the study . Hypoxia had a more prominent influence on (201)Tl uptake, which displayed a reduction of approximately 60% in N(2) gas at 120 min, than on (99m)Tc-MIBI uptake . On the other hand, N(2) gas induced an increase of 170% in (99m)Tc-HL91 uptake at 120 min, indicating the hypoxic condition of cells . The results of in vitro assays employing the T24 cell line were similar to those obtained with the LS180 cell line . Hydralazine treatment markedly reduced (99m)Tc-MIBI and (201)Tl accumulation in LS180 xenografts; moreover, intratumoural pO(2) decreased from 14.5 +/- 6.6 mmHg to 7.6 +/- 6.2 mmHg . (99m)Tc-HL91 accumulation in xenografts was markedly increased by hydralazine . In conclusion, hypoxia reduced accumulation of (99m)Tc-MIBI and (201)Tl in tumour cells . Accordingly, hypoxia may be an important factor in terms of the interpretation of scintigraphic findings obtained with these tracers for pre-therapeutic prediction of tumour response to treatment . Furthermore, the enhanced (99m)Tc-HL91 accumulation in hypoxic tumour cells indicates the usefulness of this tracer in this regard.

Laryngoscope, 2002 Jul, 112(7 Pt 1), 1183 - 9
The superior turbinate as a source of functional human olfactory receptor neurons; Lane AP et al.; OBJECTIVES: The function of human olfactory receptor neurons (ORNs) remains incompletely understood, in part because of the difficulty of obtaining viable olfactory tissue for study . During endoscopic sphenoidotomy, a portion of the superior turbinate is often removed to achieve wide and safe access to the sphenoid sinus . The purpose of this study was to determine whether functional olfactory mucosa could be obtained from such superior turbinate tissue . STUDY DESIGN/METHODS: Superior turbinate tissue was resected from 4 patients undergoing transnasal endoscopic approaches to the sphenoid sinus . The gross appearance of the turbinate mucosa was normal at the time of surgery . The specimens were placed directly into cold cell culture media and transferred to the laboratory . A portion of the mucosa was fixed and embedded for histology and immunohistochemistry . The remaining tissue was enzymatically dissociated, and the resulting cell suspension was either prepared for immediate calcium imaging or placed into cell culture . Cultured ORNs underwent calcium imaging after several weeks to assess their ability to respond to odorants . RESULTS: Histologic analysis of superior turbinate tissue revealed the presence of patchy olfactory neuroepithelium staining positive for olfactory marker protein . Acutely dissociated ORNs were capable of generating calcium responses to odorant mixtures . ORNs could be maintained in mixed culture and retained their ability to respond to odorants . CONCLUSIONS: Superior turbinate tissue removed during endoscopic sphenoidotomy can provide a valuable source of human olfactory neuroepithelium for functional or histologic study . Superior turbinate tissue yields stem cells and immature neurons capable of differentiating into ORNs that retain many of their functional characteristics even after growth in culture.

J Biol Chem, 2002 Nov 1, 277(44), 41987 - 2002 Epub 2002 Aug 06.
Cell cycle-regulated gene expression in Arabidopsis; Menges M et al.; Regulated gene expression is an important mechanism for controlling cell cycle progression in yeast and mammals, and genes involved in cell division-related processes often show transcriptional regulation dependent on cell cycle position . Analysis of cell cycle processes in plants has been hampered by the lack of synchronizable cell suspensions for Arabidopsis, and few cell cycle-regulated genes are known . Using a recently described synchrony system, we have analyzed RNA from sequential samples of Arabidopsis cells progressing through the cell cycle using Affymetrix Genearrays . We identify nearly 500 genes that robustly display significant fluctuation in expression, representing the first genomic analysis of cell cycle-regulated gene expression in any plant . In addition to the limited number of genes previously identified as cell cycle-regulated in plants, we also find specific patterns of regulation for genes known or suspected to be involved in signal transduction, transcriptional regulation, and hormonal regulation, including key genes of cytokinin response . Genes identified represent pathways that are cell cycle-regulated in other organisms and those involved in plant-specific processes . The range and number of cell cycle-regulated genes show the close integration of the plant cell cycle into a variety of cellular control and response pathways.

