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 u