Cell suspensions

Proc Natl Acad Sci U S A, 1999 Dec 7, 96(25), 14246 - 51
Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells; Oxley D et al.; Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development . We recently have shown that two AGPs, NaAGP1 (from Nicotiana alata styles) and PcAGP1 (from Pyrus communis cell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor . However, paradoxically, both AGPs were buffer soluble rather than membrane associated . We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs . This GPI anchor has the minimal core oligosaccharide structure, D-Manalpha(1-2)-D-Manalpha(1-6)-D-Manalpha(1-4)-D-GlcN -inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial beta(1-4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid . The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D . The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways.

Masui, 1999 Nov, 48(11), 1194 - 201
{Measurements of extracellular water volume by bioelectrical impedance analysis during perioperative period of esophageal resection}; Tatara T et al.; Segmental bioelectrical impedance analysis (BIA) was conducted in five patients who underwent esophageal resections . Resistance values fitted at zero frequency (R0) in each body segment (arm, trunk and leg) were determined before the induction of anesthesia, at the end of surgery and on the second or third postoperative day . Extracellular water volume (ECW) in each body segment was estimated using the equation derived from the cell suspension theory . ECW in whole body was obtained from the sum of each body segment . R0 in trunk and leg significantly decreased at the end of surgery compared to the values before the induction of anesthesia (P < 0.05) . The change ratio of R0 in trunk before the induction of anesthesia was significantly lower at the end of surgery than that in arm (P < 0.05), resulting from the most striking fluid accumulation in the trunk . Postoperatively, R0 in all body segments, however, appeared to decrease similarly compared to the values before the induction of anesthesia, suggesting the redistribution phenomena of extracellular water among body segments . The correlation (r = 0.90, P < 0.001) and good agreement {bias = 0.01 (L)} between net fluid balances and estimates of ECW changes in whole body suggest that BIA allows close monitoring of tissue hydration during perioperative period by providing estimates of ECW in body segments.

J Appl Microbiol, 1999 Oct, 87(4), 546 - 56
A study of dextran production from maltodextrin by cell suspensions of Gluconobacter oxydans NCIB 4943; Mountzouris KC et al.; This study investigated dextran synthesis from a commercial maltodextrin substrate using cell suspensions of G . oxydans NCIB 4943 as catalysts . Experiments were arranged according to a central composite statistical design . The effects of substrate concentration (10-100 g l-1), cell concentration (0.32-32.0 g wet weight l-1), time of reaction (8-48 h) and pH (3.5-5.5), each at three levels, on dextran yield and dextran molecular weight (MW), were investigated . Response surface methodology was used to assess factor interactions, and empirical models describing the two responses were fitted . Most of the variance in dextran yield could be explained by the fitted model (R2 = 0.96) . Dextran yield ranged from 1.21 to 41.69% . The presence of significant negative quadratic effects of cell concentration and time indicated that dextran yield reached a plateau and thus, optimum levels of cell concentration and time could be identified to maximize dextran yield . Dextran MW ranged from 6.6 to 38 kDa and was characterized by the significant interactions of reaction time with substrate concentration and cell concentration . The model, however, could account for only 60% of the variance in dextran MW . Possible reasons for this are discussed.

Brain Res Bull, 1999 Nov 1, 50(4), 275 - 81
Impact of a preceding striatal excitotoxic lesion and treatment with ciliary neurotrophic factor on striatal graft survival; Petersen A et al.; The survival of grafted embryonic striatal tissue, dissected from the lateral ganglionic eminence, depends on the status of the host striatum . We found significantly larger volumes of surviving graft tissue and of striatal-like tissue (P-zone) within the graft, when the host striatum had been subjected to an excitotoxic lesion prior to transplantation surgery . Concomitantly the numbers of surviving grafted cells, assessed in both cresyl violet-stained sections and in sections stained with an immunohistochemical marker for striatal neurons, increased as compared to when graft tissue was placed in an intact unlesioned striatum . Finally, we examined the impact of treatment of the donor tissue with ciliary neurotrophic factor (CNTF) on graft survival . CNTF has previously been shown to protect striatal neurons against excitotoxic insults both in vitro and in vivo, but it did not improve striatal graft survival when added to the cell suspension prior to implantation.

Brain, 1999 Dec, 122 ( Pt 12), 2321 - 35
Primary CA1 and conditionally immortal MHP36 cell grafts restore conditional discrimination learning and recall in marmosets after excitotoxic lesions of the hippocampal CA1 field; Virley D et al.; Common marmosets (Callithrix jacchus, n = 18) were trained to discriminate between rewarded and non-rewarded objects (simple discriminations, SDs) and to make conditional discriminations (CDs) when presented sequentially with two different pairs of identical objects signifying reward either in the right or left food well of the Wisconsin General Test Apparatus . After bilateral N-methyl-D-aspartate (0.12 M) lesions through the cornu ammonis-1 (CA1) field (7 microl in five sites), marmosets showed profound impairment in recall of CDs but not SDs, and were assigned to lesion only, lesion plus CA1 grafts and lesion plus Maudsley hippocampal cell line, clone 36 (MHP36) grafts groups matched for lesion-induced impairment . Cell suspension grafts (4 microl, 15-25 000 cells/microl) of cells dissected from the CA1 region of foetal brain at embryonic day 94-96, or of conditionally immortalized MHP36 cells, derived from the H-2Kb-tsA58 transgenic mouse neuroepithelium and labelled with {3H}thymidine, were infused at the lesion sites . The lesion plus MHP36 grafts group was injected five times per week with cyclosporin A (10 mg/kg) throughout testing . Lesion, grafted and intact control marmosets (n = 4-5/group) were tested on recall of SDs and CDs learned before lesioning and on acquisition of four new CDs over a 6-month period . Lesioned animals were highly impaired in recall and acquisition of CD tasks, but recall of SDs was not significantly disrupted . Both grafted groups of marmosets showed improvement to control level in recall of CDs . They were significantly slower in learning the first new CD task, but mastered the remaining tasks as efficiently as controls and were substantially superior to the lesion-only group . Visualized by Nissl staining, foetal grafts formed clumps of pyramidal-like cells within the denervated CA1 field, or jutted into the lateral ventricles . MHP36 cells, identified by beta-galactosidase staining and autoradiography, showed neuronal and astrocytic morphology, and were distributed evenly throughout the CA1 region . The results indicate that MHP36 cell grafts are as functionally effective as foetal grafts and appear to integrate into the host brain in a structurally appropriate manner, showing the capacity to differentiate into both mature neurons and glia, and to develop morphologies appropriate to the site of migration . These findings, which parallel the facilitative effects of foetal and MHP36 grafts in rats with ischaemic CA1 damage, offer encouragement for the development of conditionally immortal neuroepithelial stem cell lines for grafting in conditions of severe amnesia and hippocampal damage following recovery from cardiac arrest or other global ischaemic episodes.

Plant Physiol Biochem, 1999 Nov, 37(11), 869 - 874
Chlorogenic acid in a Nicotiana plumbaginifolia cell suspension; Gillet F et al.; A phenylpropanoid compound has been characterized in a Nicotiana plumbaginifolia cell suspension . This compound has been isolated and purified by semi-preparative reverse phase-high performance liquid chromatography . Its structure has been identified by NMR spectroscopy as 5-O-caffeoylquinic acid, which is chlorogenic acid (CA) . The influence of culture conditions on the accumulation of this metabolite by N . plumbaginifolia cell suspensions has been studied . Darkness strongly inhibits the CA accumulation . Moreover, it has been shown that feeding experiments with caffeic acid had a deleterious effect upon the CA content . This one was not influenced by a supplementation with quinic acid.

Bioessays, 1999 Dec, 21(12), 1061 - 8
How I learned to love carrots: the role of the cytoskeleton in shaping plant cells; Lloyd C; The carrot cell suspension was originally used because it provided a model system for studying directional cell expansion - a key process in plant morphogenesis . Early immunofluorescence studies of plant microtubules, using these cells, provided hints that the cortical array of microtubules was dynamic and this was later confirmed by microinjection studies on plant epidermal cells . A nonfixation approach for detecting F-actin was then developed on these cells and showed that, unlike animal cells, actin filaments remained associated with the nucleus throughout division and could have a role in aligning the plane of cell division . Currently, we are using detergent-extracted carrot cytoskeletons for isolating microtubule-associated proteins (MAPs) . I discuss how MAPs may be involved in the oriented deposition of cellulose in the cell wall .

Mech Ageing Dev, 1999 Oct 1, 110(1-2), 1 - 12
Age-related alteration of intracellular ATP maintenance in the cell suspensions of mice cerebral cortex; Joo HJ et al.; Neurological alteration in the aging brain has been suggested to be due to, in part, a declined cellular energy metabolism . In order to understand age-related alteration of intracellular ATP maintenance, the present in vitro study measured change of intracellular adenosine triphosphate (ATP) content in cell suspensions of cerebral cortex isolated from male ICR mice aged 2 days (infant), 8 weeks (young adult) and 12 months (aged) under several different conditions, using the chemiluminescence technique . Among the three different ages, significant decrease of intracellular ATP content by oxygen deprivation for 15 min was observed in the cell suspensions of cerebral cortex from 12-month-old mice (P < 0.05) . When cell suspensions of 8-week cerebral cortex were incubated with or without glucose (0-60 min), intracellular ATP content decreased in a time-dependent manner under both conditions, but depletion rate was significantly high in the glucose-free condition . This decrease was maximally restored by adding 1 mM glucose as tested . In addition, the ability for intracellular ATP maintenance in the presence or absence of glucose was age-dependently different . The rank order of difference of intracellular ATP content between with and without glucose was 3 months > 12 months > 2 days . The highest decrease of intracellular ATP content by incubation without glucose was observed in the 12-month samples . Sodium cyanide (100 microM) produced a gradual ATP depletion in cerebral cortex suspended from 2-day-old mice, but rapid change in both 8-week and 12-month samples . Combination of cyanide and iodoacetate (3.5 mM) rapidly depleted the intracellular ATP content in all age groups tested . These results suggest that the aging process in the cerebral cortex of mice is accompanied by alteration of maintenance of intracellular ATP homeostasis under a given condition, and this may be associated with pathological change of overall mechanisms involved in the development of neuronal disease in the senescent brain.

Cell Transplant, 1999 Sep-Oct, 8(5), 489 - 99
Lack of effect of short-term depletion of plasma complement C3 on the survival of syngeneic dopaminergic neurons following grafting into the intact rat striatum; Khorooshi MH et al.; Metabolically compromised cells may be subject to complement-mediated cytotoxicity . The aim of this study was to clarify to what extent plasma complement C3 might contribute to the low survival (5-20%) of grafted dopaminergic neurons . The survival of intrastriatal cell suspension grafts of syngeneic dopaminergic, tyrosine hydroxylase (TH)-containing neurons was compared in rats subjected to short-term i.v . treatment with 1) cobra venom factor (CVF), or 2) placebo treatment . Depletion of plasma complement C3 by CVF was confirmed by crossed immunoelectrophoresis . With 159 +/- 37 (mean +/- SEM) TH-immunoreactive and 154 + /- 40 TH mRNA-expressing neurons in the CVF-treated rats (n = 9), and 117 +/- 34 TH-immunoreactive and 160 +/- 49 TH mRNA-expressing neurons in placebo rats (n = 6), the CVF treatment did not increase the survival of the grafted dopaminergic neurons . Similarly, CVF had no apparent effect on the astroglial, microglial, or oligodendroglial cell response within and around the graft . The data indicate that depletion of plasma complement C3 at the time of grafting has no effect on the long-term survival of syngeneic ventral mesencephalic dopaminergic neuronal grafts.

Cytometry, 1999 Jun 1, 36(2), 102 - 11
Evaluation of membrane physiology following fluorescence activated or magnetic cell separation; Seidl J et al.; BACKGROUND: Over the past decade, cell separation technology has become an important tool in various fields of cell biology allowing for the analysis or subsequent cultivation of specific cell subsets . The objective of the present study was to evaluate if the established sorting techniques fluorescence-activated (FACS) and magnetic cell separation (MACS) affect cell membrane physiology in order to define the most non-perturbing application for the separation of tumor and stromal cells . MATERIALS AND METHODS: Membrane physiology was monitored in single cell suspensions of adherently grown BT474 breast tumor cells and N1 normal skin fibroblasts using flow cytometry . Cell membrane integrity was evaluated by propidium iodide (PI) staining . Microviscosity within the lipophilic membrane layer was determined by a monomer/excimer method utilizing pyrene decanoic acid, membrane potential measurements were carried out using the fluorescence indicator DiBAC4(3), and Annexin-V-staining reflected transversal membrane asymmetry, and an altered phospholipid distribution . RESULTS: Not only the number of preparative cycles prior to cell separation but also the sort conditions during FACS resulted in loss of membrane integrity of a certain cell fraction . If these PI-positive cells were excluded from further analysis, neither MACS nor FACS affected membrane microviscosity while a clear hyperpolarization in both cell types after MACS resulting from exposure to the ferromagnetic matrix of the depletion column and the inhomogeneous magnetic field was shown . In addition, cell sorting of BT474 tumor cells by MACS and FACS was accompanied by the generation of an Annexin-V-positive/PI-negative cell fraction with altered phospholipid distribution . Data were discussed with regard to the sort-induced "stress" conditions such as exposure to hydrodynamic forces or magnetic fields . CONCLUSIONS: Both separation procedures modify cell membrane with neither technique being physiologically preferable for subsequent analysis or recultivation of the sorted cells.

Blood Cells Mol Dis, 1999 Jun-Aug, 25(3-4), 170 - 9
Adenine nucleotide synthesis in human erythrocytes depends on the mode of supplementation of cell suspension with adenosine; Komarova SV et al.; In suspensions of washed human erythrocytes, adenosine added in a single dose to concentrations of 0.1-10.0 mmol/l suspension was deaminated at rates ranging from 10 to 50 mmol/l cells h . The sum of adenosine, inosine, and hypoxanthine concentrations in the suspension, as well as the intracellular concentration of ATP, remained constant . In the presence of 25-50 mmol/l orthophosphate, addition of a single dose of adenosine into erythrocyte suspension increased the ATP concentration by up to 280% of the initial level . If the initial adenosine concentrations were greater than 5 mmol/l suspension, ATP increased independently of adenosine concentration to the level determined only by the concentration of orthophosphate . After orthophosphate was returned to its initial level, ATP in erythrocytes began to decrease . In the presence of coformycin, erythrocytes utilised adenosine at a rate of 0.2-0.3 mmol/l cells h . Their adenylate pool increased at a rate of 0.10-0.16 mmol/l cells h for several hours, but intracellular ATP increased only slightly . The energy charge of cells decreased significantly from 0.86 +/- 0.05 (control) to 0.82 +/- 0.06 . Adenosine continuously pumped into erythrocyte suspensions at rates of 0.02-5.0 mmol/l cells h for several hours caused the adenylate pool of erythrocytes and intracellular ATP to increase synchronously at a rate of 0.02-0.35 mmol/l cells h . The energy charge of these erythrocytes increased significantly up to 0.91 +/- 0.03 . After pumping of adenosine was stopped, the intracellular ATP and the adenylate pool began to decrease, returning sometimes to the initial level in 2-3 h.

J Biol Chem, 1999 Dec 3, 274(49), 34699 - 705
Characterization of the cryptogein binding sites on plant plasma membranes; Bourque S et al.; Cryptogein is a 98-amino acid proteinaceous elicitor of tobacco defense reactions . Specific binding of cryptogein to high affinity binding sites on tobacco plasma membranes has been previously reported (K(d) = 2 nM; number of binding sites: 220 fmol/mg of protein) . In this study, biochemical characterization of cryptogein binding sites reveals that they correspond to a plasma membrane glycoprotein(s) with an N-linked carbohydrate moiety, which is involved in cryptogein binding . Radiation inactivation experiments performed on tobacco plasma membrane preparations indicated that cryptogein bound specifically to a plasma membrane component with an apparent functional molecular mass of 193 kDa . Moreover, using the homobifunctional cross-linking reagent disuccinimidyl suberate and tobacco plasma membranes incubated with (125)I-cryptogein, we identified, after SDS-polyacrylamide gel electrophoresis and autoradiography, two (125)I-cryptogein linked N-glycoproteins of about 162 and 50 kDa . Similar results were obtained using Arabidopsis thaliana and Acer pseudoplatanus plasma membrane preparations, whereas cryptogein did not induce any effects on the corresponding cell suspensions . These results suggest that either cryptogein binds to nonfunctional binding sites, homologues to those present in tobacco plasma membranes, or that a protein involved in signal transduction after cryptogein recognition is absent or inactive in both A . pseudoplatanus and A . thaliana.

FEBS Lett, 1999 Sep 24, 458(3), 349 - 53
Indomethacin-induced G1/S phase arrest of the plant cell cycle; Ehsan H et al.; In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway . To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied . Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S . These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells.

Br J Cancer, 1999 Oct, 81(4), 638 - 46
Characterization of a novel transplantable orthotopic rat bladder transitional cell tumour model; Xiao Z et al.; An animal tumour model that mimics the human counterpart is essential for preclinical evaluation of new treatment modalities . The objective of this study was to develop and characterize such a model . To accomplish this, the established AY-27 rat bladder transitional cell carcinoma (TCC) cell line was transplanted orthotopically into Fischer CDF344 female rats . AY-27 TCC cells were grown in monolayer cell culture and instilled intravesically as single cell suspensions into bladders that had been conditioned with mild acid washing . Tumour growth was assessed weekly by subjecting the rats to magnetic resonance imaging (MRI) . At intervals following implantation and MRI tumour detection, the animals were sacrificed for necropsy, histological examination and immunocytochemical studies . Flow cytometry was also performed for detection of Fas or Fas-ligand expression on AY-27 cells . The overall tumour establishment was 95% (97/102 rats) at 12-50 days, while in a subgroup of animals sacrificed at 16 days, 80 out of 82 animals (97%) developed TCC, the majority of which was superficial . Tumour stage was assessed by gross pathology and light microscopy . Histological examination of the tumour specimens confirmed the presence of grade II-III TCC . Immunocytochemistry confirmed that the tumour model maintained the features of TCC . The changes seen on MRI correlated well with the extent of tumour invasion identified histologically . Patchy carcinoma in situ could be detected histologically 12-13 days post-inoculation, and progressed to papillary tumour or invasive disease thereafter . Neither Fas nor Fas-ligand was expressed on AY-27 cells . The orthotopic AY-27 TCC model is highly reproducible and is ideal for preclinical studies on experimental intravesical therapies.

Biomed Mater Eng, 1999, 9(3), 171 - 8
Microprocessor-controlled freezing device for cryopreservation of cell samples; Djuric D et al.; A freezing device for cryopreservation of blood mononuclear cells has been developed . The device is microcontroller operated, allowing cell freezing by a fully automatic, unattended process . To ensure optimum preservation, the temperature in the cell suspension uniformly decreases from room temperature to -100 degrees C and then the samples are transferred to long-term storage . The performance of the device has been tested using both physiological solution and a sample of cell suspension . The control of temperature variation of cell suspension in the entire temperature range has been realised with an accuracy better than +/- 0.1% . The viability of cells recovered from the frozen samples was 95% . The nitrogen consumption for one cycle of cryopreservation was 1.51 . In addition to the fully automatic mode, the manual and semi-automatic modes are available for research purposes . The device has been designed using low cost and widely used electronic components and materials, it is compact and simple to operate.

Appl Microbiol Biotechnol, 1999 Oct, 52(4), 516 - 23
Production of functional human alpha 1-antitrypsin by plant cell culture; Terashima M et al.; Recombinant human alpha 1-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form . This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein . Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice alpha-amylase gene Amy3D . The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose . It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically . Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans . This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT . The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity . Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT . The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT . Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded . The productivity of rice suspension cells expressing rAAT was 4.6-5.7 mg/g dry cell . Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins.

Biotechnol Bioeng, 1999, 66(2), 114 - 21
Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release; Steward N et al.; The death of Medicago sativa L . cells cultivated in a batch culture was investigated by measuring both the appearance of intact dead cells determined on the basis of the trypan blue (TB) dye exclusion, and the release of the cytoplasmic esterase activity into the culture medium upon cell death . Taking into account the strong instability of this released esterase activity, the total dead cell and lysed cell densities have been estimated . A mechanism for cell death and lysis is proposed and the specific rates of cell growth, death and lysis estimated . The specific rate of appearance of TB dead cells was low and essentially constant (0.25 day(-1)) during the first 8 days of the batch culture, and then increased above 1.5 day(-1) after 2 weeks of cultivation . Whereas no lysis occurred during the first seven days, this phenomenon occurred during the second period and accounted for about 20% of the total cell death by the end of the process . Thus, the viability determined by the trypan blue exclusion method appeared to be invalid after 7 days of culture . When lysis of viable cells is taken into consideration, the specific growth rate was significantly increased and growth was shown to continue for a further 8 days . Increased sensitivity of the cells to shear stresses and consequent cell lysis could be the result of a 35% increase in the cell size

J Immunol, 1999 Nov 15, 163(10), 5211 - 8
Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity; Shimizu J et al.; This study shows that removal of a T cell subpopulation can evoke effective tumor immunity in otherwise nonresponding animals . Elimination of CD25-expressing T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, elicited potent immune responses to syngeneic tumors in vivo and eradicated them . The responses were mediated by tumor-specific CD8+ CTLs and tumor-nonspecific CD4-8- cytotoxic cells akin to NK cells . Furthermore, in vitro culture of CD25+4+ T cell-depleted splenic cell suspensions prepared from tumor-unsensitized normal mice led to spontaneous generation of similar CD4-8- cytotoxic cells capable of killing a broad spectrum of tumors; reconstitution of CD25+4+ T cells inhibited the generation . In this culture, self-reactive CD25-4+ T cells responding to self peptides/class II MHC complexes on APCs spontaneously proliferated upon removal of CD25+4+ T cells, secreting large amounts of IL-2 . The IL-2 thus produced appeared to be responsible for the generation of CD4-8- NK cells as lymphokine-activated killer cells, because direct addition of an equivalent amount of IL-2 to the culture of CD4-8- cells generated similar lymphokine-activated killer/NK cells, whereas coculture of normal CD4-8- cells with CD25-4+ T cells from IL-2-deficient mice did not . Thus, removal of immunoregulatory CD25+4+ T cells can abrogate immunological unresponsiveness to syngeneic tumors in vivo and in vitro, leading to spontaneous development of tumor-specific effector cells as well as tumor-nonspecific ones . This novel way of evoking tumor immunity would help to devise effective immunotherapy for cancer in humans.

Plant Physiol, 1999 Nov, 121(3), 1025 - 1035
A Re-Evaluation of the Relative Roles of Two Invertases, INCW2 and IVR1, in Developing Maize Kernels and Other Tissues; Carlson SJ et al.; We have examined the relative abundance and distribution of the transcripts and protein products of a cell wall gene (Incw2) and a soluble invertase gene (Ivr1) to better understand their relative roles during maize (Zea mays L.) kernel development . In developing kernels the steady-state levels of Incw2 transcript increased dramatically from 0 to 12 d after pollination, while Ivr1 transcript, in contrast to a previous report, was undetectable . Consistent with the RNA expression data, the IVR1 protein could not be detected in kernel extracts using antisera raised to a synthetic peptide . Fractionation of the soluble form of invertase from developing kernels by isoelectric focusing and protein blots suggested that the enzyme activity was due to contamination of the cell wall invertase protein . A similar observation was made in a maize cell suspension culture in which Ivr1 RNA, but not IVR1 protein, was significantly modulated by sugars in the medium . Protein-blot analyses of the soluble enzyme activity suggested that changes in the enzyme activity are attributable to a cell wall invertase protein in the soluble fraction . Based on the collective evidence, we propose that the cell wall, but not the soluble invertase, is critical to heterotrophic sinks such as cell suspension cultures and developing kernels.

Transplantation, 1999 Oct 27, 68(8), 1153 - 60
Discordant neural tissue xenografts survive longer in immunoglobulin deficient mice; Larsson LC et al.; BACKGROUND: The immune response against discordant xenografts in the brain is incompletely understood and remains a major obstacle for future clinical applications of xenogeneic neural tissue transplants in neurodegenerative disorders . To determine the role of antibodies in the rejection process, we compared graft survival and immune reactions between immunoglobulin deficient (IgKO) and normal mice . METHODS: A cell suspension of embryonic porcine ventral mesencephalon was injected into the striatum of adult normal and IgKO mice . Graft sizes and number of infiltrating CD4- and CD8-positive lymphocytes were determined by stereological methods at 4 days and 2, 4, and 6 weeks after the transplants . Microglial accumulation was determined using the optical densitometrical method . Intraparenchymal deposition of IgG was investigated at 4 days and 2 weeks . RESULTS: The majority of IgKO mice had surviving grafts for up to 4 weeks, whereas survival was minimal in control mice beyond 4 days . Graft sizes differed significantly between IgKO and control mice at 2 weeks (P<0.01, Kruskal Wallis ANOVA, followed by Mann Whitney test) . The majority of infiltrating lymphocytes were CD4-positive in control mice but CD8-positive in IgKO mice . Microglial accumulation was strong around surviving grafts in IgKO mice at 4 weeks . Prominent staining of IgG, diffuse in the transplanted hemisphere and specific on grafted neurons, was found in control mice . CONCLUSIONS: Our results suggest that immunoglobulins play an initiating role in rejection of discordant neural xenografts . After a prolonged graft survival of approximately 4 weeks, a cellular response with a large proportion CD8-positive cells leads to rejection in IgKO mice.

Arch Toxicol, 1999 Sep, 73(7), 381 - 6
Effect of dental materials on gluconeogenesis in rat kidney tubules; Reichl FX et al.; The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) as well as mercuric chloride (HgCl(2)) and methylmercury chloride (MeHgCl) on gluconeogenesis was investigated in isolated rat kidney tubules . From starved rats kidney tubules were prepared and isolated by digestion with collagenase . 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 3.3 +/- 0.2 nmol/mg . per min (mean +/- SEM, n=21) . Relative rates of glucose formation were obtained by expressing individual rates as a 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 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), <10 (MeHgCl), and <10% (HgCl(2)) . Values of 50% effective concentration (EC(50)) were calculated from fitted curves . EC(50) values were (mmol; mean +/- SEM; n=4): HEMA, 17.7 +/- 2.9; TEGDMA, 1.8 +/- 0.2; MeHgCl, 0.018 +/- 0.0005; and HgCl(2), 0 . 0016 +/- 0.0005 . The toxic effect of HgCl(2) was approximately 1000 or 10 000 higher than that of the dental composite components TEGDMA or HEMA, respectively.

C R Acad Sci III, 1999 Sep, 322(9), 743 - 8
Glutamine synthetase activity in Solanaceous cell suspensions accumulating alkaloids or not . 13C NMR and enzymatic assay; Mesnard F et al.; The metabolism of labelled pyruvate followed by 13C NMR and the measure of glutamine synthetase (GS) showed, according to previous results, a high activity of this enzyme in suspension cells of Nicotiana plumbaginifolia . This activity could derive glutamate from the alkaloid synthesizing pathways . However, a recent work showed that the rate of the GS gene transcription was inversely proportional to the Gln/Glu ratio . The measures of Gln and Glu concentrations in Nicotiana plumbaginifolia cells revealed that high GS activity correlates with the weak value of Gln/Glu ratio . Therefore, the hypothesis of GS dysfunctioning for the non-biosynthesis of alkaloids in N . plumbaginifolia suspension cells can be discarded . This conclusion is strengthened by the results obtained when using a GS inhibitor.

Appl Environ Microbiol, 1999 Nov, 65(11), 5035 - 41
Oxidation of methyl halides by the facultative methylotroph strain IMB-1; Schaefer JK et al.; Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr . Consumption of >100 microM MeBr by cells grown on glucose, acetate, or monomethylamine required induction . Induction was inhibited by chloramphenicol . However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr . Glucose-grown cells were able to readily oxidize {(14)C}formaldehyde to (14)CO(2) but had only a small capacity for oxidation of {(14)C}methanol . Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for {(14)C}MeBr oxidation than did cells without preincubation . Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr . No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds . Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K(s) values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively . These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO(2) . The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.

Biofizika, 1999 Jul-Aug, 44(4), 708 - 13
{Kinetics of decrease in erythrocyte filterability under the effect of ephazol}; Dubniskii VZ et al.; The effect of palladium-containing complex Ephazol on the filtration rate of erythrocyte suspensions through nuclear filters was studied by the constant-pressure filtration method . It was shown that the filterability of red blood cells incubated with ephazol decreased . If the time necessary for a fixed volume of red blood cell suspension to pass through a filter was plotted against the time of incubation with Ephazol or against its initial concentration, the curves typical of autoaccelerated processes were obtained . From analysis of kinetic models, it was concluded that the effects observed are due to the nonlinear dependence of the filtration rate w on the rate at which an erythrocyte passes through a pore and the influence of Ephazol on the distribution of erythrocytes with respect to w . Several models describing changes in the distribution of erythrocytes with respect to w in the presence of Ephazol and possible mechanisms relating the filtration kinetics to the incubation parameters are discussed.

J Nat Prod, 1999 Oct, 62(10), 1395 - 8
Isolation of labeled 9-dihydrobaccatin III and related taxoids from cell cultures of taxuscell cultures of taxus canadensis elicited with m; Ketchum RE et al.; Cell suspension cultures of Taxus canadensis rapidly produced paclitaxel (1) and other taxoids in response to elicitation with methyl jasmonate . Three of these taxoids, of potential value in the synthesis of taxoid analogues, have been isolated from cell cultures of Taxus canadensis and identified as 13-acetyl-9-dihydrobaccatin III (2), baccatin VI (3), and 9-dihydrobaccatin III (4) . Of these metabolites, 9-dihydrobaccatin III (4) has not been isolated from any Taxus species, whereas 13-acetyl-9-dihydrobaccatin III (2) and baccatin VI (3) have been isolated from a number of natural sources . 2D NMR techniques, mass spectrometry, and partial synthesis were used to rigorously elucidate the structure and stereochemistry of these natural products.

J Cancer Res Clin Oncol, 1999 Nov, 125(11), 609 - 14
Repair of potentially lethal damage by total and quiescent cells in solid tumors following a neutron capture reaction; Masunaga S et al.; Purpose: We analyzed the time-course of changes in the sensitivity of total (proliferating + quiescent and quiescent (Q) cell populations within solid tumors in situ following a neutron capture reaction and compared it with that after gamma-ray irradiation . Methods: After continuous labeling of proliferating cells with BrdU for 5 days, mice bearing SCC VII tumors received thermal neutron irradiation with or without a (10)B-labeled compound (sodium {(10)B}borocaptate, BSH, or DL-p-{(10)B}boronophenylalanine, BPA), or gamma-ray irradiation . From 5 min to 72 h after treatment, tumors were excised, minced, and trypsinized . Cell suspensions were incubated for 48 h with the cytokinesis blocker cytochalasin-B . The micronucleus frequency for BrdU-unlabeled cells, Q cells at treatment, was then determined by immunofluorescence staining for BrdU . The micronucleus frequency for total cells was obtained from tumors that had not been pretreated with BrdU labeling . The sensitivity was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (micronucleus frequency) . Results: Overall, Q cells showed greater repair capacities than total cells . gamma-Ray irradiation and neutron irradiation with BPA induced larger repair capacities in each cell population . In contrast, thermal neutron irradiation without a (10)B-labeled compound induced the smallest repair capacity in both cell populations . The use of a (10)B-labeled compound, especially BPA, widened the difference in sensitivity between total and Q cells, resulted in an increase in repair capacity in both cell populations, and made the repair patterns of the two cell populations look like those induced by gamma-ray irradiation . Conclusion: Differences in sensitivity and repair patterns following the neutron capture reaction were thought to depend on differences in the distribution of the (10)B-labeled compound between the proliferating and Q cell populations.

Clin Cancer Res, 1999 Oct, 5(10 Suppl), 3232s - 3242s
Radioimmunotherapy of small volume disease of colorectal cancer metastatic to the liver: preclinical evaluation in comparison to standard chemotherapy and initial results of a phase I clinical study; Behr TM et al.; At the time of surgery, occult metastases (micrometastases) are present in more than 50% of colorectal cancer patients, and the liver is the most frequent site of apparent metastatic disease . Frequently, adjuvant chemotherapy is unable to prevent tumor recurrence . Thus, novel therapeutic strategies are warranted . The aim of this study was to establish a model of human colon cancer metastatic to the liver of nude mice, to assess, in this setting, the therapeutic efficacy of radioimmunotherapy (RAIT) compared to standard chemotherapy and to evaluate, in a Phase I/II trial, the toxicity and therapeutic efficacy of RAIT in colorectal cancer patients with small volume disease metastatic to the liver . Multiple liver metastases of the human colon cancer cell line GW-39 were induced by intrasplenic injection of a 10% tumor cell suspension . Whereas controls were left untreated, therapy was initiated on day 10 or 20 after tumor inoculation with the 131I-labeled, low affinity anticarcinoembryonic antigen (anti-CEA) monoclonal antibody (MAb), F023C5 (Ka = 10(7) liters/mol), or the high-affinity anti-CEA MAb, MN-14 (Ka = 10(9) liters/mol), or chemotherapy (5-fluorouracil/leucovorin (folinic acid) versus irinotecan) at their respective maximum tolerated doses (MTDs) . Twelve colorectal cancer patients with small volume disease metastatic to the liver (all lesions < or = 2.5 cm) were entered into a mCi/m2-based Phase I dose escalation study with 131I-labeled humanized version of MN-14, hMN-14 . The patients were given single injections, starting at 50 mCi/m2 and escalating in 10-mCi/m2 increments . The MTD was defined as the dose level at which < or = 1 of 6 patients develop grade 4 myelotoxicity . In the mice, untreated controls died from rapidly progressing hepatic metastases at 6-8 weeks after tumor inoculation . The life span of mice treated with 5-fluorouracil/leucovorin was prolonged for only 1-3 weeks, whereas irinotecan led to a 5-8-week prolongation . In contrast, at their respective MTDs, the 131I-labeled low-affinity anti-CEA MAb, F023C5, led to a 20% permanent cure rate, and the high affinity MAb, MN-14, led to an 80% permanent cure rate, when therapy was initiated at 10 days after tumor inoculation . In the 20-day-old tumor stage, although it prolonged life, 131I-F023C5 was unable to achieve cures, whereas 131I-MN-14 was still successful in 20% . Histologically, no remaining viable tumor cells could be demonstrated in these animals surviving > 6 months . In patients, the MTD was reached at 60 mCi/m2 of hMN-14 (at 70 mCi/m2, two of three grade 4 myelotoxicities) . Of 11 assessable patients, 2 had partial remissions (corresponding to an objective response rate of 18%), and 5 (45%) had minor/mixed responses or experienced stabilization of previously rapidly progressing disease . These data suggest that in small volume disease, RAIT may be superior to conventional chemotherapy . Antibodies of higher affinity seem to be clearly superior . The clinical response rates in patients with small volume disease are encouraging, being comparable to the response rates of conventional chemotherapeutic regimens but with fewer side effects . Ongoing studies will show whether treatment at the MTD will further improve therapeutic results.

J Investig Dermatol Symp Proc, 1999 Sep, 4(2), 169 - 72
Molecular mechanisms involved in the migration of epidermal dendritic cells in the skin; Nakamura K et al.; The murine epidermis contains two types of dendritic cells (DC), Thy-1+ dendritic epidermal T cells (DETC) and Langerhans cells . In this review, we introduce our data obtained using a skin organ culture system to examine the migratory capacity of DETC and Langerhans cells into the epidermis . DETC or Langerhans cells were depleted by topical application ofclobetazole propionate (CP) solution onto the murine ears . CP-treated or untreated ear skin was co-cultured with syngeneic (semi-syngeneic, or allogenic, in experiments with Langerhans cells) epidermal cell suspension . We found (i) that donor DETC or Langerhans cells migrated into the CP-treated epidermis as well as into untreated epidermis, (ii) that leukosialin Ly48 recognized by monoclonal antibody S11 and TNF-alpha strongly inhibited donor Langerhans cell migration into the epidermis . We mention other molecules that may participate in the migration of Langerhans cells such as chemotactic cytokines, monocyte chemoattractant protein (MCP)-1, TGF-beta and skin-homing molecule, cutaneous lymphocyte-associated antigen (CLA) on Langerhans cells.

Glia, 1999 Nov, 28(2), 156 - 65
Effects of schwann cell suspension grafts on axon regeneration in subacute and chronic CNS traumatic injuries; Stichel CC et al.; In a previous study, we have shown that microtransplanted Schwann cell suspensions foster structural recovery of the acutely transected postcommissural fornix . The emphasis of the present study was to examine whether subacutely and chronically injured axons also demonstrate significant responsiveness to implanted Schwann cells . Microinjected suspensions of cultured Schwann cells i) elicited a growth response and attracted axons in a subacute and chronic traumatic lesion but ii) failed to stimulate regrowth of the postcommissural fornix projection at any nonacute postlesion stage . In conclusion, the single intervention strategy of Schwann cell microimplantation is not sufficient to ensure regeneration of the subacutally or chronically transected postcommissural fornix . The use of Schwann cells as stimulators of axon regrowth depends on the neuronal cell type and the appropriate postinjury time point .

Jpn J Physiol, 1999 Aug, 49(4), 379 - 87
Automatic measurement of the red cell oxygen dissociation curve identical with the whole blood curve; Mawjood AH et al.; Automatic measurement of the entire oxygen dissociation curve (ODC) of blood and hemoglobin provides a useful means for evaluating their gas-transport function . The automatic oxygenation apparatus previously developed by Imai et al . (1970, 1981), which uses a polarographic determination of partial pressure of oxygen and a spectrophotometric determination of oxygen saturation of hemoglobin, has mostly been used for the measurement of accurate ODCs of hemoglobin solution . However, it was not suitable for red cell suspension because a significant noise was superimposed on the absorbance signal due to light-scattering by red cells . In the present study, we have overcome this problem by using an integrating sphere for the photometric system . Through extensive tests we found the optimal experimental conditions for obtaining the red cell oxygenation data that were identical with the whole blood data with respect to the position (oxygen affinity) and shape (sigmoid character) of the ODC and its pH-dependence (the Bohr effect) . The accuracy was higher than that of commercially available automatic apparatuses such as the "Hemox-Analyzer" (Technical Consulting Service) and "Hem-O-Scan" (Aminco) . Thus, our method provides an easy and convenient means for obtaining accurate ODCs mimicking the whole blood ODCs from one drop of whole blood . An application of our method to the effect of blood storage on ODC is presented, demonstrating the usefulness of our method.

J Neurosci Methods, 1999 Sep 15, 91(1-2), 101 - 7
A chemiluminescent catecholamine assay: its application for monitoring adrenergic transmitter release; Israel M et al.; A chemiluminescent procedure for measuring catecholamines (dopamine, norepinephrine, epinephrine) is described . It is based on the observation that lactoperoxidase catalyses both the oxidation of catecholamines, and the chemiluminescent reaction of luminol with their oxidation product . The assay has been adapted for continuously monitoring the release of catecholamines from adrenergic tissues, from cell suspensions and from cells loaded in culture with dopamine.

Br J Haematol, 1999 Sep, 106(4), 1033 - 6
Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) DNA sequences are absent in leukapheresis products and ex vivo expanded CD34+ cells from multiple myeloma patients; De Greef C et al.; Recently it was reported that Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) infects bone marrow (BM) dendritic cells (DC) in multiple myeloma (MM) patients and therefore might play a role in MM development . Because of the use of myeloid growth factors like GM-CSF and G-CSF for the mobilization of peripheral blood progenitor cells (PBPC), the subsequent increase of DC precursors might imply a risk for KSHV contamination in PBPC grafts . Therefore, in this study leukapheresis products and ex vivo cultured CD34+ cell suspensions were analysed . KSHV DNA could not be amplified in any of them.

Plant Physiol, 1999 Oct, 121(2), 535 - 43
Ascorbate biosynthesis in Arabidopsis cell suspension culture; Davey MW et al.; The biosynthesis of L-ascorbic acid (L-AA) in an Arabidopsis (L.) Heynh . cell suspension culture was studied by quantifying the effects of incubation with a range of potential biosynthetic precursors, analogs, and inhibitors on the intracellular levels of reduced and oxidized forms of L-AA . Our results support the recently published biosynthetic pathway of L-AA from L-galactose (G.L . Wheeler, M.A . Jones, N . Smirnoff {1998} Nature 393: 365-369), but suggest that Arabidopsis cell suspension culture simultaneously contains two other routes leading to L-AA . The possible physiological significance of these alternate routes is discussed.

Mol Gen Genet, 1999 Sep, 262(2), 239 - 49
Four mismatch repair paralogues coexist in Arabidopsis thaliana: AtMSH2, AtMSH3, AtMSH6-1 and AtMSH6-2; Ade J et al.; By using degenerate oligonucleotides based on the sequence homology between known MutS homologues, three MSH cDNAs belonging to the MSH2, MSH3 and MSH6 families, as defined in eukaryotes, have been isolated from Arabhidopsis thaliana (ecotype Columbia) . Genomic sequences for two of these genes (AtMSH2 and AtMSH6-2) were also isolated and determined, whereas the genomic sequence of AtMSH3 was obtained through the Arabidopsis sequencing project, as was the sequence of a second, distinct AtMSH6 homologue (AtMSH6-1) . Comparative analysis of the AtMSH2 Landsberg erecta genomic sequence (reported here) and the previously described AtMSH2 Columbia allele revealed several polymorphisms, including the presence of a small, transposon-like element in the 3' untranscribed region of the former allele . Arabidopsis is the first organism to show such divergence of two AtMSH6 genes; the divergence is strongly supported by sequence data and phylogenetic analysis . Southern analysis revealed that the three genes we have isolated exist as single copies, and genetic mapping indicated that AtMSH2 and AtMSH6-2 both reside on chromosome III . Finally, expression of these three genes could only be observed in suspensions of A . thaliana cells . Such a cell suspension divides actively after subculture, and the AtMSH genes are most strongly expressed at this stage.

Blood, 1999 Oct 15, 94(8), 2819 - 26
Early maturation of T-cell progenitors in the absence of glucocorticoids; Sacedon R et al.; In the present work, we demonstrated that both fetal liver and thymic T-cell precursors express glucocorticoid receptors (GRs) indirectly suggesting a role for glucocorticoids (GCs) in the earliest events of T-cell differentiation . To evaluate this issue, we analyzed the thymic ontogeny in the progeny of adrenalectomized pregnant rats (Adx fetuses), an in vivo experimental model, which ensures the absence of circulating GCs until the establishment of the fetal hypothalamus-pituitary-adrenal (HPA) axis . In the absence of maternal GCs, T-cell development was significantly accelerated, the process being reversed by in vivo GC replacement . Mature single positive thymocytes (both CD4 and CD8) appeared in 16-day old fetal Adx thymus when in the control fetuses, most thymocytes still remained in the double-negative (DN) CD4(-)CD8(-) cell compartment . In addition, emigration of T-cell receptor (TcR)alphabeta positive cells to the spleen also occurred earlier in Adx fetuses than in control ones . In vitro recolonization of cultured deoxiguanosine-treated mouse fetal thymus lobes with 13-day-old fetal liver cell suspensions from both Adx and control fetuses demonstrated changes in the developmental capabilities of fetal liver T-cell precursors from embryos grown in the absence of GCs . Furthermore, a precocious lymphoid colonization of the thymic primordium from Adx fetuses was evidenced by ultrastructural analysis of both Adx and Sham early thymus . Both findings accounted for the accelerated T-cell differentiation observed in Adx fetuses . Together, these results support a role for GCs not only in the thymic cell death, but also in the early steps of T-cell differentiation.