Am J Physiol Endocrinol Metab, 2002 Sep, 283(3), E545 - 55
Diets enriched in sucrose or fat increase gluconeogenesis and G-6-Pase but not basal glucose production in rats; Commerford SR et al.; High-fat (HFD) and high-sucrose diets (HSD) reduce insulin suppression of glucose production in vivo, increase the capacity for gluconeogenesis in vitro, and increase glucose-6-phosphatase (G-6-Pase) activity in whole cell homogenates . The present study examined the effects of HSD and HFD on in vivo gluconeogenesis, the catalytic and glucose-6-phosphate translocase subunits of G-6-Pase, glucokinase (GK) translocation, and glucose cycling . Rats were fed a high-starch control diet (STD; 68% cornstarch), HSD (68% sucrose), or HFD (45% fat) for 7-13 days . The ratio of 3H in C6:C2 of glucose after 3H2O injection into 6- to 8-h-fasted rats was significantly increased in HSD (0.68 +/- 0.07) and HFD (0.71 +/- 0.08) vs . STD (0.40 +/- 0.10) . G-6-Pase activity was significantly higher in HSD and HFD vs . STD in both intact and disrupted liver microsomes . HSD and HFD significantly increased the amount of the p36 catalytic subunit protein, whereas the p46 glucose-6-phosphate translocase protein was increased in HSD only . Despite increased nonglycerol gluconeogenesis and increased G-6-Pase, basal glucose and insulin levels as well as glucose production were not significantly different among groups . Hepatocyte cell suspensions were used to ascertain whether diet-induced adaptations in glucose phosphorylation and GK might serve to compensate for upregulation of G-6-Pase . Tracer-estimated glucose phosphorylation and glucose cycling (glucose <--> glucose 6-phosphate) were significantly higher in cells isolated from HSD only . After incubation with either 5 or 20 mM glucose and no insulin, GK activity (nmol . mg protein(-1) . min(-1)) in digitonin-treated eluates (translocated GK) was significantly higher in HSD (32 +/- 4 and 146 +/- 6) vs . HFD (4 +/- 1 and 83 +/- 10) and STD (9 +/- 2 and 87 +/- 9) . Thus short-term, chronic exposure to HSD and HFD increase in vivo gluconeogenesis and the G-6-Pase catalytic subunit . Exposure to HSD diet also leads to adaptations in glucose phosphorylation and GK translocation.

J Immunol Methods, 2002 Sep 15, 267(2), 109 - 17
Immunofluorescent surface labelling, flow sorting and culturing of putative epidermal stem cells derived from small skin punch biopsies; van Rossum MM et al.; Basal keratinocytes of human epidermis strongly express the cell surface glycoprotein beta(1)-integrin, and putatively harbour epidermal stem cells . Selective sorting and culturing of keratinocyte stem cells forms the basis for studies on the role of these cells as targets for therapeutic intervention and gene therapy . Here we have studied variables which affect cell surface labelling for beta(1)-integrin, flow sorting and subsequent culturing of beta(1)-integrin-positive and beta(1)-integrin-negative keratinocytes . Keratinocytes were derived from small human skin punch biopsies (3 or 4 mm in diameter), and we tested a number of variables such as choice of proteolytic enzyme for cell isolation, cell concentration, fixation, storage of fixed cell suspensions and labelling conditions . In contrast to thermolysin treatment for cell isolation, trypsin treatment left most cell surface beta(1)-integrin molecules intact . Ethanol and paraformaldehyde fixation interfered with beta(1)-integrin detection, and unfixed cells gave the best results . Optimisation of all the individual steps resulted in a labelling protocol for reproducible staining and sorting of the cells . Sorted cells were seeded in 96-well plates (300 cells/well) and colonies were obtained in more than 50% of the wells with beta(1)-integrin-positive keratinocytes . In plates with beta(1)-integrin-negative cells, only 10% of the wells contained keratinocyte colonies . Flow sorted keratinocytes obtained by trypsin formed numerous colonies in cell culture experiments . In cell suspensions obtained with thermolysin, only sparse colonies were formed . We conclude that our methodology permits the use of small human tissue samples for cell labelling and sorting, while preserving the clonogenic potential.

Haematologica, 2002 Aug, 87(8), 851 - 4
HLA typing strategies in a cord blood bank; Laurenti L et al.; BACKGROUND AND OBJECTIVES: Although widely used, serological typing of HLA loci does not always produce uequivocal results . This may be particularly the case for cord blood since samples may be of small volume and poor quality, and contaminated . DESIGN AND METHODS: We typed 220 cord blood units (CBU) for HLA class I antigens using the serological technique . For those samples giving doubtful results we repeated the HLA typing by polymerase chain reaction with sequence specific primers (PCR-SSP) . RESULTS: Results were satisfactory for 181 samples (82.3%) . For the remaining 39 (17.7%) we had a doubtful antigen assignment for A locus in 9/39 cases (23.1%) and for B locus in 22/39 cases (56.4%) . Eight of the 39 samples (20.5%) could not be analyzed by serology due to the high mortality of the cell suspension . Using PCR-SSP we obtained clear definition of class I antigens in all cases . All CBU were typed for HLA class II alleles by PCR-SSP with clear results in 100% of cases . INTERPRETATION AND CONCLUSIONS: In our experience, PCR-SSP can resolve the limitations of serology but, at the moment, it cannot substitute the latter in routine practice . The best strategy, in cord blood typing, is to perform both serological and molecular typing in order to obtain an accurate and clear result.

Exp Hematol, 2002 Aug, 30(8), 862 - 9
Liver and marrow of adult mdr-1a/1b(-/-) mice show normal generation, function, and multi-tissue trafficking of primitive hematopoietic cells; Uchida N et al.; OBJECTIVE: Several lines of evidence suggest that expression of two ABC transporters (Abcg2/Bcrp1 and mdr-1a/b) and the related abilities to efflux Hoechst 33342 (Hst) and Rhodamine-123 (Rho) are features of primitive hematopoietic cells in adult bone marrow . Here we sought to determine the phenotypic and hematopoietic properties of the Hst-effluxing "side" population (SP) cells present in the liver of adult normal mice and whether these might be altered in mdr-1a/1b(-)(/-) mice . MATERIALS AND METHODS: Single-cell suspensions of liver (and sometimes bone marrow) were stained with Hst, separated into SP and non-SP fractions, and analyzed for hematopoietic cell-surface marker expression and functional activity in standard in vitro and in vivo (transplantation) assays . RESULTS: SP cells constituted approximately 1-2% of adult liver cell suspensions and were phenotypically and functionally heterogeneous, even within the approximately 20-25% that expressed CD45 . The latter included some lineage marker-positive (lin(+)) cells, less than 15% of all in vitro hematopoietic colony-forming cells in the adult liver and more than 90% of cells identified as long-term culture-initiating cells or in vivo repopulating cells . Interestingly, primary mice reconstituted for greater than or equal to 1 year with adult liver SP cells contained derivative primitive hematopoietic cells in their livers . No differences were seen between +/+ and mdr-1a/1b(-)(/-) mice except for a loss of Rho efflux ability by lin(-)mdr-1a/1b(-)(/-) SP cells . CONCLUSION: Adult murine liver contains a spectrum of hematopoietic cells that are phenotypically and functionally similar to those in the marrow and their generation and properties appear unaffected by a lack of mdr-1a/1b.