Eur J Oral Sci, 1999 Oct, 107(5), 338 - 43
Chemosensitivity testing of oral cancer cells treated with a p185neu-specific agent; Werkmeister R et al.; The amplification and overexpression of the erbB-2 oncogene and its involvement in tumorigenesis makes this receptor an appropriate target for specific agents directed towards tumor cells . The purpose of this study was to evaluate the in vitro effect of the bacterially produced recombinant immunotoxin scFv(FRP5)-ETA on the protein synthesis and adenosine triphosphate (ATP) reduction in oral squamous cell carcinoma (OSCC) cells . This agent recognizes the erbB-2 receptor and inhibits protein synthesis in receptor-overexpressing cells . OSCC cells were selected for this study, and amplification and expression levels of the erbB-2 receptor were determined . Cell suspensions were cultured for 6 d with various concentrations of scFv(FRP5)-ETA (1-1000 ng/ml) . A431 and MDA-MB468 cell lines were used as controls . Chemosensibility of tumor cells was measured by {3H}leucine incorporation assay and by an ATP luminescence assay . In OSCC cells with amplification and overexpression of erbB-2 inhibition, up to 92% of protein synthesis and 90% of ATP reduction was observed when cells were exposed to 1,000 ng/ml immunotoxin . In OSCC cells showing a deletion of erbB-2 and in erbB-2-negative MDA-MB468 cells, protein synthesis was inhibited by 22% and 8%, respectively . These results indicate that the effectiveness of a recombinant immunotoxin targeting erbB-2 receptors in OSCC cells depends on the level of erbB-2 amplification and expression, that it is highly specific for tumor cells expressing these receptors, and that a dose-dependency can be observed.

Eur J Gastroenterol Hepatol, 1999 Aug, 11(8), 839 - 43
Increased release of interleukin-6 by oesophageal mucosa in children with reflux oesophagitis; Corrado G et al.; OBJECTIVE: To evaluate the release of interleukin-6 (IL-6) by oesophageal mucosa and to establish the serum levels of IL-6 and C-reactive protein (CRP), and plasma fibrinogen in children with reflux oesophagitis . DESIGN: In a prospective study, IL-6 release by tissue fragments obtained from oesophageal biopsies was determined and serum IL-6 and CRP as well as plasma fibrinogen were analysed . METHODS: The study population comprised ten children with reflux oesophagitis, diagnosed on the basis of 24 h oesophageal pH monitoring and endoscopy with biopsies . Ten children with recurrent abdominal pain were studied for comparative purposes . Biopsy tissue fragments were processed to obtain a cell suspension and the release of IL-6 was determined in culture medium . Serum IL-6 levels were measured by ELISA, serum CRP by turbidimetry, and plasma fibrinogen by spectrophotometry . RESULTS: Oesophageal cells obtained from reflux oesophagitis patients synthesize and release in vitro a significantly higher amount of IL-6 than controls (71.26+/-19.5 versus 31.67+/-8.02 pg/10(6) cells; P<0.01) . Serum IL-6, serum CRP and plasma fibrinogen levels were not statistically different between patients with reflux oesophagitis and controls . CONCLUSIONS: These results suggest a short-term action of IL-6 since its effects could be exerted only in the microenvironment of the oesophageal mucosa.

Radiat Med, 1999 Jul-Aug, 17(4), 259 - 64
Potentially lethal damage repair by total and quiescent tumor cells following various DNA-damaging treatments; Masunaga S et al.; After continuous labeling of proliferating (P) cells with 5-bromo-2'-deoxyuridine (BrdU) for 5 days, SCC VII tumor-bearing mice received various kinds of DNA-damaging treatments: gamma-ray irradiation, tirapazamine (TPZ, hypoxia-specific cytotoxin) administration, or cisplatin injection . From 0.5 to 72 hr after treatment, tumors were excised, minced, and trypsinized . Single tumor cell suspensions were incubated for 48 hr with a cytokinesis-blocker, cytochalasin-B . Then, the micronucleus (MN) frequency for BrdU-unlabeled cells, quiescent (Q) cells at treatment, was determined using immunofluorescence staining for BrdU . The MN frequency for total (P+Q) cells was obtained from tumors that were not pretreated with BrdU labeling . The sensitivity to each DNA-damaging treatment was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency) . Treatment with gamma-rays or cisplatin resulted in a larger MN frequency in total cells than in Q cells . In contrast, TPZ treatment produced a smaller MN frequency in total cells than in Q cells . Regardless of the treatment used, Q cells showed greater repair capacities than total cells . However, TPZ caused much smaller repair capacity in both total and Q cells, compared with gamma-rays or cisplatin . Gamma-rays and cisplatin produced similar repair patterns . Differences in sensitivity between total and Q cells and repair patterns of the two cell populations were thought to depend on differences between the two cell populations in the toxicity of the DNA-damaging treatment and distribution pattern of the anticancer agent.

Br J Pharmacol, 1999 Sep, 128(2), 472 - 8
The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant mu-opioid receptors and SH-SY5Y cells; Harrison C et al.; 1 Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands for the mu-opioid receptor . The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and {Ca2+}i levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant mu-opioid receptors . 2 E-1 displaced {3H}-diprenorphine ({3H}-DPN) binding in CHO micro and SH-SY5Y membranes with pKi values of 8.02+/-0.09 and 8.54+/-0.13 respectively . E-2 displaced {3H}-DPN binding in CHOmu and SH-SY5Y cells with pKi values of 7.82+/-0.11 and 8.43+/-0.13 respectively . E-1/E-2 bound weakly to CHOdelta and CHOkappa membranes, with IC50 values of greater than 10 microM . 3 In CHOmu cells, E-1/E-2 inhibited forskolin (1 microM) stimulated cyclic AMP formation with pIC50 values of 8.03+/-0.16 (Imax = 53.0+/-9 . 3%) and 8.15+/-0.24 (Imax = 56.3+/-3.8%) respectively . In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72+/-0.13 (Imax=46.9+/-5.6%) and 8.11+/-0.31 (Imax = 40.2+/-2.8%) respectively . 4 E-1/E-2 (1 microM) increased {Ca2+}i in fura-2 loaded CHOmu cell suspensions in a thapsigargin sensitive and naloxone reversible manner . Mean increases observed were 106+/-28 and 69+/-6.7 nM respectively . In single adherent cells E-1/E-2 (1 microM) increased {Ca2+}i with a mean 340/380 ratio change of 0.81+/-0.09 and 0.40+/-0.08 ratio units respectively . E-1/E-2 failed to increase intracellular calcium in CHOdelta, CHOkappa and SH-SY5Y cells . 5 These data show that E-1/E-2 bind with high affinity and selectivity to mu-opioid receptors and modulate signal transduction pathways typical of opioids . This provides further evidence that these two peptides may be endogenous ligands at the mu-opioid receptor.

Eur J Neurosci, 1999 Sep, 11(9), 3073 - 81
Differential effects of Bcl-2 overexpression on fibre outgrowth and survival of embryonic dopaminergic neurons in intracerebral transplants; Schierle GS et al.; The causes of death of transplanted neurons are not known in detail, but apoptotic mechanisms involving caspase activation are likely to play a role . We examined whether overexpression of the anti-apoptotic protein Bcl-2 may enhance the survival of dopaminergic {tyrosine hydroxylase (TH)-immunoreactive} grafted neurons . For this purpose, we prepared cells from embryonic day 13 ventral mesencephalon (VM) of mice overexpressing human Bcl-2, or from their wild-type littermates . The bcl-2 transgene was strongly expressed in these cells, and resulted in protection of neuronal cultures from death triggered by serum deprivation or exposure to staurosporine . To model pretransplantation stress more closely in vitro, we stored dissociated embryonic mesencephalic cells for 8 h in the same type of medium used for intracerebral transplantation . This resulted in massive cell death as quantified by lactate dehydrogenase (LDH) release, and increased DNA fragmentation . Although this cell loss was strongly reduced by a caspase inhibitor, Bcl-2 had no significant protective effect . Finally, mesencephalic cell suspensions were xenografted into the striatum of immunosuppressed hemiparkinsonian rats . Neither the survival of TH-immunopositive transplanted neurons nor the functional recovery of the rats was improved by Bcl-2, although the Bcl-2 protein was strongly expressed in transgenic grafts 5 weeks after implantation, and dopaminergic fibre outgrowth from the grafts was significantly improved . These data suggest that cell death in neuronal transplants involves apoptotic mechanisms that can bypass negative regulation by Bcl-2.

J Pediatr Surg, 1999 Sep, 34(9), 1378 - 84
Immunoscintigraphy of xenotransplanted hepatoblastoma with iodine 131-labeled anti-alpha-fetoprotein monoclonal antibody; Fuchs J et al.; BACKGROUND/PURPOSE: Hepatoblastoma (HB) is the most common primary malignant liver tumor affecting infants and young children . The alpha-fetoprotein level is elevated in 95% of all children with hepatoblastoma . Therefore, it is of interest to assess targeting of the HB marker alpha-fetoprotein by antibody imaging . In this pilot study, the authors investigated the radioimmunoscintigraphy of xenotransplanted HB in nude mice utilizing an anti-alpha-fetoprotein antibody . METHODS: HB cell suspensions from tumors of 3 children were transplanted subcutaneously into nude mice NMRI (nu/nu) . A total of 200 microg of intact anti-alpha-fetoprotein antibody was injected intravenously into 8 animals from each HB . Before injection, the monoclonal antibody was labeled with iodine (I) 131 (specific activity of 75 MBq/mg, labeling yield of 95%) using the conventional iodogen method . Planar scintigraphic images of anesthetized mice in posterior views were acquired with a gamma camera immediately after injection, and after 1, 2, 3, 7, and 14 days . The biodistribution data were obtained by killing and dissecting animals, and the activity in the tissues was measured in a gamma counter . The alpha-fetoprotein levels in the animals' sera were recorded 15 days after imaging and were compared with the control group . RESULTS: A total of 66% of the hepatoblastomas could be detected by scintigraphy . Within 24 hours, the mean specific tumor uptake in nude mice hepatoblastomas with a volume of over 1,000 mm3, was 14% per injected dose (+/-3.9%) . The biological half-life of the labeled antibody complex in the tumor was 3.86 (+/-0.84) days . Thyroid uptake of free I-131 was 2.85% per injected dose (+/-1.5%) reflecting the deiodination of the labeled antibody complex . CONCLUSIONS: The results show the possibility of imaging xenotransplanted hepatoblastoma with 131I-labeled anti-alpha-fetoprotein and may, in the future, determine tumor recurrence and extension, and thereby improve the prognosis of advanced HBs.

AIDS Res Hum Retroviruses, 1999 Sep 20, 15(14), 1305 - 14
The dendritic cell-T cell milieu of the lymphoid tissue of the tonsil provides a locale in which SIV can reside and propagate at chronic stages of infection; Hu J et al.; Previous studies described the presence of numerous human immunodeficiency virus (HIV)-positive cells within and just beneath the mucosal surfaces of the tonsillar tissue of HIV-1-infected individuals . The virus-positive cells were most abundant in the dendritic cell (DC)-T cell rich areas of the lymphoepithelia lining the crypts, and consisted of multinucleated syncytia that contained DCs . This suggested that such cells within the tonsillar tissue might represent a site for chronic virus replication in infected individuals . Using the simian immunodeficiency virus (SIV)-macaque system, we chose to study further the viral distribution within the tonsillar tissue of animals infected via the vaginal route 8-10 months earlier . Our initial studies demonstrated that in situ hybridization (ISH)-positive DCs and T cells could be identified within the genital mucosa and draining lymph nodes of these infected animals even at this chronic stage of infection . Here we specifically examined the distal mucosa-associated lymphoid tissues of the tonsil . ISH-positive cells were mostly restricted to the DC-rich T cell areas of the underlying lymphoid tissue . However, T cells were the most commonly infected cell type and virus-positive cells were rarely found within the epithelia . In isolated cell suspensions, ISH-positive lymphocytes were often tightly associated with ISH-negative DCs, although few ISH-positive lymphocytes were often tightly associated with ISH-negative DCs, although few ISH-positive DCs could be identified within these clusters . Therefore, the naturally occurring DC-T cell milieu of the lymphoid tissue of the tonsil provides a locale in which SIV can reside and propagate on a chronic basis, even many months after the animals were infected by virus crossing the genital mucosa.

Plant J, 1999 Sep, 19(5), 509 - 19
Cloning and expression of a cDNA encoding betanidin 5-O-glucosyltransferase, a betanidin- and flavonoid-specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae; Vogt T et al.; Based on protein sequence data and RT-PCR, a full length cDNA encoding betanidin 5-O-glucosyltransferase (5-GT) was obtained from a cDNA library of Dorotheanthus bellidiformis (Burm.f.) N.E.Br . (Aizoaceae) . 5-GT catalyses the transfer of glucose from UDP-glucose to the 5-hydroxyl group of the chromogenic betanidin . Betanidin and its conjugates, referred to as betacyanins, are characteristic fruit and flower pigments in most members of the Caryophyllales, which fail to synthesise anthocyanins . The 5-GT cDNA displayed homology to previously published glucosyltransferase sequences and exhibited high identity to sequences of several inducible glucosyltransferases of tobacco and tomato (Solanaceae) . The open reading frame encodes a polypeptide of 489 amino acids with a calculated molecular mass of 55.24 kDa . The corresponding cDNA was expressed in Escherichia coli . The recombinant protein displayed identical substrate specificity compared to the native enzyme purified from D . bellidiformis cell suspension cultures . In addition to the natural substrate betanidin, ortho-dihydroxylated flavonols and flavones were glycosylated preferentially at the B-ring 4'-hydroxyl group . 5-GT is the first enzyme of betalain biosynthesis in plants, of which the corresponding cDNA has been cloned and expressed . The results are discussed in relation to molecular evolution of plant glucosyl- transferases.

Br J Surg, 1999 Sep, 86(9), 1180 - 4
Impact of laparoscopic colonic resection on tumour growth and spread in an experimental model; Gutt CN et al.; BACKGROUND: The influence of surgical manipulation and carbon dioxide pneumoperitoneum on intraperitoneal tumour growth and port-site metastasis during laparoscopic colon resection is still unknown . METHODS: Some 33 male WAG/Rij rats were randomized into three experimental groups: a laparoscopy group with carbon dioxide pneumoperitoneum (n = 11), a gasless laparoscopy group (n = 11) and a laparotomy group (n = 11) . After transanal injection of a tumour cell suspension (1 x 106 CC 531 cells) into the distal colon, a colon segment resection and an end-to-end anastomosis (laparoscopy; intra-abdominal technique) were performed . Tumour growth was scored semiquantitatively 24 days after the operation . Data were analysed by the Kruskal-Wallis test . RESULTS: The tumour indices from the four locations with the greatest tumour growth were significantly decreased in the laparoscopy group with carbon dioxide pneumoperitoneum compared with the gasless laparoscopy and laparotomy groups (P < 0.01) . Port-site metastases were significantly decreased in the carbon dioxide pneumoperitoneum group compared with the gasless laparoscopy group (P = 0.05) . CONCLUSION: A full laparotomy incision promotes greater tumour growth than does carbon dioxide pneumoperitoneum . Surgical manipulation stimulates local tumour spread more than the establishment of a carbon dioxide pneumoperitoneum.

Eur J Pharmacol, 1999 Aug 27, 379(2-3), 237 - 42
The effect of C-terminal truncation of the recombinant delta-opioid receptor on Ca2+i signaling; Harrison C et al.; We have previously shown a stimulatory coupling of the recombinant delta-opioid receptor to phospholipase C leading to production of inositol (1,4,5) triphosphate {Ins(1,4,5)P3} that is affected by truncation of the C-terminus of the receptor . Using a C-terminal mutant of the delta-opioid receptor lacking the final 37 amino acids (CHOdelta37), we examined its coupling to intracellular calcium ion concentration ({Ca2+}i) compared to the full length wild type receptor (CHOdeltaWT) in transfected Chinese hamster ovary (CHO) cells . D-{Pen2,5}enkephalin (DPDPE) mediated increases in {Ca2+}i were measured fluorimetrically in fura-2 loaded whole cell suspensions . DPDPE produced time- and concentration-dependent increases in {Ca2+}i in CHOdeltaWT and CHOdelta37 . In both cell types the DPDPE simulated increase in {Ca2+}i was naloxone reversible and pertussis toxin and thapsigargin sensitive . Removal of the C-terminus resulted in a rightward shift of the Ca2+ release concentration-response curve {pEC50 = 8.43 +/- 0.13 and 6.08 +/- 0.25 for CHOdeltaWT and CHOdelta37, respectively} . These data indicate that the C-terminus of the recombinant delta-opioid receptor is important in {Ca2+}i coupling and may be attributed to the effect of C-terminus truncation on phospholipase C coupling reported previously.

J Am Soc Nephrol, 1997 Apr, 8(4), 530 - 4
Inhibition of IMCD 11 beta-hydroxysteroid dehydrogenase type 2 by low pH and acute acid loading; Nolan PJ et al.; Mineralocorticoid receptors in the inner medullary collecting duct (IMCD) are protected from glucocorticoid binding by an enzyme, 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) . To study the role of 11 beta-HSD2 in acid-base homeostasis, 11 beta-HSD2 activity was measured in rat IMCD-enriched cell suspensions . Homogenates of cell suspensions were incubated in buffers ranging in pH from 6.00 to 8.15 in the presence of 1 microCi of 3H-corticosterone (CS) and 400 microM NAD+ . Enzyme activity was expressed as the amount of 3H-CS converted to 3H-11-dehydrocorticosterone (DHCS) . IMCD 11 beta-HSD2 activity at pH 6.5 was 49% of activity at pH 7.5; 22.5 versus 11.0 fmol/microgram of protein per h . Experiments also were performed on intact cell suspensions at pH 7.5 and 6.5 . There was a 42% inhibition in the IMCD cell suspension conversion rate of 3H-CS to 3H-11-DHCS at pH 6.5; 13.1 versus 7.6 fmol/microgram per h (P < 0.005) . In cell suspensions at pH 7.5, 1-day acid loading caused a 26% inhibition in conversion rate, 13.2 versus 9.9 fmol/microgram per h (P < 0.05), when compared with controls . These results suggest that during acute metabolic acidosis, IMCD 11 beta-HSD2 is inhibited and may allow access to the mineralocorticoid receptors by glucocorticoids.

Oral Microbiol Immunol, 1999 Jun, 14(3), 143 - 52
Cloning of Prevotella intermedia loci demonstrating multiple hemolytic domains; Beem JE et al.; A gene bank was created from Prevotella intermedia strain 27 chromosomal DNA, and a clone was isolated that conferred the expression of two separate modes of hemolytic activity in recombinant Escherichia coli . The original recombinant hemolytic strain (EB34) contained plasmid, pEB34, with a 5.6-kb insert from Sau 3 AI-digested P . intermedia strain 27 chromosomal DNA cloned into the Bam HI site of pUC18 . EB34 and deletion subclones were tested for expression of hemolytic activity in a standard tube assay, measuring lysis of erythrocytes spectrophotometrically as a function of hemoglobin release . Cell suspensions of EB34 demonstrated a dose-dependent hemolytic activity, inhibitable by proteases, and heat treatment but not dependent on calcium ions, and not inhibitable by osmoprotectants . Cell-free lysates also demonstrated a heat inhibitable, dose dependent hemolytic activity . Sub-cloning experiments localized the hemolytic region of the insert to a 3.9-kb fragment under direction of the lac promoter . Sequence analysis of the entire insert revealed the presence of multiple open reading frames (1 to 3) in this region which correlated to different forms of hemolytic expression, such that subclones containing all open reading frames 1 to 3 demonstrated strong hemolytic phenotype on blood plates and in the tube assay . Subclones containing only ORF1 demonstrated hemolysis on plates, but not in the tube assay . Subclones containing only open reading frames 2 and 3, but not ORF1 demonstrated hemolysis in the tube assay but not on plates . Homology searches of DNA and protein databases have not revealed significant homologies with reported hemolysins or proteins in any of the open reading frames.

Nat Toxins, 1999, 7(2), 71 - 9
Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin; Igarashi T et al.; Maitotoxin (MTX), a putative Ca(2+) channel activator produced by the dinoflagellate Gambierdiscus toxicus showed extremely potent hemolytic and ichthyotoxic activities . Hemolysis of 1% mouse blood cell suspension in saline occurred at 15 nM of MTX . The activity was enhanced six-fold in the presence of 10 microM of Ca(2+) and completely blocked by EDTA2Na, indicating its dependency on external Ca(2+) . The MTX-induced hemolysis was little affected by L-type Ca(2+) channel blockers (diltiazem, nifedipine, verapamil) but was strongly inhibited by calmodulin blockers (prenylamine and chlorpromazine) or a phospholipase A2 inhibitor (quinacrine) . MTX was mimicked by a calcium ionophore, calcimycin . Based on these results, a series of cellular events triggered by MTX were presumed to occur in the following sequence: increased Ca(2+) entry in cells, activation of calmodulin, promotion of phospholipase A2 activity, and finally destruction of cell membrane resulting from hydrolysis of membrane lipids . The sensitivity of blood cells to MTX varied significantly, dependent on the animal sources . Nucleated blood cells of carps and chickens were 100 times more resistant than those of mammals . LC(50) of MTX to freshwater fish Tanichthys albonubes in Ca(2+) free media (pH 8) was 5 nM but was markedly lowered to 3 pM by raising pH to 8 and increasing Ca(2+) concentration to 2 mM . In a marine environment MTX was 2000 times more toxic to fish than 42-di-hydrobrevetoxin-B (PbTx-3), one of the best known ichthyotoxins of red-tide origins .

Int J Cancer, 1999 Oct 29, 83(3), 393 - 400
Targeting HER-2/neu for active-specific immunotherapy in a mouse model of spontaneous breast cancer; Cefai D et al.; The identification of tumor-associated antigens has led to increased interest in vaccination strategies to treat and/or prevent cancer . This study examined the feasibility of active-specific immunotherapy against the breast-tumor antigen HER-2/neu using a HER-2/neu transgenic (rNeu-TG) mouse model . rNeu-TG mice develop spontaneous breast tumors after pregnancy, indicating that they fail to mount an effective immune response against rNeu . Allogeneic fibroblasts expressing HER-2/neu were used as a cell-based vaccine . Vaccination induced a rNeu-specific anti-tumor immune response that prevented tumor formation of transplanted breast-tumor cells, and also protected mice from spontaneous tumor formation . Both T-cell-mediated and humoral immune responses were detectable in vaccinated mice . Vaccination also protected tumor-bearing mice from a challenge with cell suspensions isolated from spontaneous tumors, indicating that rNeu-TG mice are not tolerant to rNeu, even after spontaneous tumor formation . However, established spontaneous tumors themselves were never affected . This observation correlated with T-cell infiltrations in the injected but not in the established spontaneous tumor . Thus, allogeneic fibroblasts are efficient vaccine vectors to prime a specific immune response against an over-expressed tumor antigen . Moreover, our results suggest striking differences in the immunological requirements for the rejection of an established vs . a transplanted tumor .

Haemostasis, 1999 Sep, 29(1), 41 - 9
Recent advances in platelet-polymorphonuclear leukocyte interaction; de Gaetano G et al.; Epidemiological evidence suggests a positive correlation between the number of PMN and the risk of ischemic vascular disease . The observation that activated PMN induce platelet activation my provide some biological plausibility to the role of PMN in thrombogenesis . Between other PMN products, cathepsin G, a protease released during PMN activation, is a potent platelet agonist . However, the antiproteinases present in plasma could virtually abolish its activity . Indeed it was shown that, when PMN were stimulated after interaction with platelets in mixed cell population, P-selectin-mediated platelet-PMN adhesion may result in the formation of a sequestered microenvironment in which cathepsin G activity is protected by antiproteases . P-selectin-mediated adhesion was also shown to facilitate the transcellular metabolism of arachidonic acid, resulting in increased production of both thromboxane B2 and leukotriene C4 . PMN adhesion to activated platelets in mixed cell suspensions subjected to high shear rate can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step followed by an adhesion-strengthening interaction mediated by the beta(2)-integrin Mac-1 . Moreover, an intermediate tyrosine-kinase-dependent signal regulating beta(2)-integrin adhesiveness is required . Indeeed activated platelets express not only P-selectin but also different beta(2)-integrin ligands including fibrinogen and ICAM-2 . Some of the functional responses elicited by P-selectin on PMN could be prevented by specific antibody to the P-selectin glycoprotein ligand-1, indicating that this adhesive receptor is able to transduce an 'outside-in' signal when engaged by the ligand . By using activated platelets, P-selectin-expressing CHO cells and soluble recombinant P-selectin, P-selectin was shown to trigger protein tyrosine phosphorylation in PMN and the tyrosine kinase-dependent function of Mac-1 . In conclusion, adherence of activated platelets to PMN may be a key event in the sequence of thrombus formation . The recognition of the essential contribution of PMN beta(2)-integrins in addition to P-selectin in platelet-PMN adhesion provides an additional evidence to the broad range of function and mechanisms in which PMN integrins are involved and may be potential targets for pharmacological intervention.

J Rheumatol, 1999 Sep, 26(9), 1869 - 76
Increased HLA-DR and CD44 antigen expression in the gut: evidence of extraarticular immunological activity in rheumatoid arthritis; Abuzakouk M et al.; OBJECTIVE: To examine the gastrointestinal (GI) immune system in rheumatoid arthritis (RA) for evidence of activation . METHODS: Duodenal biopsies from 25 patients with RA were obtained by endoscopy . Single cell suspensions from the epithelial layer and lamina propria were prepared . Flow cytometry was used to examine the expression of CD4, CD8, T cell receptor-gammadelta (TCR-gammadelta), TCR-alphabeta, HLA-DR, CD44, and interleukin 2 receptor on gut T lymphocytes . Fifteen disease control (DC) individuals and 6 patients with osteoarthritis (OA) taking longterm nonsteroidal antiinflammatory drug (NSAID) therapy were also investigated . Peripheral blood T lymphocytes from all individuals were examined for the expression of these surface molecules . RESULTS: HLA-DR expression was significantly increased on intraepithelial lymphocytes (IEL) and enterocytes from patients with RA (n = 13) compared with the 2 control groups (p<0.01) . Immunohistochemistry also revealed increased expression of HLA-DR on enterocytes from patients with RA . RA IEL (n = 6) expressed significantly higher levels of CD44 (p<0.02) . In the lamina propria, a small but significant gammadelta T lymphocyte population (mean 5.5%, range 2-12%) was detected in rheumatoid factor positive RA patients (n = 8) compared with RF negative RA patients (n = 8, mean 2%, range 0.4-6%; p<0.01) and the disease control group (n = 15, mean 2%, range 0.5-5%; p<0.01) . None of these changes were detectable in peripheral blood lymphocytes from patients with RA . CONCLUSION: This study demonstrates evidence of activation of specific components of the GI immune system in RA . Peripheral blood T lymphocytes from patients with RA did not show increased expression of activation markers, suggesting that changes in the RA GI tract are not systemic but localized . Moreover, these changes appear to be independent of NSAID therapy.

Vision Res, 1999 Aug, 39(17), 2817 - 32
Enhanced retinal longwave sensitivity using a chlorophyll-derived photosensitiser in Malacosteus niger, a deep-sea dragon fish with far red bioluminescence; Douglas RH et al.; Through partial bleaching of both visual pigment extracts and cell suspensions we show that the deep-sea stomiid Malacosteus niger, which produces far red bioluminescence, has two visual pigments within its retina which form a rhodopsin/porphyropsin pigment pair with lambda max values around 520 and 540 nm, but lacks the very longwave sensitive visual pigments (lambda max > 550 nm) observed in two other red light producing stomiids . The presence of only a single opsin gene in the M . niger genome was confirmed by molecular and cladistic analysis . To compensate for its apparently reduced longwave sensitivity compared to related species, the outer segments of M . niger contain additional pigments, which we identify as a mixture of defarnesylated and demetallated derivatives of bacteriochlorophylls c and d, that are used as a photosensitiser to enhance its sensitivity to longwave radiation.

Biol Reprod, 1999 Oct, 61(4), 927 - 34
A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules; Johnson L et al.; For many infertile couples, intracytoplasmic germ cell/spermatozoon injection into unfertilized eggs may be their only hope for producing their own biological children . Thus far, success with injection of pre-spermatozoan germ cells such as round spermatids has not been as great as that of spermatozoon injection . This could be due in part to the difficulty of identifying younger (less mature) male germ cells in testicular biopsy dispersions . To improve the identification of various types of live, dispersed, human testicular cells in vitro, a comparative study of the morphological characteristics of human spermatogenic germ cells in vitro or in situ within seminiferous tubules was conducted . Live human testicular tissue was obtained from an organ-donating, brain-dead person with a high density of various germ cells . A cell suspension was obtained by enzymatic digestion, and cells were cultured for 3 days in an excessive volume (100-fold medium:cells; v:v) of HEPES-TC 199 medium at 5 degrees C and observed live with Nomarski optics (interference-contrast microscopy) . For comparative purposes, testes from ten men obtained at autopsy were fixed, embedded in epoxy resin, sectioned at 20 microm, and observed unstained by Nomarski optics . This approach allowed comparison of morphological characteristics of individual germ cells seen in vitro or in situ in the human testis . In both live and fixed preparations from control men with varied daily sperm production rates, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells . The size, shape, and chromatin pattern of nuclei, and the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece of germ cells are characteristically seen in live cells in vitro and in those cells observed in the fixed seminiferous tubules . Hence, this comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells, to be used by scientists and technical staff in infertility clinics when selecting specific germ cells from a testicular aspirate or enzymatically digested biopsy.

Br J Cancer, 1999 Sep, 81(1), 114 - 21
A quantitative polymerase chain reaction-enzyme immunoassay for accurate measurements of human papillomavirus type 16 DNA levels in cervical scrapings; Jacobs MV et al.; A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings . The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and beta-globin DNA . The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region . The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines . Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples . We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings . Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach.

J Chromatogr A, 1999 Aug 20, 853(1-2), 381 - 9
Direct measurement of ascorbic acid biosynthesis in Arabidopsis cell suspension culture using capillary electrophoresis; Davey MW et al.; We describe procedures to directly measure the biosynthesis of vitamin C (L-ascorbic acid, L-AA) in crude extracts of an Arabidopsis thaliana cell suspension culture by capillary electrophoresis . Optimal conditions have been established for the quantitation of L-AA formed by the oxidation of three different substrates: L-galactose, L-galactono-1,4-lactone, and L-gulono-1,4-lactone . We also demonstrate that L-galactono-1,4-lactone dehydrogenase activity does not require exogenous cofactor . The minimal sample handling requirements, the high selectivity, and short analysis times represent significant advantages over existing protocols.

Cytometry, 1999 Oct 1, 37(2), 147 - 55
Phenotyping of epidermal dendritic cells: clinical applications of a flow cytometric micromethod; Wollenberg A et al.; BACKGROUND: The differential diagnosis of inflammatory skin diseases is largely based on the patient's history and the morphological analysis of the skin lesion . Laboratory data, such as serum IgE-level and prick and patch tests, may be helpful but do not assess individual lesions . The assumption of our approach is that each individual lesion is associated with a specific microenvironment and that the immunophenotype of the two epidermal dendritic cell populations, Langerhans cells (LC) and inflammatory dendritic epidermal cells (IDEC), reflects this environment in a disease-specific manner . METHODS: A flow cytometric micromethod was developed to directly analyze individual inflammatory human skin lesions . Crude epidermal single cell suspensions were prepared by trypsinization, stained for three-color analysis with different monoclonal antibodies and the vital stain 7-amino-actinomycin-D, and finally analyzed on a single laser equipped FACScan flow cytometer . RESULTS: With a limited set of cell surface markers, such as FcepsilonRI, FcgammaRII/CD32, CD1b and CD36, highly specific diagnostic criteria for atopic dermatitis and inflamed human skin could be established . CONCLUSIONS: Phenotyping of epidermal dendritic cells is a useful procedure helpful in differential diagnosis of inflammatory skin diseases .

Biotechnol Bioeng, 1999 Nov 5, 65(3), 247 - 57
A new coiled hollow-fiber module design for enhanced microfiltration performance in biotechnology; Luque S et al.; The microfiltration performance of a novel membrane module design with helically wound hollow fibers is compared with that obtained with a standard commercial-type crossflow module containing linear hollow fibers . Cell suspensions (yeast, E . coli, and mammalian cell cultures) commonly clarified in the biotechnology industry are used for this comparison . The effect of variables such as transmembrane pressure, particle suspension concentration, and feed flow rate on membrane performance is evaluated . Normalized permeation fluxes versus flow rate or Dean number behave according to a heat transfer correlation obtained with centrifugal instabilities of the Taylor type . The microfiltration performance of this new module design, which uses secondary flows in helical tubes, is significantly better than an equivalent current commercial crossflow module when filtering suspensions relevant to the biotechnology industry . Flux and capacity improvements of up to 3.2-fold (constant transmembrane pressure operation) and 3.9-fold (constant flux operation), respectively, were obtained with the helical module over those for the linear module .

Am J Physiol, 1999 Sep, 277(3 Pt 1), C572 - 9
Regulation of intestinal tyrosine phosphorylation and programmed cell death by peroxovanadate; Scheving LA et al.; Cell suspensions of ileal mucosa undergo a rapid and synchronized form of programmed cell death when cultured in a simple medium at 37 degrees C . Because tyrosine phosphorylation of proteins plays a crucial role in the signal transduction of many cellular processes, we examined its role in intestinal programmed cell death by use of immunoblot and immunohistochemical methods . We observed a 50-70% reduction in tyrosine phosphorylation during the initial 10 min of intestinal epithelial cell culture . We hypothesized that the inhibition of protein tyrosine phosphatases would increase protein tyrosine phosphorylation in these suspensions and decrease programmed cell death . A strong inhibitor of these phosphatases (peroxovanadate) but not a weaker one (sodium orthovanadate) abolished the DNA fragmentation/laddering normally seen in dying enterocytes . Peroxovanadate enhanced protein tyrosine phosphorylation of many intestinal proteins, dramatically increasing the dually phosphorylated and active form of mitogen-activated protein kinase . Immunohistochemistry revealed a particularly high level of increased tyrosine phosphorylation in the intestinal crypts in peroxovanadate-treated mucosa . Kinetic studies indicated that the pivotal time for protein tyrosine phosphatase inhibition occurred within 5 min of ex vivo culture, precisely when protein tyrosine phosphorylation declined . Our data suggest that tyrosine kinase inactivation or tyrosine phosphatase activation may initiate intestinal epithelial cell death.

J Virol, 1999 Oct, 73(10), 8571 - 7
Prevalence of varicella-zoster virus DNA in dissociated human trigeminal ganglion neurons and nonneuronal cells; LaGuardia JJ et al.; Previous analyses using in situ hybridization alone or together with PCR have yielded conflicting results regarding the cell type in which latent varicella-zoster virus (VZV) resides . We separated human trigeminal ganglia (TG) into neuronal and nonneuronal fractions, followed by primary and nested PCR to quantitate VZV DNA at the single cell level . Both TG from each of eight cadavers were dissociated and separated into neuronal and nonneuronal cell suspensions by differential filtration . Analysis of the neuron fraction (5,000 neurons per sample) revealed VZV DNA in 9 of 16 samples, with copy numbers ranging from 1 to 12, whereas only 2 of 16 nonneuronal cell samples were positive for VZV DNA, with 1 copy each . Further analysis of 10 samples of 100 neurons and the corresponding nonneuronal cell fractions from each TG of a single subject revealed VZV DNA in 3 of 10 samples of the left TG (range, 2 to 5 copies) and in 1 of 10 samples of the right TG (2 copies) but in none of the 20 nonneuronal cell fractions . These data indicate that latent VZV DNA is present primarily, if not exclusively, in neurons, at a frequency of two to five copies per latently infected neuron.

FEBS Lett, 1999 Sep 17, 458(2), 204 - 8
An early salicylic acid-, pathogen- and elicitor-inducible tobacco glucosyltransferase: role in compartmentalization of phenolics and H2O2 metabolism; Chong J et al.; Treatment of tobacco cell suspension cultures with a fungal elicitor of defense responses resulted in an early accumulation of the phenylpropanoid glucosyltransferase TOGT, along with the rapid synthesis and secretion of scopolin, the glucoside of scopoletin . Elicitor-triggered extracellular accumulation of the aglycone scopoletin and of free caffeic and ferulic acids could only be revealed in the presence of diphenylene iodonium, an inhibitor of extracellular H2O2 production . Our results strongly support a role for TOGT in the elicitor-stimulated production of transportable phenylpropanoid glucosides, followed by the release of free antioxidant phenolics into the extracellular medium and subsequent H2O2 scavenging.

J Cancer Res Clin Oncol, 1999 Aug-Sep, 125(8-9), 461 - 74
Galectins-1 and -3 and their ligands in tumor biology . Non-uniform properties in cell-surface presentation and modulation of adhesion to matrix glycoproteins for various tumor cell lines, in biodistribution of free and liposome-bound galectins and in their expression by breast and colorectal carcinomas with/without metastatic propensity; Andre S et al.; Protein (lectin)-carbohydrate (cellular glycoconjugate) recognition is operative in biochemical information transfer . Galectins constitute a family of endogenous galactoside-binding lectins with conserved features in the binding site . The members of this lectin category are assumed to be involved in cell adhesion and growth regulation . To assess to what extent the different modes of binding-site presentation and/or carbohydrate fine-specificities will affect aspects of galectin behavior, homodimeric cross-linking galectin-1 and monomeric chimeric galectin-3, with its collagenase-sensitive stalk linked to the carbohydrate-recognition domain, were investigated . Cell-surface expression of the two galectins and accessible galectin-binding sites on various tumor cell lines was ascertained by FACScan analysis . In particular, ligand accessibility for the two galectins differed for the tested cell line types . Binding of tumor cells to laminin and plasma or placental fibronectin was generally reduced by treatment of cells or matrix with galectins . Galectin-3 was more efficient than galectin 1 at impairing laminin's potency as matrix . Cell binding of galectin-1, on the other hand, proved on average more effective for blocking cell association to fibronectins after its preincubation with cell suspensions . Differences were also apparent in the biodistribution of the galectins, where an avian homolog of galectin- served as the control to distinguish effects of spatial and sugar-binding features . Histopathological analysis of lymph-node-negative and -positive breast and colorectal carcinomas (n = 180 including 60 metastatic lesions) indicated a correlation of either increased galectin-1 binding and reduced galectin-3 expression or reduced binding of both galectins with the occurrence of lymph node lesions . Together with data on the heparin-binding lectin, revealing reduced expression to be associated with a positive lymph-node status in the breast cancer group, these results can be interpreted to reflect cell-type-dependent requirements of galectin ligand presentation during the metastatic cascade . By introducing mammalian lectins to lectin-histochemical studies, the detection of quantitative differences in glycosylation brings an understanding of its cell biological significance one step closer.

Bull Cancer, 1999 Jul-Aug, 86(7-8), 685 - 91
{Standardization and quality control in the evaluation of proliferation parameters in T1T2, N0N1, M0 breast cancer: multicentric retrospective study II . DNA-ploidy and S-phase fraction}; Chassevent A et al.; As part of a clinical research project, proliferative parameters were studied in primary breast cancer: standardization and technical validation of thymidine kinase (TK), thymidylate synthase (TS) and protein tyrosine kinase (PTK) are described . A total of 633 frozen tumor specimens, available in four institutions, was analyzed in three flow cytometry laboratories for DNA content and percentage of S-phase cells (%S) measurement . 1) The standardization step consisted in developing a common protocol for sample preparation; then, common cell suspensions were analyzed in order to perform an inter-laboratory control . Objective guidelines were elaborated to interpret DNA histograms in breast carcinoma . 2) DNA-aneuploidy was observed in 61% of cases of the retrospective series . Compared with DNA-aneuploid tumors, mean %S was significantly lower in case of DNA-diploidy (respectively: 6.4% and 2.2%, p < 0.001) . When compared between the four institutions, %S distributions did not differ significantly . 3) %S is strongly correlated with TK, TS and PTK and high percentages were also observed in high grade tumors or tumor without hormone receptors . These results show that a standardization in using flow cytometers and DNA software allows multicenter studies.

J Neurosci Methods, 1999 Jul 1, 89(1), 17 - 24
In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits; Keilhoff G et al.; In vivo predegeneration of peripheral nerves is presented as a convenient and effective method to obtain activated Schwann cells and an enhanced cell yield following in vitro cultivation . The experiments conducted in rats were aimed at clinical use in gaining Schwann cell suspensions for filling artificial conduits in order to bridge peripheral nerve gaps . The rat sciatic nerve used as a model was transected distally to the spinal ganglia . Predegeneration in vivo was allowed to take place for 1, 2, 3 and 4 days and up to 1, 2 and 3 weeks . The nerve was then resected and prepared for cell cultivation . Schwann cells cultivated from the contralateral untreated nerve served as control . Immunostaining for S100, nerve growth factor receptor and the adhesion molecules N-cadherin and L1 was used to characterize the general state of the cultures . Viability was assessed by fluorescein fluorescence staining, and the proliferation index was determined by bromodeoxyuridine-DNA incorporation . The Schwann cells from predegenerated nerves revealed an increased proliferation rate compared to the control, whereas fibroblast contamination was decreased . Best results were obtained 1 week after predegeneration.

J Neurosci Methods, 1999 Jul 1, 89(1), 1 - 8
Separation of dorsal and ventral dopaminergic neurons from embryonic rat mesencephalon by buoyant density fractionation: disassembling pattern in the ventral midbrain; Silverman WF et al.; The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine neurons, are organized into dorsal and ventral 'tiers' with regard to their ontogeny, efferent projections and their relative position in the various mesencephalic sub-nuclei . We have employed buoyant density fractionation to separate the dopaminergic neurons of the two compartments and compare their subsequent phenotype development with respect to their expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway . Using immunocytochemistry, separately and combined with in situ hybridization, we demonstrate here that sedimentation of cell suspensions from E19 rat ventral mesencephalon on 5-step Percoll gradients produces cell fractions enriched in ventral and dorsal tier DA neurons, respectively.

Plant J, 1999 Aug, 19(3), 321 - 31
Yariv reagent treatment induces programmed cell death in Arabidopsis cell cultures and implicates arabinogalactan protein involvement; Gao M et al.; Arabinogalactan proteins (AGPs) are a family of highly glycosylated, hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development . (beta-D-glucosyl)3 and (beta-D-galactosyl)3 Yariv phenylglycosides, commonly known as Yariv reagents, specifically bind AGPs in a non-covalent manner . Here (beta-D-galactosyl)3 Yariv reagent was added to Arabidopsis thaliana cell suspension cultures and determined to induce programmed cell death (PCD) by three criteria: (i) DNA fragmentation as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) of DNA 3'-OH groups; (ii) inter- nucleosomal DNA fragmentation as visualized by genomic Southern blotting; and (iii) structural changes characteristic of PCD including cytoplasmic shrinkage and condensation, chromatin condensation and nuclear membrane blebbing . These findings implicate AGP involvement in PCD in plants, presumably by perturbation of AGPs located at the plasma membrane-cell wall interface.

Plant J, 1999 Aug, 19(3), 297 - 307
MAP kinase activation by hypoosmotic stress of tobacco cell suspensions: towards the oxidative burst response?
Cazale AC, Droillard MJ, Wilson C, Heberle-Bors E, Barbier-Brygoo H, Lauriere C.
Hypoosmotic stress activates a phosphorylation-dependent oxidative burst . In-gel kinase assays were performed to characterize the protein kinases that could be implicated in osmoregulation and in the activation of the oxidative burst . Hypoosmotic stress activated several kinases among which 50 and 46 kDa proteins displayed mitogen-activated protein kinase (MAP kinase) properties . They phosphorylated myelin basic protein in the absence of calcium, were recognized by antibodies directed against human MAP kinases, and were phosphorylated on tyrosine . Immunoprecipitation with an antibody directed against the tobacco MAP kinase Ntf4 showed that at least one of the activated kinases would be Ntf4-like . Apigenin, a MAP kinase and cyclin-dependent kinase inhibitor which prevents the hypoosmotically induced oxidative burst (Cazale et al . 1998; Plant Physiol . 116, 659-669), inhibited these kinases in vitro suggesting that they may play a role in the activation of the oxidative burst . Like the oxidative response, activation of the kinases depended on extracellular calcium influx and protein kinases sensitive to staurosporine and 6-DMAP . However, kinase activation did not depend on effluxes through anion channels or on the oxidative burst . Two-dimensional in-gel kinase assays revealed the presence of three protein kinases with an apparent molecular mass of 50 kDa and one of 46 kDa, all four being activated by hypoosmotic stress . The same kinases were also activated by oligogalacturonides and salicylic acid, underlying the importance of these MAP kinases as common components of different signaling pathways triggered by different extracellular stimuli.

Biorheology, 1998 Jul-Oct, 35(4-5), 263 - 79
Wall shear stress in backward-facing step flow of a red blood cell suspension; Gijsen FJ et al.; An experimental investigation of the wall shear stress distribution downstream of a backward-facing step is carried out . The wall shear stress distribution was determined by measuring the deformation of a gel layer, attached to the wall downstream of the step . Speckle pattern interferometry was applied to measure the deformation of the gel layer . The measured deformation, combined with the properties of the gel layer, served as an input for a finite element solid mechanics computation to determine the stress distribution in the gel layer . The wall shear stress, required to generate the measured deformation of the gel layer, was determined from these computations . A Newtonian buffer solution and a non-Newtonian red blood cell suspension were used as measuring fluids . The deformation of the gel layer was determined for a Newtonian buffer solution to evaluate the method and to obtain the properties of the gel layer . Subsequently, the wall shear stress distribution for the non-Newtonian red blood cell suspension was determined for three different flow rates . The inelastic non-Newtonian Carreau-Yasuda model served as constitutive model for the red blood cell suspension . Using this model, the velocity and wall shear stress distribution were computed by means of a finite element fluid mechanics computation . From the comparison between the numerical and the experimental results, it can be concluded that wall shear stresses, induced by the red blood cell suspension, can be modeled accurately by employing a Carreau-Yasuda model.

Cytobios, 1999, 98(388), 77 - 94
Influence of noradrenaline on the respiratory status of Rana balcanica red cell suspension under normoxia, hypoxia and hypercapnia: alpha 1-receptor involvement; Kaloyianni M et al.; The effect of normoxia, hypoxia and hypercapnia on the extracellular pH, partial pressure carbon dioxide (pCO2), partial pressure oxygen (pO2) and HCO3- levels after noradrenaline treatment of Rana balcanica erythrocytes, was investigated . Noradrenaline caused a significant reduction of the extracellular pH which may have been due to the activation of red blood cell Na+/H+ exchange . Significant falls in the partial extracellular pressure of CO2 and O2 were evident . The initial reduction in extracellular pCO2 and pO2 was followed by a rise reflecting the desensitization of the Na+/H+ exchange after 15 min of hormone stimulation . Both hypercapnia and hypoxia increased the magnitude of these changes in relation to normoxia, although the greatest changes were observed under hypercapnic conditions . The involvement of alpha 1 receptors in regulating the concentration of respiratory gases after catecholamine stimulation was demonstrated . It is suggested that these responses increased the effectiveness of gas transfer over the respiratory surfaces.

Arch Immunol Ther Exp (Warsz), 1999, 47(3), 161 - 8
Tumor infiltrating lymphocytes in HLA+ and HLA- laryngeal cancer--quantitative approach; Dworacki G et al.; In search of factors governing the accumulation of tumor infiltrating lymphocytes (TIL), frozen sections from fresh surgical specimens of laryngeal carcinoma (n = 36) were tested by alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for monomorphic determinants of HLA class I and class II expression on tumor cells and for the distribution of lymphoid cells bearing CD differentiation antigens . Cell subsets were quantitated in two tumor compartments, tumor mass and tumor stroma, by computer-assisted image analysis . In a portion of examined samples lymphoid cell suspension was isolated from cancerous tissues and assessed by flow cytometry . It has been found that T cells, localized mostly in tumor stroma, were predominant cell population in the tumor microenvironment . Their ability to penetrate tumor mass but not tumor stroma, by CD8+ T cells in particular, but also by natural killer (NK) cells, was associated with HLA class I antigen expression on tumor cells . In flow cytometric analysis activated T lymphocytes (CD3+DR+) were abundant in HLA+ tumors as compared to HLA- ones . In 4 year follow up of 20 patients the mortality was higher in HLA- group but the data were not statistically significant . These results show that HLA class I expression on tumor cells favor penetration of cytotoxic lymphoid cells into tumor mass, at least in the laryngeal cancer.

J Bone Miner Res, 1999 Sep, 14(9), 1562 - 9
Development and characterization of a human in vitro resorption assay: demonstration of utility using novel antiresorptive agents; James IE et al.; A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the alphavbeta3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase . The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human alphavbeta3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80) . Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue . These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype . The cryopreserved cells can be cultured on bovine cortical bone for 24-48 h and resorption can be measured by either confocal microscopy or biochemical assays . The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds . In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay . Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles . This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.

Eur J Biochem, 1999 Aug, 263(3), 686 - 94
Identification and characterization of cDNA clones encoding hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase from tobacco; Farmer MJ et al.; The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported . The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv . Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum . The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences . cDNA clones tht4 and tht11 differ only by their 5' leader and 3' UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively . The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa . The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants . In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases . The THT clones hybridized to a 0.95-kb mRNA from elicited tobacco cell-suspension cultures and also to a mRNA of similar size from wound-healing potato tubers . The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues . Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome . Inhibition experiments using amino-acid-specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity . In the course of these experiments THT was also found to be inhibited by (2-hydroxyphenyl) amino sulfinyl acetic acid 1,1-dimethylethyl ester, an irreversible inhibitor of cinnamyl alcohol dehydrogenase.

Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10512 - 7
Sugars modulate an unusual mode of control of the cell-wall invertase gene (Incw1) through its 3' untranslated region in a cell suspension culture of maize; Cheng WH et al.; We show here that a cell-wall invertase encoded by the Incw1 gene is regulated at both the transcriptional and posttranscriptional levels by sugars in a heterotrophic cell suspension culture of maize . The Incw1 gene encoded two transcripts: Incw1-S (small) and Incw1-L (large); the size variation was attributable to different lengths in the 3' untranslated region . Both metabolizable and nonmetabolizable sugars induced Incw1-L RNA apparently by default . However, only the metabolizable sugars, sucrose and D-glucose, were associated with the increased steady-state abundance of Incw1-S RNA, the concomitant increased levels of INCW1 protein and enzyme activity, and the downstream metabolic repression of the sucrose synthase gene, Sh1 . Conversely, nonmetabolizable sugars, including the two glucose analogs 3-O-methylglucose and 2-deoxyglucose, induced greater steady-state levels of the Incw1-L RNA, but this increase did not lead to either an increase in the levels of the INCW1 protein/enzyme activity or the repression of the Sh1 gene . We conclude that sugar sensing and the induction of the Incw1 gene is independent of the hexokinase pathway . More importantly, our results also suggest that the 3' untranslated region of the Incw1 gene acts as a regulatory sensor of carbon starvation and may constitute a link between sink metabolism and cellular translation in plants.

J Dermatol Sci, 1999 Sep, 21(1), 23 - 6
High frequency of DNA aneuploidy detected by DNA flow cytometry in Bowen's disease; Kawara S et al.; To detect DNA aneuploidy in Bowen's disease, we investigated DNA flow cytometric analysis . Single cell suspensions were prepared from 18 fresh samples histopathologically diagnosed as solitary Bowen's disease and analyzed by DNA flow cytometry . In 16 (89%) of 18 lesions, DNA aneuploidy was demonstrated with a single aneuploid peak . DNA indices ranged from 1.29 to 1.74 . The incidence of DNA aneuploidy in Bowen's disease is higher than those of cutaneous squamous cell carcinoma, which was 25-80% in the previous reports . Therefore, in Bowen's disease . DNA aneuploidy may not imply a good marker for characteristics of non-melanoma skin cancer . A single aneuploid peak commonly observed in Bowen's disease suggests that this disease consists of the monoclonal proliferation of keratinocytes containing abnormal DNA content.

Pharm Res, 1999 Aug, 16(8), 1266 - 72
Transcellular and lipophilic complex-enhanced intestinal absorption of human growth hormone; Wu SJ et al.; PURPOSE: To evaluate the transcellular mechanism of novel enhancers absorption enhancement of human growth hormone (hGH), by examining the involvement of a P-glycoprotein-like efflux system, changes in membrane fluidity, and membrane damage . METHODS: Caco-2 cell monolayers were grown on Snapwell filter supports and placed in a side-by-side diffusion apparatus . Transport in both the apical to basolateral (AP to BL) and basolateral to apical (BL to AP) direction was measured at different temperatures and in the presence of potential inhibitors . Fluorescence anisotropy measurement was used to measure membrane fluidity . The fluorescence anisotropy of DPH- and TMA-DPH-labeled cell suspensions was measured at room temperature . LDH (a measure of cytosolic lactate dehydrogenase) leakage assay was used to evaluate cytotoxicity . RESULTS: The bi-directional transepithelial fluxes of hGH in the presence of these novel enhancers across Caco-2 cells showed marked asymmetry . Average permeability coefficient values obtained in the apical to basolateral (AP to BL) direction were lower than those of the reverse (BL to AP) direction . On the other hand, the fluxes for hGH alone were symmetric . When P-gp-like efflux inhibitors were included in the transport medium, the permeability coefficient value of BL to AP direction was significantly decreased while the transport was increased in the reverse direction in the presence of novel enhancers . In addition, lowering the temperature to 25 degrees C completely eliminated the asymmetry of hGH transport in the presence of novel enhancers . It was also shown by fluorescence anisotropy that these novel enhancers alone only slightly increased membrane fluidity . On the other hand, upon addition of hGH to the novel enhancers, the cell membrane showed a dramatic change as compared to treatment with novel enhancers alone . The results from the LDH assay showed that the novel enhancers and/or hGH did not cause cell damage, at least up to 1 hour, and the damage seen at the 2 hour point is also much lower than other known enhancers . CONCLUSIONS: This study shows that human growth hormone alone cannot be transported across Caco-2 cells, except in small quantities, by passive diffusion, but in the presence of novel enhancers, human growth hormone permeation is substantial . In addition, the asymmetry of transport of the complexed hGH appears to be due to a P-gp-like efflux system . Assuming that the present substrate specificity of the P-gp-like efflux system shows the same preference for hydrophobic molecules as p-gp, the present work also indirectly shows that human growth hormone has become more lipophilic in the presence of these novel enhancers . Furthermore, membrane fluidity data also supports the premise that these novel enhancers interact and stabilize hGH, to make them more hydrophobic and easier to be transported through cell membranes.

Planta, 1999 Jul, 209(1), 33 - 44
Purification, characterization, and immunolocalization of hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase from opium poppy; Yu M et al.; A development-specific and elicitor-inducible acyltransferase {hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110)] that catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to hydroxyphenethylamines was purified 988-fold to apparent homogeneity from opium poppy (Papaver somniferum L.) cell-suspension cultures . The purification procedure, which resulted in a 6.8% yield, involved hydrophobic interaction and anion-exchange chromatography, followed by affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent . Purified THT had an isoelectric point of 5.2, a native molecular mass of approximately 50 kDa, and consisted of two apparently identical 25-kDa subunits as determined by two-dimensional polyacrylamide gel electrophoresis . The purified enzyme was able to synthesize a variety of amides due to a relatively low specificity for cinnamoyl-CoA derivatives and hydroxyphenethylamines . The best substrates were feruloyl-CoA (VK(m)(-1)13.4 mkat g(-1) M(-1)) and tyramine (VK(m)(-1)6.57 mkat g(-1) M(-1)) . The THT activity increased during development of opium poppy seedlings, occurred at high levels in roots and stems of mature plants, and was induced in cell-suspension cultures after treatment with a pathogen-derived elicitor . Immunoblot analysis using THT mouse polyclonal antibodies did not always show a correlation between THT polypeptide and enzyme activity levels . For example, despite low THT activity in leaves, an abundant 25-kDa immunoreactive polypeptide was detected . Immunohistochemical localization showed that THT polypeptides occur in cortical and xylem parenchyma, immature xylem vessel elements, root periderm, anthers, ovules, and the inner layer of the seed coat, but are most abundant in phloem sieve-tube members in roots, stems, leaves, and anther filaments.

AIDS, 1999 Aug 20, 13(12), 1503 - 9
Sampling lymphoid tissue cells by ultrasound-guided fine needle aspiration of lymph nodes in HIV-infected patients . Swiss HIV Cohort Study; Bart PA et al.; OBJECTIVE: To establish the feasibility of using ultrasound-guided lymph node needle aspiration as a means to obtain lymphoid tissue cells for the determination of a series of immunologic and virologic measures in HIV-infected patients . DESIGN: First, a comparison of the characteristics of cell populations obtained by simultaneous needle aspiration and standard excisional biopsy in six patients . Second, use of lymph node needle aspiration to assess longitudinally T-cell subset changes in patients initiating highly effective antiretroviral treatment . METHODS: T-cell subsets (CD4 and CD8) and percentage Ki67+ cycling T cells were measured in lymph node cell populations harvested by ultrasound-guided aspiration or standard biopsy by flow cytometry . Cellular RNA content was assessed by a modification of the Roche Amplicor HIV-1 Monitor test . RESULTS: CD4 and CD8 T-cell percentage and HIV RNA cell content of lymph node cell suspensions obtained from the simultaneous performance of ultrasound-guided needle aspiration and excisional biopsy in the same patients were correlated (n = 6) . Among the 87 aspiration sessions reported here, mononuclear cell suspensions were obtained in 100% of the sessions, in numbers ranging between 4x10(4) to 6.7x10(6) cells (median: 7x10(5)) . This limited number of cells did not allow to perform all type of analyses in all patients . By prioritizing the cells for the determination of T-cell subsets and proliferation rate, this approach was instrumental for demonstrating the normalization of the T-cell subset ratio and the kinetic of normalization of proliferating rates of CD4 and CD8 T cells, as well as the decrease in HIV-1 viral load in the lymph node following HAART initiation . CONCLUSION: Ultrasound-guided aspiration appears to be a non-invasive and ad libitum, safe and repeatable procedure for the longitudinal monitoring of changes in lymph nodes.

Cell Prolif, 1994 Jun, 27(6), 321 - 32
Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques; Dorr W et al.; A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ({3H}dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue . Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phosphatase anti-alkaline-phosphatase technique, the peroxidase-anti-peroxidase method, and an indirect method with a polyclonal peroxidase-conjugated secondary antibody yielded results similar to standard autoradiography . Preparation of single cell suspensions for flow cytometry was not successful . A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a G0/G1 peak nor a G2 + M peak was observed in DNA histograms . A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring . Both S-phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations . However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry . The FCM S-phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S-phase cells . In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.

Lab Invest, 1999 Aug, 79(8), 993 - 1005
Suppression of telomerase activity as an indicator of drug-induced cytotoxicity against cancer cells: in vitro studies with fresh human tumor samples; Faraoni I et al.; Telomerase is a ribonucleoprotein complex with reverse-transcriptase activity responsible for telomere reconstitution . High telomerase activity was found in cancer cells, but not in differentiated homologous nonmalignant tissues . We demonstrated previously that the disappearance of telomerase activity is a reliable marker of tumor cell killing in human cancer cell lines . We have investigated the possibility of evaluating chemosensitivity of neoplastic cells of different origin {ovary, lung, breast, gastrointestinal, skin (melanoma)} obtained from cancer patients, by measuring residual telomerase activity after drug treatment in vitro . Using the classical telomeric repeat amplification protocol ("TRAP") assay based on polymerase chain reaction, we examined telomerase activity of untreated or drug-treated tumor cell suspensions, derived from the processing of surgical specimens . Feasibility and reproducibility of the assay were evaluated according to various parameters, including drug concentration, time of in vitro culture, and type of tumor . The results indicated that the assay is highly sensitive and reproducible, and can be performed using surgical specimens in a reasonable percentage of cases, ranging from 40% (breast cancer) to 100% (ovarian cancer) . Moreover, the assay provides comparable results using a wide range of tumor cells, and the presence of normal cells does not interfere with the results . Prolonged tumor cell culture is not required because the assay can be completed within 24 to 72 hours after sample collection . In conclusion, the present investigation provides the technical bases for future studies to evaluate whether this assay would be able to predict patient's response to antitumor agents.

J Nutr, 1999 Sep, 129(9), 1688 - 91
Carnitine import to isolated hepatocytes and synthesis are accelerated in pivalate-treated rats; Nakajima H et al.; To investigate the effect of pivalate on carnitine import and carnitine synthesis in the liver, we measured carnitine uptake in isolated rat hepatocytes with L-{(14)C} carnitine and concentrations of free carnitine, gamma-butyrobetaine and acylcarnitines using tandem mass spectrometry . Hepatocytes from rats treated with 20 mmol/L of pivalate for 4 wk had greater L-{(14)C} carnitine uptake than those of unsupplemented rats after 5, 10, 30 and 90 min . Addition of 1 mmol/L of pivalate or 1 mmol/L of pivaloylcarnitine to control cell suspensions did not affect L-{(14)C} carnitine uptake . The K(m) values for L-{(14)C} carnitine uptake for pivalate-treated rats were significantly lower than control (2.9 +/- 0.7 mmol/L for pivalate-treated rats, 6.2 +/- 1.1 mmol/L for controls) . The concentration of free carnitine was not reduced in the liver of pivalate-treated rats, whereas the concentrations of acetylcarnitine and gamma-butyrobetaine were significantly lower than controls . In the heart and muscle the concentration of free carnitine was significantly lower and that of gamma-butyrobetaine was higher than controls . These results suggest that carnitine transport from plasma into the liver and synthesis in the liver are accelerated in rats with secondary carnitine deficiency induced by the administration of pivalate.

Cryobiology, 1999 Aug, 39(1), 13 - 28
A parametric study of freezing injury in AT-1 rat prostate tumor cells; Smith DJ et al.; The connection between thermal history and cell injury in single AT-1 cells is studied systematically through a two-level, four-parameter (2(4)) experiment . The four parameters considered are cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR) . Cryosurgically relevant high and low values of each parameter are chosen (CR, 5 to 50 degrees C/min; ET, -20 to -80 degrees C; HT, 0 to 15 min; TR, 20 to 200 degrees C/min) to maximize applicability of the results to cryosurgery; it is important to note that any conclusions drawn from the results are valid only for the range of parameter values studied . AT-1 cell suspensions are frozen in a controlled way on a directional solidification stage, and viability is assessed postthaw with a live/dead assay using the fluorescent dyes calcein-AM and propidium iodide to indicate live and dead cells, respectively . The parameters which most significantly affect short-term survival outcome are determined through calculation of the individual parameter effect values (E) according to the factorial experimental design guidelines . In addition, any synergy between two parameters in determining short-term survival outcome is revealed by calculation of the interaction value for those parameters (I) . The results suggest that survival is most significantly affected by variation in end temperature and hold time, and the only significant parameter interaction found is between these two parameters . The analysis further suggests that survival depends nonlinearly on the thermal parameters, based on calculation of the survival curvature (C) in the parameter ranges studied . These results are discussed within the context of previously proposed mechanisms of cellular injury during freezing . Although coupling between several mechanisms is possible, single mechanisms which may explain the survival results include slow-cooling injury mechanisms such as solute effects injury, dehydration-induced membrane instabilities, and volume-catalyzed nucleation of intracellular ice .

Biol Reprod, 1999 Sep, 61(3), 764 - 75
Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents; Devireddy RV et al.; Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs) . Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution . Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs . Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume . By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined . The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg){cpa} = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp){cpa} = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg){cpa} = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp){cpa} = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99) . These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice . The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs . This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice . As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media . The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.

Immunol Invest, 1999 Jul, 28(4), 235 - 46
Optimisation of a technique for isolating lymphocyte subsets from human endometrium; Flynn L et al.; Human endometrium is a rich source of lymphocytes which may have unique immunoregulatory functions . The aim of this study was to compare current procedures for endometrial tissue disaggregation, and optimise a method for isolation of endometrial lymphocytes . Tissue was obtained from 41 women undergoing elective hysterectomy or dilation and curettage (D&C) for reasons of benign non-endometrial pathology . Specimens were exposed to reduction/chelation, mechanical or enzymatic disruption . Optimal single cell suspensions of high yields (mean 8.8 x 10(6) range 3.5-18 x 10(6)lymphs) and good viability (60%) were obtained, using a combination of collagenase IV (200 U/ml) and DNase I (35 U/ml) . Suspensions were further purified by density gradient centrifugation . Multi-colour flow cytometry was used for analysis of endometrial lymphocyte subsets . Cell suspensions were stained with mAbs specific for CD3, CD4, CD8, CD56, CD45 and CD14, and it was clearly shown that the developed method had no effect on surface glycoprotein expression . Phenotypic analysis revealed consistent populations of endometrial large granular lymphocytes (CD56+CD3-) 54.16%, and T-cells (CD3+) 37.73% . This technique was applicable to the characterisation of T-cell populations, including CD8+ (56.6%), CD4+ (44.0%), and particularly smaller populations of CD4+CD8+(3.56%), CD4-CD8-(3.34%) and CD56+(6.3%) due to it's sensitivity . In conclusion, optimised enzymatic digestion, in combination with flow cytometry provides an effective method for phenotypic examination of small endometrial lymphocyte subpopulations.

Plant Cell, 1999 Aug, 11(8), 1537 - 52
Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway
Rasmussen S, Dixon RA.
3H-l-Phenylalanine is incorporated into a range of phenylpropanoid compounds when fed to tobacco cell cultures . A significant proportion of (3)H-trans-cinnamic acid formed from (3)H-l-phenylalanine did not equilibrate with exogenous trans-cinnamic acid and therefore may be rapidly channeled through the cinnamate 4-hydroxylase (C4H) reaction to 4-coumaric acid . Such compartmentalization of trans-cinnamic acid was not observed after elicitation or in cell cultures constitutively expressing a bean phenylalanine ammonia-lyase (PAL) transgene . Channeling between PAL and C4H was confirmed in vitro in isolated microsomes from tobacco stems or cell suspension cultures . This channeling was strongly reduced in microsomes from stems or cell cultures of transgenic PAL-overexpressing plants or after elicitation of wild-type cell cultures . Protein gel blot analysis showed that tobacco PAL1 and bean PAL were localized in both soluble and microsomal fractions, whereas tobacco PAL2 was found only in the soluble fraction . We propose that metabolic channeling of trans-cinnamic acid requires the close association of specific forms of PAL with C4H on microsomal membranes.

Cancer Res, 1999 Aug 1, 59(15), 3677 - 81
Tumor radiosensitization by sustained intratumoral release of bromodeoxyuridine; Doiron A et al.; We have previously reported that the use of the polymer bis(p-carboxyphenoxy)propane-sebacic acid (20:80) for intratumoral delivery of cis-platinum in a mouse tumor model (RIF-1) potentiated the effects of acute and fractionated radiation . This mode of drug delivery seems particularly applicable to the administration of radiosensitizing drugs because an optimum concentration of radiosensitizer can be maintained in the tumor over the prolonged period required for fractionated radiation treatment . We have now investigated, in the same tumor model, radiosensitization by the thymidine analogue bromodeoxyuridine (BrdUrd) . BrdUrd (20%, w/w) was incorporated into bis(p-carboxyphenoxy)propane-sebacic acid (20:80) and polymer rods containing the drug implanted in the RIF-1 tumor . Preliminary in vitro studies of the rate of release of BrdUrd from the polymer showed an initial rapid loss over 24 h, followed by a slower release extending over the next 5 days . In experiments in which tumor cells, which had incorporated BrdUrd in vivo from implanted polymer, were excised and a single cell suspension irradiated in vitro radiosensitization indicative of BrdUrd incorporation was associated mainly with an increase in the alpha constant for the linear quadratic model of cell survival . Radiosensitization was seen for tumor cells harvested between 5 and 10 days after polymer implant, a finding that is consistent with results of experiments in which the percentage of cells that had incorporated BrdUrd were measured by flow cytometry at various times after polymer/BrdUrd implant . The proportion of tumor cells positive for BrdUrd was 40-50% between 3 and 8 days after polymer implant . When tumors were irradiated in situ and response measured in terms of tumor growth delay (TGD), radiosensitization was not seen for an acute dose of 16.5 Gy . In contrast, significant radiosensitization was seen for fractionated treatments when polymer/BrdUrd was implanted 3 days before the first radiation dose . For a dose of 5 x 6 Gy, TGD was increased from 22 days for radiation alone to 27 days for radiation plus polymer implant . For 10 x 6 Gy fractions, TGD increased from 45-77 days for those mice in whom the tumor eventually regrew, whereas for 25% of the mice in this group the tumor volume was reduced to a point where it was no longer detectable and there was no recurrence for at least 120 days after treatment.

Methods Find Exp Clin Pharmacol, 1999 Jul-Aug, 21(6), 391 - 3
In vivo establishment of T98G human glioblastoma; Rubenstein M et al.; Human derived T98G glioblastoma has long been utilized as an in vitro model for epidermal growth factor receptor (EGFR)-mediated growth regulation . Recently, T98G has been employed to develop new types of therapy directed at limiting EGFR expression such as by administration of antisense oligonucleotides directed against EGFR encoding mRNA . A major limitation to extending this model for in vivo application is that T98G implanted s.c . or intracerebrally has been reported not to grow in nude mice . In an effort to extend this model to permit in vivo studies, we evaluated the use of Matrigel and orthotopic (intracranial) implantation techniques . When equal volumes of Matrigel were mixed with T98G cell suspensions, tumors developed at both flank and orthotopic locations . Four groups of nude mice were inoculated into the flanks with either 10(5), 10(6), 4 x 10(6) or 10(7) T98G cells in a 150 microliters total volume with Matrigel . In 1/5, 3/5, 1/5 and 1/3 mice receiving 10(5), 10(6), 4 x 10(6) and 10(7) cells, respectively, tumors developed 11, 15, 15 and 15 weeks, respectively, following inoculation . Out of 4 mice inoculated orthotopically (intracranially into the frontal lobe) with only 4 x 10(4) cells and Matrigel, 2 developed tumors . However, all mice (4/4) inoculated orthotopically with 4 x 10(5) cells in a 10 microliters total volume with Matrigel developed tumors . Two were identified histologically following a scheduled sacrifice at 36 and 60 days and two more at 103 and 118 days after sacrifice following abnormal behavior . The best tumor establishment efficacy combined orthotopic implantation of 4 x 10(5) T98G cells with Matrigel . These techniques permit the use of T98G glioblastoma as an in vivo model for new forms of therapy.

Arch Androl, 1999 Jul-Aug, 43(1), 67 - 71
Effects of vasoactive intestinal peptide on human sperm motility; Siow Y et al.; Sperm flagellar activity is modulated by cAMP . In target tissues, vasoactive intestinal peptide (VIP) stimulates adenyl cyclase activity, which elevates intracellular cAMP levels and activates protein kinase activity . This study investigated the effects of VIP on motility of sperm from 17 subjects . Motile activities, monitored before (0 min, baseline) and for 40 min after incubation with VIP (0.2 microgram/mL cell suspension), were analyzed by computer-assisted semen analysis . The data (mean +/- SEM) are expressed as percentages of baseline values and changes were compared by trend analysis for interval level measures by repeated measures analysis of variance orthogonal polynominal contrasts . The addition of VIP significantly increased motile sperm concentration (110 +/- 17% {10 min}, 132 +/- 15% {20 min}, 152 +/- 18% {30 min}, 125 +/- 18% {40 min}; p < .02) and sperm with rapid straight-line motility (V > 25 microns/s) (167 +/- 20%, 174 +/- 19%, 173 +/- 23%, 141 +/- 16%; p < .02) . Mean track speed (micron/s) was increased (125 +/- 12%, 134 = 9%, 129 +/- 12% and 126 +/- 12%; p < .02), while mean progressive velocity, amplitude of head displacement, and beat frequency were not affected by VIP . These results indicate that VIP stimulates sperm motile activity by cAMP-mediated phosphorylation of axonemal proteins.

Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 652 - 7
Multi-site modulation of flux during monolignol formation in loblolly pine (Pinus taeda); Anterola AM et al.; Loblolly pine (Pinus taeda L.) cell suspension cultures secrete monolignols when placed in 8% sucrose/20 mM KI solution, and these were used to identify phenylpropanoid pathway flux-modulating steps . When cells were provided with increasing amounts of either phenylalanine (Phe) or cinnamic acid, cellular concentrations of immediate downstream products (cinnamic and p-coumaric acids, respectively) increased, whereas caffeic and ferulic acid pool sizes were essentially unaffected . Increasing Phe concentrations resulted in increased amounts of p-coumaryl alcohol relative to coniferyl alcohol . However, exogenously supplied cinnamic, p-coumaric, caffeic, and ferulic acids resulted only in increases in their intercellular concentrations, but not that of downstream cinnamyl aldehydes and monolignols . Supplying p-coumaryl and coniferyl aldehydes up to 40, 000-320,000-fold above the detection limits resulted in rapid, quantitative conversion into the monolignols . Only at nonphysiological concentrations was transient accumulation of intracellular aldehydes observed . These results indicate that cinnamic and p-coumaric acid hydroxylations assume important regulatory positions in phenylpropanoid metabolism, whereas cinnamyl aldehyde reduction does not serve as a control point .

Zhongguo Yao Li Xue Bao, 1999 Jan, 20(1), 10 - 4
Effects of superoxide anion on intracellular Ca2+ concentration in rabbit pulmonary arterial smooth muscle cells; Wang YP et al.; AIM: To study the effect of superoxide anion on the Ca2+ homeostasis in smooth muscle cells isolated from the rabbit pulmonary artery . METHODS: Intracellular Ca2+ concentration ({Ca2+}i) was investigated using cell suspension of freshly isolated smooth muscle cells from rabbit pulmonary artery (PASMC) . Fura-2 fluorescent ratio obtained at 340 nm and 380 nm wave lengths was measured as an indicator of {Ca2+}i . RESULTS: ATP 30 mumol.L-1 induced a transient increase in the ratio (Ca2+ transient) . Thapsigargin, an inhibitor of sarcoplasmic Ca2+ ATPase, induced a phasic increase in the ratio due to Ca2+ leak from intracellular store sites, but not the sustained increase, thereby suggesting the absence of Ca2+ release-activated Ca2+ entry (CRAC) mechanism in PASMC . When PASMC were exposed to superoxide anion by the pretreatment with xanthine and xanthine oxidase (X/XO) for 30 min, sustained component of ATP-induced Ca2+ transient was elevated . The ratios at 5 and 10 min after ATP application (delta ratio5 min and delta ratio10 min) were increased from 0.091 +/- 0.022 to 0.149 +/- 0.048 (P < 0.05) and from 0.021 +/- 0.020 to 0.117 +/- 0.047 (P < 0.01), respectively . But, thapsigargin-induced {Ca2+}i transient was not affected by X/XO . CONCLUSION: Superoxide anion makes ATP-induced Ca2+ transient sluggish, and does not affect Ca2+ leak pathway in PASMC.

Biochimie, 1999 Jun, 81(6), 663 - 8
Involvement of plasma membrane proteins in plant defense responses . Analysis of the cryptogein signal transduction in tobacco; Lebrun-Garcia A et al.; Cryptogein, a 98 amino acid protein secreted by the fungus Phytophthora cryptogea, induces a hypersensitive response and systemic acquired resistance in tobacco plants (Nicotiana tabacum var Xanthi) . The mode of action of cryptogein has been studied using tobacco cell suspensions . The recognition of this elicitor by a plasma membrane receptor leads to a cascade of events including protein phosphorylation, calcium influx, potassium and chloride effluxes, plasma membrane depolarization, activation of a NADPH oxidase responsible for active oxygen species (AOS) production and cytosol acidification, activation of the pentose phosphate pathway, and activation of two mitogen-activated protein kinase (MAPK) homologues . The organization of the cryptogein responses reveals that the earliest steps of the signal transduction pathway involve plasma membrane activities . Their activation generates a complex network of second messengers which triggers the specific physiological responses . This study may contribute to our understanding of plant signaling processes because elicitors and a variety of signals including hormones, Nod factors, light, gravity and stresses share some common transduction elements and pathways.

Clin Exp Metastasis, 1999 May, 17(3), 265 - 70
Development of a high metastatic orthotopic model of human renal cell carcinoma in nude mice: benefits of fragment implantation compared to cell-suspension injection; An Z et al.; In this study we compared the metastatic rate of human renal cell carcinoma SN12C in two orthotopic nude mouse models: surgical orthotopic implantation (SOI) of histologically intact tumor tissue and cellular orthotopic injection (COI) of cell suspensions in the kidney . The primary tumors resulting from SOI were larger and much more locally invasive than primary tumors resulting from COI . SOI generated higher metastatic expression than COI . The differences in metastatic rates in the involved organs (lung, liver, and mediastinal lymph nodes) were 2-3 fold higher in SOI compared to COI (P < 0.05) . Median survival time in the SOI model was 40 days, which was significantly shorter than that of COI (68 days) (P < 0.001) . Histological observation of the primary tumors from the SOI model demonstrated a much richer vascular network than the COI model . Lymph node and lung metastases were larger and more cellular in the SOI model compared to COI . We conclude that the tissue architecture of the implanted tumor tissue in the SOI model plays an important role in the initiation of primary tumor growth, invasion, and distant metastasis . This study directly demonstrates that the implantation of histologically intact tumor tissue orthotopically allows accurate expression of the clinical features of human renal cancer in nude mice . This model should be of value in cancer research and antimetastatic drug discovery for renal cancer, a currently very poorly responding malignancy.

Am J Reprod Immunol, 1999 Jul, 42(1), 49 - 57
Antigen-presenting cells in the human female reproductive tract: analysis of antigen presentation in pre- and post-menopausal women; Fahey JV et al.; PROBLEM: To determine whether cells in the female reproductive tract (FRT) are functionally capable of presenting antigen to T cells . METHOD OF STUDY: Analysis was done by determining the proliferation of purified autologous T cells to antigen, following co-incubation with non-proliferating cell suspensions isolated from the uterus and prepared by enzymatic digestion of reproductive tract tissues from hysterectomy patients with benign disease . RESULTS: All uterine preparations analyzed were functionally capable of presenting antigen; the ability to present antigen was independent of pre- and post-menopausal status . In contrast, some, but not all, tissues from the ovary, Fallopian tube, cervix, and vagina were capable of presenting antigen . CONCLUSION: These results suggest that the human FRT is an inductive site for immune responses . Regulation of antigen presentation in the reproductive tract may be important for protection against sexually transmitted diseases.

FEMS Microbiol Lett, 1999 Jul 15, 176(2), 291 - 9
Bioconversion of pyrimidine by resting cells of quinoline-degrading bacteria; Fetzner S; Nine quinoline-degrading bacterial strains were tested for their ability to hydroxylate pyrimidine . All strains converted pyrimidine to uracil via pyrimidine-4-one in a cometabolic process . Quinoline 2-oxidoreductases (QuinORs) were the catalysts of fortuitous pyrimidine hydroxylation . Whereas in most strains the activity of the QuinOR towards pyrimidine was very low compared to its activity towards quinoline, QuinOR in crude extracts from Comamonas testosteroni 63 showed a specific activity of 64 (mU mg protein)-1 with pyrimidine as substrate, compared to a specific activity of 237 (mU mg protein)-1 towards the intrinsic substrate quinoline . Resting cells of Comamonas testosteroni 63 rapidly converted pyrimidine almost stoichiometrically to uracil, which accumulated in the cell suspension . Using an adsorbent resin, uracil was prepared from the supernatant of Comamonas testosteroni 63 resting cells with a yield of > 98%.

J Magn Reson, 1999 Aug, 139(2), 258 - 72
Permeability coefficients from NMR q-space data: models with unevenly spaced semi-permeable parallel membranes; Kuchel PW et al.; The NMR "q-space" experiment conducted on water provides information on the sizes of repeated structures on the micrometer-length scale in heterogeneous samples, including cell suspensions or tissues . Under some circumstances these plots display coherence peaks, and it has been implied theoretically that the position of the peaks will vary with the rate of molecular exchange across the membranes . This has been demonstrated (qualitatively) with human erythrocytes in suspension . Thus, in the quest for a quantitative approach to the interpretation of such data, we address here the "inverse problem," namely the estimate of the permeability coefficient of membranes from q-space experiments . The present work describes theoretical predictions of q-space plots from molecules diffusing in a simple system of parallel semi-permeable membranes arranged with separations that alternate between two different values; this was designed to (loosely) mimic the intra- and extracellular compartments in a suspension of cells or a tissue . The development of the theory was facilitated by symbolic computation, and the analysis of synthetic data was shown to be achievable by the use of a three-layer back-propagation artificial neural network .

Eur J Oral Sci, 1999 Jun, 107(3), 194 - 201
Macrophages, dendritic cells and T lymphocytes in rat buccal mucosa and dental pulp following 5-fluorouracil treatment; Bultzingslowen I et al.; Cancer chemotherapeutic drugs may affect immunocompetent cells of oral soft tissues, causing an impaired capacity to induce immune defence reactions . This study was designed to investigate changes in the number of macrophages, dendritic cells and T lymphocytes in the oral mucosa and dental pulp following treatment with the antineoplastic agent 5-fluorouracil (5-FU) . Rats were given 5-FU (30 mg/kg or 50 mg/kg) i.v . on days 0, 1, 2, 5, 6 and 7 . The number of cells in buccal epithelium and dental pulp expressing ED2, MHC class II, or CD2 molecules was analyzed following immunohistochemical peroxidase staining . Major histocompatibility complex (MHC) class II molecules were analyzed in epithelial sheets and in epithelial cell suspensions by flow cytometry . Increasing concentrations of 5-FU changed the morphology of the epithelial Langerhans cells with a reduced dendritic appearance as the most prominent feature . At 50 mg/kg of 5-FU, the oral epithelium detached from the connective tissue at the basement membrane . MHC class II molecule-expressing cells were reduced in number in the lamina propria of the buccal mucosa and in the dental pulp after both low and high dose of 5-FU, but only after high dose in the epithelium . The number of ED2- and CD2-expressing cells in the dental pulp was only slightly reduced by 5-FU treatment at both low and high dose, while these cells decreased in number in the oral mucosa . The varying sensitivity to 5-FU by macrophages, dendritic cells, and T cells depending on the tissues in which they reside may be due to differences in cell origin or differences in antigenic load.

Appl Microbiol Biotechnol, 1999 Jun, 51(6), 765 - 72
Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining; Gorenflo V et al.; The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acid were investigated as film castings and in living cells respectively after staining with Nile red . All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm . This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry . The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 micrograms Nile red is necessary for total staining of 1.0 mg granules . The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations . These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids.

Histochem J, 1999 Mar, 31(3), 181 - 94
Detection of oxidant producing-sites in glutaraldehyde-fixed human neutrophils and eosinophils stimulated with phorbol myristate acetate; Kobayashi T et al.; The aim of the present study was to detect oxidant-producing sites, and to elucidate their dynamic reorganization in human polymorphonuclear leukocytes (PMNs) fixed with glutaraldehyde which preserves cell structure . In biochemical analyses, the detectable O2- generation in unfixed PMNs upon stimulation with phorbol 12-myristate 13-acetate (PMA) in the presence of cytochalasin B was characterized by a lag period of approximately 10 sec followed by O2- production . The maximal rate reached was 3.18+/-0.07 nmol/min/l x 10(6) cells (mean+/-S.D.; n = 4) after 30 sec of stimulation . PMNs exposed to PMA and cytochalasin B followed by fixation with glutaraldehyde generated O2- without a lag period at a rate of 0.35+/-0.05 n mol/min/l x 10(6) cells (mean+/-S.D.) by the addition of NADPH as substrate to the cell suspension . In the cytochemical assays, we employed both cells exposed to PMA and cytochalasin B, and then fixed with glutaraldehyde followed by incubation in the cytochemical reaction medium (pre-fixed cells) and cells incubated in the medium containing PMA and cytochalasin B followed by fixation with glutaraldehyde (post-fixed cells) . Oxidant reaction in the pre-fixed cells was detected by the addition of NADPH and FAD to the reaction medium . No oxidant-reaction product was seen in pre-fixed cells stimulated for 10 sec whereas the oxidant reaction was visualized in intracellular compartments of pre-fixed PMNs stimulated for 20 sec . The fact that the pre-fixed PMNs stimulated for 30 sec showed increased numbers of oxidant-producing structures compared to those seen in the pre-fixed cells stimulated for 20 sec, demonstrates that the amount of the reaction product and the number of oxidant-producing intracellular compartments increases between 20 and 30 sec after start of stimulation with PMA . These cytochemical results using the pre-fixed cells coincided with the findings obtained from the biochemical assays in the pre-fixed cells exposed to PMA and cytochalasin B . The oxidant reaction was observed in elongated tubular structures that were arranged in a radial fashion, and were associated with the plasma membrane in the pre-fixed PMNs, whereas post-fixed PMNs exhibited slender spherical or rod-shaped structures of various lengths . The present results indicate that the pre-fixed PMNs can be employed for elucidating the dynamic reorganization of oxidant-producing sites in human PMNs.

Planta, 1999 Jun, 208(4), 574 - 82
Cloning and expression of an arginine decarboxylase cDNA from Vitis vinifera L . cell-suspension cultures; Primikirios NI et al.; Arginine decarboxylase (ADC; EC 4.1.1.19) is a key enzyme in one of the two pathways to putrescine . We present the first ADC cDNA from a woody perennial plant species, the grapevine (Vitis vinifera L.), which exhibits 70-80% homology with other dicot ADCs . The effects of ammonium, nitrate, and putrescine on ADC specific activity, soluble polyamine levels, ADC-mRNA, endogeneous arginine and ornithine, and arginase specific activity were investigated in suspension cultures of grapevine cells . The addition of NH4+ to cells cultured in NH4(+)-free medium, resulted in a 4-fold increase in ADC activity and concomitantly in a 4-fold increase in putrescine and a 3-fold decrease in arginine . During this period ornithine increased and arginase activity followed a reverse pattern of changes compared with ADC . In contrast, the addition of NO3- did not markedly affect ADC activity, putrescine, arginine and ornithine, but transiently increased arginase activity . The addition of putrescine caused a 4-fold decrease in ADC activity and increased arginine, ornithine and arginase activity . The changes in ADC specific activity were not accompanied by analogous changes in the ADC transcript levels . These results further support the view that ADC regulation is not exhibited, at least for the factors considered in this work, at the transcriptional level.