Plant Cell Physiol, 2002 Jul, 43(7), 778 - 84
Elicitor activity of cerebroside, a sphingolipid elicitor, in cell suspension cultures of rice; Umemura K et al.; Cerebrosides, compounds categorized as glycosphingolipids, were found to occur in a wide range of phytopathogens as novel elicitors and to induce the effective disease resistance for rice plants in our previous study . Here, we showed that cerebroside elicitors lead to the accumulation of phytoalexins and pathogenesis-related (PR) protein in cell suspension cultures of rice with the structural specificity similar to that for the rice whole plants . This elicitor activity of the cerebroside was greater than jasmonic acid (JA) and chitin oligomer (which is known to be an elicitor for cell suspension cultures of rice) . Treatment of cell suspension cultures with cerebroside and chitin oligomer resulted in a synergetic induction of phytoalexins, suggesting that cerebroside and carbohydrate elicitors, such as glucan and chitin elicitor, enhance the defense signals of rice in vivo . Induction of phytoalexins by the treatment with cerebroside elicitor was markedly inhibited by LaCl(3) and GdCl(3), Ca(2+ )channel blockers . It is possible that Ca(2+) may be involved in the signaling pathway of elicitor activity of cerebroside.

Phytochemistry, 2002 Aug, 60(8), 795 - 8
Galloylated catechins and stilbene diglucosides in Vitis vinifera cell suspension cultures; Decendit A et al.; Suspension cultures of Vitis vinifera were found to produce catechins and stilbenes . When cells were grown in a medium inducing polyphenol synthesis, (-)-epicatechin-3-O-gallate, dimeric procyanidin B-2 3'-O-gallate and two resveratrol diglucosides were isolated, together with a new natural compound that was identified as cis-resveratrol-3,4'-O-beta-diglucoside by spectroscopical methods.

Cryo Letters, 2000 Jan, 21(1), 19 - 24
In-field behaviour of banana plants (Musa AA sp) obtained after regeneration of cryopreserved embryogenic cell suspensions; Cote F et al.; This study describes the in-field behavior of bananas (Musa AA sp.) obtained after regeneration of cryopreserved embryogenic cell suspensions . Observations were focused on the classical vegetal development descriptors . We observed no significant differences between the cryopreserved-derived plants and the control plants with respect to the plant height and circumference, the number of leaves, the number of fruits, the fruit length, the fruit diameter and weight, the bunch weight and the date of harvest . During the first culture cycle, 2 out of 11 descriptors analyzed were however found to be different between the control and the cryopreserved suspensions derived plants . These were the number of nodal clusters of the inflorescence (usually called hands) and the date of flowering . These differences were, however, quite minor as the two cases together amounted to only 2 % of the control value . During the second cycle of culture, no significant difference between the two groups of plants was found whatever the parameter analysed . These results suggest that, with the experimental conditions of the study, there is no difference at the agronomic level between plants produced from cryopreserved embryogenic cell suspensions and control plants.

Plant Mol Biol, 2002 Sep, 50(1), 111 - 27
Characterization of cis-acting element involved in cell cycle phase-independent activation of Arath;CycB1;1 transcription and identification of putative regulatory proteins; Planchais S et al.; Progression through the cell cycle is driven by cyclin-dependent kinases (CDKs) whose activity is controlled by regulatory subunits called cyclins . The expression of cyclins is subject to numerous controls at multiple levels, not least at the level of transcription . As a first step to unravel the mechanisms that regulate expression of B-cyclins in plants, we undertook the identification of the required promoter elements of the Arath;CycB1;1 gene . A detailed analysis of different promoter fragments consisted in analysing their ability to mediate cell cycle-dependent transcriptional oscillations of the gus reporter gene in transformed BY-2 cell lines . We showed that different promoter regions took part in transcriptional activation . Furthermore, 202 bp upstream of the ATG were sufficient to induce M-phase-specific expression . This region contains an 18 bp sequence including a Myb-binding core (AACGG) which is able to activate reporter gene without leading to M-phase-specific expression . Electrophoretic mobility shift assays showed that this 18 bp sequence specifically binds protein complexes from Arabidopsis cell suspension enriched either in G1 or G2 phase . Furthermore, the Myb core, AACGG, was characterized as necessary for the binding of proteins . DNA affinity purification of the complexes bound to the 18 bp sequence allowed the isolation of three different complexes and two proteins from these complexes were identified by mass spectrometry analyses . A new putative Myb transcription factor and a hypothetical protein, HYP containing with a leucine zipper and Myc-type dimerization domains were identified . When over-expressed in plants, HYP factor is able to trans-activate the expression of gus reporter gene downstream from the -202 promoter fragment as well as the endogenous CycB1;1 gene.

Cancer Gene Ther, 2002 Aug, 9(8), 681 - 6
Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression; Zhou JH et al.; There has been no reliable orthotopic model available to visualize the growth of human superficial bladder cancer over time and to evaluate the efficacy of intravesical therapies . We have developed a novel approach to accomplish this task by generating human superficial bladder tumor cells to stably express high levels of green fluorescent protein (GFP) in vivo . Superficial bladder tumors were produced in athymic mice by intravesical instillation . In our initial studies tumors were quantitated by image analysis at a single time point, and the results compared to the estimation of the percentage of GFP cells present using flow cytometry after obtaining single cell suspensions of normal and tumor cells in the same bladder . A high correlation between the two methods was seen . Therefore, in subsequent studies, approximately 1 week after the intravesical instillation of the GFP expressing cancer cells a small incision was made to expose the bladder . The anterior, posterior, and lateral images of each bladder were captured to visualize GFP-expressing tumors . The ratio of green fluorescence pixel area, which represented the tumor burden, to the total area of the bladder was then calculated . A similar procedure was performed at 2, 3, and 4 weeks after instillation of the tumor cells . Using this procedure tumor progression over time could be measured in each mouse . By using this approach, it will now be possible to monitor the initial tumor sizes in the bladder of each mouse and then to evaluate the efficacy of various intravesical therapy protocols including intravesical gene therapy alone or in combination with other treatment modalities.

Ophthalmic Surg Lasers, 2002 Jul-Aug, 33(4), 340 - 2
Use of an oil-hydraulic microinjection pump for subretinal infusions; Weichel J et al.; The injection of cell suspensions or drugs into the subretinal space is a new promising option of vitreoretinal surgery for the treatment of degenerative retinal disorders . We used a manual oil-hydraulic microinjection pump to subretinally inject suspensions of retinal pigment epithelial cells in Royal College of Surgeons rats and in patients suffering from age-related macular degeneration with geographic atrophy . The histological examination of the treated rat eyes showed that cell suspensions could be placed precisely in the subretinal space . Intra- and postoperative outcome of the patients in the clinical trial revealed no retinal complications during 6 months of follow up . We suggest the oil-hydraulic microinjection pump to be a valuable instrument for controlled and precisely dosed atraumatic infusion or aspiration of small volumes of cell suspensions, fluids or drugs in vitreoretinal surgery.

Endocrinology, 2002 Aug, 143(8), 3171 - 4
Purified gonocytes from the neonatal rat form foci of proliferating germ cells in vitro; Moore TJ et al.; Due to the lack of a specific marker for gonocytes from newborn rats, isolation of these cells has proven difficult and laborious . We have found a specific cell membrane marker, the Epithelial Cellular Adhesion Molecular (Ep-CAM) that can be used to isolate these cells using antibody directed cell sorting . 4 days post partum (dpp) rat testes were enzyme treated to attain a cell suspension, which was labelled with an antibody (GZ1) against Ep-CAM and tagged with a fluorescent probe . The labelled cell suspension was run through a FACS cell sorter, from which a gonocyte suspension of >85% purity was attained . The cells remained viable in culture and proliferated actively as determined by double labelling the cells with anti-HSP90alpha (a specific germ cell marker) and anti-BrdU antibodies (after BrdU incorporation) . During culture, these cells formed chains of 2 to 4 cells and aggregates of proliferating germ cells were found after 8 days of culture.