Chem Biol Interact, 1999 Jun 1, 121(1), 99 - 115
Cryopreserved porcine hepatocyte cultures; Koebe HG et al.; Cryopreservation of freshly isolated hepatocytes is regarded the standard technique for long term storage of liver cells . Frankly, we were not successful in reproducing viability rates of about 70% of that which have been reported by most authors as results of various freezing protocols for hepatocyte suspensions . In fact, we saw mostly devastating results . We assume that intracellular ice crystal formation as well as osmotic changes during freezing and thawing of liver cells cause hazardous effects, especially on membranes of cells after enzymatic isolation, and, thus, generally result in a severe loss in number and impaired specific hepatocyte functions in subsequent culture . We tried to improve results by freezing cell cultures instead . We allowed hepatocytes to regain a more stable condition prior to storage and placed them in tissue flasks in a uniform configuration, rather than to pack cell suspensions in vials or bags under rather indefinable conditions . Porcine hepatocytes (viability 92+/-2%) were isolated from slaughterhouse organs and cultured in a double gel (sandwich) configuration . At day 3, cultures were rate controlled frozen (RCF) and stored in a cell bank for three hours (Group A) or 14 days at -80 degrees C (Group B), respectively . Non-frozen cells (NF) and cultures subjected to a linear freezing rate of -10 degrees C/min (LFR, Group C) with 3 h of storage served as controls from identical cell batches . Upon thawing, at day 2 of subsequent culture, fluorescence microscopy studies revealed a survival rate of 75+/-10% (mean+/-S.D.) in the RCF groups . Time of storage (3 h, 14 d) did not influence results . Survival in Group C was 10+/-5% . Cell integrity was measured by LDH-release, which indicated a larger damage of cells in the LFR group, and thereby resembled the morphological findings . Functional parameters, such as albumin synthesis and CYT P 450-activity were comparable to non-frozen liver cells at 48 h after thawing in the RCF groups (A + B), and showed significantly higher levels in these groups as compared to the LFR Group (C) . We recommend to freeze hepatocytes in culture in a rate controlled fashion rather than cell suspensions . This way a cell bank of cryopreserved hepatocyte cultures for batch controlled investigations can be easily obtained . This could ameliorate the availability of rare (human) cell material and might also improve the quality of data generated from in vitro experiments in hepatology or pharmacology/toxicology with liver cells from identical sources . It remains to be seen whether this technique might also be of value in hybrid bioartificial liver devices to make these systems become readily available upon clinical demand.

Food Chem Toxicol, 1999 Apr, 37(4), 319 - 26
Effects of berberine on arylamine N-acetyltransferase activity in human bladder tumour cells; Chung JG et al.; Berberine was used to determine inhibition of arylamine N-acetyltransferase (NAT) activity in human bladder tumour cells . The NAT activity was measured by HPLC assaying for the amounts of N-acetyl-2-aminofluorene (AAF) and N-acetyl-p-aminobenzoic acid (N-Ac-PABA) and remaining 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) . Two assay systems were performed, one with cellular cytosols, the other with intact bladder tumour cell suspensions . The NAT activity in human bladder tumour cells was inhibited by berberine in a dose-dependent manner, that is, the higher the concentration of berberine, the higher the inhibition of NAT activity . The values of apparent Km and Vmax calculated from cytosol NAT and intact cells were also decreased by berberine . This report is the first demonstration to show berberine did affect human bladder tumour cell NAT activity.

J Biomech Eng, 1998 Oct, 120(5), 559 - 69
Measurement of water transport during freezing in mammalian liver tissue: Part II--The use of differential scanning calorimetry; Devireddy RV et al.; There is currently a need for experimental techniques to assay the biophysical response (water transport or intracellular ice formation, IIF) during freezing in the cells of whole tissue slices . These data are important in understanding and optimizing biomedical applications of freezing, particularly in cryosurgery . This study presents a new technique using a Differential Scanning Calorimeter (DSC) to obtain dynamic and quantitative water transport data in whole tissue slices during freezing . Sprague-Dawley rat liver tissue was chosen as our model system . The DSC was used to monitor quantitatively the heat released by water transported from the unfrozen cell cytoplasm to the partially frozen vascular/extracellular space at 5 degrees C/min . This technique was previously described for use in a single cell suspension system (Devireddy, et al . 1998) . A model of water transport was fit to the DSC data using a nonlinear regression curve-fitting technique, which assumes that the rat liver tissue behaves as a two-compartment Krogh cylinder model . The biophysical parameters of water transport for rat liver tissue at 5 degrees C/min were obtained as Lpg = 3.16 x 10(-13) m3/Ns (1.9 microns/min-atm), ELp = 265 kJ/mole (63.4 kcal/mole), respectively . These results compare favorably to water transport parameters in whole liver tissue reported in the first part of this study obtained using a freeze substitution (FS) microscopy technique (Pazhayannur and Bischof, 1997) . The DSC technique is shown to be a fast, quantitative, and reproducible technique to measure dynamic water transport in tissue systems . However, there are several limitations to the DSC technique: (a) a priori knowledge that the biophysical response is in fact water transport, (b) the technique cannot be used due to machine limitations at cooling rates greater than 40 degrees C/min, and (c) the tissue geometric dimensions (the Krogh model dimensions) and the osmotically inactive cell volumes Vb, must be determined by low-temperature microscopy techniques.

J Immunol Methods, 1999 Jun 24, 226(1-2), 1 - 10
Affinity-purification of a TMV-specific recombinant full-size antibody from a transgenic tobacco suspension culture; Fischer R et al.; A TMV-specific full-size murine IgG-2b/K antibody (mAb24) was expressed in a Nicotiana tabacum cv . Petite Havana SR1 suspension culture (P9s), which was derived from a stably transformed transgenic plant (P9) . The integration of an N-terminal murine leader peptide directed the assembled immunoglobulin for secretion . However, in suspension culture, the full-size recombinant antibody, rAb24, was retained by the plant cell wall and was not present in the culture medium . rAb24 expression reached a basal level of 15 microg per gram wet cell weight, corresponding to 0.3% of the total soluble plant cell protein . The level of rAb24 could be increased three-fold by amino acid supplementation of the culture medium . For purification of the recombinant antibody from batch-cultured tobacco suspension cells, the primary plant cell wall was partially digested by enzymatic treatment . This resulted in a total release of recombinant full-size rAb24 into the extraction buffer . A three-step procedure was used to purify the immunoglobulins, starting with cross-flow filtration (step 1) followed by protein A affinity chromatography (step 2) and gel filtration as a final purification step (step 3) . This procedure gave a recovery of more than 80% of the expressed rAb24 from plant cell extracts . SDS-PAGE, IEF and immunoblot analyses demonstrated a high degree of homogeneity for the affinity-purified rAb24 . An ELISA procedure demonstrated that the specificity and affinity of the protein A affinity purified antibody was indistinguishable from its murine counterpart, indicating the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies.

Am J Physiol, 1999 Jul, 277(1 Pt 2), H413 - 6
Separation of cardiomyocytes and coronary endothelial cells for cell-specific RT-PCR; Preisig-Muller R et al.; A simple method for analyzing the differential gene expression of coronary endothelial cells and cardiac muscle cells was developed . Cells were isolated from guinea pig hearts by collagenase digestion . In the diluted cell suspension, single cardiomyocytes and capillary fragments containing 6-15 endothelial cells could be identified morphologically . A simple "cell picker" was constructed using a polyethylene pipette with a tip diameter of approximately 150 micrometers that was attached to a micromanipulator and connected to an electric miniature valve . Intermittent suction pulses (1- to 2-cm water column) were applied by opening the valve for 100-200 ms at 1-s intervals . Cardiomyocytes (800-1,000) or capillary fragments (150) were picked under visual control using an inverted microscope . The cells were transferred to a reaction tube for RNA extraction, reverse transcription (RT), and DNA amplification (RT-PCR) with gene-specific and intron-spanning primers . All PCR products were verified by sequencing . Troponin T and endothelin-1 were found to be specific markers for guinea-pig cardiac muscle cells and coronary endothelial cells, respectively.

Endocr Regul, 1998 Mar, 32(1), 1 - 8
Specific Stimulatory Effect of LH on the Synthesis of delta4 Gestagens and Androgens in Adrenocortical Cells in vitro; Szymanski Z; The aim of this study was to answer the question whether gonadotropins are able to stimulate the synthesis of delta4 gestagens and androgens in adrenals by the same way as in gonads . Adrenal cells of male guinea pigs (n=12) and adrenocortical cells of sows (n=2) were isolated with collagenase 1A and DNA-se and used in two separate experiments . Cell suspensions divided in quadruplicate number of aliquots for each test were preincubated (1 h) and then incubated (h) with high purity pLH-USDA, pLH-GPZ (this was used only in one experiment), pFSH-NIH (the residual contamination of this preparation with ACTH was not excluded) and ACTH1-24 . The concentrations of progesterone (P), 17alpha-hydroxyprogesterone (OH-P), androstenedione (A), testosterone (T) and cortisol (F) in the incubated cells were estimated by RIA . The stimulatory effect of two high purity pLH preparations on P, A and T synthesis in guinea pig adrenal cells and pig adrenocortical cells was demonstrated . Moreover, the synthesis of OH-P in pig adrenocortical cells was also stimulated . It may be concluded that these results are specific for LH, since the used pLH-USDA was deprived of any residual ACTH contamination and pLH-GPZ was chromatographically homogenous . These preparations also showed indirect evidences of the activation of steroid 3beta-hydroxysteroid dehydrogenase/isomerase (3beta HSD) which catalyses the synthesis of these four delta4 steroids from their delta5 path precursors . The LH dependent activation of this enzyme in adrenals, which was demonstrated in this work, supported the well known observations of its independence of ACTH . As high as 6-times increase of P synthesis and 2-5 times increase of OH-P synthesis under the influence of pLH in pig adrenocortical cells (consistent with the species of LH) needs the induction of the labile protein i synthesis, since the cholesterol transport into mitochondria and the extent of pregnenolone and its derivatives synthesis depends on that protein . The influence of LH on adrenal steroidogenesis indicates that adrenal cells are the target not only for ACTH but also for LH . The influence of the used pFSH specimen on adrenal steroidogenesis resembles that of ACTH, including the increase of cortisol synthesis . Due to this similarity and lack of evidences of excluding residual ACTH contamination of such pFSH specimen, these results are considered nonspecific . Thus, the problem of FSH influence on adrenal steroidogenesis is still open . Regardless of that, the presented demonstration of specific LH effect appears to be an original contribution to the basic knowledge on adrenal steroidogenesis.

Acta Ophthalmol Scand, 1999 Jun, 77(3), 247 - 54
Long-term outcome of RPE allografts to the subretinal space of rabbits; Crafoord S et al.; PURPOSE: To determine the long-term RPE allograft survival in the subretinal space using suspensions of RPE cells and atraumatic transplantation surgery . METHODS: Nineteen albino rabbits were transplanted with suspensions of pigmented RPE cells from brown rabbits . Following pars plana vitrectomy, the RPE cell suspension was injected through a small retinotomy using a glass micropipette into the subretinal space under microscopic control . No immunosuppression was used . The eyes were monitored by biomicroscopy, color fundus photography, and fluorescein angiography . Rabbits were sacrificed at 1, 3 and 6 months, respectively, and the eyes processed for light and electron microscopy, using monoclonal antibodies for identifying macrophages . RESULTS: Transplanted RPE cells were present in the subretinal space in all eyes at 6 months . There was no fluorescein leakage . Generally, the RPE allograft formed a monolayer, but focal fragmentation and disruption with dispersion of melanin pigment occurred . Foci of multilayers of cells in the subretinal space, containing large macrophages, were associated with adjacent photoreceptor damage . There was no infiltration of lymphocytes but macrophages and glial cells were contiguous to the transplant . Cells harboring intracytoplasmatic melanin pigment were observed in the neural retina . CONCLUSION: Transplantation of RPE cell suspensions to the subretinal space generally forms a monolayer that persists at 6 months . However, in areas of multilayers of RPE cells and macrophages, graft failure occurs in combination with adjacent photoreceptor damage . Graft failure is not associated with the infiltration of lymphocytes, but other mechanisms seem to occur.

J Virol Methods, 1999 Jun, 80(1), 59 - 67
Comparison of the plaque test and reverse transcription nested PCR for the detection of FMDV in nasal swabs and probang samples; Moss A et al.; In order to compare the sensitivity of assays for the diagnosis of foot-and mouth disease (FMD), a cell suspension plaque test on BHK21-CT cells and a reverse transcription nested PCR (RT-nPCR) were used to examine 485 nasal swabs and 227 probang samples obtained from FMDV-infected cattle during vaccine potency tests . In nasal swabs, FMDV could be detected by both tests before the onset of clinical symptoms . However, after two weeks p.i., FMDV was only detected routinely in the probang samples . Examination of nasal swabs revealed a higher number of infected animals using RT-nPCR than by the use of the plaque test . Both tests gave approximately equivalent results with the probang samples.

Biochimie, 1999 May, 81(5), 477 - 84
Expression of integrins of the beta1 family in thyroid cells from patients with Graves' disease in vivo and in vitro; Vitale M et al.; The expression of the beta1 family of integrins was determined in thyroid follicular cells from patients with Graves' disease (GD) . Integrin expression was quantitated by flow fluorocytometry of single cell suspensions with antibodies against the common beta1 chain and the alpha1-alpha6 subunits . Results indicated that also in thyroid glands of GD, as previously observed in nodular goiters, two follicular cell populations with different patterns of beta1 integrin expression coexist (VLAalpha3beta1 and VLAalpha1,3,5,6beta1) . The VLAalpha1,3,5,6beta1 thyrocyte population in GD was more abundant than in nodular goiters, ranging from 40 to 70% of the total follicular cells and the overall expression of the beta1 integrins was a two-fold higher . In thyrocytes from patients with GD cultured in vitro, alpha3 and alpha2 expression was regulated by cell-to-cell contact as previously described in normal thyroid cells, while the expression of alpha1, alpha5 and alpha6 was quickly lost during the culture . Our data suggest that the integrin profile of the VLAalpha1,3,5,6beta1 thyrocyte population in GD is induced by micro-environmental conditions rather than being the expression of a constitutive phenotype.

Arch Oral Biol, 1999 Jun, 44(6), 519 - 23
A technique for the study of endocytosis in human oral epithelial cells; Innes NP et al.; Fluorescently labelled latex microspheres (0.01, 0.1 and 1.0 micron dia.) were used to establish whether oral epithelial cells could exhibit an endocytic function . Oral mucosa biopsies were incubated in organ culture at 37 degrees C for 20 h with one of the three sizes of fluorescent microspheres in the medium . Tissue pieces were then disaggregated and cell suspensions analysed for cell content and viability . Evidence of endocytosis was sought by using fluorescence-activated cell scanning (FACS) and confocal microscopy to study the epithelial cell suspensions for internalization of the microspheres . Confirmation that the microspheres had been internalized and were not merely attached to the cell exterior was shown by using trypan blue quenching to extinguish extracellular fluorescence, allowing analysis of only intracellular fluorescent microspheres . Both FACS and confocal microscopy confirmed uptake of 0.01 and 0.1 micron dia . microspheres but not 1.0 micron . Endocytosis was quantitated using FACS and a dose-dependent relation between the concentration of spheres in the incubation medium and uptake was found . Internalization of microspheres of < 1.0 micron dia . and the dose-dependent uptake support a fluid-phase constitutive endocytic process.

Nahrung, 1999 Jun, 43(3), 201 - 4
Flavonols and flavones of parsley cell suspension culture change the antioxidative capacity of plasma in rats; Hempel J et al.; The flavonoid aglycones from an illuminated parsley (Petroselinum crispum (Mill.) Nym.) cell suspension culture were identified and quantified as the flavones apigenin, luteolin and chrysoeriol and the flavonols kaempferol, quercetin and isorhamnetin . Flavonoid extracts from these cultures were purified by solid phase extraction from RP C-18 phase and given by gavage to rats . Only extract from illuminated culture increased the antioxidative capacity (AOC) of blood plasma temporarily with maximum values after 1 h . It is concluded that the course of AOC reflects changes in the plasma content of flavonoids.

Mol Reprod Dev, 1999 Aug, 53(4), 422 - 33
Spermatogonia of rainbow trout: I . Morphological characterization, mitotic activity, and survival in primary cultures of testicular cells; Loir M; Prerequisites of developing in vitro studies for a better understanding of the control mechanisms underlying the proliferation and differentiation of spermatogonia (Go) in the teleost testis are: (1) to be able to identify the different types of Go; (2) to maintain in culture the structural relationships occurring in situ between the various testicular cell types, as intact as possible; and (3) to know how the Go survive and proliferate in culture for several days . After very gentle homogenization of trout testes treated with collagenase, a cell suspension containing mainly spermatocysts (one or several Sertoli cells enclosing one Go or a clone of germ cells) and clusters of myoid cells and Leydig cells was seeded in culture onto a laminin plus fibronectin coating . After 4.5-6 days in culture, then staining with May-Grunwald and Giemsa reagents, the determination of the nuclear and cellular size of the various Go and of the number of Go present in clones has enabled the identification of two types of large Go, in pairs or alone (Go A) and six successive types of smaller Go (Go B) . Cell viability determination by staining with Rhodamine 123/propidium iodide (PI)/Hoechst 33342 and with FITC-Annexin V/PI indicated that after 5-7 days in culture, all the somatic cells and most of the Go were viable . Only some of the Go, mainly among the most differentiated ones, underwent apoptosis, as it was the case for a number of spermatocytes and spermatids increasing with the time in culture . Brdu labelling and 3H-Thymidine (3H-Tdr) incorporation indicated that the proliferative activity of Go was at a maximum after 4.5 days in culture and that the response to at least two molecules (QAYL-IGF-I and GTH-I) remained unchanged between 3 and 6 days . As only very scarce somatic cells from immature/spermatogenetic testes synthesized DNA up to 6 days in culture, the measurement of 3H-Tdr incorporation by cells from such testes reliably reflected synthesis of DNA by only the Go (and eventually also by primary spermatocytes when they are present) . In conclusion, this study provides information allowing a detailed analysis of the events related with the mitotic phase of spermatogenesis in the trout and it establishes that primary cultures of testicular cells carried out in the reported conditions represent a useful tool to develop an analysis of the mechanisms participating in the control of this phase .

Crit Care Med, 1999 Jun, 27(6), 1191 - 4
Accuracy of methemoglobin measurements: comparison of six different commercial devices and one manual method; Dotsch J et al.; OBJECTIVE: During nitric oxide inhalation, methemoglobinemia needs to be monitored . We compared six commercially available instruments and one manual method for methemoglobin measurements . In addition, we studied whether and to what degree methylene blue interferes with methemoglobin measurements . DESIGN: In vitro methodologic study . SETTING: Research laboratory in a university hospital . PATIENTS: Five healthy volunteers from whom red blood cells were obtained . INTERVENTIONS: Methemoglobinemia was generated in a red blood cell suspension by nitric oxide; methemoglobin was measured with six commercial instruments and one manual photometric method to calculate variation coefficients and to determine the differences between the devices . Methemoglobin was measured with and without the addition of methylene blue with two instruments . Measurements were performed immediately after the addition of methylene blue . MEASUREMENTS AND MAIN RESULTS: All six commercially available instruments had variation coefficients of <0.1 at methemoglobin concentrations of 5%, whereas the manual photometric method did not reach a variation coefficient of <0.1 at 8% of methemoglobin . Apart from two devices that measured slightly but significantly higher methemoglobin levels, all instruments measured similar values of methemoglobin when the same samples were determined simultaneously . Higher concentrations of methylene blue (10, 40, 100 microM) reduced substantially the apparent concentrations of methemoglobin . Interference by methylene blue was most pronounced at low methemoglobin levels . CONCLUSIONS: With some limitations, all commercial instruments that were tested performed adequately for the monitoring of methemoglobinemia . Methylene blue interferes with the methemoglobin measurements in a dose-dependent manner.

J Nat Prod, 1999 Jun, 62(6), 906 - 8
Patulosides A and B, novel xanthone glycosides from cell suspension cultures of Hypericum patulum; Ishiguro K et al.; Two new xanthone glycosides, patuloside A (3-beta-D-glucopyranosyloxy-1,5,6-trihydroxy-9H-xanthene-9-one, 1) and patuloside B {3-(2-O-alpha-L-rhamnopyranosyl-beta-D-glucopyranosyl)oxy-1,5, 6-trihydroxy-9H-xanthene-9-one, 2}, have been isolated from cell suspension cultures of Hypericum patulum . Their structures were elucidated by spectral techniques.

Phytochemistry, 1999 Jul, 51(5), 651 - 6
Production of 13C-labelled anthocyanins by Vitis vinifera cell suspension cultures; Krisa S et al.; The use of plant cell cultures for producing isotopically (13C) labelled phenolic substances is reported . Vitis vinifera cells synthesize high levels of anthocyanins when they are cultured in a polyphenol synthesis-inducing medium . Three major anthocyanin monoglucosides found in red wine were identified in grape cells: cyanidin-3-O-beta-glucoside, peonidin-3-O-beta-glucoside, and malvidin-3-O-beta-glucoside . Kinetic study of the intracellular level of phenylalanine and its metabolites showed that it is preferable to add this precursor to grape cell suspensions after the 5th day of culture, i.e . at the beginning of the exponential growth phase . After adding phenylalanine to the culture medium, its uptake was complete and the accumulation of anthocyanins in grape cells was stimulated . Incorporation of {1-13C}-phenylalanine into anthocyanins was measured by means of 13C satellites in the proton NMR spectrum . The maximal rate of 13C enrichment anthocyanins obtained with this technique reached 65% . The production of 13C labelled phenolic compounds was undertaken in order to investigate their absorption and metabolism in humans.

Ann Hematol, 1999 May, 78(5), 223 - 31
Human acute myeloblastic leukemia-ascites model using the human GM-CSF- and IL-3-releasing transgenic SCID mice; Fukuchi Y et al.; To generate an appropriate model for human acute myeloblastic leukemia (AML), we have successfully established a human hematopoietic growth factor-dependent AML cell line (TF-1 and UT-7/GM)-ascites model using human granulocyte-macrophage colony-stimulating factor (hGM-CSF)- and human interleukin 3 (hIL-3)-releasing transgenic (Tg)-SCID mice . When 1 x 10(7) cells of TF-1, a human erythroleukemia cell line, were transplanted into the peritoneum of irradiated Tg-SCID mice (TF-1 ip/Tg-SCID mice), TF-1 cells grew in both the single cell suspension form (asTF-1) and solid form in ascites and invaded various tissues: lungs, liver, pancreas, and genitals, 3-6 weeks following transplantation . Subsequently, 0.5-1 x 10(7) cells of UT-7/GM, a subline of the UT-7 human megakaryoblastic leukemia cell line, grown in the back of hGM-CSF Tg-SCID mice after subcutaneous inoculation, were transplanted into the peritoneum of other irradiated hGM-CSF Tg-SCID mice . After 4 weeks, UT-7/GM cells (asUT-7/GM) also grew in the same manner as TF-1 cells in hGM-CSF Tg-SCID mice . Analysis of the cells from the peritoneum and tissues by PCR amplifying ALU and human GM-CSF receptor beta sequences and by immunohistochemical staining using anti-human CD45 revealed that they possessed the original characteristics of the parental cells . To confirm the usefulness of this human AML-ascites model, experimental treatment of AML cells grown in these mice was carried out with a differentiation inducer, delta-aminolevulinic acid (deltaALA), which induces hemoglobin synthesis for TF-1 in vitro and is thus regarded as an anti-leukemia drug candidate . Unexpectedly, growth promotion of TF-1 cells was observed in the treated TF-1 ip/hIL-3 Tg-SCID mice without differentiation to erythroid cells after treatment with delta-ALA (5 mM) for 7 days . These results indicate that Tg-SCID mice can support the growth of human hematopoietic growth factor-dependent AML cell lines which are usually rejected by SCID mice, without modification of the parental cell characteristics . In addition, this Tg-SCID leukemia-ascites model may become a useful preclinical tool for estimation of drug efficacy in vivo, since the drug candidate which was promising in vitro did not act in the same manner in vivo.

Syst Appl Microbiol, 1999 May, 22(2), 161 - 8
Transformation of Escherichia coli in foodstuffs; Bauer F et al.; The plasmid transfer by transformation of Escherichia coli in 12 foods was investigated under conditions commonly found in processing and storage of food . Transformation occurred in all foods with frequencies of at least 10(-8) when a simplified standard transformation protocol with non-growing cells was applied . Higher rates (ca . 10(-7)) were found in milk, soy drink, tomato and orange juice . Furthermore, E . coli became transformed at temperatures below 5 degrees C, i.e . under conditions highly relevant in storage of perishable foods . In soy drink this condition resulted in frequencies which were even higher than those determined after application of a temperature shift to 37 degrees C . The transformation of cells growing in milk and carrot juice at a constantly kept temperature of 37 degrees C provides evidence for the potential of E . coli to become transformed naturally . With purified DNA frequencies were determined in these substrates of ca . 2.5 x 10(-7) and 2.5 x 10(-8), respectively . Similar frequencies were also obtained in milk containing the crude nucleic acids of homogenised cell suspensions of E . coli (pUC18) . Moreover, the release of plasmid DNA from E . coli during food processing and the subsequent uptake of this DNA by growing E . coli cells was shown to take place after homogenisation in milk indicating a horizontal plasmid transfer by transformation of E . coli.

Pigment Cell Res, 1999 Jun, 12(3), 164 - 74
An ex vivo study of congenital pigmented nevi in epidermal reconstructs; Bessou-Touya S et al.; In order to study morphologic and functional characteristics of pigment cells in congenital pigmented nevi, autologous or heterologous reconstructs have been made using normal keratinocytes and nevus cells from the dermal-epidermal junction or from the dermis . All these cells, keratinocytes and nevus cells, were used as cell suspensions immediately after dissociation from the tissues or after subsequent brief cultivation in a serum-free medium . Reconstructed epidermis were cultured for 15 days at the air-liquid interface with or without ultraviolet (UV) B exposure . The reconstructs were examined macroscopically (formation of hyperpigmented macules), histologically (pigment cell nesting) and ultrastructurally (pigment structure and transfer) . Typical nesting of nevus cells was observed in the dermal-epidermal junction or in the superficial dermis associated with macroscopically detectable small pigmented macules . UVB exposure induced an upward migration of nevus cells in the suprabasal layers of the epidermis . This tissue model can be considered as an excellent system for the ex vivo reproduction of pigmented nevi and as an assay of the sensitivity of nevus cells towards UVB irradiation.

Pigment Cell Res, 1999 Jun, 12(3), 147 - 63
Genetic and epigenetic control of the proliferation and differentiation of mouse epidermal melanocytes in culture; Hirobe T et al.; Serum-free culture of epidermal cell suspensions from neonatal skin of mice of strain C57BL/10JHir (B10) showed that alpha-melanocyte-stimulating hormone (alpha-MSH) was involved in regulating the differentiation of melanocytes by inducing tyrosinase activity, melanosome formation, and dendritogenesis . Dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) similarly induced the differentiation of melanocytes . On the other hand, DBcAMP induced the proliferation of epidermal melanocytes in culture in the presence of keratinocytes . Basic fibroblast growth factor (bFGF) was also shown to stimulate the sustained proliferation of undifferentiated melanoblasts in the presence of DBcAMP and keratinocytes . These results suggest that the proliferation and differentiation of mouse epidermal melanoblasts and melanocytes in culture are regulated by the three factors; namely, cAMP, bFGF, and keratinocyte-derived factors . Moreover, serum-free primary culture of mouse epidermal melanocytes derived from B10 congenic mice, which carry various coat color genes, showed that the coat color genes were involved in regulating the proliferation and differentiation of mouse epidermal melanocytes by controlling the proliferative rate, melanosome formation and maturation, and melanosome distribution.

Med Oncol, 1999 Apr, 16(1), 46 - 52
Role of macrophage colony-stimulating factor in the differentiation and expansion of monocytes and dendritic cells from CD34+ progenitor cells; Kamps AW et al.; The present study focused on whether it is possible to expand monocytic cells from CD34+ progenitor cells by using macrophage colony-stimulating factor (M-CSF) in the absence and presence of mast cell growth factor (MGF) and IL-6 . It was demonstrated that CD34+ cells differentiate without expansion to functional mature monocytic cells in the presence of M-CSF or combinations of M-CSF plus IL-6 and MGF . A different response pattern was observed for the number of clonogenic cells . The addition of IL-6 or both IL-6 and MGF to M-CSF containing cultures resulted in significant higher numbers of colony-forming unit-macrophage (CFU-M) as tested in clonogenic and 3H-thymidine assays . Furthermore, M-CSF plus both IL-6 and MGF appeared to be the most potent combination to preserve the monocytic precursor in cell suspension culture assays . These results indicate that IL-6 and MGF in conjunction with M-CSF affect CD34+ cells especially at precursor level without distinct effect on the more mature stages . Secondly we studied whether M-CSF is only critical for the monocytic lineage or also affects dendritic cell (DC) development . Indeed, we were able to culture CD83+ DC from CD34+ progenitor cells in the presence of M-CSF in conjunction with TNF-alpha, IL-4, and MGF although their absolute number is almost threefold lower than the number of CD83+ cells yielded from GM-CSF plus TNF-alpha, IL-4, and MGF stimulated CD34+ cells.

Transfusion, 1999 Jun, 39(6), 599 - 607
Photodynamic sterilization of red cells and its effect on contaminating white cells: viability and mechanism of cell death; Moor AC et al.; BACKGROUND: Phthalocyanines are useful sensitizers for photodynamic sterilization of red cell concentrates . Various lipid-enveloped viruses can be inactivated with only limited red cell damage . Because white cells are involved in the immunomodulatory effects of blood transfusions, the study of the effect of photodynamic treatment on these cells is imperative . STUDY DESIGN AND METHODS: White cell-enriched red cell suspensions were photodynamically treated with either the hydrophobic Pc4 (HOSiPcOSi-(CH3)2(CH2)3N(CH3)2) or water-soluble aluminum phthalocyanine tetrasulfonate (AIPCS4) under virucidal conditions . Viability of white cell subpopulations on Days 0, 1, and 4 after treatment was determined by fluorescence-activated cell sorting by flow cytometric analysis of propidium iodide uptake . Apoptosis induction was studied by DNA ladder formation and staining for an early marker of apoptosis (annexin V) . RESULTS: Treatment with Pc4 causes a significant decrease in cell viability of all white cells, as shown by prodidium iodide uptake . Monocytes and granulocytes are the most sensitive, and lymphocytes are relatively more resistant . Some of the cells die by apoptosis, which is induced within 30 minutes after treatment . Treatment with AIPCS4 damages only monocytes; other cell populations are not affected . CONCLUSIONS: Physicochemical properties of the photosensitizers partly determine their effect on white cells . Differences in intracellular localization are likely to be responsible for the effects observed.

Exp Eye Res, 1999 Jun, 68(6), 671 - 8
Evaluation of the human choroidal melanoma rabbit model for studying microcirculation patterns with confocal ICG and histology; Mueller AJ et al.; The aim of this study was to develop consistently focal elevated choroidal masses of human choroidal melanoma in immunosuppressed rabbits and to correlate the visualization of prognostically significant microcirculation patterns from confocal indocyanine green angiography with histologic microcirculation patterns . A human choroidal melanoma cell line (OCM1) was implanted in the choroid of 40 rabbit eyes using three different techniques: transscleral choroidal injection of a cell suspension, injection of a cell suspension in a surgically induced cyclodialysis cleft, and implantation of solid tumor fragments in a surgically induced cyclodialysis cleft . The rabbits were immunosuppressed with daily injections of Cyclosporin A to prevent host versus graft reaction . The eyes were studied weekly with indirect ophthalmoscopy and fundus photography to monitor tumor growth and indocyanine green angiography using a confocal scanning laser ophthalmoscope to identify microcirculation patterns in vivo and correlate these findings with the histologic demonstration of tumor microcirculation patterns . A tumor mass was identified by indirect ophthalmoscopy in 16 of the 40 implanted rabbit eyes (40%) . Each of these tumors was confirmed histologically to represent a focal elevated choroidal mass . All 16 elevated choroidal masses grow in eyes in which solid tumor fragments were implanted . In total, a melanoma was identified histologically in 28 of the implanted 40 eyes (70%) . In addition to the 16 eyes where the melanoma appeared as a focal elevated choroidal mass, 4 eyes contained a focal elevated mass in the sclera and 8 eyes contained a flat choroidal tumor . Histologically, microcirculation patterns were identified only in the 16 eyes with focal elevated choroidal masses . Confocal indocyanine green angiography imaged microcirculation patterns in 13 of these 16 eyes (81%) . The surgical implantation of small solid fragments of human choroidal melanoma in immunosuppressed rabbit eyes provides the best method to consistently obtain focal elevated choroidal masses . These focal elevated choroidal masses resemble booth the localization and the growth pattern of choroidal melanomas in humans . In addition, they also contain microcirculation patterns similar to those seen in humans that are detectable with confocal indocyanine green angiography . The use of indocyanine green angiography with this animal model may be especially useful in designing and evaluating anti-microcirculation treatments directed at uveal melanoma .

Am J Reprod Immunol, 1999 Apr, 41(4), 286 - 92
Effect of serum on mouse granulated metrial gland cell cytotoxicity; Stewart IJ et al.; PROBLEM: To examine factors affecting mouse granulated metrial gland cell cytotoxicity . METHOD OF STUDY: Chromium-release assays using mouse metrial gland cell suspensions targeted against Wehi 164 fibrosarcoma cells were used . Modulation of cellular cytotoxicity was investigated using an antibody, anti-asialo-GM1, to attempt depletion of effector cells and sera to determine if inhibitory factors are present in blood in vivo . RESULTS: No reduction in cellular cytotoxicity was found following treatment of effector cells with asialo-GM1 antibody, but there was a reduction following treatment with asialo-GM1 antibody plus guinea pig serum and with guinea pig serum alone . Sera from pregnant, male, and SCID mice also reduced the level of cytotoxicity by metrial gland cell suspensions . CONCLUSIONS: The reduction in cytotoxicity in the presence of serum indicates that there are factors in the blood which are inhibitory to granulated metrial gland cell cytotoxic activity in vivo . The resistance of mouse metrial gland cells to asialo-GM1 antibody depletion is supportive of a natural cytotoxic (NC) lineage for this cell type.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 1998 Mar, 12(2), 90 - 3
{Experimental study of the effect on growth of Schwann cell from chitin and chitosan in vitro}; Kuang Y et al.; In order to study the effect of chitin and chitosan on the growth of Schwann cell (SC) of rats in vitro, the SC was isolated from sciatic nerve and brachial plexus of new-born rats . After the enzymatic and mechanical dissociation, the cell suspension was vaccinated on chitin membrane and chitosan fluid-coated glass coverslips . Then, the growth of SC was examined at 1, 3, 7 days after culture under light microscope and scanning electron microscope . The results showed that 94 percent of the cell grown from was SC and only 6% was fibroblast (FB), while that of the control SC 71% and FB 29% in population . The number of SC in chitosan suspension was more than that in chitin . Therefore, the conclusion was that the chitin and chitosan was histocompatible to SC, and chitosan suspension was superior to chitin, and both could inhibit the growth of fibroblast.

Zhonghua Wai Ke Za Zhi, 1997 Jan, 35(1), 19 - 21
{Intraparenchymal and intraventricular transplantation of fetal substantia nigra cell suspension in rat model of Parkinson's disease}; Yang H et al.; The grafts of fetal substantia cell suspension grafts could bring into a behavioral effect and characteristics of histology and immunocytochemistry in rat models of PD after the grafts were transplanted either into the lateral ventricles or directly into the striatum . It was found that the intraparenchymal tromsplantation of fetal nigral cell suspensions not only was beneficial to reinnervation between host and grafts, but also produced more complete and steady effects than the intraventricular transplantation . We concluded that the restoration of nervous functional deficits of PD rats is likely to depend on both extensive dopaminergic reinnervation throughout the grafted sites and on closer integration of the grafts with the host striatal circuitry.

Ann N Y Acad Sci, 1999 Apr 30, 872, 233 - 40; discussion 240-2
Ex vivo expansion and genetic marking of primitive human and baboon hematopoietic cells; Medin JA et al.; The achievement of positive outcomes in many clinical protocols involving hematopoietic stem cells (HSCs) has been handicapped by the limited numbers of marrow repopulating cells available to actually bring about therapy . This insufficiency has been especially problematic in stem cell transplantation and gene therapy . A number of studies have been initiated to attempt expansion of HSCs, mainly by manipulation of key cytokines in cell suspension cultures . Unfortunately, these expansion methods usually lead to altered properties in the amplified cells, mainly by reducing their self-renewal and multi-lineage differentiative potentials . Here we discuss our ongoing work, utilizing a unique endothelial cell line that supports primitive hematopoiesis, to attempt to generate expansion of primate HSCs that retain their elementary properties . Genetic marking of early hematopoietic cells to facilitate tracking will be mentioned as will the development and employment of assay systems designed to evaluate the long-term functional attributes of the expanded cells.

Bull Acad Natl Med, 1999, 183(1), 143 - 55; discussion 156-8
{Early nuclear modifications related to cellular activation: from histology to molecular cytopathology (1967-1997)}; Pompidou A; The nuclear chromatin structure and function are altered as soon as the first steps of cellular activation induced by membrane stimulation . Different studies carried on lymphatic tissues as well as isolated cell suspensions (lympho-monocytes) demonstrate that an early and transitory chromatin dispersion, visualised by nuclear refringency (1967), is related to the genome derepression allowing DNA transcription mechanism . This is linked to the expression of c-fos proto-oncogene and that of specific proteins synthesis . This occurs before c myc expression and could play a role in the regulation of surface antigens expression . Independent from the induction of cell proliferation, but related to cell differentiation, the early and transitory chromatin dispersion as well as the influence of c-fos related to proteic system activator (AP1) is discussed in referring to recent studies (1997), as well as the regulation factors of the chromatin filament structure.

Pediatr Surg Int, 1999, 15(3-4), 238 - 9
T-lymphocyte subsets in the contralateral testis after unilateral blunt testicular trauma in pre-pubertal mice; Sharma RB et al.; Although spermatozoa express antigens, they normally do not produce an immunological response because of the blood-testis barrier and the predominance of CD8(+) T-lymphocytes in the rete testis . Unilateral blunt testicular trauma (UBTT) has been reported to decrease fertility . The present study was designed to evaluate the sub-populations of T-lymphocytes in mice with testicular trauma . Twenty male mice aged 20 days were randomized into control and test groups . At about 70 days of age the contralateral testis was harvested, cell suspensions were prepared, and immunofluorescence staining was performed for detection of CD4(+ )and CD8(+) T-lymphocytes by flow-cytometry . The ratio of CD8(+) and CD4(+) lymphocytes was significantly higher (P < 0.001) in the control mice compared to the UBTT group (1.3 +/- 0.3 vs 0.5 +/- 0.01) . The results suggest that UBTT alters the CD8(+)/CD4(+) ratio in the contralateral testis, which may have an important bearing on the pathogenesis of infertility in cases of testicular injury.

Osteoarthritis Cartilage, 1999 Jan, 7(1), 15 - 28
Articular cartilage repair: are the intrinsic biological constraints undermining this process insuperable?
Hunziker EB.
This article reviews the experimental and clinical strategies currently in use or under development for the treatment of articular cartilage lesions . The vast majority of protocols under investigation pertain to the treatment of full-thickness defects (i.e., those which penetrate the subchondral bone and trabecular-bone spaces) rather than partial-thickness ones (i.e., those which are confined to the substance of articular cartilage tissue itself) . This bias probably reflects the circumstance that partial-thickness defects do not heal spontaneously whereas full-thickness ones below a critical size do, albeit transiently . And it is, of course, a seemingly easier task to manipulate a process which is readily set in train than it is to overcome an induction-problem which Nature herself has not solved . Indeed, the reasons for this inert state of partial-thickness defects have only recently been elucidated, and these are briefly discussed . However, the main body of this review deals with the various transplantation concepts implemented for the repair of full-thickness defects . These fall into two broad categories: tissue-based (entailing the grafting of perichondrial, periosteal, cartilage or bone-cartilage material) and cell-based (utilizing chondroblasts, chondrocytes, periochondrial cells or mesenchymal stem cells) . Cell-based systems are further subdivided according to whether cells are transplanted within a matrix (biodegradable, non-biodegradable or synthetic) or free in suspension . Thus far, the application of cell suspensions has always been combined with the grafting of a periosteal flap . The strengths and weaknesses of each concept are discussed.

Brain Res Dev Brain Res, 1999 Jun 8, 115(1), 1 - 8
GABA induces proliferation of immature cerebellar granule cells grown in vitro; Fiszman ML et al.; The presence of GABA and its receptors early in rodent nervous system development has lead to speculation on the role of this transmitter system in neuroblast proliferation, migration and differentiation . We studied the effect of GABA and GABA agonists on immature cerebellar granule cell proliferation and survival . Cerebellar granule cell suspensions were obtained from 6-8-day-old rats and grown in culture for up to 7 days in serum-containing or serum-free medium . The addition of GABA (0.1-100 microM) or muscimol (0.01-10 microM) 2 h after inoculation and harvested 22 h later, lead to an increase in 3H-thymidine incorporation over control samples with the correspondent increase in granule cells number assayed 48 h later . The effect on cell proliferation exerted by GABAA agonists was blocked by MgCl2 and nifedipine, as well as by the chloride channel blocker, picrotoxin (50 microM), and the GABAA receptor specific blocker, bicuculline (50 microM) . The increase on cell proliferation induced by GABA also was blocked by PD98059 (75 microM), a specific inhibitor of the mitogen-activated protein kinase kinase (MAPKK) . GABAA receptor-mediated proliferation was consistently seen in cells inoculated in serum-containing medium supplemented with 25 mM KCl but not seen in serum-free medium, with 5 mM or 25 mM KCl . The presence of serum did not enhance the survival of cerebellar granule cells grown for 7 days in either 5 mM or 25 mM KCl . Additionally, neither GABA nor muscimol applied from day 2 to day 7 in vitro affected cell survival in any culture condition . We conclude that GABA and GABAA receptor agonists influence granule cell proliferation but not survival and that this effect is mediated by a calcium influx via voltage-dependent calcium channel activation, with a subsequent activation of the MAPK cascade .