J Surg Res, 2002 Jul, 106(1), 124 - 30
Genetic detection of liver micrometastases that are undetectable histologically; Ikawa K et al.; BACKGROUND: Predicting liver metastasis from colon cancer is essential for improving its prognosis . We studied to what extent genetic detection of cancer cells in the resected liver tissue can predict the incidence of macroscopic liver metastasis with a similar mouse model to clinical colorectal cancer that causes a several decade percentage of metachronous hepatic metastases after resection of the primary lesions . MATERIALS AND METHODS: A LS174T human colorectal cancer cell suspension was injected into the spleens of nude mice . One to 10 days after splenic injection, 3 x 3 mm of liver tissue was removed, and a splenectomy was performed . Liver tissue was used for genetic detection and histological examination . Five weeks after splenic injection, the number of macroscopic metastases on the surface of the liver was counted . RESULTS: Eight of the 45 cases were positive for tumor cells in liver tissue genetically, while only 1 was positive for tumor cells histologically . Macroscopic liver metastases were seen 5 weeks after splenic injection in 11 of 37 (29.7%) cases negative for tumor cells genetically and in 8 of 8 (100%) cases positive for tumor cells genetically . Five or more metastases were seen in 3 of 37 (8.1%) cases negative for tumor cells genetically and in 7 of 8 (87.5%) cases positive for tumor cells genetically . CONCLUSIONS: The cases which were positive for tumor cells in liver tissue genetically at the time of splenectomy had more significantly macroscopic liver metastases some weeks later than the cases negative for tumor cells . This study suggests that if micrometastasis was detected genetically, the development of metachronous macroscopic liver metastasis could be predicted.

J Neurosci Res, 2002 Aug 1, 69(3), 382 - 96
Comparison of fresh and cryopreserved porcine ventral mesencephalon cells transplanted in A rat model of Parkinson's disease; Jacoby DB et al.; To evaluate whether cryopreservation of porcine ventral mesencephalon cells influences graft survival and function in vivo, we have transplanted either freshly prepared or cryopreserved cells into the striatum of 6-hydroxydopamine-lesioned rats . A single cell suspension of porcine ventral mesencephalon cells from the same isolation either was stored at 4 degrees C and transplanted the next day or was cryopreserved for 4 weeks in liquid nitrogen vapor . The cryopreserved cells were then rapidly thawed, rinsed, and transplanted in the same manner as the fresh cells, with the same dose of viable cells . All animals received daily injections of cyclosporin A to prevent xenograft rejection . To monitor graft function, amphetamine-induced rotation was measured every 3 weeks between 6 and 15 weeks posttransplantation . After sacrifice at 15 weeks posttransplantation, histological methods were used to compare fresh cell and cryopreserved cell transplants with respect to graft survival, differentiation and integration, and host immune response . Cryopreserved cells were found to be either equivalent or in some cases superior to fresh cells with respect to rotational correction, graft survival, graft volume, numbers of graft-derived dopaminergic neurons, and host immune responses . In conclusion, the results indicate that it is feasible to cryopreserve porcine ventral mesencephalon cells for long-term storage of cells prior to transplantation in an animal model of Parkinson's disease .

J Cell Physiol, 2002 Sep, 192(3), 315 - 26
Role of leukemia inhibitory factor in the regulation of the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture; Hirobe T; Mouse epidermal melanoblasts/melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast/melanocyte-proliferation medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF) . Leukemia inhibitory factor (LIF) supplemented to the medium from initiation of primary culture increased the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes . Pure cultured primary melanoblasts or melanocytes were further cultured with the medium supplemented with LIF from 14 days (keratinocyte depletion) . LIF stimulated the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes in the absence of keratinocytes . Moreover, anti-LIF antibody supplemented to the medium from initiation of primary culture inhibited the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes . These results suggest that LIF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF .

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