Exp Neurol, 1999 Jun, 157(2), 259 - 67
Partial restoration of striatal GABAA receptor balance by functional mesencephalic dopaminergic grafts in mice with hereditary parkinsonism; Stasi K et al.; Levels of inhibitory amino acid receptors were studied in the weaver (wv/wv) mouse model of dopamine (DA) deficiency after unilateral intrastriatal transplantation of fetal mesencephalic cell suspensions . Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM {3H}GBR 12935 . The average increase in {3H}GBR 12935 binding in grafted dorsal striatum compared to nongrafted wv/wv striatum was 60% 3 months after grafting . Autoradiography of 8 nM {3H}flunitrazepam and 12 nM {3H}muscimol binding was carried out to visualize the distribution of GABAA receptors in +/+ mice and in recipient weaver mutants . A 17% increase in {3H}flunitrazepam binding and a 20% increase in {3H}muscimol binding was found in the nongrafted dorsal striatum of weaver mutants compared to +/+ . The functional mesencephalic grafts had a partial normalizing effect on both {3H}flunitrazepam and {3H}muscimol binding in the dorsal striatum of the weaver recipients . The normalization brought about by the grafts was around 20% for {3H}flunitrazepam binding and more than 40% for {3H}muscimol binding . The results are discussed in the context of the important interaction between the converging glutamatergic corticostriatal and DAergic nigrostriatal pathways in controlling the functional GABAergic output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency .

Plant Physiol, 1999 Jun, 120(2), 371 - 82
Isolation of tobacco isoperoxidases accumulated in cell-suspension culture medium and characterization of activities related to cell wall metabolism; de Marco A et al.; All of the most important guaiacol-type peroxidase (POX) isoforms accumulated in the culture medium of BY-2 tobacco (Nicotiana tabacum L . cv Bright Yellow 2) cells have been isolated . Five basic and two acidic isoforms were found . The four major isoforms (B2, B3, P1, and P2), all strongly basic, have been purified to homogeneity and partially sequenced . B2 and B3 are new isoforms showing high homology to only one POX isolated so far . Amino acid sequencing and specific activities indicated that basic isoPOXs constitute two pairs of strictly related isoforms (P1/P2 and B2/B3) . Their specific activities measured in the presence of different substrates, as monolignols and NAD(P)H, indicated possible specialized functions in cell wall metabolism . Only P-type POXs were able to oxidize indoleacetic acid . Variations in pH could play a regulatory role by changing the relative contribution of different isoforms to total POX activity . Apart from cell culture medium, polyclonal antibodies obtained against P1 and P2 detected P1 in roots and in lower parts of stems . Immunocytochemical labeling indicated that P-type POXs were expressed in stem phloem and in phloem and epidermal cells of roots.

J Neurooncol, 1999 Mar, 42(1), 59 - 67
Morphometrical characterization of two glioma models in the brain of immunocompetent and immunodeficient rats; Saini M et al.; Although several glioma models exist, systematic morphometrical studies on such experimental tumors are lacking . The purpose of this study was the quantitative assessment of how rat strains, cell lines, injection techniques and location affect tumors reproducibility and histopathological features . Glioma cells were implanted in 3 brain locations, with different injection techniques (free hand, stereotactic, water-tight device), variable volumes, cell concentrations and infusion rates . Tumors were developed from 2 rat glioma cell lines (9L and C6) in immunocompetent (Wistar and Fischer 344) and immunodeficient rats (New Zealand) . Animals underwent daily neurological examination . At the scheduled time the tumors were macro and microscopically evaluated and a quantitative morphometrical analysis was performed . C6 gliomas appeared very infiltrative and irregularly shaped; 9L gliomas showed, by using the same injection technique, a grossly regular shape . Margins at the tumor-brain interface were macroscopically demarcated in the immunocompetent rats . In the nude rats, 9L tumors appeared microscopically more infiltrative, although regularly shaped, with a closer morphological resemblance to human gliomas . The implantation in the frontal area, anterior to the nucleus caudatus (3 mm anterior the coronal suture) gave reproducible tumor shape and size, no hydrocephalus and no early neurological deterioration . The use of a stereotactic technique or of a water-tight device, small volume (< 10 microl) of cell suspension, low infusion rate were useful to reduce morbidity and to improve data reproducibility . No difference in morbidity and mortality were observed in immunocompetent and immunodeficient rats . The 9L glioma model with stereotactic implantation constitutes a good option for reliable morphometrical evaluation of tumor growth . We propose a location for tumor implantation anterior to the nucleus caudatus . This produced the longest symptom-free survival.

Acta Cytol, 1999 May-Jun, 43(3), 393 - 9
Optimal prefixation of cells to demonstrate apoptosis by the TUNEL method; Kiyozuka Y et al.; OBJECTIVE: To determine the optimal fixation method for cultured human ovarian cancer cell line SHIN-3 to document cisplatin-induced apoptosis by the terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling (TUNEL) assay . STUDY DESIGN: Cisplatin-treated cancer cell suspensions were (1) fixed in 4% buffered formalin for 10 minutes (BF group); (2) treated with 2% Carbowax in 50% ethanol (CW) for 10 minutes and then fixed in 100% ethanol for one hour (CW + ET); or (3) treated with CW for 10 minutes and then fixed in 4% buffered formalin for one hour (CW + BF) . Cell morphology, adhesion to the glass slides and TUNEL reactivity were compared among the three groups . The effects of prolonged prefixation of cell suspensions in CW and of the postfixation of cell smears in BF for one, three and seven days were also examined . RESULTS: CW + BF treatment yielded satisfactory cell morphology, minimum cell loss and an excellent TUNEL reaction . However, prolonged prefixation in CW resulted in cell shrinkage . CONCLUSION: CW + BF treatment can be widely recommended for use with cytologic preparations for the TUNEL assay.

Gastroenterology, 1999 Jun, 116(6), 1342 - 7
Characterization of the inhibitory effect of boiled rice on intestinal chloride secretion in guinea pig crypt cells; Mathews CJ et al.; BACKGROUND & AIMS: When rice is incorporated into oral rehydration therapy for patients with secretory diarrhea, clinical outcomes improve . We have shown that a factor purified from boiled rice (RF) blocks the secretory response of intestinal crypt cells to adenosine 3',5'-cyclic monophosphate (cAMP) . Now we report that the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the cellular target for this rice inhibitor . METHODS: We used RF, the same previously described extract prepared from boiled rice, to assess chloride channel activation in vitro, measuring (1) cell volume regulation of guinea pig intestinal crypt epithelial cell suspensions using standard Coulter counter technology, (2) transepithelial chloride current in monolayers of T84 cells mounted in Ussing chambers, and (3) whole-cell and single-channel currents using the patch-clamp technique in cells transfected to express CFTR . RESULTS: RF inhibited activation by cAMP of CFTR chloride channels in all experimental preparations; RF did not block volume-stimulated Cl- secretion, suggesting that its effect might be specific for CFTR chloride channels . RF inhibited transepithelial cAMP-stimulated Cl- current in T84 cells and inhibited forskolin (i.e., cAMP)-induced current in cells transfected with CFTR . Excised patch and single-channel patch-clamp recordings supported the view that the response was a direct effect on CFTR rather than on cAMP signal transduction . CONCLUSIONS: RF exerts a specific inhibitory effect on CFTR chloride channels, blocking activation from the luminal surface of the cell and reversing established activation . Many major diarrheal states are based on cAMP-induced CFTR activation, leading to excessive gut secretion; our findings could have clinical relevance.

J Membr Biol, 1999 May 15, 169(2), 103 - 9
Electric field pulses can induce apoptosis; Hofmann F et al.; Injection of electric field pulses of high intensity (kV/cm) and short duration (microsecond range) into a cell suspension results in a temporary increase of the membrane permeability due to a reversible electric breakdown of the cell membrane . Here we demonstrate that application of supercritical field pulses between 4 . 5 and 8.1 kV/cm strength and 40 microsec duration induce typical features of apoptosis in Jurkat T-lymphoblasts and in HL-60 cells including DNA fragmentation and cleavage of the poly(ADP ribose) polymerase . Apoptosis induction did not depend on the presence of any particular electrolyte in the extracellular medium . However, no apoptosis was observed in solutions without a minimum amount of salt . Apoptotic DNA fragmentation was prevented by the caspase inhibitor zVAD.

Cell Transplant, 1999 Jan-Feb, 8(1), 111 - 29
Pig fetal septal neurons implanted into the hippocampus of aged or cholinergic deafferented rats grow axons and form cross-species synapses in appropriate target regions; Deacon T et al.; The anatomical specificity of axon growth from fetal pig septal xenografts was studied by transplanting septal cells from E30-35 pig fetuses into cholinergic deafferented (192-IgG-saporin-infused) rats or into aged rats (> 18 months) . Cell suspensions (100,000 cells/microl) were injected bilaterally into the dorsal and ventral hippocampus of immunosuppressed rats (10 mg/kg/day cyclosporine A) . To assess axonal growth and synapse formation, acetylcholinesterase histochemistry, an antibody to choline acetyltransferase (ChAT), and three pig-positive/rat-negative antibodies: bovine 70kD neurofilament (NF70), human low-affinity NGF receptor (hNGFr), and human synaptobrevin (hSB) were used . In rats with surviving grafts at 6 months, NF70 axonal labeling was more extensive than either ChAT or hNGFr labeling . All three markers demonstrated graft axons extending selectively through the hippocampal CA fields and the molecular layer of the dentate gyrus . Graft axons did not extend into adjacent entorhinal cortex or neocortex . The distribution of pig hSB-positive synapses correlated with AChE-positive fiber outgrowth in to the host . Electron microscopic analysis of hSB-immunostained hippocampal sections revealed pig presynaptic terminals in contact with normal rat postsynaptic structures in the CA fields and the dentate gyrus . These data demonstrate target-appropriate growth of pig cholinergic axons and the formation of cross-species synapses in the deafferented or aged rat hippocampus.

Cell Transplant, 1999 Jan-Feb, 8(1), 103 - 9
Purification of adrenal chromaffin cells increases antinociceptive efficacy of xenotransplants without immunosuppression; Michalewicz P et al.; We have found that immunosuppression is necessary for the survival of xenogeneic adrenal medullary transplants . Because chromaffin cells are essentially nonimmunogenic, it is likely that the highly immunogenic "passenger" cells in the transplant preparation bring about rejection . This article describes a procedure that produces an essentially pure preparation of chromaffin cells for transplantation . Bovine adrenal medullary cells were isolated and differentially plated, resulting in a semipurified preparation of chromaffin cells . Ferromagnetic beads were added to the cell suspension, some of which were phagocytized by endothelial cells, which allowed their removal by exposure to a magnet . The remaining cells were then exposed to ferromagnetic beads coated with isolectin B4 from Griffonia simplicifolia and once again to a magnetic field . The "semipurified" preparation contained approximately 90% chromaffin cells, whereas the "highly purified" preparation was > 99.5% chromaffin cells as determined immunohistochemically . The immunogenicity of the two cell preparations was assessed in vitro by determining their capacity to evoke lymphocyte proliferation . Rat spleen lymphocytes were mixed with either a highly purified or semipurified population of bovine chromaffin cells . The results of this assay demonstrated that the highly purified preparation was a much weaker stimulant of lymphocyte proliferation than was the semipurified preparation and may demonstrate better graft survival in vivo . Transplantation via intrathecal catheter of either 80,000 or 250,000 cells from the highly or partially purified preparations onto the lumbar spinal cord of nonimmunosuppressed and non-nicotine-stimulated rats produced a cell number-dependent antinociception for both A(delta) and C fiber-mediated thermonociception at 6 days after transplantation . After 6 days and up to 28 days, only the "highly purified" preparation showed antinociception . These results suggest that nearly complete purification of bovine chromaffin cells minimizes immunorejection of xenogeneic transplants of these cells.

Cell Transplant, 1999 Jan-Feb, 8(1), 11 - 23
{3H}CNQX and NMDA-sensitive {3H}glutamate binding sites and AMPA receptor subunit RNA transcripts in the striatum of normal and weaver mutant mice and effects of ventral mesencephalic grafts; Mitsacos A et al.; Levels of excitatory amino acid receptors were studied in the weaver mouse model of DA deficiency after unilateral intrastriatal transplantation of E12+/+ mesencephalic cell suspensions . Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM {3H}GBR 12935 (average increase in grafted dorsal striatum compared to nongrafted side, 60%) . Autoradiography of 80 nM {3H}CNQX and 100 nM NMDA-sensitive {3H}glutamate binding was carried out to visualize the topography of non-NMDA and NMDA receptors, respectively, in +/+ mice and in recipient weaver mutants 3 months after grafting . Increases of 30% or more were found for {3H}CNQX binding in the dorsal nongrafted weaver striatum compared to +/+, and a further 6-9% increase in grafted weaver compared to nongrafted side . The added increase of non-NMDA receptors in the transplanted striatum might be explained by a presence of such receptors on DA presynaptic endings of graft origin . A 20% increase in NMDA-sensitive {3H}glutamate binding was measured in the dorsal nongrafted weaver striatum compared to +/+ . NMDA-sensitive {3H}glutamate binding in the transplanted side of weaver mutants tended to be slightly higher in all areas of the striatal complex compared to the nongrafted side, without reaching conventional levels of statistical significance . Using in situ hybridization histochemistry with synthetic 32p labeled oligonucleotide probes, we investigated RNA transcripts encoding the four AMPA receptor subunits . RNA transcripts in the striatum are seen with a decreasing signal intensity in the following order: GluRB > GluRA > GluRC > GluRD . The weaver caudate-putamen shows a 12% increase in GluRA subunit mRNA compared to +/+, whereas mesencephalic neuron transplantation leads to slight increases (3%) in the levels of GluRB mRNA in the nucleus accumbens . The results are placed in the context of the important interaction between the converging glutamatergic corticostriatal and the DAergic nigrostriatal pathways in controlling the functional output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.

Pol Merkuriusz Lek, 1999 Feb, 6(32), 104 - 6
{Flow cytometry: diagnostic, therapeutic and prognostic usefulness in solid tumors}; Iwaszkiewicz-Pawlowska A et al.; Flow cytometry is a new technique measuring several optical parameters of particular cells in cell suspension . These parameters are: optical density, light scatter and fluorescence emission . Optical assay conjugated with digital data processing permit the precise characterisation of chosen cellular subpopulations . The optical signs transformed by photomultipliers as analogue signals are presented as descriptive cytograms . Digitally converted signals analysed by multichannel analyser are stored as histigrams . Flow cytometry is widely used in the diagnostics of blood neoplastic diseases and, in a less routine manner, in the diagnostics of solid tumours . The latter requires accurate disaggregation of tumour tissue to obtain homogenous cell suspension . Different, more or less efficient, mechanical or enzymatic disaggregation techniques are used to prepare appropriate cell suspensions . Flow cytometry DNA analysis with simultaneous determination of several protein proliferation factors are heavily used in research and clinical laboratories to follow tumour cell proliferation, efficacy of cancer therapy and finally as prognostic factors . Although these assays yield promising results in some specific tumours, their value and accuracy seems still equivocal.

Folia Neuropathol, 1999, 37(1), 1 - 9
Survival of cryopreserved fetal substantia nigra implanted into the rat brain in the two forms and estimation of the host brain reaction against graft; Kosno-Kruszewska E; A fetal, cryopreserved ventral mesencephalic rat tissue was grafted into striatum of 143 healthy adult rats, applying two different forms, solid tissue block or cell-suspension . The fetal tissue was preserved in liquid nitrogen for 30-80 days . For transplantation stereotaxic placement technique was employed . The controls were subjected to sham transplantation . The survival of transplanted dopaminergic cells in rat striatum was evaluated by means of histological and immunocytochemical methods (TH-tyrosine hydroxylase) 1, 3, 7, 14 and 21 days after transplantation . The host cellular reaction against the graft and sham-lesion was examined . Glial fibrillary acidic protein (GFAP) was used for the visualization of astroglial reaction and ferritin for microglia . The occurrence of T-lymphocytes was also assessed using W3/13 antibodies . Electron microscopy was utilized as well in the evaluation . It was found that fetal cells of cryopreserved rat mesencephalon transplanted into adult rat striatum in two different models survive and mature similarly . The host cellular reaction against the graft was nonspecific and similar to that found in the control groups . Over a posttransplantation period of 21 days no graft rejection was observed in any of the experimental groups.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1999 Mar, 122(3), 297 - 306
The eicosanoid generating capacity of isolated cell populations from the gills of the rainbow trout, Oncorhynchus mykiss; Holland JW et al.; Rainbow trout gill filaments generated a wide range of eicosanoid products following calcium ionophore challenge . The putative lipoxygenase products were separated by reverse phase high performance liquid chromatography (RP-HPLC), while prostanoids were quantified by enzyme immunoassay . Three main monohydroxy compounds containing conjugated dienes were observed after RP-HPLC namely 12-(S) hydroxyeicosatetraenoic acid (12-HETE), 12-(S) hydroxyeicosapentaenoic acid (12-HEPE) and 14-(S) hydroxydocosahexaenoic acid (14-HDHE), derived from endogenous arachidonic, eicosapentaenoic and docosahexaenoic acids, respectively . Their identification was confirmed by mass spectrometry . A further five compounds containing conjugated trienes were also observed but in lesser amounts . One of these products was identified as 8,15-dihydroxyeicosatetraenoic acid (8,15-DiHETE) based on its UV spectrum, co-elution with authentic standard on RP-HPLC and mass spectrometry . Overall, the generation of these products suggests the presence of 12- and possibly 15-lipoxygenase activities in trout gill acting on endogenous sources of fatty acid . To determine if the various cell types in trout gill had differing eicosanoid generating potential, gills were disrupted and the resultant cell suspensions separated by density gradient centrifugation . Following this three bands were formed on the gradients and the cell populations from these were characterised using periodic acid Schiff's (PAS) reactivity for mucosubstances, haematoxylin and eosin staining, and immunoreactivity with both monoclonal and polyclonal antibodies . The first band consisted of polygonal cells and other more minor cell types, the second cell band contained mainly polygonal and PAS-positive goblet epithelial cells, while the third band consisted of mainly erythrocytes . There were significant differences in the eicosanoid generating potential of the isolated cells, with cells from the second band generating significantly more 12-HETE and 8,15-DiHETE than those from both the first band and unfractionated populations . The eicosanoid generating activity of the trout gill epithelial cell line, RTG-W1, was also elucidated . It proved to be a modest generator of eicosanoids in that only low levels of thromboxane B2 and prostaglandin E2 were detected while no lipoxygenase products were observed.

Anal Biochem, 1999 Jun 1, 270(2), 195 - 200
A microplate assay for DNA damage determination (fast micromethod); Batel R et al.; A rapid and convenient procedure for DNA damage determination in cell suspensions and solid tissues on single microplates was developed . The procedure is based on the ability of commercially available fluorochromes to interact preferentially with dsDNA in the presence of ssDNA, RNA, and proteins at high pH (>12.0), thus allowing direct measurements of DNA denaturation without sample handling or stepwise DNA separations . The method includes a simple and rapid 40-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 12.4 after NaOH addition . The time course and the extent of DNA denaturation is followed in a microplate fluorescence reader at room temperature for less than 1 h . The method requires only 30 ng DNA per single well and could conveniently be used whenever fast analysis of DNA integrity in small samples has to be done, e.g., in patients' lymphocytes after irradiation or chemotherapy (about 3000 cells per sample), in solid tissues or biopsies after homogenization (about 25 microg tissue per well), or in environmental samples for genotoxicity assessment .

Bioelectromagnetics, 1999, Suppl 4, 21 - 39
A quarter century of in vitro research: a new look at exposure methods; Guy AW et al.; The specific absorption rate (SAR) distributions in radio frequency-exposed solutions containing suspended or plated cells in vessels used for in vitro research were calculated by the finite-difference-time-domain method, graphed in color, and statistically analyzed in terms of uniformity for application to research on safety of wireless devices . The uniformity of SAR was quantified by visual inspection of colored plots, histograms, means, standard deviations, and maximums for the cell suspensions exposed in test tubes, Petri dishes, and rectangular flasks . Exposure sources included plane waves, transverse electromagnetic (TEM) cells, and striplines used at frequencies of 837, 2450, or 3,000 MHz . The results demonstrated that the most nonuniform SARs for plated or suspended cells in solution occurred for exposures of test tubes and rectangular flasks with plane waves, polarized for maximal absorption . The most uniform SARs for a layer of cells occurred for exposure of Petri dishes oriented for weakest coupling to the fields in a TEM cell . Additional improvement in uniformity was found to be possible by restricting the edge of the layer of cells from being too near the edges of the dish . It was not possible to achieve satisfactory uniformity in the SAR in cell suspensions exposed in standard vessels to any of the sources . The best but not satisfactory SAR uniformity was observed for cells suspended in the lowest 1-ml volume of the liquid contained in a test tube exposed at the bottom in a TEM cell . Experimental measurements of average SAR by temperature change for this case varied from 18% higher to 26% lower than finite difference time domain-derived values . The most uniform SAR distribution for cell suspensions in nonstandard containers was found for a rectangular slab configuration exposed in a stripline with the plates separated from the media by a thin layer of insulation . It is possible to experimentally implement this model by placing a fluid-filled thin-wall rectangular container tightly between the plates of a stripline.

J Pharmacol Toxicol Methods, 1998 Oct, 40(3), 137 - 43
Application of microcalorimetry for recording basal metabolic and Na+, K+-ATPase activity in LLC-PK1 cells, a model for the renal tubular epithelial cell; Xie Y et al.; In the present study we have employed a microcalorimetric procedure to measure the heat generated by a porcine renal tubule cell line (LLC-PK1) and its Na+, K+-ATPase . Microplates with an area of 2.2 cm2 were found to be optimal in terms of producing sufficient heat and a steady-state power curve . We compared the rate of heat production by cells in suspension and on monolayers and found a much lower value in suspension, that is, 1.42+/-0.2 versus 2.54+/-0.19 microW/microg DNA . Ouabain, the specific Na+, K+-ATPase inhibitor, caused a reduction in this heat output . The maximal inhibition in cell suspensions was 40% and remained unchanged with as much as 100 microM ouabain, the highest concentration tested . With cells cultured on microplates, ouabain in the concentration interval 0.1-3 microM caused a 25% inhibition of heat output . With 25-100 microM ouabain, a 50% inhibition was observed and at higher concentrations, no further inhibition occurred . Furthermore, upon removal of ouabain, full recovery of the Na+, K+-ATPase was observed, a process that could easily be monitored by using cell monolayers cultured on microplates.

Haematologica, 1999 May, 84(5), 397 - 404
Clinical and molecular follow-up by amplification of the CDR-III IgH region in multiple myeloma patients after autologous transplantation of hematopoietic CD34+ stem cells; Martinelli G et al.; BACKGROUND AND OBJECTIVE: Autologous blood stem cell transplantation (ABSCT) using chemotherapy-induced mobilization of peripheral blood stem cells (PBSC) is being increasingly used in the treatment of multiple myeloma (MM) . We report the clinical and molecular follow-up of 10 MM patients who underwent autologous stem cell transplantation with peripheral blood selected CD34+ cells, as support therapy following a myeloablative conditioning regimen . DESIGN AND METHODS: The CDR-III coding region of the IgH gene was studied by a) consensus PCR applied to 8 MM patients, or b) by direct sequencing of PCR product generated by family-specific primers in the remaining two patients (who became immunofixation analysis (IF) negative) . In this case, two patient-specific primers were generated, thus obtaining a high PCR assay sensitivity and specificity (ASO PCR) . RESULTS: Seven patients are alive: 4 of them have serum M protein assessable by IF, while 1 was not a secretor and 2 converted from serum IF positivity to negativity 6 and 12 months after ABSCT . Three patients died: 1 from disease progression and 2 from infective complications during clinical remission . The molecular analysis during the follow-up showed that the bone marrow samples from the two patients who obtained IF negativity were persistently PCR positive for the presence of rearranged CDR-III region . Moreover, despite the remarkable reduction of myeloma burden, a minimal level of residual myeloma cells was still detectable by molecular analysis . INTERPRETATION AND CONCLUSIONS: These results confirm that although positive selection of CD34+ cells markedly reduces the contamination of myeloma cells from apheresis products by up to 3 log, and provides a cell suspension capable of restoring normal hematopoiesis after ablative conditioning regimen, it does not abrogate myeloma cell contamination in most of the apheresis products.

Haematologica, 1999 May, 84(5), 390 - 6
In vitro growth and quantification of early (CD33-/CD38-) myeloid progenitor cells: stem cell factor requirement and effects of previous chemotherapy; Ferrero D et al.; BACKGROUND AND OBJECTIVE: All culture systems exploring the early (pre-CFU) hematopoietic compartment are generally complex, time-consuming and unsuitable for routine application . The aim of our study was to develop a stroma-free culture system to quantify early bone marrow (BM) myeloid progenitor cells . DESIGN AND METHODS: Low density, progenitor cell enriched BM cells underwent a double cytotoxic treatment with CD38 and CD33 monoclonal antibodies + rabbit complement, which depleted 99% of CFU-GM and BFU-E . Then they were cultured, both in agar and in limiting-dilution liquid culture, in the presence of 5637 cell line supernatant (containing GM-CSF, G-CSF and interleukin 1 ), stem cell factor (SCF) and interleukin 3 (IL3) . RESULTS: The largest number (median 14.9 on 1x10(5) cells) and size (>50,000 cells) of myelomonocytic cell clones from CD33Eth /CD38Eth progenitors was reached after 3-4 weeks of liquid culture . SCF, but not IL3, was essential for that growth . The frequency of CD33-/ CD38- progenitors grown in liquid culture was approximately three times greater than the LTC-IC frequency in the same cell suspension . An average 93% of CD33-/CD38- progenitors displayed HLA-DR antigens and 43% generated secondary CFU-GM . In the BM of 9/10 patients, previously exposed to chemotherapy, CD33-/CD38- progenitor frequency was quite low (median 0.9 on 1x10(5) cells), in spite of normal cellularity and morphology and sustained disease remission . INTERPRETATION AND CONCLUSIONS: CD33-/CD38- progenitors can be grown and quantified in a stroma-free culture system in a relatively short time . The test can reveal long-lasting, subclinical BM damage induced by chemotherapy and could also be valuable for estimating the amount of early myeloid progenitors for transplantation purposes.

Neurosci Lett, 1999 Apr 16, 265(2), 79 - 82
The potentiation of amphetamine-induced hyperlocomotion by fimbria-fornix lesions in rats is abolished by intrahippocampal grafts rich in serotonergic neurons; Balse E et al.; Three-month old Long-Evans female rats were submitted to aspirative lesions of the fimbria-fornix and intrahippocampal grafts of a cell suspension prepared from a region of the fetal brain including the septum and the diagonal band of Broca (rich in cholinergic neurons) or the raphe (rich in serotonergic neurons) . A group of lesioned rats was grafted with both suspensions mixed . Lesion-only and sham-operated rats served as controls . Four months after the lesions, all rats were tested daily for locomotor activity in their home cage, 1 day without being injected, 2 days with an injection of NaCl and 5 days with an injection of 1 mg/kg (i.p.) d-amphetamine . The effects of the lesions and grafts were assessed by measuring the accumulation of {3H}-choline or {3H}-5-hydroxytryptamine (5-HT) by hippocampal slices, and the electrically-evoked release of tritium . Amphetamine injections produced hyperlocomotion which was potentiated by the lesion . This lesion-induced potentiation was also found in rats with septal grafts, but not in those with raphe or co-grafts . The uptake and electrically-evoked release of {3H}-acetylcholine or {3H}-5-HT were reduced in hippocampal slices from lesion-only rats . In rats which received grafts of septal cells or co-grafts, but not in those with raphe grafts, uptake and release of {3H}-acetylcholine were close to normal . Uptake and release of {3H}-5-HT were close to normal in rats with raphe grafts or with co-grafts, but not in those with septal grafts . Altogether, these data suggest that damage to the serotonergic afferents of the hippocampus might play some role in the potentiation of amphetamine-induced hyperlocomotion associated with fimbria-fornix lesions.

Cardiovasc Res, 1999 Jan, 41(1), 246 - 54
Rabbit mononuclear leukocytes cause contraction of isolated aorta by the release of serotonin; Hart-Favaloro JL et al.; OBJECTIVE: The aim of this study was to examine the vasoactive effects of rabbit isolated mononuclear leukocytes, and to identify the mediators responsible for those vasoactive effects . METHODS: Mononuclear leukocytes (MNLs) were isolated from New Zealand White rabbit whole blood, suspended at 5 x 10(7) cells/ml and incubated for 30 min at 37 degrees C . This cell suspension, or the cell-free supernatant from this suspension, were then examined for vasoactive effects in rabbit isolated thoracic aorta . RESULTS: Both the MNL suspension and the cell-free supernatant from this suspension caused endothelium-independent contraction of aortic rings, both from resting tension and when pre-contracted . The MNL suspension caused a significantly greater contraction than the MNL supernatant under all conditions . The contractions to the MNL product were significantly inhibited by the 5-HT2 receptor antagonist ketanserin (0.1 microM), but not by the alpha 1-adrenoceptor antagonist prazosin (10 microM) . High-performance liquid chromatography (HPLC) analysis showed that the MNL supernatant contained serotonin (5-hydroxytryptamine, 5-HT) at an average concentration of 5 microM . CONCLUSIONS: We conclude that MNLs cause contraction of rabbit isolated aortic rings by the release of 5-HT.

Circ Res, 1999 May 14, 84(9), 1020 - 31
Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes; Vila-Petroff MG et al.; Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels . We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1-loaded adult cardiac myocytes . Whereas a high concentration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 micromol/L) significantly attenuated contraction amplitude by 24.4+/-4.5% (without changing the Ca2+ transient or total cAMP), a low concentration of SNAP (1 micromol/L) significantly increased contraction amplitude (38+/-10%), Ca2+ transient (26+/-10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein) . The negative contractile response of 100 micromol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 micromol/L); the positive contractile response of 1 micromol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ, 10 micromol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 micromol/L), as well as by the NO scavenger oxyhemoglobin . Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 micromol/L) of SNAP (AC, 18% to 20% above basal activity) . We conclude that NO can regulate both AC and GC in cardiac myocytes . High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+ . Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.

Zhongguo Ying Yong Sheng Li Xue Za Zhi, 1997 Nov, 13(4), 319 - 21
{The protective effect of SOD on injury of bone marrow hematopoietic progenitor cells of mice under 4 degrees C storage}; Liu X et al.; The protective effect of Cu, Zn, SOD on bone marrow hematopoietic progenitor cells of mice, under 4 degrees C storage was studied . The results showed, if 1.65 U or 0.165 U/ml SOD was added to the cell suspension for 3 days at 4 degrees C, the production rate of CFU-GM, CFU-E, BFU-E, CFU-Meg and CFU-Mix were 6.2, 2.6, 2.9, 4.0 and 5.1 times that of control group (without SOD) respectively . The survival rate of hematopoietic progenitor cells increased obviously . The mechanism of SOD effect is supposed to prevent hematopoietic progenitor cells from death, instead of the regulating proliferation . It may be closely related to the antioxidation of SOD which scavenges the superoxide free radicals.

Phytochemistry, 1999 Apr, 50(7), 1099 - 109
Purification, partial amino acid sequence and structure of the product of raucaffricine-O-beta-D-glucosidase from plant cell cultures of Rauwolfia serpentina; Warzecha H et al.; Plant cell suspension cultures of Rauwolfia produce within 1 week approximately 250 nkat/l of raucaffricine-O-beta-D-glucosidase . A five step procedure using anion exchange chromatography, chromatography on hydroxylapatite, gel filtration and FPLC-chromatography on Mono Q and Mono P delivered in a yield of 0.9% approximately 1200-fold enriched glucosidase . A short protocol employing DEAE sepharose, TSK 55 S gel chromatography and purification on Mono Q gave a 5% recovery of glucosidase which was 340-fold enriched . SDS-PAGE showed a Mr for the enzyme of 61 kDa . The enzyme is not glycosylated . Structural investigation of the enzyme product, vomilenine, demonstrated that the alkaloid exists in aqueous solutions in an equilibrium of 21(R)- and 21(S)-vomilenine in a ratio of 3.4:1 . Proteolysis of the pure enzyme with endoproteinase Lys C revealed six peptide fragments with 6-24 amino acids which were sequenced . The two largest fragments showed sequences, of which the motif Val-Thr-Glu-Asn-Gly is typical for beta-glucosidases . Sequence alignment of these fragments demonstrated high homologies to linamarase from Manihot esculenta (81% identity) or to beta-glucosidase from Prunus avium (79% identity) . Raucaffricine-O-beta-D-glucosidase seems to be a new member of the family 1 of glycosyl hydrolases.

J Immunol, 1999 May 15, 162(10), 5949 - 56
Fibroblast-like synoviocytes from rheumatoid arthritis patients have intrinsic properties of follicular dendritic cells; Lindhout E et al.; The production of IgG rheumatoid factors in the inflamed synovium of many patients with rheumatoid arthritis (RA) implies that local sites exist where plasma cell precursors undergo isotype switching and affinity maturation by somatic mutation and selection . Lymphonodular infiltrates of the synovium-containing germinal centers (GCs), are candidates to fulfill such function in the rheumatoid patient . It has been suggested that these GCs are organized around, obviously ectopic, follicular dendritic cells (FDCs) . The present study attempts to find out whether these putative FDCs 1) are specific for RA, 2) have the same phenotype and functional capacity as FDCs in lymphoid organs, and 3) may locally differentiate from fibroblast-like synoviocytes (FLS) . Synovial biopsies from patients with RA versus non-RA, yet arthritic backgrounds, were compared . Cells with the FDC phenotype were found in both RA and non-RA tissues as well as in single cell suspensions thereof . When FLS were cultured in vitro, part of these cell lines could be induced with IL-1beta and TNF-alpha to express the FDC phenotype, irrespective of their RA or non-RA background . By contrast, the FDC function, i.e., stable binding of GC B cells and switching off the apoptotic machinery in B cells, appeared to be the prerogative of RA-derived FLS only . The present data indicate that FDC function of FLS in RA patients is intrinsic and support the idea that synovial fibroblast-like cells have undergone some differentiation process that is unique for this disease.

Bioelectrochem Bioenerg, 1999 Feb, 48(1), 17 - 25
Chinese hamster ovary cells sensitivity to localized electrical stresses; Vernhes MC et al.; Application of an external electric field on a cell suspension induces an alteration in the membrane structure giving free access to the cell cytoplasm . Under mild pulsation conditions, permeabilization is a reversible process which weakly affects cell viability while drastic electrical conditions lead to cell death . The field pulse must be considered as a complex stress applied on the cell assembly . This study is a systematic investigation of the stress effects of field strength, pulse duration and number of pulses, at given joule energy . The loss in cell viability is not related to the energy delivered to the system . At a given joule energy, a strong field during a short cumulated pulse duration affects more viability than using a weak field associated with a long cumulated pulsation . At a given field strength and for a given cumulated pulse duration an accumulation of short pulses is also observed to be very damaging for cells . A control by the delay between the pulses suggests a memory effect . The field effect appears also to be vectorial in line with the known asymmetry of the membrane organization . These results suggest that processes at a cellular level are involved, either an activation of cell death or damage in cellular functions.

Life Sci, 1999, 64(15), 1329 - 37
In vitro evaluation of nanoparticles spleen capture; Demoy M et al.; After intravenous injection, the main part of nanoparticles trapped by the spleen are concentrated in the marginal zone . The first step of this capture is the adhesion of the particles to the marginal zone macrophages . As classical techniques of cell suspension preparation did not allow to isolate without damage these actively capturing cells, tightly bound to a well-developed reticular meshwork, we designed a tissue slice incubation method, in order to study in vitro the interaction of nanoparticles with these particular macrophages, in conditions close to in vivo . In a serum supplemented medium, this in vitro model was able to give similar uptake profile than after intravenous injection of nanoparticles thus proving its validity . Surprisingly, no significant decrease of nanoparticles capture was observed when the medium was depleted from complement, immunoglobulins or proteins affine for heparin, while substitution of serum by purified albumin allowed a near optimal uptake . Addition of competitive ligands for lectin-like receptors did not show any clear inhibition of spleen capture . On the other hand, the scavenger receptor blocking agents, such as maleylated albumin or polyinosinic acid, induced a strong reduction of the spleen nanoparticles uptake . Thus, this paper proposes an in vitro binding assay as a reliable method to investigate the spleen capture of a large variety of nanoparticulate drug carriers . It is also a useful methodology to highlight the interactions between spleen cells and nanoparticles . The data obtained suggest that capture of nanoparticles depends on a multifactorial and complex phenomenon involving for a part albumin and the scavenger receptor.

Artif Cells Blood Substit Immobil Biotechnol, 1999 May, 27(3), 255 - 61
Culture of cells at perfluorocarbon-aqueous interfaces; Lowe KC et al.; Protoplasts (wall-less cells) isolated enzymatically from leaf tissues of Manihot esculenta, Passiflora edulis and Petunia parodii, and from cell suspensions of Oryza sativa, Passiflora giberti, Petunia hybrida and Salpiglossis sinuata, were cultured for up to 35 d at an interface between the inert, oxygen-gassed perfluorocarbon (PFC) liquid, perfluorodecalin, overlaid with liquid or semi-solidified aqueous media . The maximum increase in mitotic division, as assessed by initial plating efficiency (IPE) occurred with protoplasts of O . sativa, which showed a 4-fold increase above the control over 35 d . Similar, but less pronounced increases in IPE of 90-103% occurred with S . sinuata, P . giberti and P . parodii following culture with oxygenated PFC . The least responsive species was M . esculenta, where the mean IPE after 25 d was increased by 33% over control . For those totipotent protoplast systems (e.g . P . edulis, P . giberti, O . sativa and P . parodii) phenotypically normal plants were regenerated following initial culture with oxygenated PFC . The advantages of such an interface system include (1) ease of sterilisation of the PFC by autoclaving, (2) the recycleability and, hence, recovery of the PFC, thereby offsetting the high initial costs, and (3) the ability to aspirate cells at the interface.

Anticancer Res, 1999 Jan-Feb, 19(1A), 409 - 12
In vitro toxicity of taxol based anticancer drug combinations on human hemopoietic progenitors; Pannacciulli I et al.; The sequence dependency of the interaction of taxol with other anticancer drugs is of clinical importance, and may be due to pharmacokinetic changes and/or to inherent differences in the sensitivity of target normal or cancer cells . This study presents results on the in vitro interaction of taxol with doxorubicin, cisplatin, etoposide and vinorelbine in alternate sequences on human hemopoietic progenitors (CFU-GM) . Peripheral blood mononuclear non adherent cells were exposed to IC50 of Taxol for 24 hours and then, for 1 hour to IC50 of each of the other drugs . In a second set of experiments the reverse sequence was applied . The cell suspension was subsequently cultured to assay the growth of CFU-GM . A strong sequence dependency characterizes the combination taxol-vinorelbine, while for the other combinations the order of sequence appears to have little impact on in vitro toxicity on CFU-GM . Comparing results on CFU-GM with that obtained in vitro with the same combination sequences on cancer cell lines some remarkable differences show up . Studies on a normal human myeloid line may therefore have a place in preclinical evaluation of sequence of anticancer drug combinations.

Infect Immun, 1999 May, 67(5), 2428 - 32
Immune response to Nocardia brasiliensis antigens in an experimental model of actinomycetoma in BALB/c mice; Salinas-Carmona MC et al.; Nine- to twelve-week-old BALB/c mice were injected in footpads with 10(7) CFU of a Nocardia brasiliensis cell suspension . Typical actinomycetoma lesions, characterized by severe local inflammation with abscess and fistula formation, were fully established by day 28 after infection . These changes presented for 90 days, and then tissue repair with scar formation slowly appeared, with complete healing after 150 days of infection . Some animals developed bone destruction in the affected area . Histopathology showed an intense inflammatory response, with polymorphonuclear cells and hyaloid material around the colonies of the bacteria, some of which were discharged from draining abscesses . Sera from experimental animals were analyzed by Western blotting, and immunodominant antigens P61 and P24 were found as major targets for antibody response . Anti-P24 immunoglobulin M (IgM) isotype antibodies were present as early as 7 days, IgG peaking 45 days after infection . Lymphocyte proliferation with spleen and popliteal lymph node cells demonstrated thymidine incorporation at 7 days after infection, the stimulation index decreasing by day 60 . Levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) were determined by enzyme-linked immunosorbent assay in the sera of infected animals . The circulating levels of IFN-gamma increased more than 10 times the basal levels; levels of IL-4, IL-6 and IL-10 also increased during the first 4 days of infection.

Hum Gene Ther, 1999 Apr 10, 10(6), 983 - 93
Immunotherapy of metastatic malignant melanoma by a vaccine consisting of autologous interleukin 2-transfected cancer cells: outcome of a phase I study; Schreiber S et al.; We performed a phase I trial to evaluate the safety and tolerability of repeated skin injections of IL-2-transfected autologous melanoma cells into patients with advanced disease . Cell suspensions, propagated from excised metastases, were IL-2 gene transfected by adenovirus-enhanced transferrinfection and X-irradiated prior to injection . Vaccine production was successful in 54% of the patients . Fifteen patients (37%) received two to eight skin vaccinations of either 3 x 10(6) (intradermal) or 1 x 10(7) (half intradermal, half subcutaneous) transfected melanoma cells per vaccination (secreting 140-17,060 biological response modifier program units of IL-2/10(6) cells/24 hr) . Analyses of safety and efficacy were carried out in 15 and 14 patients, respectively . Overall, the vaccine was well tolerated . All patients displayed modest local reactions (erythema, induration, and pruritus) and some experienced flu-like symptoms . Apart from newly appearing (4 of 14) and increasing (5 of 14) anti-adenovirus and newly detectable anti-nuclear antibody titers (1 of 15), recipients developed de novo or exhibited increased melanoma cell-specific delayed-type hypersensitivity (DTH) reactions (8 of 15) and vitiligo (3 of 15) and showed signs of tumor regression (3 of 15) . This supports the idea of a vaccine-induced or -amplified anti-cancer immune response . None of the patients exhibited complete or partial regressions, but five of them experienced periods of disease stabilization . Three of these individuals received more than the four planned vaccinations and their mean survival time was 15.7 +/- 3.5 months as compared to 7.8 +/- 4.6 months for the entire patient cohort . These data indicate that IL-2-producing, autologous cancer cells can be safely administered to stage IV melanoma patients and could conceivably be of benefit to patients with less advanced disease.

Exp Neurol, 1999 May, 157(1), 58 - 68
The transplantation of human fetal neuroretinal cells in advanced retinitis pigmentosa patients: results of a long-term safety study; Das T et al.; The purpose of this study was to determine the long-term safety of transplanting human fetal neuroretinal cells (14 to 18 week gestational age) into a series of patients with advanced retinitis pigmentosa (RP) . After obtaining informed consent, both hosts and mothers of donors were screened for transmissible diseases . Pre- and postoperative clinical exams, visual acuity, electroretinograms, and fluorescein angiograms were performed and visual field testing was attempted in each case . Surgically, an anterior approach through pars plana ciliaris was used . A retinotomy was performed in the paramacular area and a two-function cannula was introduced into the subretinal space to deliver a suspension of donor cells . The cell suspension carried approximately 4000 cells/microl; the volume injected did not exceed 150 microl . The patients were examined for periods ranging from 12 to 40 months posttransplantation . To date, no evidence of inflammation, infection, or overt rejection of the graft was noted in the host eye, neither was any change observed in the contralateral, unoperated eye . In conclusion, neuroretinal cells were injected into the subretinal space of 14 patients with advanced RP with no clinical appearance of detrimental effects at the time of surgery or up to 40 months postinjection except in 1 patient who developed retinal detachment . This sets the stage for a phase II clinical trial to determine the possible beneficial effects of this procedure in patients blinded by degenerative retinal disease .

Anal Biochem, 1999 May 1, 269(2), 230 - 5
Flow injection analysis of binding reaction between fluorescent lectin and cells; Oda Y et al.; A fluorometric binding assay for lectin and yeast cells using the avidin-biotin system was previously reported (Y . Oda, M . Kinoshita, and K . Kakehi, Anal . Biochem . 254, 41-48, 1997) . However, the true amount of bound lectin could not be determined by this method due to difficulty in determination of the number of bound biotin molecules . In the present study, we have developed a method for assaying the binding reaction between fluorescent lectin and cells using a flow injection technique, which allows estimation of the amount of lectin bound to cells . An aliquot of the cell suspension was directly analyzed by injection into a flow injection system after the binding between the fluorescently labeled lectin and cells . The labeled lectins showed good linearity, at least over a range of 20-1000 ng as the injected amount . The intrinsic fluorescence of the labeled lectins did not change upon the binding . The binding reaction of the hydroxycoumarin-labeled lectins with yeast cells was rapid and reached an equilibrium state within 10 min . Scatchard analysis showed that Saccharomyces cerevisiae cells contained approximately 1 . 3-1.6 x 10(8) binding sites per cell for Concanavalin A, Lycoris radiata agglutinin, and Tulipa gesneriana lectin with affinity constants of 3.2-4.7 x 10(6) M-1 . The present method was applied to the study of binding between lectins and bacteria and mouse spleen cells . The assay method described here is highly sensitive and will be an alternative to assays using lectins labeled with radioisotopes . The procedure is quite simple and can be completed within 1 h .

Hum Reprod, 1999 Apr, 14(4), 1022 - 7
Immature germ cell separation using a modified discontinuous Percoll gradient technique in human semen; Gandini L et al.; The difficulty of identifying immature germ cells in unstained, fresh semen has led most laboratories to use the broad definition 'round cells' to indicate cells other than spermatozoa, thus grouping together both leukocytes and immature germ cells . This is also the case in research andrology, where very little attention has been given to immature germ cells in the semen apart from some rare exceptions, such as the attempts to study meiosis . Here we report on the use of a discontinuous Percoll gradient method modified to enable the best separation possible of immature germ cells from the other cells found in the ejaculate, in order to obtain a cellular suspension free of spermatozoa . Our technique (intra-assay variation in duplicates < 10%) demonstrated a high immature germ cell concentration in gradient fractions with 30% to 45% Percoll with a small contamination (1.5-6%) of leukocytes, confirmed by May-Grunwald-Giemsa staining, immunofluorescence and cytofluorimetry . The concentrations of immature germ cells ranged from zero in obstructive azoospermia to 2.0 x 10(6)/ml in oligozoospermia and genital tract infection . The purified immature germ cell suspensions obtained can be useful for diagnostic and research purposes.

Clin Chim Acta, 1999 Mar, 281(1-2), 1 - 17
Quantitative acylcarnitine profiling in fibroblasts using {U-13C} palmitic acid: an improved tool for the diagnosis of fatty acid oxidation defects; Ventura FV et al.; A method was developed for the investigation of mitochondrial fatty acid beta-oxidation in cultured fibroblasts . Monolayer cultures were incubated without foetal calf serum with commercially available {U-13C} palmitic acid and L-carnitine for 96 h . The acylcarnitines produced by the cells were extracted from the cell suspension and analysed either by quantitative stable isotope dilution gas chromatography chemical ionization mass spectrometry, or by fast atom bombardment mass spectrometry . Characteristic acylcarnitine profiles were obtained for all the different enzyme deficiencies investigated, with the exception of carnitine palmitoyltransferase II deficiency and carnitine/acylcarnitine carrier deficiency which showed similar patterns . Comparison between this method and the 3H-myristate and 3H-palmitate tritium release assays revealed that the method described here is superior, allowing unequivocal identification of patients.

FEBS Lett, 1999 Apr 1, 448(1), 135 - 40
Native acridone synthases I and II from Ruta graveolens L . form homodimers; Lukacin R et al.; Acridone synthase II cDNA was cloned from irradiated cell suspension cultures of Ruta graveolens L . and expressed in Escherichia coli . The translated polypeptide of Mr 42,681 revealed a high degree of similarity to heterologous chalcone and stilbene synthases (70-75%), and the sequence was 94% identical to that of acridone synthase I cloned previously from elicited Ruta cells . Highly active recombinant acridone synthases I and II were purified to apparent homogeneity by a four-step purification protocol, and the affinities to N-methylanthraniloyl-CoA and malonyl-CoA were determined . The molecular mass of acridone synthase II was estimated from size exclusion chromatography on a Fractogel EMD BioSEC (S) column at about 45 kDa, as compared to a mass of 44 +/- 3 kDa found for the acridone synthase I on Superdex 75 . Nevertheless, the sedimentation analysis by ultracentrifugation revealed molecular masses of 81 +/- 4 kDa for both acridone synthases . It is proposed, therefore, that the acridone synthases of Ruta graveolens are typical homodimeric plant polyketide synthases.

Planta, 1999 Mar, 208(1), 12 - 8
Maize glutathione-dependent formaldehyde dehydrogenase: protein sequence and catalytic properties; Wippermann U et al.; Glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1.2.1.1) has been purified 3900-fold from maize cell-suspension cultures to a specific activity of 4.68 mumol (mg protein)-1 min-1 . The homogeneous enzyme consisted of two identical subunits with a molecular mass of 42 kDa, and an isoelectric point of 5.8 . Eight tryptic peptides were sequenced and gave a perfect fit to the protein sequence derived from maize Fdh cDNA (J . Fliegmann and H . Sandermann, 1997, Plant Mol Biol 34: 843-854) . There was 62% identity with the eucaryotic FDH consensus sequence . Michaelis constants of approx . 20 microns (formaldehyde), approx . 50 microns (glutathione) and approx . 31 microns (NAD+) were determined for the maize enzyme as well as for FDH partially purified from dog lung . Besides S-hydroxymethylglutathione, pentanol-1, octanol-1, and omega-hydroxy-fatty acids served as substrates for both FDH preparations . The unusual substrate specificity indicates that FDH may be involved in the detoxification of long-chain lipid peroxidation products.

Dis Colon Rectum, 1999 Jan, 42(1), 10 - 5
Influence of cytotoxic agents on intraperitoneal tumor implantation after laparoscopy; Neuhaus SJ et al.; PURPOSE: Recent experimental studies suggest that laparoscopic surgery for abdominal malignancy may be associated with increased tumor implantation . This study investigated the influence of cytotoxic agents (administered intraperitoneally or intramuscularly) on implantation of a tumor cell suspension after laparoscopic surgery in an experimental model . METHODS: Thirty-three Dark Agouti rats underwent laparoscopy with CO2 insufflation and instillation of a tumor cell suspension into the abdominal cavity . Rats were randomly allocated to one of the following study groups (9 rats in the control group, 6 rats in all other groups): 1) control (no intraperitoneal instillation); 2) intraperitoneal normal saline (0.9 percent); 3) intraperitoneal povidone-iodine (Betadine to normal saline 1:10 dilution); 4) intraperitoneal methotrexate (2 doses of 0.125 mg/kg body weight in normal saline administered 24 hours apart); 5) intramuscular injection of 2 doses of 0.125 mg/kg body weight administered 24 hours apart (no intraperitoneal agent) . Rats were killed 7 days after the procedure, and the peritoneal cavity and port sites were examined for the presence of tumor . RESULTS: A significant reduction in tumor implantation and port-site metastases was observed in all treatment groups (povidone-iodine and intramuscular and intraperitoneal methotrexate) . CONCLUSIONS: This study suggests that tumor implantation after laparoscopic surgery and port-site metastases might be prevented by the intraperitoneal or systemic administration of cytotoxic agents . Further studies are needed to determine whether these findings can be applied to clinical practice.

Oral Oncol, 1999 Jan, 35(1), 86 - 92
Fluid phase endocytosis within buccal mucosal cells of alcohol misusers; Axford SE et al.; The structure of the oral mucosa is now well characterised, although studies on oral epithelial cell function have received less attention . The aims of this study were to see whether endocytosis could be demonstrated in cells from oral smears and if so, to assess the effect of chronic high alcohol intake on such uptake . Buccal mucosal smears were collected from 135 patients (91 non- or social drinkers, and 44 patients with harmful alcohol use) . Name, age, sex, and alcohol history (for alcohol problem patients) were recorded . Cell suspensions were incubated in a solution of bovine serum albumin (BSA)-coated fluorescently labelled latex microspheres (0.02 micron diameter) in Ham's F-10 culture medium for 1 h at 37 degrees C as a marker of fluid phase endocytosis . Uptake of microspheres was confirmed by confocal microscopy, and mean endocytosed fluorescence levels determined by flow cytometry . A repeat smear from 11 of the alcohol patients was taken 9-14 days later . Endocytosis was significantly reduced in both male (P < 0.01) and female (P < 0.01) alcohol problem patients compared to controls . Units of alcohol consumed and cigarettes smoked per day did not show a dose-response correlation with endocytosis in the alcohol problem patients . Apparent abstinence from alcohol had no further effect on endocytic uptake at days 9-14 . This study shows that normal oral squamous cells removed as buccal smears readily endocytose fluorescent microspheres and that this capacity can be affected by alcohol . Chronic high alcohol intake would appear to down regulate endocytosis in buccal cells even up to 14 days of abstinence . This may have implications for the pathogenesis of oral mucosal disorders in long-term users.

Biorheology, 1998 Jan-Feb, 35(1), 69 - 87
Effect of nonaxisymmetric hematocrit distribution on non-Newtonian blood flow in small tubes; Das B et al.; Hematocrit distribution and red blood cell aggregation are the major determinants of blood flow in narrow tubes at low flow rates . It has been observed experimentally that in microcirculation the hematocrit distribution is not uniform . This nonuniformity may result from plasma skimming and cell screening effects and also from red cell sedimentation . The goal of the present study is to understand the effect of nonaxisymmetric hematocrit distribution on the flow of human and cat blood in small blood vessels of the microcirculation . Blood vessels are modeled as circular cylindrical tubes . Human blood is described by Quemada's rheological model, in which local viscosity is a function of both the local hematocrit and a structural parameter that is related to the size of red blood cell aggregates . Cat blood is described by Casson's model . Eccentric hematocrit distribution is considered such that the axis of the cylindrical core region of red cell suspension is parallel to the axis of the blood vessel but not coincident . The problem is solved numerically by using finite element method . The calculations predict nonaxisymmetric distribution of velocity and shear stress in the blood vessel and the increase of apparent viscosity with increasing eccentricity of the core.

Biorheology, 1998 Jan-Feb, 35(1), 53 - 68
The kinetics of thrombin- and SFLLRN-induced aggregation of human platelets in flow through tubes; Goldsmith HL et al.; The kinetics of aggregation of human platelets activated by alpha-thrombin (0.17-0.35 nM) and the hexapeptide SFLLRN (2-10 microM) was studied in plasma-free washed cell suspensions undergoing Poiseuille flow at 37 degrees C using a previously described double infusion technique . Platelet-rich Tyrodes, prepared from venous blood by multiple centrifugation, and agonist were rapidly mixed in a small chamber and the suspension flowed through various lengths of 1.19 and 0.76 mm diameter polyethylene tubing at mean transit times t from 0.2 to 43 s and mean tube shear rates <G> = 41.9, 335, and 1335 s-1 . Effluent was collected in 0.5% glutaraldehyde and single cells and aggregates in the volume range 1-10(5) micron 3 counted and sized using an aperture impedance counter . The rate and extent of aggregation with thrombin increased with increasing {thrombin} and <G>, and although characterized by a small initial lag time, exhibited a very rapid growth of aggregates to macroscopic size, >> 10(5) micron 3, at low and moderate shear rates . With SFLLRN, the initial lag times were appreciably longer, but subsequently aggregates also rapidly grew to macroscopic size . We hypothesize that the initial lag time is due to the time required for sufficient secretion and surface organization of ligands such as vWF (known to be released by the platelet) to occur, in order for cross-bridging of the GPIIb-IIIa receptors on adjacent platelets to take place . It appears that thrombin, which, at the low concentrations used, primarily activates the platelet via binding to the GPIb alpha receptor, can more rapidly facilitate secretion of the ligand than SFLLRN, which activates the cell via binding to the seven transmembrane domain receptor.

J Physiol Pharmacol, 1999 Mar, 50(1), 75 - 87
Effects of nitroglycerin on energy metabolism of rat reticulocytes; Maletic SD et al.; Nitric oxide (NO) in many cells inactivates aconitase and mitochondrial respiratory chain, and influenced glyceraldehyde 3-phosphate dehydrogenase activity . The aim of this study was to evaluate role of nitroglycerin (NTG), a widely used NO donor, on energy metabolism of rat reticulocytes . Rat reticulocyte rich red blood cell suspensions containing 70-100% of reticulocytes, were aerobically incubated without (control) or in the presence of different concentrations of (a) NTG (0.1, 0.25, 0.5, 1.0, 1.5 mmol/l), (b) 8-Br-cGMP (0.1, 0.5, 1.0 mmol/l) and (c) NaNO2 and NaNO3 (1 mmol/l) . NTG in dose- and time-dependent manner decreased total (p>0.05; EC50 = 0.78+/-0.05 mmol/l) and coupled (p<0.05; EC50 = 0.50+/-0.04 mmol/l) and increased uncoupled oxygen consumption (p<0.05: EC50 = 0.36+/-0.01 mmol/l) . They were accompanied by stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.001 EC50 = 0.53 and 0.53 mmol/l, respectively) . Levels of all glycolytic intermediates in the presence of NTG indicate stimulation of HK-PFK, GA3PDH and PK activity . NTG significantly decreased ATP level, which accompanied by increased ADP and AMP levels . However, level of total adenine nucleotides (TAN) was significantly lower, which was consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level; p<0.05) . Stimulation of glycolysis accompanied with inhibition of the OxP, activation of HK-PFK, decrease of ATP and simultaneous rise of ADP and AMP levels, all together represent an example of Pasteur effect occurring in NTG-treated reticulocytes . In rat reticulocytes under steady state conditions 93% of overall energy was produced by OxP, but only 7% by glycolysis . Due to decrease of coupled oxygen consumption in the presence of NTG, ATP production via OxP was significantly diminished . Simultaneous increase of glycolytic ATP production is not enough to provide constant either ATP production or concentration . Calculated mean ATP-turnover time was prolonged even for 45% in the presence of 1.5 mmol/l NTG . Metabolic effects of NTG were not mimic by exogenous 8-Br-cGMP, NaNO2 or NaNO3, which indicate that NTG induced a) inhibition of coupled respiration and b) stimulation of glycolysis in rat reticulocytes are mediated by NO as an effector molecule.

Graefes Arch Clin Exp Ophthalmol, 1999 Apr, 237(4), 326 - 35
Cellular response in rabbit eyes after human fetal RPE cell transplantation; Gabrielian K et al.; BACKGROUND: This study was carried out to study inflammatory and proliferative cellular responses in the rabbit eye after subretinal transplantation of human fetal retinal pigment epithelial (HFRPE) cells . METHODS: 5-Bromo-2-deoxyuridine (BrdU)-labeled HFRPE cells were injected subretinally into rabbit eyes at three different concentrations . Macrophage, glial, and proliferative responses of the eye tissues were studied by immunohistochemistry and light microscopy at different times after the surgery . RESULTS: In transplanted eyes, the HFRPE cells were distributed irregularly either as multilayers or monolayers . In eyes receiving high-density cell suspensions, retinal breaks were seen . No retinal breaks were noted in the eyes receiving low-density HFRPE cell suspensions . The highest intensity of inflammatory response was seen at 4-14 days after surgery, with greater expression in transplanted eyes receiving high-density cell suspensions . The host cellular response was characterized initially by local infiltration of the retina and subretinal space by macrophages and glial cells . After day 14, a decline in the number of donor cells was noted in all eyes . At later stages the host cellular response was characterized mainly by local choroidal thickening and infiltration by inflammatory cells . Proliferative response was expressed mainly by retinal cells . CONCLUSION: Initial inflammatory and proliferative responses after the xenogenic human to rabbit HFRPE cell transplantation were expressed by retinal cells with later involvement of the choroid . Our results showed a decline in the number of donor cells starting from day 14 after the transplantation . This may suggest a possibility of rejection . The initial quantity of injected cells may be critical for the intensity of the immune and inflammatory responses.

Int J Radiat Biol, 1999 Mar, 75(3), 379 - 85
Poloxamine 1107 sealing of radiopermeabilized erythrocyte membranes; Hannig J et al.; PURPOSE: Lipid peroxidation-mediated permeabilization of cell membranes following intense ionizing irradiation is well documented . This form of membrane radiopermeabilization leads to rapid exhaustion of cellular high-energy compounds, resulting in the acute onset of cellular necrosis . Strategies to reverse the process of necrosis and preserve cell viability require membrane sealing . This report documents the relative efficacy of Poloxamine 1107, a non-ionic surfactant, compared with other polymers, in sealing radiopermeabilized cell membranes . MATERIALS AND METHODS: Isolated erythrocytes were exposed to 600 Gy 60Co irradiation at a dose rate of 1.3 Gy/s . Different polymer compounds were added 10 min later to the irradiated cell suspensions . At 2 h later the haemoglobin content in the supernatants was determined spectrophotometrically . RESULTS: Compared with the non-treated irradiated control, Poloxamine 1107 significantly reduced the leakage of haemoglobin from irradiated erythrocytes . Poloxamer 188 and dextran at equal concentrations had no significant reverse effect on the irradiation-mediated increased membrane permeability . The amount of haemoglobin released from irradiated erythrocytes was inversely related to the Poloxamine 1107 concentration . CONCLUSIONS: This study demonstrates the capability of Poloxamine 1107 to seal radiopermeabilized cell membranes . Thus, surfactants such as Poloxamine 1107 might be useful as a therapeutic agent in the treatment of high-dose radiation injuries since cellular necrosis due to metabolic exhaustion following radiopermeabilization of their membranes might be prevented.

Br J Surg, 1999 Mar, 86(3), 400 - 4
Experimental study of the effect of intraperitoneal heparin on tumour implantation following laparoscopy; Neuhaus SJ et al.; BACKGROUND: Conclusions drawn from clinical reports of port site metastases following laparoscopic resection of intra-abdominal malignancy are now supported by a burgeoning experimental literature which suggests that laparoscopy promotes tumour metastasis to wounds . This study investigated the effect of intraperitoneal blood and heparin on the incidence of tumour cell implantation and port site metastasis . METHODS: Twenty-four Dark Agouti rats underwent laparoscopy with carbon dioxide insufflation and the instillation of a tumour cell suspension and/or blood into the peritoneal cavity . Rats were allocated randomly to one of the following study groups (six rats per group): (1) controls; (2) intraperitoneal blood (2 ml blood introduced from a syngeneic donor rat); (3) intraperitoneal heparin; (4) intraperitoneal blood and heparin . Rats were killed 7 days after the procedure, and the peritoneal cavity and port sites were examined for the presence of tumour . RESULTS: Tumour implantation and port site metastases were reduced by the intraperitoneal administration of heparin, but increased by the presence of intraperitoneal blood . CONCLUSION: The results of this study suggest that tumour implantation following laparoscopy is promoted by the presence of intraperitoneal blood and that this effect may be reduced by the use of intraperitoneal heparin.

Int J Hyperthermia, 1999 Jan-Feb, 15(1), 7 - 16
Modification of tirapazamine-induced cytotoxicity in combination with mild hyperthermia and/or nicotinamide: reference to effect on quiescent tumour cells; Masunaga S et al.; C3H/He and Balb/c mice bearing SCC VII or EMT6/KU tumours received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days to label all proliferating (P) cells . The tumours were locally heated at 40 degrees C for 60 min and/or the tumour-bearing mice received intraperitoneal injection of nicotinamide, and then tirapazamine (TPZ) was injected intraperitoneally . Sixty minutes after TPZ injection, the tumours were excised, minced and trypsinized . The tumour cell suspensions were incubated with cytochalasin-B (a cytokinesis-blocker), and the micronucleus (MN) frequency in cells without BrdU labelling (quiescent (Q) cells) was determined using immunofluorescence staining for BrdU . The MN frequency in total (P+Q) tumour cells was determined from the tumours that were not pretreated with BrdU . The cytotoxicity of TPZ was evaluated in terms of the frequency of induced micronuclei in binuclear tumour cells (= MN frequency) . In both tumour systems, the MN frequencies of Q cells were greater than those of total tumour cell populations . Mild heat treatment elevated the MN frequency in total and Q cells in both tumour systems, but the effect was more marked in Q cells . In total cells, mild heat treatment increased the MN frequency in EMT6/KU tumour cells more markedly than in SCC VII tumour cells . In contrast, in both tumour systems, nicotinamide decreased the MN frequency in both cell populations, with a greater influence on the total cells . The combination of TPZ and mild heat treatment may be useful for sensitizing tumour cells in vivo, including Q cells.

Phytochemistry, 1999 Mar, 50(5), 763 - 74
Purification and characterization of acetyl coenzyme A: 10-hydroxytaxane O-acetyltransferase from cell suspension cultures of Taxus chinensis; Menhard B et al.; An O-acetyltransferase that catalyzes the regiospecific acetylation of a range of taxanes possessing an unsubstituted 10-hydroxyl group was detected and purified to apparent electrophoretic homogeneity from a cytosolic fraction of Taxus chinensis cell cultures . The purification involved negative calcium phosphate adsorption, sephadex desalting, DEAE, AcA44 chromatography, HighQ, CHT II, HiTrap Blue, Phenylsepharose and Mimetic Green purification steps . The purified acetyltransferase was found to be a monomeric protein of 71 +/- 1.5 kDa that is highly regio- and stereospecific towards the 10 beta-hydroxyl group of the taxane molecule and is also active towards 10-desacetylbaccatine III . The acetyltransferase reaction had a pH optimum of 9.0 with halfmaximal activities at pH 6.8 and 10.8, respectively . The temperature optimum was at 35 degrees C and the isoelectric point at 5.6 . The apparent K(m) values for 10-desacetyltaxuyunnanine C and acetyl CoA were 23 and 61 microM, respectively . The turnover rate for the enzyme using both substrates was 0.2 mol mol-1 of enzyme . The kinetic optimum was determined to be Kcat/K(m) = 8.7 s-1 L M-1.

Exp Neurol, 1999 Mar, 156(1), 205 - 8
Addition of fresh blood to intrastriatal grafts of embryonic mesencephalon into the hemiparkinsonian rat does not impair the survival of grafted dopaminergic neurones; Zietlow R et al.; Cell transplantation therapy for Parkinson's patients, although seen to bring benefit to some patients during first clinical trials, remains impracticable on a large scale, in part because of the poor survival of the dopaminergic neurones transplanted . The loss of dopaminergic neurones occurs rapidly over the first 1-2 days after transplantation, in response to factors intrinsic to the host brain . Here we investigated whether contamination of the grafted cell suspension with blood during the transplantation procedure may be one factor responsible for the poor survival of DA neurons within the graft, possibly through factors such as free iron or complement . 6-Hydroxydopamine lesioned rats were grafted with 2 microl suspension of dissociated E14 ventral mesencephalon to which 1 microl blood or 1 microl grafting medium was added . After 6 weeks, there was no significant difference in the number of surviving DA neurones in the two groups . We conclude that contamination of grafts with blood is not a major factor responsible for the extensive death of dopaminergic neurones within them .

Drugs Exp Clin Res, 1998, 24(5-6), 247 - 52
The influence of glucose, succinate, pH of the medium and higher temperature on the cytotoxic activity of the preparation Ukrain; Todor IN et al.; Cells of ascitic forms of Ehrlich's carcinoma and sarcoma 37 transplanted into C57BI/6 mice were used to study the influence of glucose, succinate, and pH of the medium on the cytotoxic activity of the preparation Ukrain . Viability of cells was estimated by the method of intravital staining with tripan blue after the incubation of cell suspension in the presence of the preparation (1.05 x 10(-5) mol and 1.69 x 10(-4) mol) . It was established that glucose (5 mmol) reduces the cytotoxic activity of Ukrain, while succinate (5 mmol) increases it . The activity of the preparation was practically absent at pH 6.1-6.7 of the incubation medium and was at a maximum at pH 7.3-8.0 . A temperature of 41.5 degrees C has no influence on the effectiveness of Ukrain.

Haematologica, 1999 Mar, 84(3), 212 - 7
Evaluation of trisomy 12 by fluorescence in situ hybridization in peripheral blood, bone marrow and lymph nodes of patients with B-cell chronic lymphocytic leukemia; Liso V et al.; BACKGROUND AND OBJECTIVE: Trisomy 12 is the most common numerical chromosomal aberration in patients with B-cell chronic lymphocytic leukemia (B-CLL) . Fluorescence in situ hybridization (FISH) has improved the detection of this cytogenetic abnormality and has made detection possible in all phases of the cell cycle . The presence of the trisomy 12 positive (+12) cell population has generally been investigated in leukemic cells obtained from the peripheral blood of CLL patients . To ascertain whether trisomy 12 is expressed homogeneously in cells of different hemopoietic tissues, we applied FISH to lymph node, peripheral blood and bone marrow samples obtained simultaneously from 23 untreated B-CLL patients . DESIGN AND METHODS: Twenty-three newly diagnosed patients with B-CLL, 15 in stage B and 8 in stage C, were included in the present study . Peripheral blood smears, bone marrow aspirate smears and lymph node touch imprints were collected from each patient at diagnosis . Cytologic preparations were examined by light microscopy in order to assess the lymphocyte morphology . Immunophenotyping was performed by cytofluorimetric analysis of the peripheral blood, bone marrow and lymph node mononuclear cell suspensions . The diagnosis was supported in all cases by histologic findings in bone marrow biopsy and lymph node biopsy specimens . Fluorescence in situ hybridization was performed on smears of blood and aspirated bone-marrow and lymph node touch imprints obtained by fresh tissue apposition . RESULTS: In 6 of the 23 cases (26%) trisomy 12 was clearly present in all tissues examined . A comparative analysis of the three different hemopoietic tissues was performed . A higher percentage of leukemic CD5+CD23+ cells was detected in lymph nodes than in peripheral blood and bone marrow . A significantly higher proportion of trisomic cells was observed in lymph nodes samples than in peripheral blood or bone marrow smears of trisomy 12 positive CLL patients . INTERPRETATION AND CONCLUSIONS: Several previous reports show that only a proportion of malignant B-CLL cells carry trisomy 12 when analyzed by interphase FISH . The higher proportion of +12 cells in lymph nodes than in peripheral blood or bone marrow of CLL patients with trisomy 12 could reflect different cell distributions in different tissues, or lymph node specific tropism, or proliferative advantage in selected tissue . At present, the role of trisomy 12 in the pathogenesis of lymphoproliferative disorders is unclear.

Biotechnol Bioeng, 1999 Apr 5, 63(1), 122 - 6
On-line monitoring of the progress of infection in Sf-9 insect cell cultures using relative permittivity measurements; Zeiser A et al.; The use of on-line relative permittivity (epsilon') measurements for monitoring cultures of Sf-9 cells was evaluated in a batch culture and a batch infected with a baculovirus expressing beta-galactosidase . It was found that viable cell density and volume essentially accounted for all the variation in epsilon' in both non-infected and synchronously infected cultures, indicating that the epsilon' of a cell suspension was sensitive only to changes in the viable cell population . Additionally the parameter provided clearly defined signposts of the progress of the infection .

Biotechnol Bioeng, 1999 Jan 5, 62(1), 97 - 105
The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate; Ketchum RE et al.; Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate . By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported . The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle . The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day . Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation . Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture . Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures . The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel . Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor .

Biotechnol Bioeng, 1998 Dec 20, 60(6), 768 - 70
Time course of SDS-alkaline lysis of recombinant bacterial cells for plasmid release; Ciccolini LA et al.; SDS-alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction . The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number . The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time .

Biotechnol Bioeng, 1998 Jun 20, 58(6), 595 - 604
The effects of turbulent jet flows on plant cell suspension cultures
MacLoughlin PF, Malone DM, Murtagh JT, Kieran PM.
Cell suspensions of Morinda citrifolia were subjected to turbulent flow conditions in a submerged jet apparatus, to investigate their hydrodynamic shear susceptibility . The suspensions were exposed to repeated, pressure-driven passages through a submerged jet . Two nozzles, of 1 mm and 2 mm diameter, were employed . Average energy dissipation rates were in the range 10(3)-10(5) W/kg and cumulative energy dissipation in the range 10(5)-10(7) J/m3 . System response to the imposed conditions was evaluated in terms of suspension viability (determined using a dye exclusion technique) and variations in both chain length distribution and maximum chain length . Viability loss was well-described by a first-order model, and a linear relationship was identified between the specific death rate constant and the average energy dissipation rate . This relationship was consistent with results obtained using the same suspension cultures in a turbulent capillary flow device . Morphological measurements indicated that exposure to the hydrodynamic environment generated in the jet resulted in a significant reduction in both the average and maximum chain lengths, and the reduction in the maximum chain length was identified as an appropriate measure of sustained damage . Analysis of both viability and chain length in terms of cumulative energy dissipated revealed good agreement with results reported by other authors for morphologically different plant cell systems .

Biotechnol Bioeng, 1998 Jun 5, 58(5), 515 - 28
Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry; Yanpaisan W et al.; Flow cytometry was used to measure cell cycle parameters in Solanum aviculare plant cell suspensions . Methods for bromodeoxyuridine (BrdU) labeling of plant nuclei were developed so that cell cycle times and the proportion of cells participating in growth could be determined as a function of culture time and conditions . The percentage of cells active in the cell cycle at 25 degrees C decreased from 52% to 19% within 7.6 d of culture; presence of a relatively large proportion of non-active cells was reflected in the results for culture growth . While the maximum specific growth rate of the suspensions at 25 degrees C was 0.34 d-1 (doubling time: 2.0 d), the specific growth rate of active cells was significantly greater at 0.67 d-1, corresponding to a cell cycle time of 1.0 d . A simple model of culture growth based on exponential and linear growth kinetics and the assumption of constant cell cycle time was found to predict with reasonable accuracy the proportion of active cells in the population as a function of time . Reducing the temperature to 17 degrees C lowered the culture growth rate but prolonged the exponential growth phase compared with 25 degrees C; the percentage of cells participating in the cell cycle was also higher . Exposure of plant cells to different agitation intensities in shake flasks had a pronounced effect on the distribution of cells within the cell cycle . The proportion of cells in S phase was 1.8 times higher at a shaker speed of 160 rpm than at 100 rpm, while the frequency of G0 + G1 cells decreased by up to 27% . Because of the significant levels of intraculture heterogeneity in suspended plant cell systems, flow cytometry is of particular value in characterizing culture properties and behavior .

Hum Reprod, 1999 Feb, 14(2), 388 - 94
Flow cytometric method to isolate round spermatids from mouse testis; Lassalle B et al.; The purpose of this study was to isolate pure populations of round spermatids from mouse testis by flow cytometry followed by cell sorting . Cell suspensions from mouse testis were enriched in germ cells by centrifugation on a discontinuous Percoll gradient, then analysed using a FACScalibur flow cytometer measuring the cell size and density . A large and well-delimited population of cells (R1) expected to contain round spermatids was observed on the dot plot diagram . Sorted R1 cells were very homogeneous in size (approximately 11 microns) and displayed the characteristic cytological aspect of round spermatids . Spermatid-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of R1 cells using primers for protamine 2 gene (PRM2) and SP-10 . A positive signal for SP-10 was obtained with a single cell using nested primers . The 5.5 kb transcript of c-kit, which is not expressed in spermatids, was not detected by nested RT-PCR, excluding a contamination with spermatogonia . Our results clearly established that flow cytometry followed by cell sorting allows the isolation of a highly homogeneous population of round spermatids from the testis.

Chin J Physiol, 1998 Dec 31, 41(4), 181 - 8
Nifedipine, verapamil and diltiazem block shock-wave-induced rises in cytosolic calcium in MDCK cells; Jan CR et al.; Nifedipine and verapamil have been shown previously to protect against renal function alterations induced by shock wave lithotripsy (SWL) in humans and rats; however, the mechanism is unclear . This study was aimed to examine whether these drugs could protect cultured kidney cells following shock wave exposure (SWE) . The effect of nifedipine, verapamil and diltiazem on Madin Darby canine kidney (MDCK) cells following SWE was examined by determining the release of glutamate oxalactate transferase (GOT) and lactate dehydrogenase (LDH) in cell suspensions; and also cytosolic Ca2+ concentration ({Ca2+}i) . Immediately after SWE, there was a transient release of GOT and LDH (16% and 4 fold, respectively) . In contrast, {Ca2+}i measured within 1-6 hr after SWE gradually increased by 15-156% . The Ca2+ entry blockers (1 or 10 microM) failed to inhibit the enzyme release; however, they abolished the progressive rises in {Ca2+}i . The Ca2+ entry blockers may protect the cells from damage of SWE via maintaining a low resting {Ca2+}i.

J Control Release, 1999 Apr 19, 58(3), 289 - 301
Flow cytometric and optical microscopic evaluation of poly(D, L-lactide-co-glycolide) microspheres phagocytosis by pig alveolar macrophages; Torche AM et al.; The phagocytosis of fluorescent poly(D,L-lactide-co-glycolide) microspheres by fresh and frozen pig alveolar macrophages was investigated by optical microscopy on adherent cell culture and by flow cytometry with cell suspension . The kinetic of phagocytosis was studied on a 360 min period as a function of the ratio between microspheres and macrophages (MS:AM ratio from 1:1 to 10:1) . No difference of phagocytosis between fresh and frozen macrophages was observed whatever the MS:AM ratio following flow cytometric evaluation while a significant phagocytosis pattern was noticed following optical microscopic evaluation for the highest ratio . The intensity of phagocytosis was dependent on the duration of incubation and dependent, but not proportionally, to the MS:AM ratio showing that the highest efficiency was obtained with the MS:AM ratio of 1:1 . Flow cytometry analysis has shown a correlation between cell population and fluorescent events suggesting that phagocytosis of nonfluorescent antigen-loaded particles with different characteristics could be investigated.

Br J Cancer, 1999 Mar, 79(7-8), 1121 - 6
Mediastinal lymph node metastasis model by orthotopic intrapulmonary implantation of Lewis lung carcinoma cells in mice; Doki Y et al.; This study is designed to establish a pulmonary tumour model to investigate the biology and therapy of lung cancer in mice . Current methods for forming a solitary intrapulmonary nodule and subsequent metastasis to mediastinal lymph nodes are not well defined . Lewis lung carcinoma (LLC) cell suspensions were orthotopically introduced into the lung parenchyma of C57/BL6 mice via a limited skin incision without thoracotomy followed by direct puncture through the intercostal space . The implantation process was performed within approximately 50 s per mouse, and the operative mortality was less than 5% . Single pulmonary nodules developed at the implanted site in 93% of animals and subsequent mediastinal lymph node metastasis was observed in all mice that formed a lung nodule after intrapulmonary implantation . The size of tumour nodule and the weight of mediastinal lymph node increased in a time-dependent manner . The mean survival time of mice implanted successfully with LLC cells was 21+/-2 days (range 19-24 days) . Histopathological analysis revealed that no metastatic tumour was detectable in the mediastinal lymph nodes on day 11, but metastatic foci at mediastinal lymph nodes were clearly observed on days 17 and 21 after implantation . Other metastases in distant organs or lymph nodes were not observed at 21 days after the implantation . Comparative studies with intrapleural and intravenous injections of LLC cells suggest that the mediastinal lymph node metastasis by intrapulmonary implantation is due to the release of tumour cells from the primary nodule, and not due to extrapulmonary leakage of cells . An intravenous administration of cis-diamine dichloro platinum on day 1 after tumour implantation tended to suppress the primary tumour nodule and significantly inhibited lymph node metastasis . Thus, a solitary pulmonary tumour nodule model with lymph node metastasis approximates clinical lung cancer and may provide a useful basis for lung cancer research.

Br J Cancer, 1999 Mar, 79(7-8), 1085 - 9
Radiosensitization of hypoxic tumour cells by S-nitroso-N-acetylpenicillamine implicates a bioreductive mechanism of nitric oxide generation; Janssens MY et al.; The radiosensitizing activity of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide (NO) donor, was assessed in a model of non-metabolic hypoxia achieved in an atmosphere of 95% nitrogen-5% carbon dioxide . A 10 min preincubation of hypoxic EMT-6 cells (10 x 10(6) ml(-1)) with 0.1 and 1 mM SNAP before radiation resulted in an enhancement ratio of 1.6 and 1.7 respectively . The level of spontaneous NO release, measured by a NO specific microsensor, correlated directly with the concentration of SNAP and was enhanced 50 times in the presence of cells . Dilution of the cell suspension from 10 to 0.1 x 10(6) ml(-1) resulted in a 16-fold decline in NO release, but only a twofold decrease in radiosensitization was observed . Preincubation of hypoxic cells with SNAP for 3 min up to 30 min caused an increasing radiosensitizing effect . Extended preincubation of 100 min led to the loss of radiosensitization although the half-life of SNAP is known to be 4-5 h . Taken together, these observations suggest that SNAP generates NO predominantly by a bioreductive mechanism and that its biological half-life is unlikely to exceed 30 min . The lack of correlation between free NO radical and radiosensitizing activity may reflect a role of intracellular NO adducts which could contribute to radiosensitization as well.

Gan To Kagaku Ryoho, 1999 Mar, 26(4), 497 - 502
{Experimental study on intraperitoneal administration of 5-fluorouracil for liver metastasis in comparison with intravenous administration}; Maruyama M et al.; We studied the effects of 5-fluorouracil intraperitoneal administration using mouse liver metastasis model . We inoculated 50 microliters Colon26 cell suspension into the spleen and resected it 15 min after cell inoculation under general anesthesia with Ketamine . Control group (n = 7) had no treatment . The intraperitoneal (i.p.) group (n = 8) and intravenous (i.v.) group (n = 7) underwent the treatment on the 2nd and 4th day after the operation . Experimental chemotherapies consisted of 1.5 ml 5-fluorouracil solution (50 mg/kg) for i.p . group and 0.2 ml 5-fluorouracil solution (50 mg/kg) for i.v . group . On the 14th day after the cell implantation, necropsies were performed . Deposits on mouse livers were counted and the mouse livers weighted . Counting of metastatic liver deposits revealed the number of deposits in the control group was 25.6 +/- 12.9, against 2.9 +/- 1.9 and 16.0 +/- 15.6, in the i.p . and i.v . group, respectively . Significant differences in the number of liver deposits were obtained between the control group and i.p . group, and between i.p . group and i.v . group (p < 0.05) . The mean liver weight (mg)/mouse body weight (g) were 76.3 +/- 24.7 in the control group, 54.3 +/- 4.7 in the i.p . group and 60.0 +/- 12.7 in the i.v . group . A significant difference was observed only between the control group and the i.p . group (p < 0.05) . I.p . administration of 5-fluorouracil was superior to i.v . administration for control of the liver metastasis . Moreover, the side effect by 5-fluorouracil i.p . treatment was milder than by i.v . therapy . We confirmed the effectiveness of 5-fluorouracil intraperitoneal chemotherapy for the potential liver metastasis and liver micrometastasis . Intraperitoneal chemotherapy is also useful for peritoneal seeding . We think intraperitoneal chemotherapy is a recommendable administration route for gastrointestinal malignancies.

J Dent Res, 1999 Mar, 78(3), 751 - 8
Langerhans cells from oral epithelium are more effective in stimulating allogeneic t-cells in vitro than Langerhans cells from skin epithelium; Hasseus B et al.; Dendritic cells, such as Langerhans cells (LC), in different ectodermal compartments may have different functional capabilities . The present study was undertaken to compare oral Langerhans cells (LC) with those of the epidermis in terms of their ability to co-stimulate T-cells in vitro . A Mixed Epithelial Cell Lymphocyte Reaction (MELR) and a mitogen-driven (concanavalin A) T-cell proliferation assay were used . In both assays, LC in a crude cell suspension of freshly isolated oral epithelial cells were found to be five times more effective in mediating T-cell proliferation than freshly isolated epidermal LC . Twenty-four-hour cell culture at 37 degrees C enhanced the T-cell response in the MELR compared with cells cultured at 4 degrees C . This applied to both skin and oral epithelial cells . Oral and skin epithelial cell suspensions depleted of LC lost the capacity to stimulate allogeneic T-cells . Incubation of the epithelial cell suspensions with recombinant Granulocyte/Macrophage-Colony Stimulating Factor (rGM-CSF) did not enhance the co-stimulating capacity of the LC . Titration of different numbers of oral and skin LC to T-cells showed that skin LC were never able to reach more than 44% of the maximal stimulatory capacity of oral LC . Data show that oral LC are more efficient than skin LC in providing co-stimulatory signals to T-cells, suggesting a difference in functional capacity between the two cell populations.

Artif Cells Blood Substit Immobil Biotechnol, 1999 Mar, 27(2), 163 - 9
Haemoglobin (Erythrogen)-enhanced post-thaw growth of cryopreserved cells; Azhakanandam K et al.; Supplementation of semi-solid R2 culture medium with a commercial bovine haemoglobin (Hb) solution (Erythrogen) at 1:50-1:500 (v:v), had beneficial effects on the growth, following cryopreservation, of cells of the Indica rice, Oryza sativa cv . Pusa Basmati 1 . The mean absorbance, as assessed by triphenyl tetrazolium chloride reduction, of rice cells at 8 d post-thawing, was increased by up to 60% (P < 0.05), compared to cells recovered in the absence of Hb . Erythrogen (1:50-1:500 v:v) promoted an increase in biomass, of up to 25% over control (P < 0.05), at 24 d post-thawing . Cell suspensions, re-established by transfer to liquid medium of cells initially thawed and cultured with Erythrogen for 24 d, exhibited increased (up to 2-fold) growth rates over a subsequent 20-d period, compared to cells recovered without Hb.

Photochem Photobiol, 1999 Mar, 69(3), 306 - 16
Can cellular phototoxicity be accurately predicted on the basis of sensitizer photophysics?
Aveline BM, Redmond RW.
The phototoxicity of three structurally related photosensitizers (PS), deuteroporphyrin IX (DP) and monobromo (Br-DP) and dibromo (Br2-DP) derivatives, was studied in murine L1210 leukemia cells . These compounds were chosen on the basis of heavy-atom-induced differences in triplet yield, phi T, and lifetime, tau T, and used as tools to test a model for phototoxicity based on photophysical parameters . All three porphyrins were found to localize preferentially in the plasma membrane of L1210 cells by confocal fluorescence microscopy . A poor correlation was observed between the measured photodynamic efficacies of these PS and a model using photophysical parameters determined by laser flash photolysis in homogeneous solution . However, an excellent correlation was obtained when the same parameters measured directly in the cells were used . The biological microenvironment of the porphyrins in cells induces significant changes in the photophysics of the PS . Reduction in fluorescence yield, phi F, and phi T observed for Br2-DP in cell suspensions arises from self association of the molecule due to increased hydrophobicity and high local concentrations . The photophysical model was also tested for its ability to handle variations in the oxygen dependence of cellular phototoxicity of these PS . The good correlation achieved between laser flash photolysis data determined in cells and the measured phototoxicity under air, 1.5% and 0.5% O2-saturated conditions, proves the intermediacy of singlet oxygen . This study gives further credence to the direct use of photophysical techniques to elucidate photochemical mechanisms in biological media while highlighting the potential pitfalls of using solution data to predict photosensitizing potential.

J Exp Biol, 1999 Apr, 202 (Pt 8), 965 - 75
CO2 excretion and postcapillary pH equilibration in blood-perfused turtle lungs
Stabenau EK, Heming TA.
Turtles possess a significant postcapillary CO2 partial pressure (PCO2) disequilibrium between arterial blood and alveolar gas . There are several possible explanations for this blood disequilibrium including a slow rate of erythrocyte physiological anion shift (Cl-/HCO3- exchange) or inaccessibility of plasma HCO3- to red blood cell or pulmonary carbonic anhydrase . The present study characterized the contribution of erythrocyte anion exchange and pulmonary and erythrocyte carbonic anhydrase to CO2 excretion and, hence, to postcapillary CO2-HCO3--H+ equilibration in blood-perfused turtle (Pseudemys scripta) lungs . Turtle lungs perfused in situ with red cell suspensions containing inhibitors of erythrocyte anion exchange and/or pulmonary and red cell carbonic anhydrase produced significant postcapillary blood PCO2 and pH disequilibria, while no disequilibria were measured when lungs were perfused with control red cell suspensions . Erythrocyte anion exchange and pulmonary intravascular carbonic anhydrase contributed 11 % and 9 %, respectively, to CO2 excretion during single-pass perfusion, whereas red cell and pulmonary carbonic anhydrase contributed 32 % to the measured CO2 excretion . The lack of a measurable PCO2 disequilibrium during perfusion with control erythrocyte suspensions in this study suggests that alternative mechanisms may be responsible for the arterial-lung PCO2 disequilibrium measured during breathing or diving episodes in turtles.

Immunobiology, 1999 Feb, 200(1), 49 - 61
A novel 26 kilodalton antigen expressed on the surface membrane of activated T cells; Bank I et al.; We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) 1B10, raised against an activated gammadelta T cell clone . Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb 1B10 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC) . After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B10+ . 1B10 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) alphabeta+ and gammadelta+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were 1B10+ . In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb 1B10 . Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb 1B10 from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively . In conclusion, mAb 1B10 recognizes a novel <<late>> appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.

J Clin Endocrinol Metab, 1999 Mar, 84(3), 1017 - 21
Impact of architectural disruption on adrenocortical steroidogenesis in vitro; Hines GA et al.; The adrenal cortex is an architecturally complex tissue, with cellular zonation thought to determine steroidogenesis . The impact that disruption of this tissue's architecture has on steroidogenesis in vitro, particularly adrenal androgen (AA) production, is unclear . We hypothesized that the extent of architectural disruption during tissue preparation would impact the study results . To test this hypothesis, we compared adrenocortical steroidogenesis in freshly prepared tissue slices, minces, and cell suspensions . Normal human adrenals (n = 5, three males and two females, age range 17-43 yr) were obtained at the time of organ donation . The three adrenal tissue preparations were incubated in serum-free medium with 10 microM pregnenolone substrate +/- 1 microM ACTH . The production of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, and cortisol in the media were measured by radioimmunoassay . Initial time course intubations using adrenals from a single donor generally demonstrated that minces and suspensions had a greater steroid production compared with slices . In another series of 6-hr incubations using adrenals from four donors, production of dehydroepiandrosterone sulfate was found to be quite sensitive to architectural disruption, i.e . slices less than minces less than suspensions (0.88 vs . 2.1 vs . 3.0 microg/gm tissue, respectively, P < 0.0001) . Alternatively, cortisol and androstenedione production was higher in minces compared with slices or suspensions (25.6 vs . 37.7 vs . 18.7 ng/gm tissue, P < 0.0028, and 254 vs . 709 vs . 456 ng/gm tissue, P < 0.0042, respectively) . Production of dehydroepiandrosterone was apparently not significantly affected by the type of tissue preparation (28.2 vs . 22.2 vs . 31.2 ng/gm tissue, P < 0.297, respectively) . It is unlikely that generalized tissue disruption alone accounted for the observed differences, as the trends among tissue preparations were not consistent among steroids . We conclude that the type of tissue preparation of fresh adrenal tissue impacts significantly on steroidogenesis in vitro.

Biochim Biophys Acta, 1999 Feb 10, 1430(1), 25 - 38
Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures; Prior A et al.; A cDNA encoding a plant-type APS reductase was isolated from an axenic cell suspension culture of Catharanthus roseus (Genbank/EMBL-databank accession number U63784) . The open reading frame of 1392 bp (termed par) encoded for a protein (Mr=51394) consisting of a N-terminal transit peptide, a PAPS reductase-like core and a C-terminal extension with homology to the thioredoxin-like domain of protein disulfide isomerase . The APS reductase precursor was imported into pea chloroplasts in vitro and processed to give a mature protein of approximately 45 kDa . The homologous protein from pea chloroplast stroma was detected using anti:par polyclonal antibodies . To investigate the catalytical function of the different domains deleted par proteins were purified . ParDelta1 lacking the transit sequence liberated sulfite from APS (Km 2.5+/-0.23 microM) in vitro with glutathione (Km 3+/-0.64 mM) as reductant (Vmax 2.6+/-0.14 U mg-1, molecular activity 126 min-1) . ParDelta2 lacking the transit sequence and C-terminal domain had to be reconstituted with exogenous thioredoxin as reductant (Km 15 . 3+/-1.27 microM, Vmax 0.6+/-0.014 U mg-1) . Glutaredoxin, GSH or DTT were ineffective substitutes . ParDelta1 (35.4%) and parDelta2 (21 . 8%) both exhibited insulin reductase activity comparable to thioredoxin (100%) . Protein disulfide isomerase activity was observed for parDelta1.

Cytometry, 1999 Mar 1, 35(3), 260 - 6
Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue; Leers MP et al.; BACKGROUND: Flow cytometry of single-cell suspensions prepared by enzymatic digestion from formalin-fixed, paraffin-embedded tissue suffers from several major drawbacks . The most important factors that influence the results are the high and unpredictable coefficients of variation (CVs) of the G0/G1 peak in the DNA histogram and reduction of propidium iodide (PI) intercalation with DNA, resulting from protein cross-linking by formalin . METHODS: In this study we introduce a heating step (2 h incubation in citrate solution at 80 degrees C) prior to a brief pepsin digestion of tissue sections in the protocol for DNA content analysis of formalin-fixed and paraffin-embedded tissue . This new method is compared with established methods for the preparation of cell suspensions from frozen and paraffin-embedded tissues with respect to cell yield, DNA histogram resolution, DNA dye saturation kinetics, cell cycle parameters, and antigen retrieval in various epithelial and nonepithelial tissues . RESULTS: The recovery of single cells from the paraffin sections was doubled by the heat treatment step, while the limited time of proteolysis resulted in decreased cell debris . Furthermore, an increased fraction of cells became cytokeratin-positive, while these immunocytochemically stained cells also exhibited a higher mean fluorescence intensity . The DNA histograms prepared from cell suspensions obtained according to this new protocol showed a significantly improved resolution, leading to a better identification of peridiploid cell populations . Heat pretreatment of paraffin-embedded archival tissue sections showed PI saturation kinetics similar to, or even better than, those of fresh unfixed tissues, independent of duration of fixation . CONCLUSIONS: This new method, making use of routinely available antigen retrieval principles, thus allows high-resolution DNA analysis of routinely fixed and paraffin-embedded tissue samples . Using external reference cells, inter- and intralaboratory standardization of DNA histograms can be achieved.

Plant Mol Biol, 1999 Jan, 39(1), 83 - 93
Positive-negative selection and T-DNA stability in Arabidopsis transformation; Gallego ME et al.; We have analysed the application of positive-negative selection for the selection of homologous recombination interactions between the chromosome and a T-DNA molecule after transformation of plant cells . Two different genomic loci in a cell suspension of Arabidopsis thaliana were chosen to study gene targeting events . One was the chalcone synthase (CHS) gene present as a single copy and the second an hemizygous chromosomally inserted T-DNA containing the hpt gene, conferring resistance to hygromycin, flanked by CHS sequences . The target lines were transformed with replacement-type T-DNA vectors which contained a positive selectable marker flanked by the regions of the CHS gene and a negative selectable marker to counter-select random insertions . As negative marker we used the Escherichia coli codA gene encoding cytosine deaminase, conferring upon the cells sensitivity to 5-flourocytosine (5-FC) . Doubly selected transformants represent 1-4% of the primary transformed cells . Targeting events were not found at the chalcone synthase locus nor at the artificial hpt locus in a total of 4379 doubly selected calli, corresponding to at least 109,475 individual primary transformants . We show by PCR and Southern analysis that the 5-FC resistance in the majority of these cells is associated with substantial deletions of the T-DNA molecule from the right-border end.

J Biomech Eng, 1999 Feb, 121(1), 28 - 34
Molding of deep polydimethylsiloxane microstructures for microfluidics and biological applications; Folch A et al.; Here we demonstrate the microfabrication of deep (> 25 microns) polymeric microstructures created by replica-molding polydimethylsiloxane (PDMS) from microfabricated Si substrates . The use of PDMS structures in microfluidics and biological applications is discussed . We investigated the feasibility of two methods for the microfabrication of the Si molds: deep plasma etch of silicon-on-insulator (SOI) wafers and photolithographic patterning of a spin-coated photoplastic layer . Although the SOI wafers can be patterned at higher resolution, we found that the inexpensive photoplastic yields similar replication fidelity . The latter is mostly limited by the mechanical stability of the replicated PDMS structures . As an example, we demonstrate the selective delivery of different cell suspensions to specific locations of a tissue culture substrate resulting in micropatterns of attached cells.

J Chemother, 1999 Feb, 11(1), 34 - 9
Antifungal activity of voriconazole (UK-109,496), fluconazole and amphotericin B against hematogenous Candida krusei infection in neutropenic guinea pig model; Ghannoum MA et al.; Voriconazole (UK-109,496) is a new triazole with in vitro activity against a wide spectrum of fungi including yeasts intrinsically resistant to fluconazole such as Candida krusei . In this study the efficacy of voriconazole was compared to amphotericin B and fluconazole in a neutropenic guinea pig model of hematogenously disseminated C . krusei infection . In guinea pigs, neutropenia was established by using cyclophosphamide (intraperitoneally, i.p., 100 mg/kg on day 1 and 4), and dexamethasone (orally, 2 mg/kg/day, for 8 days) . Neutropenic guinea pigs were infected with 0.5 ml of yeast cell suspension (1 x 10(8) CFU) intravenously . Challenged animals were treated with antifungals starting 1 h postinfection for 7 days . The animals were divided into five groups: untreated control, amphotericin B (1 mg/kg i.p . on alternate days), fluconazole (20 mg/kg orally twice daily), and voriconazole (two groups: 5 and 10 mg/kg orally twice daily) groups . Guinea pigs were sacrificed 1 day after the last treatment . Brain, liver, and kidneys were removed and weighed, tissues were homogenized and fungal burden determined by serial quantitative counts . Voriconazole at dosages of 5 or 10 mg/kg b.i.d . was shown to be significantly more efficacious than either amphotericin B or fluconazole in eradicating C . krusei from brain, liver and kidney tissue . These data indicate that voriconazole could be efficacious for the treatment of infections caused by fluconazole-resistant Candida, such as C . krusei.

Immunol Invest, 1999 Jan, 28(1), 29 - 41
Phenotyping of immune cell infiltrates in breast and colorectal tumours; Toomey D et al.; White cell infiltration of solid tumors is an important prognostic indicator in malignant disease . Although macrophage infiltration is associated with good outcome in colorectal cancer, a high macrophage content is associated with poor prognosis in breast cancer . Suppressor macrophages prevent T cell activation in normal tissues such as mucosal linings exposed to continuous antigenic challenge . Interleukin 10 (IL-10), an immunosuppressive cytokine, inhibits macrophage co-stimulation of T cells . Suppressor macrophage numbers, T cell numbers and T cell activation status were assessed in cell suspensions obtained from fresh specimens of breast and colorectal tumours and matched normal tissues . IL-10 production by both malignant and matched normal tissue was also assessed . This study identified elevated numbers of suppressor macrophages in breast tumors compared to matched normal breast tissue . Colorectal tumors did not contain significant numbers of these cells . Although T cell numbers are increased in breast tumors, these cells do not appear to be fully activated, as assessed by major histocompatibility complex class II and Interleukin 2 receptor expression . In contrast, T cells in colorectal tumors exhibit greater expression levels of these markers . Breast tumors produce significantly higher levels of IL-10 than normal breast tissue whereas IL-10 levels in colorectal tumors are similar to normal colon tissue . Our findings of high suppressor macrophage numbers, high levels of IL-10 and poorly activated T cells in breast tumors compared to low suppressor macrophage numbers, low IL-10 and fully activated T cells in colorectal tumors may explain why high macrophage content is associated with poor prognosis in breast cancer and good prognosis in colorectal malignancy.

J Orthop Res, 1999 Jan, 17(1), 121 - 9
Effect of seeding duration on the strength of chondrocyte adhesion to articular cartilage; Schinagl RM et al.; Chondrocyte adhesion to cartilage may play an important role in the repair of articular defects by maintaining cells in positions where their biosynthetic products can contribute to the repair process . The objective of this in vitro study was to determine the effect of the duration of seeding time on the ability of chondrocytes to resist detachment from cartilage when subjected to mechanical perturbation (fluid-induced shear stress) . Suspensions of adult bovine articular chondrocytes were prepared from primary, high-density monolayer cultures and infused into a parallel-plate shear-flow chamber where they settled onto 50-microm-thick sections of bovine articular cartilage at a density of approximately 20,000 cells/cm2 . The chondrocytes were seeded and allowed to attach to the cartilage surface for specific durations (5-40 minutes) in medium including 10% serum at 22 degrees C, after which the cells were exposed to fluid flow-induced shear stresses (6-90 Pa) . The fraction of detached cells at each shear stress was calculated from microscopic images . Shear stress was applied for 1 minute because this length of time was sufficient to induce steady-state cell detachment . Increasing the duration of cell seeding led to a more firm attachment of chondrocytes to cartilage . After 9 minutes of seeding, 50% cell detachment was induced by gravitational force alone . After 40 minutes of seeding, 50% detachment required 26 Pa of shear stress . Extrapolation of the data to account for the effect of repeated applications of cell suspensions to an individual cartilage substrate indicated that for a freshly prepared cartilage section, 50% detachment was induced by gravity after 25 minutes of seeding and by 2.3 Pa of shear stress after 40 minutes of seeding . The increase in resistance to shear stress-induced cell detachment with increasing seeding duration suggests that it may be beneficial to allow chondrocytes to stabilize in the absence of applied load for some time after chondrocyte transplantation for cartilage repair in vivo.

J Immunol, 1999 Mar 1, 162(5), 2946 - 55
Differential regulation of eosinophil chemokine signaling via CCR3 and non-CCR3 pathways; Sabroe I et al.; To investigate eosinophil stimulation by chemokines we developed a sensitive assay of leukocyte shape change, the gated autofluorescence/forward scatter assay . Leukocyte shape change responses are mediated through rearrangements of the cellular cytoskeleton in a dynamic process typically resulting in a polarized cell and are essential to the processes of leukocyte migration from the microcirculation into sites of inflammation . We examined the actions of the chemokines eotaxin, eotaxin-2, monocyte chemoattractant protein-1 (MCP-1), MCP-3, MCP-4, RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and IL-8 on leukocytes in mixed cell suspensions and focused on the responses of eosinophils to C-C chemokines . Those chemokines acting on CCR3 induced a rapid shape change in eosinophils from all donors; of these, eotaxin and eotaxin-2 were the most potent . Responses to MCP-4 were qualitatively different, showing marked reversal of shape change responses with agonist concentration and duration of treatment . In contrast, MIP-1alpha induced a potent response in eosinophils from a small and previously undescribed subgroup of donors via a non-CCR3 pathway likely to be CCR1 mediated . Incubation of leukocytes at 37 degrees C for 90 min in the absence of extracellular calcium up-regulated responses to MCP-4 and MIP-1alpha in the majority of donors, and there was a small increase in responses to eotaxin . MIP-1alpha responsiveness in vivo may therefore be a function of both CCR1 expression levels and the regulated efficiency of coupling to intracellular signaling pathways . The observed up-regulation of MIP-1alpha signaling via non-CCR3 pathways may play a role in eosinophil recruitment in inflammatory states such as occurs in the asthmatic lung.

Immunol Lett, 1999 Feb, 65(3), 143 - 51
Immunoglobulin variable regions usage by B-lymphocytes infiltrating a human breast medullary carcinoma; Kotlan B et al.; Breast medullary carcinoma are heavily infiltrated by B-lymphocytes and associated with a good prognosis despite their high histological grade . We investigated the Ig repertoire of B-lymphocytes infiltrating one such tumour . A single cell suspension was obtained from a tumor specimen by enzymatic digestion . VH, Vkappa, and Vlambda regions were amplified by RT-PCR using mixtures of primers optimized to maximize the diversity of the PCR products . They were then cloned and sequenced . Analysis of 9 VH, 5 Vkappa, and 10 Vlambda sequences using the Kabat database indicated that several VH and VL region subgroups (I, II and III) are expressed by B-lymphocytes infiltrating this tumor . The analysis of CDR3 regions also showed a variability, although some VH and VL clones exhibited identical or nearly identical sequences . Thus, the B-cell infiltration observed in this breast medullary carcinoma does not reflect a monoclonal proliferation and represents an oligoclonal or a polyclonal B-cell proliferation.

Med Pediatr Oncol, 1999 Mar, 32(3), 209 - 15
Paclitaxel: an effective antineoplastic agent in the treatment of xenotransplanted hepatoblastoma; Fuchs J et al.; BACKGROUND: Hepatoblastoma is an uncommon liver tumor of infancy and early childhood . Though most patients with nonmetastatic hepatoblastomas can be cured by defining surgical strategies and chemotherapy regimes, new drugs are needed for children with advanced hepatoblastomas . The activity of paclitaxel as a new antineoplastic agent with limited experience in pediatric oncology was studied in a xenograft model . PROCEDURE: Hepatoblastoma cell suspensions from three children were transplanted subcutaneously into nude mice NMRI (nu/nu) . One of the primary tumors was an embryonal multifocal hepatoblastoma, whereas the other tumors were embryonal/fetal hepatoblastomas localized on a liver lobe . After 4 weeks, xenografted tumor sizes reached 50-100 mm3 . The xenografted tumors resembled their originals histologically and produced high levels of alpha-fetoprotein . The efficiency of paclitaxel at equitoxic doses was analyzed . RESULTS: Paclitaxel produced an effect in all three hepatoblastomas . There was a significant reduction of tumor volume (P < 0.001) and alpha-fetoprotein levels after chemotherapy (P < 0.0001) . The proliferation activity of the tumor cells corresponded with these results . Histologically, after treatment with paclitaxel the tumor regression was 35%-49% . The mechanism of paclitaxel action could be demonstrated by light microscopy immunohistochemistry and electron microscopy . CONCLUSIONS: The preliminary results in phase I trials of solid tumors in children and the results of this study suggest that paclitaxel in phase II studies can now be entertained for patients with hepatoblastoma.

Dig Dis Sci, 1999 Feb, 44(2), 364 - 71
Expansion conditions for early hepatic progenitor cells from embryonal and neonatal rat livers; Brill S et al.; Long-term primary cultures were established from fetal or neonatal livers by using cell suspensions depleted of red blood cells and by culturing the cells in hormonally defined medium containing dimethyl sulfoxide . Two distinct populations of hepatic progenitor cells were evident in the cultures, based on morphology, proliferative ability, and liver-specific gene expression . Most colonies consisted of immature hepatic progenitors: small, blastlike cells, weakly expressing alpha-fetoprotein, albumin, and gamma-glutamyltranspeptidase, and showing evidence of proliferation as measured by bromodeoxyuridine incorporation . At the perimeter of these colonies of immature cells and forming some colonies by themselves were more mature hepatic progenitor cells: larger cells, with increased cytoplasmic to nuclear ratios, little proliferation, and strongly expressing albumin, alpha-fetoprotein, and gamma-glutamyltranspeptidase . The latter two proteins were localized to the bile canalicular membranes of these cells . Glycogen deposits were present in the mature cells from day 14 embryos after eight days of culture . Thus, DMSO treatment of hepatic parenchymal progenitors provides a novel system for studies of liver development.

Protein Expr Purif, 1999 Mar, 15(2), 188 - 95
Processing of preproricin in transgenic tobacco; Sehnke PC et al.; The plant protein toxin ricin has found widespread application as a potential therapeutic agent for many human diseases and in disease-model systems such as those involving apoptosis . Genetic engineering and expression of the complete two-polypeptide chain toxin have only been possible in plants, specifically in transgenic tobacco carrying the preproricin gene under the control the cauliflower mosaic virus 35S promoter . Production of modified ricin for altered controllable activity and/or fusion therapeutics to target delivery requires knowledge of the heterologous processing that occurs when preproricin is expressed in tobacco . Here, recombinant ricin from transgenic tobacco was purified using lectin affinity chromatography and characterized using various biochemical and biophysical techniques . Coomassie blue staining of an SDS-PAGE gel of lactose-agarose purified material identified predominant proteins of 30 and 35 kDa molecular weight . Western analysis using anti-ricin a- and b-chain antibodies confirmed the expression and purification of recombinant ricin, with identical protein banding profiles to that of authentic castor-bean-derived ricin . High-resolution gel filtration chromatography characterized the lactose binding complex as a 66-kDa native molecular weight protein which could be separated into 30- and 35-kDa proteins upon incubation with the reducing agent dithiothreitol . N-terminal sequencing of the recombinant ricin a-chain revealed that an equimolar ratio of two alternately processed peptides was present, which varied by an additional amino acid derived from the signal peptide . Similar analysis of ricin b-chain again identified two forms of this polypeptide as well; however, full-length ricin b-chain and b-chain missing the first alanine residue were present at 11:1 molar ratios . Transgenic tobacco plants expressing ricin were used to develop a stable cell suspension culture system from callus induced with the growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine . Double sandwich enzyme-linked immunosorbent assay using anti-ricin b-chain antibodies and Western analysis identified soluble ricin in the media of the cultures, indicating that cell cultures provide a safe and simple means to produce properly processed recombinant ricin .

J Immunol Methods, 1999 Feb 1, 223(1), 27 - 36
A flow cytometric procedure for the quantification of cell adhesion in complex mixtures of cells; Bono MR et al.; We present a simple non-radioactive cytometry-based assay that permits the simultaneous quantitation of cell adhesion of distinct subsets of cells contained in a mixture without any previous fractionation . The procedure is simple and highly reproducible and has the advantage of confining the quantitation of cell adhesion to live cells only . This new approach is based on counting the absolute number of cells . This is done by adding known numbers of distinguishable beads to the cell suspension and counting beads and cells in a cytometer . Quantitation of adhesion is accomplished by counting each subpopulation of cells before and after the adhesive process . To illustrate this methodology we determined adhesion of Ramos cells to monolayers of endothelial cells and its inhibition by specific antibodies . Also, we determined adhesion to endothelial cells of B lymphocytes and subsets of T lymphocytes present in a preparation of unfractionated human mononuclear cells . The results presented here demonstrate that the new assay has the required properties to be used in the quantitation of cell adhesion.

Clin Cancer Res, 1999 Feb, 5(2), 417 - 23
Combination interferon-alpha2a and 13-cis-retinoic acid enhances radiosensitization of human malignant glioma cells in vitro; Malone C et al.; We investigated the individual and combined effects of cis-retinoic acid (CRA) and/or IFN-alpha (IFN) and/or radiation therapy (RT) against a human glioma cell line (American Type Culture Collection; U373MG) to evaluate the possible radiosensitization properties of these agents in vitro . Glioma cells were incubated for 24 h in 96-well plates (2 x 10(2) cells/well) in standard culture medium . Sets of U373 (n = 12) were exposed to CRA (3 x 10(6) microM), IFN (25 units/ml), CRA plus IFN, or standard culture medium . After an additional 24 h of incubation, the U373 cells were subjected to increasing radiation doses (up to 16 Gy) . Glioma cells were harvested 92 h after irradiation, and cell survival curves were determined from {3H}thymidine incorporation data (over the last 24 h) . The experiment was repeated for both the untreated control group and the combined CRA/IFN group . To verify the {3H}thymidine assays, a clonogenic assay was also performed . Single cell suspensions of U373 cells were plated out in six-well plates (n = 3) . After chemical and RT treatment, colonies of 50 cells or more were counted, and cell survival curves were generated as fractions of nonirradiated controls . The amount of RT (in Gy) that would cause a 50% survival fraction (lethal dose 50 or LD50) was calculated from the survival curves by regression analysis . The following LD50s were obtained: {table: see text} The results showed that for both the {3H}thymidine incorporation assay and the clonogenic assay, the combination of IFN/CRA rendered U373 cells more susceptible to ionizing radiation than the untreated control or either single agent alone.

FEBS Lett, 1999 Jan 29, 443(3), 317 - 20
Gibberellic acid stabilises microtubules in maize suspension cells to cold and stimulates acetylation of alpha-tubulin; Huang RF et al.; Gibberellic acid is known to stabilise microtubules in plant organs against depolymerisation . We have now devised a simplified cell system for studying this . Pretreatment of a maize cell suspension with gibberellic acid for just 3 h stabilised protoplast microtubules against depolymerisation on ice . In other eukaryotes, acetylation of alpha-tubulin is known to correlate with microtubule stabilisation but this is not established in plants . By isolating the polymeric tubulin fraction from maize cytoskeletons and immunoblotting with the antibody 6-11B-1, we have demonstrated that gibberellic acid stimulates the acetylation of alpha-tubulin . This is the first demonstrated link between microtubule stabilisation and tubulin acetylation in higher plants.

Z Gastroenterol, 1998 Dec, 36(12), 1021 - 6
Helicobacter pylori induces apoptosis in mucosal lymphocytes in patients with gastritis; Reinacher-Schick A et al.; BACKGROUND: Previous studies suggest that Helicobacter pylori (H . pylori) induces apoptosis and compensatory hyperproliferation in gastric epithelial cells possibly explaining the carcinogenic capacity of the bacteria . The aim of this study was to measure the effect of H . pylori on apoptosis of gastric lymphoid cells in view of the development of gastric lymphoma . METHODS: 16 H . pylori-positive and 19 H . pylori-negative individuals were enrolled . Single cell suspensions were prepared from antral biopsies and apoptosis was measured by staining with the TUNEL-assay and the fluorochrome Hoechst 33342 . Lymphocyte subsets were simultaneously identified by immunocytochemistry . RESULTS: The apoptotic index of all gastric mucosal cells was significantly higher in H . pylori-positive mucosa compared to negative controls . Additionally, H . pylori-infected patients showed a significant increase in apoptosis of mucosal B-lymphocytes . Apoptosis of T cells and plasma cells was unaffected by H . pylori . CONCLUSION: H . pylori induces apoptosis in mucosal B cells which might be important in the development of gastritis and possibly B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT).

Development, 1999 Mar, 126(6), 1305 - 15
Mechanisms of GDF-5 action during skeletal development; Francis-West PH et al.; Mutations in GDF-5, a member of the TGF-beta superfamily, result in the autosomal recessive syndromes brachypod (bp) in mice and Hunter-Thompson and Grebe-type chondrodysplasias in humans . These syndromes are all characterised by the shortening of the appendicular skeleton and loss or abnormal development of some joints . To investigate how GDF-5 controls skeletogenesis, we overexpressed GDF-5 during chick limb development using the retrovirus, RCASBP . This resulted in up to a 37.5% increase in length of the skeletal elements, which was predominantly due to an increase in the number of chondrocytes . By injecting virus at different stages of development, we show that GDF-5 can increase both the size of the early cartilage condensation and the later developing skeletal element . Using in vitro micromass cultures as a model system to study the early steps of chondrogenesis, we show that GDF-5 increases chondrogenesis in a dose-dependent manner . We did not detect changes in proliferation . However, cell suspension cultures showed that GDF-5 might act at these stages by increasing cell adhesion, a critical determinant of early chondrogenesis . In contrast, pulse labelling experiments of GDF-5-infected limbs showed that at later stages of skeletal development GDF-5 can increase proliferation of chondrocytes . Thus, here we show two mechanisms of how GDF-5 may control different stages of skeletogenesis . Finally, our data show that levels of GDF-5 expression/activity are important in controlling the size of skeletal elements and provides a possible explanation for the variation in the severity of skeletal defects resulting from mutations in GDF-5.

Br J Dermatol, 1998 Dec, 139(6), 984 - 91
Calcipotriol inhibits the proliferation of hyperproliferative CD29 positive keratinocytes in psoriatic epidermis in the absence of an effect on the function and number of antigen-presenting cells; Jensen AM et al.; The aim of this study was to elucidate some of the possible mechanisms of action of the vitamin D analogue calcipotriol in vivo . Calcipotriol is finding increasing use in the treatment of psoriasis, but the primary target cell in vivo has not yet been identified . We treated psoriatic patients and healthy volunteers with calcipotriol and placebo ointment for 4 and 7 days, and obtained epidermal cell suspensions from treated areas . Epidermal cells were cocultured with autologous T cells, isolated from peripheral blood, in the absence or the presence of a classical antigen or a superantigen . In both psoriatic and normal skin, calcipotriol treatment did not alter the capacity of epidermal antigen-presenting cells to stimulate the proliferation of autologous T cells, either in the absence or in the presence of exogenous antigen . Epidermal cell suspensions were analysed further by staining for infiltrating leucocytes (CD45+) and Langerhans cells (CD1a+) . Flow cytometric analysis showed that calcipotriol did not alter the number of CD45+ cells or Langerhans cells in psoriatic skin . These results indicate that calcipotriol does not alter either the number of the function of epidermal antigen-presenting cells in psoriatic epidermis . In contrast, we found that calcipotriol significantly inhibited the proliferation of epidermal cells isolated from psoriatic skin after in vivo treatment, as determined by propidium iodide staining and flow cytometry . More specifically, we stained for CD29+ keratinocytes and found an even more significant reduction in proliferative capacity . This cell type contains the population of hyperproliferative keratinocytes in psoriatic epidermis . In conclusion, calcipotriol seems to act via an inhibitory effect on hyperproliferative basal keratinocytes of psoriatic epidermis, rather than via an effect on infiltrating leucocytes, including antigen-presenting cells.

Biochim Biophys Acta, 1999 Jan 11, 1448(3), 390 - 402
A redox-dependent, G-protein-coupled phospholipase A of the plasma membrane is involved in the elicitation of alkaloid biosynthesis in Eschscholtzia californica; Roos W et al.; In cultured cells of California poppy formation of benzophenanthridine alkaloids can be triggered by a yeast elicitor preparation independently of the hypersensitive reaction . A plasma membrane (PM) bound phospholipase A (PLA) is likely to play a role in the signalling process: PLA activity was detectable in individual cells, cell suspensions and PM vesicles with the fluorogenic phospholipid bis-BODIPY FL C11-PC and was sensitive to known inhibitors of PLA2 . In microscopic assays, enzyme activity increased after elicitor contact of cells that were pretreated with non-saturating concentrations of PLA2 inhibitors . In PM vesicles a PLA2-like protein as well as G alpha- and G beta-proteins were detected immunologically . Anti-G alpha or anti-G beta antisera or mastoparan stimulated PLA activity thus indicating a G-protein-controlled enzyme . Elicitation of alkaloid production was sensitive to aristolochic acid and enhanced by PLA2 products such as lysophosphatidylcholine and linolenic acid . Pretreatment of the cells with the artificial electron acceptors hexabromoiridate(V) or ferricyanide(III) reversibly abolished the effect of subsequent elicitation and reduced the activity of PLA both in intact cells and in PM vesicles . It appears, therefore, that PLA2 is a point of interference of redox control with the signal path.

J Exp Med, 1999 Feb 15, 189(4), 711 - 8
312-nanometer ultraviolet B light (narrow-band UVB) induces apoptosis of T cells within psoriatic lesions; Ozawa M et al.; Narrow-band (312 nm) ultraviolet B light (UVB) is a new form of therapy for psoriasis, but its mechanism of action is unknown . In a bilateral comparison clinical study, daily exposure of psoriatic plaques to broad-band UVB (290-320 nm) or 312-nm UVB depleted T cells from the epidermis and dermis of psoriatic lesions . However, 312-nm UVB was significantly more depleting in both tissue compartments . To characterize the mechanism of T cell depletion, assays for T cell apoptosis were performed on T cells derived from UVB-irradiated skin in vivo and on T cells irradiated in vitro with 312-nm UVB . Apoptosis was induced in T cells exposed to 50-100 mJ/cm2 of 312-nm UVB in vitro, as measured by increased binding of fluorescein isothiocyanate (FITC)-Annexin V to CD3(+) cells and by characteristic cell size/granularity changes measured by cytometry . In vivo exposure of psoriatic skin lesions to 312-nm UVB for 1-2 wk also induced apoptosis in T cells as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction in tissue sections, by binding of FITC-Annexin V to CD3(+) T cells contained in epidermal cell suspensions, and by detection of apoptosis-related size shifts of CD3(+) cells . Induction of T cell apoptosis could be the main mechanism by which 312-nm UVB resolves psoriasis skin lesions.

J Appl Toxicol, 1999 Jan-Feb, 19(1), 1 - 6
Effects of ibuprofen on arylamine N-acetyltransferase activity in human colon tumor cells; Chung JG et al.; The inhibition of arylamine N-acetyltransferase (NAT) activity by ibuprofen was determined in a human colon tumour (adenocarcinoma) cell line . Two assay systems were employed, one with cellular cytosols (9000 g supernatant) and the other with intact colon tumour cell suspensions . The NAT activity in a human colon tumour cell line was inhibited by ibuprofen in a dose-dependent manner in both systems, i.e . the greater the concentration of ibuprofen in the reaction, the greater the inhibition of NAT activities in both systems . The data also indicated that ibuprofen decreases the apparent Km and Vmax of NAT enzyme from human colon tumour cells in both systems examined . This report is the first demonstration to show that ibuprofen affects human colon tumour cell NAT activity.

Biosci Biotechnol Biochem, 1998 Nov, 62(11), 2223 - 5
Pectins in extracellular polysaccharides from a cell-suspension culture of Mentha; Maruyama K et al.; Pectin constituents, which were about 70 w/w% of extracellular polysaccharides (ECP) from a cell-suspension culture of Mentha, were purified by gel filtration chromatography, and their sugar composition and linkage were investigated . Two major constituents identified were (1-->3)-linked galactan carrying arabinosyl residues on C-6 and (1-->4)-alpha-linked galacturonan partially interspersed with (1-->2)-linked rhamnosyl resides . Acetylated or methylated pectins were not identified on 1H-NMR analysis.

Diagn Cytopathol, 1999 Feb, 20(2), 99 - 104
Fine-needle aspiration biopsy using a newly-developed pencil-grip syringe holder; Tao LC et al.; Until now, commercially available syringe holders for fine-needle aspiration (FNA) were designed to be held in a pistol-grip manner . A newly developed, pencil-grip syringe holder, the Tao Aspirator, was tested . The device is equipped with a release button for automatically drawing back the syringe plunger and a regulating knob for adjusting negative pressure for the aspiration . After direct smears were made for on-site examination, the remaining aspirated material was collected by rinsing the needle and syringe with CytoRich red fixative . Hettich cytocentrifuge preparations were then prepared . The quality of the first 150 FNA specimens procured by this device and prepared with liquid fixation was evaluated in terms of adequacy of specimen, amount of obscuring blood, preservation of cells, and ease of screening and interpretation . These 150 specimens included 32 from thyroids; 34 from breasts; 40 from lymph nodes; 24 from subcutaneous nodules; and 20 from salivary glands . There were no unsatisfactory specimens . In Hettich preparations, red blood cells were lysed, making interpretation easier . All cellular elements and tissue fragments were adequately fixed, showing excellent cellular morphology . Specimens fixed in liquid fixative yielded uniform cell suspensions, resulting in cytocentrifuge preparations with evenly distributed cells, and so the screening was also easier . The aspiration techniques using pistol-grip and pencil-grip FNA syringe holders were also compared in terms of control in tissue sampling, ease of use, and safety . The pencil-grip syringe holder allowed greater tactile sensation of the texture of the lesion, and enabled the operator to use a single hand to place a needle into a target lesion with minimal error . This device placed the hand relatively close to the needle tip while the hand was in a position of natural function, imparting more control in tissue sampling . It was more easily manipulated, and could prevent dripping when cystic fluid was aspirated . Specimen collection using the Tao Aspirator and processing with liquid fixation in addition to direct smear preparations allowed the laboratory to consistently produce adequate cytologic preparations and cell blocks.

Vox Sang, 1999, 76(1), 22 - 6
Efficiency of leukocyte removal by filters made of superfine glass fiber membranes; Zou Y et al.; BACKGROUND AND OBJECTIVES: To demonstrate the application of leukocyte removal filters made of a new type of filter material - superfine glass fiber - for depleting leukocytes in SAGM red cell suspensions and preventing nonhemolytic transfusion reactions . MATERIALS AND METHODS: The extent of leukocyte depletion and red cell recovery was based on cell counts . Trace leukocytes were counted in a 50-microl Nageotte counting chamber or by using a flow cytometer . The chemical stability of the glass fiber membranes was studied by plasma emission spectrometer and by measuring the ion content and weighing nonvolatile matter in water extract . The structural stability of the glass fiber membranes was studied by a micropore-filter membrane method . RESULTS: Leukocyte removal filters made of superfine glass fiber membranes removed more than 99.0% of leukocytes in SAGM red cell suspensions prepared from 400 ml whole blood . Red cell recovery exceeded 90%, and the total number of residual leukocytes was less than 5x10(6) . A water extract of the glass fiber membranes contained only traces of Si4+ and Ca2+ and less than 2 mg/100 ml of nonvolatile matter . No broken or loose fibers were found in the filters . Scanning electron microscopy showed that the web structure of the glass fiber membranes was instrumental in trapping and holding leukocytes . CONCLUSION: A filter made of glass fiber membranes is effective in leukocyte depletion.

J Nucl Med, 1999 Jan, 40(1), 184 - 91
Imaging of apoptosis (programmed cell death) with 99mTc annexin V; Blankenberg FG et al.; Apoptosis (programmed cell death) is a critical element in normal physiology and in many disease processes . Phosphatidylserine (PS), one component of cell membrane phospholipids, is normally confined to the inner leaflet of the plasma membrane . Early in the course of apoptosis, this phospholipid is rapidly exposed on the cell's outer surface . Annexin V, an endogenous human protein, has a high affinity for membrane-bound PS . This protein has been labeled with fluorescein and has been used to detect apoptosis in vitro . We describe the use of radiolabeled annexin V to detect apoptosis in vivo . The results are compared to histologic and flow cytometric methods to identify cells and tissues undergoing apoptosis . METHODS: Annexin V was coupled to hydrazinonicotinamide (HYNIC) and radiolabeled with 99mTc . Bioreactivity of 99mTc-HYNIC annexin V was compared with fluorescein isothiocyanate (FITC)-labeled annexin V in cultures of Jurkat T-cell lymphoblasts and in ex vivo thymic cell suspensions undergoing apoptosis in response to different stimuli . In addition, the uptake of FITC annexin V and 99mTc-HYNIC annexin V was studied in heat-treated necrotic Jurkat T-cell cultures . In vivo localization of annexin V was studied in Balb/c mice injected with 99mTc-HYNIC annexin V before and after induction of Fas-mediated hepatocyte apoptosis with intravenously administered antiFas antibody . RESULTS: Membrane-bound radiolabeled annexin V activity linearly correlated to total fluorescence as observed by FITC annexin V flow cytometry in Jurkat T-cell cultures induced to undergo apoptosis in response to growth factor deprivation (N = 10, r2 = 0.987), antiFas antibody (N = 8, r2 = 0.836) and doxorubicin (N = 10, r2 = 0.804); and in ex vivo experiments on thymic cell suspensions with dexamethasone-induced apoptosis from Balb/c mice (N = 6, r2 = 0.989) . Necrotic Jurkat T-cell cultures also demonstrated marked increases in radiopharmaceutical (4000-5000-fold) above control values . AntiFas antibody-treated Balb/c mice (N = 6) demonstrated a three-fold rise in hepatic uptake of annexin V (P < 0.0005) above control (N = 10), identified both by imaging and scintillation well counting . The increase in hepatic uptake in antiFas antibody-treated mice correlated to histologic evidence of fulminant hepatic apoptosis . CONCLUSION: These data suggest that 99mTc-HYNIC annexin V can be used to image apoptotic and necrotic cell death in vivo.

Aust N Z J Surg, 1999 Jan, 69(1), 52 - 5
In vitro inhibition of tumour growth in a helium-rich environment: implications for laparoscopic surgery; Neuhaus SJ et al.; BACKGROUND: The recent results of several experimental studies have suggested that tumour implantation after laparoscopic surgery for intra-abdominal malignancy may be partly related to the chemical composition of the insufflation gas used during surgery . These studies have demonstrated that the use of helium as a laparoscopic insufflation agent for cancer surgery results in less tumour implantation and growth at port sites . To further investigate these findings, the present study was performed to compare the growth of cultured tumour cells after exposure to simulated laparoscopic environments, rich in helium, carbon dioxide (CO2), or air . METHODS: A rat mammary adenocarcinoma cell suspension was exposed to a simulated laparoscopic environment for 40 min in one of the following groups: (i) control (atmospheric air, equivalent to a 'gasless' laparoscopic environment); (ii) a CO2-rich environment; and (iii) a helium-rich environment . Cells were then cultured for 18 h and optical density readings were used to assess the number of viable tumour cells at the end of this period . The experiment was performed twice using an identical protocol to ensure consistency in the results . In a further study, pH was continuously measured using an antimony probe during a 40 min insufflation period and for 10 min after insufflation . RESULTS: Cell growth was significantly lower after incubation in the helium-rich environment compared to both the CO2 and control groups (P < 0.001) . There was a significant decrease in pH in the CO2 group which was not observed during exposure to either air or helium . CONCLUSIONS: The inhibition of tumour growth in a helium-rich environment demonstrated by this study, and the reduced incidence of port-site metastases seen in other experimental studies, suggests that the clinical use of helium as an insufflation gas may have important advantages over CO2.

Biochim Biophys Acta, 1999 Jan 18, 1444(1), 1 - 13
Molecular characterisation of plant cDNAs BnMAP4Kalpha1 and BnMAP4Kalpha2 belonging to the GCK/SPS1 subfamily of MAP kinase kinase kinase kinase; Leprince A et al.; Several yeast and mammal MAP kinase modules require, upstream of their MAP kinase kinase kinase (MAP3K), a MAP3K kinase (MAP4K) . An Arabidopsis thaliana EST clone, sharing identity to MAP4Ks from yeast and mammals, has been used to isolate cDNA clones from a Brassica napus microspore-derived embryo cDNA library . The BnMAP4Kalpha1 and BnMAP4K-alpha2 clones encode putative proteins possessing the 12 subdomains of the serine/threonine protein kinase catalytic domain . A detailed analysis showed that they belong to the GCK/SPS1 subfamily of MAP4K proteins which possess an amino terminal catalytic domain and a long carboxy terminal tail . A Southern blot analysis suggested that the two proteins are encoded by a small multigene family . Expression studies revealed the presence of BnMAP4Kalpha1 and -alpha2 transcripts in all the tissues examined; however, they are most abundant in roots, siliques and flower buds . The expression of BnMAP4Kalpha1 and -alpha2 at the three main developmental stages of microspore-derived embryos (i.e., globular/heart, torpedo and cotyledonary) was confirmed by northern blot and RT-PCR analysis . An expression analysis of the above genes using synchronised Arabidopsis thaliana cell suspensions showed that the homologues genes are cell cycle regulated.

Endothelium, 1998, 6(2), 143 - 51
A sensitive fluorometric assay for determining hydrogen peroxide-mediated sublethal and lethal endothelial cell injury; Oral HB et al.; A rapid and sensitive quantitative fluorometric assay was developed to measure the response of endothelial cells to hydrogen peroxide (H2O2) . The response of an endothelial cell-derived cell line, EA-hy-926, or human umbilical vein endothelial cells to H2O2 was determined using calcein-AM, a dye which becomes fluorescent upon cleavage by intracellular esterase(s) . The ability of the cells to take up and convert calcein-AM was measured directly in the wells of 96-well flat-bottomed tissue culture plates with cell monolayers using a computerised microplate fluorimeter, or in cell suspensions using flow cytometry . The results obtained by these techniques were compared with each other and with a standard 51Cr release cytotoxicity assay . We found that calcein-AM is a highly sensitive probe for measuring H2O2-mediated cell injury, as it can not only detect the irreversible cytotoxicity measured by 51Cr release assay, but can also distinguish sublethal and reversible injury seen at low H2O2 concentrations.

Cancer Lett, 1998 Nov 13, 133(1), 107 - 14
Characterization of muscarinic receptors in the human melanoma cell line SK-Mel-28 via calcium mobilization; Noda S et al.; In melanoma cells of primary and metastatic human melanomas muscarinic cholinergic receptors are present . Muscarinic receptors were shown to be expressed in morphogenetically active embryonic cells . Therefore, the possibility exists that in melanomas an embryonic trait is re-expressed after transformation . In the present study, we demonstrated the presence of muscarinic receptors in the human melanoma cell line SK-Mel-28 by immunofluorescence with the monoclonal antibody M 35 and characterized the receptors further by measuring calcium mobilization after muscarinic stimulation . Cell suspensions were stained with fura-2 and fluorescence was followed at 380 nm excitation in a fluorimeter cuvette . After the addition of acetylcholine or carbachol a steep decrease in fluorescence intensity indicated calcium mobilization from intracellular stores (peak reaction), which was followed by a constantly lowered fluorescence level indicating a steady influx of extracellular calcium in the presence of agonist . By quantitative evaluation, dose-response curves were obtained from which an ED of 4.3 x 10(-6) M was calculated for acetylcholine and an ED of 2.2 x 10(-5) M was calculated for carbachol . After preincubation with antagonists the dose-response curve of acetylcholine was shifted to the right . The inhibition constant of pirenzepine was calculated as 3.9 x 10(-7) M, of methoctramine as 6.8 x 10(-7) M and of 4-DAMP-mustard as 1.9 X 10(-8) M . Comparison with the data from the literature and those obtained in the chick embryo indicates that the muscarinic receptor in SK-Mel-28 melanoma cells pharmacologically behaves as the M3 type and corresponds to the embryonic muscarinic receptor characterized by us in earlier studies.

Radiat Med, 1998 Nov-Dec, 16(6), 441 - 8
Enhancement of cisplatin sensitivity of quiescent cells in solid tumors by combined treatment with tirapazamine and low-temperature hyperthermia; Masunaga S et al.; We examined the enhanced chemosensitivity of quiescent (Q) cells in solid tumors to cis-diamminedichloroplatinum (II) (cisplatin) by combined treatment with tirapazamine (TPZ) and mild heating . C3H/He and Balb/c mice bearing SCC VII and EMT6/KU tumors, respectively, received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells . TPZ was administered intraperitoneally 2 h before cisplatin injection and/or tumors were locally heated at 40 degrees C for 60 min immediately after cisplatin injection . Sixty minutes after cisplatin injection, the tumors were excised, minced and trypsinized . The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU . The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU . The sensitivity to cisplatin was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency) . Other groups of tumor-bearing C3H/He and Balb/c mice not given BrdU were injected with 195mPt-radiolabeled cisplatin . In both tumor systems, the MN frequency in Q cells was lower than that in the total cells . TPZ and mild heat treatment elevated the MN frequency in total and Q cells in both tumor systems, and to a higher extent in Q cells . The combination of TPZ and mild heat treatment increased the MN frequency more markedly than treatment with either TPZ or mild heating alone . In total tumor cells, TPZ and mild heat treatment increased the MN frequency in EMT6/KU tumor cells more markedly than in SCC VII tumor cells . 195mPt-labeled cisplatin uptake into total tumor cells was increased by mild heat treatment but not by TPZ . The cisplatin-sensitivity of Q cells was lower than that of total cells in both tumor systems . TPZ was thought to sensitize Q cells by killing the hypoxic cells without influencing tumor blood flow, and mild hyperthermia appeared to sensitize Q cells by distributing more cisplatin with an increase in blood flow in solid tumors.

Can J Physiol Pharmacol, 1998 Jun, 76(6), 676 - 83
Effects of alpha1-antagonists on production and release of aldosterone and other steroid hormones by porcine adrenocortical cells in vitro; Jager LP et al.; Quinazoline type alpha1-adrenoceptor antagonists (range 10-100 microM) inhibited aldosterone release of a cell suspension of porcine adrenocortical cells, potency order: doxazosin > prazosin > trimazosin . Phenoxybenzamine also inhibited the aldosterone release at a concentration of 100 microM . Alpha1-adrenoceptor antagonists from other chemical classes had no measurable effect on the aldosterone output from adrenocortical cells in vitro . Agonists selective for either alpha1- or beta-adrenoceptors did not affect the aldosterone release . The inhibition of the aldosterone release induced by quinazolines was similar with different substrates . The small differences between the drug-induced inhibitions could be ranked as corticosterone = progesterone > pregnenolone = deoxycorticosterone . The doxazosin (10 microM)-induced changes in the release of nine steroids indicated that quinazoline-type alpha1-antagonists interfere with enzymes of the aldosterone biogenesis pathway involved in C18-oxidation and C21beta-hydroxylation, reducing the release of both aldosterone and corticosterone . At higher concentrations (100 microM), the C21beta-hydroxylation in the cortisol biogenesis pathway is also affected, decreasing the output of cortisol and deoxycortisol, but increasing the output of progesterone and OH-progesterone . Simultaneously, the C17-oxidation and side-chain cleavage is also inhibited, decreasing the output of androstenedione . The rank order of phenoxybenzamine (100 microM)-induced inhibition of the aldosterone release with different substrates is pregnenolone > corticosterone = progesterone > deoxycorticosterone . With pregnenolone as substrate, the output of aldosterone, corticosterone, and cortisol was reduced to the same extent . The dehydroepiandrosterone, androstenedione, and progesterone release was enhanced . It seems that phenoxybenzamine is a rather selective inhibitor of the mitochondrial P450(11beta/18) enzymes.

Zentralbl Veterinarmed A, 1998 Dec, 45(10), 635 - 43
Studies on equine lipid metabolism . 1 . A fluorometric method for the measurement of lipolytic activity in isolated adipocytes of rats and horses; Breidenbach A et al.; A simple and sensitive method for direct and continuous monitoring of free fatty acid (FFA) release, by measuring the pH-sensitive change in relative fluorescence intensity of seminaphthofluorescein (SNAFL-1) is described . The method was designed to use a small number of adipocytes isolated from fat pads of rats and biopsy specimens of horses for the detection of decreasing pH in fat cell suspensions caused by released FFA into the incubation medium . Species specific differences of lipolysis were demonstrated when adipocytes of rats and horses are incubated with stimulators or inhibitors of lipolysis . Norepinephrine (NE) stimulated lipolysis in fat cells of rats whereas adipocytes of horses showed a measurable release of FFA when concomitantly incubated with NE and adenosine deaminase (ADA) or NE and 8-Phenyltheophylline (8-PT), respectively) . The incubation of equine fat cells with NE and ADA did not influence the antilipolytic response to insulin . The method described enables micro-scaled in vitro studies on lipolytic activity.

Blood, 1999 Feb 1, 93(3), 876 - 85
Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule; Evangelista V et al.; Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation . We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated beta2-integrin CD11b/CD18 . Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P approximately 110) . Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P approximately 110 . The inhibition of P approximately 110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation . Similar effects were observed when platelets were substituted by P-selectin-transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin-IgG fusion protein . CHO-P/PMN mixed-cell aggregation and P-selectin-IgG-triggered PMN/PMN aggregation as well as P approximately 110 phosphorylation were all blocked by antibodies against P-selectin or CD18 . In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein . The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system . Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered beta2-integrin-dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN . This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase-dependent mechanisms that lead to CD11b/CD18 activation in PMN . The availability of the beta2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P approximately 110.

Exp Neurol, 1999 Jan, 155(1), 22 - 30
Successful myoblast transplantation in primates depends on appropriate cell delivery and induction of regeneration in the host muscle; Skuk D et al.; Myoblast transplantation (MT) may be a potential treatment for severe recessive hereditary myopathies . The limited results of MT in clinical trials led us to improve this technique in monkeys, an animal model phylogenetically similar to humans . Three Macaca mulata monkeys were used as donors and six as receivers for MT . Myoblasts were grown in culture from muscle biopsies of adult monkeys and infected with a retroviral vector encoding the LacZ gene . Different numbers of cells (i.e., 4 x 10(6), 8 x 10(6), and 24 x 10(6) cells) were transplanted into different muscles and 8 x 10(6) cells (resuspended in a notexin solution) were injected in one muscle of four monkeys . For these transplantations, the cell suspension (in a volume of about 100 microl) was injected at 35 sites less than 1 mm apart . Two other monkeys received 100 x 10(6) myoblasts resuspended in 1 ml of HBSS or 1 ml of notexin . For these two monkeys, the myoblasts were injected at 200-250 sites within a small portion of the muscle . All monkeys were immunosuppressed with daily injections of FK506 . Four weeks after MT, the transplanted muscle portions were biopsied and the presence of beta-galactosidase-positive (beta-Gal+) muscle fibers was investigated . The number of beta-Gal+ fibers was 822 +/- 150 (site grafted with 4 x 10(6) cells), 1253 +/- 515 (8 x 10(6) cells), 1084 +/- 278 (24 x 10(6)), and 2852 +/- 1211 (notexin) . In the monkeys grafted with 100 x 10(6) myoblasts, the number of beta-Gal+ fibers was 4850 (site without notexin) and 9600 (site with notexin) . We demonstrated that a precise mechanical distribution of myoblasts into the tissue improves substantially MT in primates . The presence of notexin with the transplanted cells further increased the success of their transplantation . These are the best results obtained either with MT or gene therapy in primates and they encourage the possibility to human MT trials .

Ann N Y Acad Sci, 1998 Sep 11, 858, 235 - 44
Three-dimensional behavior of ice crystals and biological cells during freezing of cell suspensions; Ishiguro H et al.; Behavior of ice crystals and human red blood cells during extracellular-freezing was investigated in three-dimensions using a confocal laser scanning microscope(CLSM), which noninvasively produces tomograms of biological materials . Physiological saline and physiological saline with 2.4 M glycerol were used for suspension . Various cooling rates for directional solidification were used for distinctive morphology of the ice crystals . Addition of acridine orange as a fluorescent dye into the cell suspension enabled ice crystal, cells and unfrozen solution to be distinguished by different colors . The results indicate that the microscopic structure is three-dimensional for flat, cellular, and dendritic solid-liquid interfaces and that a CLSM is very effective in studying three-dimensional structure during the freezing of cell suspensions.

Cancer, 1998 Dec 25, 84(6), 366 - 74
Evidence of intrinsic differences in the light scattering properties of tumorigenic and nontumorigenic cells; Mourant JR et al.; BACKGROUND: The objective of this study was to determine whether there are intrinsic differences in the light scattering properties of tumorigenic and nontumorigenic cells from a multistep carcinogenesis model . METHODS: Wavelength-dependent and polarization-dependent light scattering properties of cell suspensions were measured . RESULTS: Statistically significant differences were found between the tumorigenic and nontumorigenic cells . CONCLUSIONS . Differences in the light scattering properties of tumorigenic and nontumorigenic cells are attributed to a change in the average size of the scattering centers on the order of a few ten of nanometers . This work is relevant to the development of noninvasive optical methods for cancer diagnosis.

J Nat Prod, 1999 Jan, 62(1), 127 - 9
Phenylpropanoid glycosides from Penstemon serrulatus; Skrzypek Z et al.; Two new phenylpropanoid glycosides named cis-martynoside (1) and cis-leucosceptoside A (3) were recognized in cell suspension cultures of Penstemon serrulatus Menz . The structures of these compounds were determined on the basis of 1H NMR spectral data.

Immunol Cell Biol, 1998 Dec, 76(6), 550 - 5
Lymphoid cell infiltration during breast cancer growth: a syngeneic rat model; Zhang XD et al.; The systematic study of potential alterations in lymphoid infiltrates during tumour growth is extremely limited in humans . Therefore, development of a model utilizing a spontaneously arising mammary adenocarcinoma in Dark Agouti rats was adopted for the study of the dynamics of lymphoid cell infiltration during tumour development . Syngeneic rats were inoculated with tumour cell suspensions and the tumours were resected from 5 to 15 days . Serial sections were immunohistochemically stained using a panel of monoclonal antibodies . Irrespective of tumour age, ED2 (macrophages) and W3/25 (CD4)-positive cells were the most prominent cell infiltrates in tumours . There were no significant differences in cell counts for any marker between 8-day and 15-day tumours . However, in 5-day tumours there were significantly fewer macrophages, OX19+ T cells, W3/25+ cells, OX8+ (CD8) cells and OX62+ dendritic cells . Interleukin-2 receptor alpha chain expression was low at all examined stages of tumour growth, indicating a lack of tumour infiltrating lymphocyte (TIL) activation and/or possible TIL anergy . B cell staining was absent in all tumours, negating the possibility of these cells mediating coregulatory signals for TIL activation in the micro-environment of established tumours . The results parallel previous immunohistochemical findings in humans, suggesting that a dysfunctional local immune response in breast cancer may be determined very early during tumour development.

Phytochemistry, 1999 Jan, 50(1), 47 - 51
Modulation of phosphatidylcholine biosynthesis in celery by exogenous fatty acids; Parkin ET et al.; The effects of C16 and C18 fatty acids on the synthesis of phosphatidylcholine were studied in Apium graveolens cell suspension cultures and postmitochondrial supernatants . When cells were exposed to exogenous oleic acid, the rate of phosphatidylcholine biosynthesis increased 1.4-fold within 5 min of the addition of the fatty acid to the culture medium . The sensitivity of microsomal CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) to saturated and unsaturated fatty acids was monitored through the addition of unesterified fatty acids to postmitochondrial supernatants . The saturated fatty acids, palmitic and stearic, appeared to have little effect on CTP:cholinephosphate cytidylyltransferase activity, whereas exposure to oleic, linoleic and cis-vaccenic acids resulted in significant increases in enzyme activity . Optimal microsomal CTP:cholinephosphate cytidylyltransferase activities were achieved by the incubation of postmitochondrial supernatants with 500 microM oleate . The exogenous fatty acids were found to be incorporated into microsomal membranes in their unesterified form . Removal of unesterified fatty acids by incubation of microsomal membranes with defatted bovine serum albumin resulted in the reduction of microsomal CTP:cholinephosphate cytidylyltransferase activity; demonstrating that the enzyme requires unesterified unsaturated fatty acids.

Adv Exp Med Biol, 1998, 454, 415 - 23
Are there significant gradients of pO2 in cells?
Grinberg OY, James PE, Swartz HM.
It is widely recognized that the intracellular oxygen tension (pO2) plays an important role in cellular function and metabolism . In experimental and theoretical consideration involving the role of the pO2 the values that are used usually are those for the pO2 at the exterior of cells, because these values can be more readily measured . Such an approach is based on the assumption that the intracellular pO2 is very similar to the extracellular pO2 because oxygen freely diffuses across cell membranes and within cells, at a rate that is similar to that in the extracellular media . For the past several years we have been developing and applying electron paramagnetic resonance (EPR) techniques to test directly whether there are intracellular gradients of pO2 and if so, where they occur and what factors determine them . We previously reported significant gradients between the average pO2 in the intracellular and extracellular compartments in cell suspensions (Glockner 1989) . More recently we developed techniques that enabled us to measure simultaneously the concentration of oxygen within a specific compartment, the phagosomes of activated macrophages, and the extracellular compartment and found gradients of up to 48 microM under some conditions (James 1995) . The precise mechanism of the intracellular-extracellular oxygen gradient remains uncertain . The possibilities include that the diffusion of oxygen is not as free as assumed (e.g . that the cell membrane can act as a barrier) and the occurrence of active transport of O2 out of the cells . We report here on the use of a simple theoretical approach to evaluate the values of three key parameters which might account for the observed intracellular-extracellular oxygen measurements: (1) oxygen consumption; (2) the diffusion coefficient of oxygen in the cytosol; (3) the solubility of oxygen in the cytosol . We used two different models for the relationship between the oxygen consuming compartment (assumed to be primarily the mitochondria) and the intracellular compartment in which the measurements were made (especially phagosomes): uniform and non-uniform distribution of the mitochondria . Using these models and consensus values from the literature, we were unable to account for the experimentally observed differences in pO2 between the intracellular and extracellular compartments . Also we found that with the variation of any one parameter we could not plausibly account for the measurements made in the phagosomal and extracellular compartments . There are at least three logical possibilities to account for these results: 1) this methodology is erroneous and/or produces artifacts in the system resulting in invalid results; 2) the observation of a gradient in oxygen concentration between these two compartments arises from significant simultaneous variations of more than one of the critical parameters which are used conventionally to calculate potential gradients in pO2; 3) there is another factor not considered in the model which accounts for the observation (e.g . active transport; significantly higher than expected barriers to oxygen diffusion in the membrane).

J Invest Dermatol, 1999 Jan, 112(1), 19 - 24
UVA-induced immune suppression through an oxidative pathway; Iwai I et al.; Although ultraviolet B (UVB) irradiation induces local immune or systemic immune suppression, depending on the dose, the immune suppression by ultraviolet A (UVA) has not been fully investigated . In this study, we investigated the effect of UVA on the immune response in vitro and in vivo . The effect of UVA on the antigen-presenting function of epidermal cells was measured in terms of antigen-specific T cell proliferation . A murine epidermal cell suspension was exposed to UVA in vitro, pulsed with trinitrobenzenesulfonic acid, and cultured with T cells prepared from syngeneic mice previously sensitized with trinitrochlorobenzene . UVA (5-20 J per cm2) suppressed the antigen-presenting function of epidermal cells in a dose-dependent manner, accompanied with suppression of the expression of costimulatory molecules on Langerhans cells . In order to investigate the effect of an antioxidant on the immune suppression, an epidermal cell suspension was irradiated with UVA in the presence or absence of glutathione . The suppressions of antigen-presenting function and ICAM-1 expression were significantly prevented by glutathione in a dose-dependent manner . Further, the effect of UVA on the immune response at the induction phase of contact hypersensitivity was evaluated in terms of lymph node cell proliferation ex vivo . UVA irradiation suppressed the endogenous proliferation of lymph node cells in trinitrochlorobenzene-painted mice, and this suppression was significantly reversed by the application of glutathione to the skin during irradiation . These results suggest that UVA-induced immune suppression may be mediated by reactive oxygen species, at least in part.

Int Immunol, 1998 Dec, 10(12), 1847 - 51
CD80 expression is decreased in hyperplastic lymph nodes of HIV+ patients; Legendre C et al.; A centrofollicular hyperplasia is present within secondary lymphoid organs during all the asymptomatic phase of the HIV disease . Although this hyperplasia has been well characterized by histological studies, the nature of the phenotypic alterations in B cell populations occurring within HIV+ lymphoid organs remains to be established . By immunohistochemistry, we thus investigated whether a particular germinal center (GC) B cell population was increased during HIV-induced hyperplasia and whether any phenotypic change was specific to HIV-1 infection . As compared to normal tonsils (three cases) and HIV- hyperplastic lymph nodes (eight patients), we observed a loss of GC polarization in all HIV+ sections (11 patients), with no more delineation between dark and light zones, as shown by Ki67, CD10, CD77, CD95 and CD86 staining . In contrast to CD86 expression which remained as intensive in HIV+ as in HIV- lymph nodes, CD80 staining was strongly decreased in GC of HIV+ lymph nodes but not in their extrafollicular zones . The loss of CD80 expression from CD19+ B cells was also observed by cytometric analysis of cell suspensions of three HIV+ patients . Although we found no evidence of an increase in a particular GC B cell subset in HIV-1-induced hyperplasia, the strong GC disorganization observed may induce impaired cell-cell interactions and thus participate in the loss of CD80 antigen . In contrast to HIV- situations where CD80 and CD86 was similarly expressed on B cells, the lower level of CD80 expression in HIV+ GC may favor Th2 T cell responses through CD86-CD28 interactions.

Biomed Tech (Berl), 1998 Nov, 43(11), 302 - 9
{A new microprocedure for continuous and non-consuming determination of cellular oxygen uptake based on fluorescence quenching}; Trubel H et al.; The oxygen uptake in a cell suspension can be measured by various methods using manometric, paramagnetic or photometric techniques, oximetry, mass spectrometry or radiospectrometry . Easy-to-apply Clark-type electrochemical (polarographic) sensors are by far the most commonly used devices in medical applications . One of their drawbacks is the fact that they consume oxygen and may cause systematic errors when measuring oxygen uptake . Since the beginning of this century, concentration dependent quenching luminescence by oxygen has been used in a number of experimental settings . Using this analytical approach it is possible to detect oxygen without consuming it . We report about a new method of assessing cellular oxygen uptake using the luminescence quenching by oxygen . In an 850 microliters oxygen-tight microchamber, a fluorescent dye (tetraphenylporphyrin) adsorbed on a monolayer of gas chromatographic beads is separated from a cell suspension by a silicone membrane . An active electrochemical electrode integrated within the chamber is used to calibrate the fluorescence signal . Fluorescence is generated by green light (wavelength lambda = 546 nm), the intensity of the emitted red fluorescent light (lambda > 630 nm) is measured with a photomultiplier tube . As the first application of this new method, the oxygen uptake of human lymphocytes was determined . The cells were prepared using a routine separating technique-gradient centrifugation in Ficoll . For methodological reasons, all experiments were carried out at a temperature of 22 degrees C . In 7 consecutive measurements, an oxygen uptake of 2.81 +/- 0.85 mmol O2/10(11) lymphocytes/h was found . In less concentrated suspensions this figure is higher--an effect known as the "crowding phenomenon"--which means that with increasing cell concentration the specific oxygen uptake rate decreases . Our values for cellular oxygen uptake are higher than those in the literature . Since most reported studies on lymphocytes were done at 37 degrees C, a larger difference between our values and those in the literature would be expected . This may in part be attributed to different cell concentrations, separation techniques, etc . Another explanation might be the fact that electrochemical oxygen sensors used by other authors consume oxygen . Cell suspensions investigated with polarographic sensors therefore need to be stirred . Slow stirring reduces the values of oxygen uptake, so that a high stirring frequency is needed to obtain correct results (so-called stirring effect of polarographic electrodes) . High stirring frequencies, however, destroy cells, which might be another factor explaining the results reported above . Our new method based on fluorescence quenching consumes no oxygen, and is therefore independent of stirring.

Nat Med, 1999 Jan, 5(1), 97 - 100
Caspase inhibition reduces apoptosis and increases survival of nigral transplants; Schierle GS et al.; Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson disease . The main practical constraints of neural grafting are the shortage of human donor tissue and the poor survival of dopaminergic neurons grafted into patients, which is estimated at 5-10% (refs . 3,4) . The required amount of human tissue could be considerably reduced if the neuronal survival was augmented . Studies in rats indicate that most implanted embryonic neurons die within 1 week of transplantation, and that most of this cell death is apoptotic . Modified peptides, such as acetyl-tyrosinyl-valyl-alanyl-aspartyl-chloro-methylketone (Ac-YVAD-cmk), that specifically inhibit proteases of the caspase family effectively block apoptosis in a plethora of experimental paradigms, such as growth factor withdrawal, excitotoxicity, axotomy, cerebral ischemia and brain trauma . Here we examined the effects of caspase inhibition by Ac-YVAD-cmk on cell death immediately after donor tissue preparation and on long-term graft survival . Treatment of the embryonic nigral cell suspension with Ac-YVAD-cmk mitigated DNA fragmentation and reduced apoptosis in transplants . It also increased survival of dopaminergic neurons grafted to hemiparkinsonian rats, and thereby substantially improved functional recovery.

Phytochemistry, 1998 Dec, 49(7), 1879 - 90
N alpha- and N epsilon-D-galacturonoyl-L-lysine amides: properties and possible occurrence in plant cell walls; Perrone P et al.; Three representatives of a novel class of amide (isopeptide) glycoconjugates have been synthesised: N alpha-D-galacturonoyl-L-lysine and N epsilon-D-galacturonoyl-L-lysine and N epsilon-D-polygalacturonoyl-L-lysine . Galacturonoyl-lysine amide bonds were labile in 2 M trifluoroacetic acid at 120 degrees and in alkali, but relatively stable in cold acid . The amide bonds were resistant to digestion by Driselase, Pronase and trypsin . The polysaccharide backbone of N epsilon-D-polygalacturonoyl-L-lysine was hydrolysed by Driselase to yield two major ninhydrin-positive compounds which were shown by 1H and 13C NMR spectroscopy to be tri- and tetra-alpha-(1-->4)-D-galacturonoyl-L-lysines . To investigate the possible natural occurrence of N-galacturonoyl isopeptide bonds, we fed cell-suspension cultures of spinach and tomato with D-{6-14C}glucuronic acid, which radio-labels pectic polysaccharides . The radioactive cell walls were digested with, sequentially, Driselase, mild acid, and proteinases . On electrophoresis at pH 2.0, several of the radioactive digestion-products were cathodic . Some of the cathodic products yielded {14C}galacturonic acid upon complete acid hydrolysis . The existence of these products is compatible with the presence of novel N-galacturonoyl isopeptide bonds, which could serve as cross-links in plant cell walls.

Gen Comp Endocrinol, 1999 Jan, 113(1), 112 - 20
Effects of five natural gonadotropin-releasing hormones on cell suspensions of marine bivalve gonad: stimulation of gonial DNA synthesis; Pazos AJ et al.; Gonadotropin-releasing hormones (GnRH) constitute a family of neuropeptides which are important regulators of reproduction in vertebrates . The effect of mammalian GnRH (mGnRH), salmon GnRH, chicken GnRH-I, chicken GnRH-II, and lamprey GnRH-I on {3H}thymidine incorporation into DNA of dissociated gonadal cells of marine bivalves has been studied . The incorporation of {3H}thymidine is linear between 1.5 and 8 h of incubation . All five GnRHs significantly increased DNA synthesis in gonial cells of Crassostrea gigas . The maximal activation was about of 135-140% above control . The activation is dose dependent, over the range 10(-11) to 10(-6) M, but is modulated by the physiological condition of the cells and the stage of sexual maturity of the gonad . mGnRH has also a mitogenic effect in dissociated mantle cells of Mytilus edulis . The effect of mGnRH is blocked by a GnRH antagonist ({D-pGlu1,D-Phe2, D-Trp3,6}GnRH, 5 x 10(-6)M) in C . gigas as well as in M . edulis, suggesting that the GnRH action in the gonad is mediated by specific receptors for GnRH or GnRH-like peptides . The existence of GnRH-immunoreactive neurons and fibers in the cerebral and pedal ganglia of M . edulis was demonstrated by immunocytochemistry . They are located principally in the anterior internal area of the cerebral ganglia, close to the cerebral commissure and in the posterior part of the pedal ganglia . The presence of GnRH-responsive cells and GnRH-like immunoreactive material suggests that peptides of the GnRH-like family are present and functional in bivalve molluscs .

Plant J, 1998 Nov, 16(3), 325 - 33
Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants; Lancien M et al.; NAD-dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling the activity of the citric acid cycle . Despite more than 30 years of work, the plant enzyme remains poorly characterized . In this paper, a molecular characterization of the plant IDH is presented . Starting from probes defined according to sequence comparisons, three full-length cDNAs named Ntidha, Ntidhb and Ntidhc encoding different IDH subunits have been isolated from a Nicotiana tabacum cell suspension library . Sequence comparisons of the tobacco IDH subunits with the E . coli NADP-dependent enzyme, and the yeast IDH1 and IDH2 subunits suggested that only IDHa had the capacity to be catalytic as IDHb and IDHc were lacking certain residues implied in catalysis . The ability of antibodies raised against the recombinant IDHa protein to preferentially cross-react with IDH2 indicated that IDHa was more closely related to IDH2 than to IDH1 . Complementation of yeast single IDH mutants showed that IDHb and IDHc could replace the function of the yeast regulatory IDH1 subunit . Although IDHa was unable to complement the IDH2 mutant, its catalytic function was revealed by the ability of two heteromeric enzymes, composed of either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yeast double mutant lacking both subunits . Expression studies at the protein and mRNA levels show that each subunit is present in both root and leaf tissues and that the three IDH genes respond in the same way to nitrate addition . Taken together, such observations suggest that the physiologically active enzyme is composed of the three different subunits . These results show for the first time that the plant IDH is heteromeric and that IDH subunit composition appears to be conserved between plant and animal kingdoms.

Plant Physiol, 1999 Jan, 119(1), 343 - 52
Distinct cyclin D genes show mitotic accumulation or constant levels of transcripts in tobacco bright yellow-2 cells; Sorrell DA et al.; The commitment of eukaryotic cells to division normally occurs during the G1 phase of the cell cycle . In mammals D-type cyclins regulate the progression of cells through G1 and therefore are important for both proliferative and developmental controls . Plant CycDs (D-type cyclin homologs) have been identified, but their precise function during the plant cell cycle is unknown . We have isolated three tobacco (Nicotiana tabacum) CycD cyclin cDNAs: two belong to the CycD3 class (Nicta;CycD3;1 and Nicta;CycD3;2) and the third to the CycD2 class (Nicta;CycD2;1) . To uncouple their cell-cycle regulation from developmental control, we have used the highly synchronizable tobacco cultivar Bright Yellow-2 in a cell-suspension culture to characterize changes in CycD transcript levels during the cell cycle . In cells re-entering the cell cycle from stationary phase, CycD3;2 was induced in G1 but subsequently remained at a constant level in synchronous cells . This expression pattern is consistent with a role for CycD3;2, similar to mammalian D-type cyclins . In contrast, CycD2;1 and CycD3;1 transcripts accumulated during mitosis in synchronous cells, a pattern of expression not normally associated with D-type cyclins . This could suggest a novel role for plant D-type cyclins during mitosis.

Arch Dermatol Res, 1998 Dec, 290(12), 688 - 95
Characteristics and regulation of the expression on interleukin 1 receptors by murine Langerhans cells and keratinocytes; Cumberbatch M et al.; There is increasing evidence that interleukin 1beta (IL-1beta), a product in murine epidermis of Langerhans cells (LC) exclusively, contributes to the initiation and regulation of LC migration in response to skin sensitization . The hypothesis is that IL-1beta induces the production by keratinocytes of tumour necrosis factor alpha (TNF-alpha) which acts in a paracrine fashion on LC to provide one signal for migration . In addition, it is believed that IL-1beta acts in an autocrine fashion on LC to provide a second, TNF-alpha-independent, signal for the initiation of this response . The viability of this hypothesis is dependent upon the availability of appropriate membrane receptors . We describe therefore experiments designed to investigate the IL-1 receptor (IL-1R) status of keratinocytes, LC and lymph node dendritic cells (DC) . Flow cytometric analyses of epidermal cell suspensions revealed at least 60% of LC positive for the type I IL-1R (IL-1RI) . In contrast, only a small proportion of keratinocytes displayed surface IL-1RI, although high intracellular expression of this receptor could be detected either by flow cytometric analysis of cells permeabilized with saponin or by immunofluorescence microscopy . Expression of the type II IL-1R (IL-1RII) was detected at relatively low levels on both LC and keratinocytes . Interestingly, DC isolated from the lymph nodes of sensitized mice displayed upregulated expression of IL-1RI and lower levels of IL-1RII compared to LC . The conclusion drawn is that the IL-1R phenotype of LC and keratinocytes under resting conditions is consistent with the proposed contribution of IL-1beta to LC migration . Furthermore, the regulation of IL-1R expression by epidermal cells and DC will undoubtedly influence the development of cutaneous immune responses.

MAGMA, 1998 Nov, 7(1), 35 - 41
Relaxation rates of blood with osmotically modified red cell volume: application of the two-compartment fast exchange model; Yu O et al.; Blood has been considered as a simplified tissue model, both physiologically and physically consisting in two compartments, extra-cellular and intra-cellular . In the physiologic condition (300 mOsm), the relaxation rates of red cell suspensions in saline increased linearly with the hematocrit in the range 0-0.80 according to Fullerton's model of fast proton exchanges between the two compartments (Fullerton GD, Potter JL, Dornbluth NC . NMR relaxation of protons in tissues and other macromolecular water solutions . Magn Reson Imaging 1982; 1:209-228) . In experiments of osmotic variations, between 200 and 900 mOsm at three constant red cell numbers in the samples, non-linear variations of relaxation rates with red cell volume were observed . In the hyperosmotic domain, the particularly high increase in blood transverse relaxation rate with the decreasing cell volume has been attributed to the progressive water-protein organization in the cellular compartment . A generalised form of the fast exchange model has been applied to extended experimental conditions of red cells, by introducing the red cell volume ratio of modified to iso-osmotic values, and the volume fraction of iso-osmotic red cells.

Bone Marrow Transplant, 1998 Dec, 22(11), 1091 - 6
Successful cryopreservation of purified autologous CD34+ cells: influence of freezing parameters on cell recovery and engraftment; Beaujean F et al.; Conventional hematopoietic stem cell cryopreservation methods use a DMSO concentration of 10% . However, cells manipulated ex vivo may require more refined freezing protocols adapted to the specific cell suspension . In this retrospective study, we evaluated the results obtained with CD34+ cells purified from peripheral blood of 39 patients on the CEPRATE SC System and frozen in 7.5% DMSO with a view to transplantation . The post-freezing recovery of progenitor cells was 89.4 +/- 27.87% for CD34+ cells, 59.13 +/- 36.93% for CFU-GM, and 53.49 +/- 40.71 for BFU-E . Neither the purity of the suspension nor the nucleated cell density during freezing was predictive of cell recovery . No difference was observed between cells stored in vials and bags . Thirty-seven patients transplanted with the concentrated CD34+ fraction received 4.46 x 10(6) CD34+ cells/kg and 33.04 x 10(4) CFU-GM/kg . The median time to granulocyte (>0.5 x 10(9)/l) and platelet (>50 x 10(9)/l) engraftment was 11 and 13 days, respectively . Only cell density and the infused number of CD34+ cells and CFU-GM were significantly related to hematological recovery . Our data suggest that purified CD34+ cells can be successfully cryopreserved in 7.5% DMSO and may represent a first step in establishing freezing parameters for selected CD34+ cells.

Clin Hemorheol Microcirc, 1998 Nov, 19(3), 205 - 17
Loading of intact rabbit erythrocytes with fluorophores and the enzyme pronase by means of electroporation; Haritou M et al.; The application of electric field pulses in cell suspensions is known to alter membrane integrity, resulting in increased membrane permeabilization . This field-induced membrane poration provides the means to load cells with a variety of external substances, useful for clinical applications . In this work, intact rabbit erythrocytes were successfully loaded with low molecular weight fluorescent probes and with the high molecular weight enzyme pronase, which has been shown to mimic the effects of insulin . Attachment of the enzyme onto the cell surface was also achieved by modifying the applied pulse parameters . Both applications were efficient and accompanied by high cell survival rates . In this way, biological carriers loaded with active substances were produced, offering the potentials for useful clinical applications, either for diagnostic or therapeutic purposes.

Immunol Lett, 1998 Dec, 64(2-3), 153 - 60
Tumor surveillance: expression of the transporter associated with antigen processing (TAP-1) in ex-vivo human tumor samples and its elevation by in vitro treatment with IFN-gamma and TNF-alpha; Nagy N et al.; We have analyzed 30 human tumor specimens (two breast, six lung, and 21 ovarian carcinomas and one malignant melanoma) for the expression of the transporter associated with antigen processing, TAP-1 . Cell suspensions judged to contain negligible contamination with non-tumor cells were tested for reactivity with the antibody directed to TAP-1 in Western blot . According to these results all lysates prepared from the tumor samples contained the protein, but with considerable quantitative variations . Tumors exposed in vitro for a short time to IFN-gamma and TNF-alpha had elevated TAP-1 levels in 12 out of 30 experiments . In accordance with previous results, the tumor cell populations were heterogeneous with regard to MHC class I expression, as analyzed by indirect immunofluorescence on viable cell suspensions . Short term in vitro treatment with IFN-gamma and TNF-alpha elevated MHC class I expression in several tumors . In several mixed cultures, cytokine treated tumor cells induced cytotoxic activity in autologous or allogeneic lymphocyte populations . In vitro treatment with IFN-gamma and TNF-alpha upregulated thus the expression of MHC class I and TAP-1 in a fraction of the tumor samples . However the potentiation of the capacity to interact with T cells induced by the cytokines was not always parallel with the changes in these two parameters . It is therefore likely that the cytokine treatment induced additional changes in the tumors which contributed to their capacity to elicit the T cell response.

Ann Transplant, 1996, 1(1), 67 - 9
Reconstitution of lymphoid tissue after vascularized bone marrow transplantation; Lukomska B et al.; We reported previously that vascularized bone marrow transplantation (VBMT) in an orthotopic hind limb graft brings about complete repopulation of bone marrow cavities in lethally irradiated syngeneic recipients within 10 days . Intravenous infusion of an equivalent volume of bone marrow cell suspension was evidently less effective . The purpose of this study was to investigate the reconstitution of immunocompetent compartments of lethally irradiated syngeneic rats after VBMT . Lewis rat hind limbs were transplanted orthotopically into irradiated recipients . Ten days after irradiation and bone marrow transplantation, bone marrow, mesenteric lymph nodes and sera from rats were harvested . Responsiveness of mesenteric lymph node lymphocytes (MLNL) to mitogens and lymphocyte proliferation in the presence of sera and bone marrow cell (BMC) culture supernatants was measured . Our studies have shown that vascularized bone marrow transplantation brings about rapid replenishment of lymphoid organs of lethally irradiated syngeneic recipients . The repopulating subsets were fully responsive to mitogens . Sera from reconstituting rats had no evident effect on proliferation of mature lymphocytes . Intravenous infusion of BMC in suspension, in a number equivalent to that grafted in hind limb transplant, was less efficient in reconstituting lymphoid tissue.

Ann Transplant, 1996, 1(1), 51 - 3
Cultured parathyroid cells allotransplantation without immunosuppression for treatment of intractable hypoparathyroidism; Tolloczko T et al.; Cultured, viable and functioning ABO compatible parathyroid cells were allografted in 18 nonimmunosuppressed patients with a postoperative hypoparathyroidism . Variable, but promising biochemical and clinical results were obtained . Clinical and biochemical observations have documented graft function up to 14 months . The mechanism of cessation of function of implanted cultured cell suspension remains unknown . Some parameters, suggest a rejection mechanism but other mechanisms can not be excluded . This suggests that some form of immune modulation may be necessary to improve further PT allograft survival in recipients off immunosuppressive therapy.

Biochemistry (Mosc), 1998 Oct, 63(10), 1144 - 7
Energetic aspects of CO oxidation in desulfovibrio desulfuricans
Tarasova NB, Zolotov AV, Petrova OE.
In cell suspension of Desulfovibrio desulfuricans B-1388, oxidation of CO as the only energy source is associated with reduction of SO42- . After a 2-h incubation of cells in 8% CO, 81% of the gas is converted . Oxidation of 1 mole CO results in formation of 0.23 mole H2S . Intracellular ATP content increases from 2.5 (control) to 8.3 nmoles/mg (during CO conversion) . Dinitrophenol inhibits sulfate reduction and CO oxidation . CO dehydrogenase was detected in cytoplasmic and membrane cell fractions (59 and 34%, respectively).

Br J Cancer, 1998 Dec, 78(12), 1661 - 8
Aberrant cytoplasmic expression of the p16 protein in breast cancer is associated with accelerated tumour proliferation; Emig R et al.; The p16 protein plays an important role in the transition of cells into the G1 phase of the cell cycle . We have studied the prevalence of p16 protein expression in breast carcinomas in a prospective series of 368 invasive and 52 non-invasive malignancies, as well as in 88 locally recurring tumours and three tumour cell lines . p16 protein expression was evaluated immunohistochemically on paraffin sections using monoclonal and polyclonal anti-p16 antibodies, and by immunoblotting of tumour cell suspensions . Tumour cell lines were also subjected to polymerase chain reaction-single strand polymorphism (PCR-SSCP) analysis and direct DNA sequencing . The results were compared with established prognostic parameters, DNA flow cytometry and p53 protein expression . In 33 (9%) invasive and two (4%) intraductal carcinomas, a cytoplasmic accumulation of the p16 protein was seen . These cases were characterized by poor histological grade of differentiation, loss of of oestrogen receptors and progesterone receptors and frequent overexpression of the p53 protein . In addition, breast carcinomas with aberrant p16 expression demonstrated a high proliferative activity, with median S-phase fractions 74% higher than in the control group and the median Ki67 fractions elevated to 75% . A genetic alteration of the p16 gene was not detectable in three analysed cell lines with cytoplasmic p16 expression applying PCR-SSCP and direct DNA sequencing . These results indicate that cytoplasmic accumulation of the p16 protein identifies a subset of highly malignant breast carcinomas with accelerated tumour proliferation and other unfavourable parameters in breast cancer . The described protein accumulation is apparently not caused by an alteration of the p16 gene.

J Reprod Immunol, 1998 Oct, 40(1), 25 - 45
Regulation by human uterine cells of PBMC proliferation: influence of the phase of the menstrual cycle and menopause; Prabhala RH et al.; To determine the influence of human uterine cells recovered at different stages of the menstrual cycle and following menopause on the proliferation of peripheral blood mononuclear cells (PBMC), whole cell suspensions of uterine tissues were co-cultured with autologous and heterologous PBMC . PBMC proliferation in response to tetanus toxoid (TT) or Con A was inhibited by uterine endometrial cells and was dependent on the phase of the menstrual cycle . Inhibition by cells from the proliferative phase was significantly greater than by cells from the secretory phase . Uterine cells isolated from post-menopausal women also inhibited proliferation of PBMC . Cell fractionation studies indicated that epithelial cells are the primary source of uterine inhibitory activity . When epithelial cells and PBMC were cultured in separate compartments, epithelial cells released a soluble factor(s) that inhibited the PBMC proliferation . These results suggest that uterine epithelial cells produce cytokines that down-regulate the proliferation of PBMC in response to antigens and mitogens . This may be important for the control of uterine immune responses, as well as the growth of the reproductive tract in preparation for implantation during the secretory phase of the menstrual cycle.

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