Clin Exp Immunol, 2001 Sep, 125(3), 376 - 82 Flow cytometric measurement of intracellular migration inhibition factor and tumour necrosis factor alpha in the mucosa of patients with coeliac disease; O'Keeffe J et al.; There is increasing evidence that proinflammatory cytokines contribute to many of the small intestinal features in coeliac disease . The aim of the study was to investigate the expression of two proinflammatory cytokines, migration inhibition factor (MIF) and tumour necrosis factor alpha (TNF-alpha) in duodenal biopsy specimens from patients with coeliac disease on a gluten-free diet and normal control subjects . A flow cytometric system was used to analyse intracellular protein levels of MIF and TNF-alpha in freshly isolated cells from duodenal biopsies taken from 12 patients with treated coeliac disease and 10 healthy control subjects . From the biopsy specimens, single cell suspensions of the epithelium and lamina propria were prepared using EDTA/DTT and enzymes . Intracellular cytokine expression was studied in intraepithelial lymphocytes (IELs), lamina propria T cells (LP T) and intestinal epithelial cells using different surface labelling antibodies . MIF protein was constitutively expressed in IELs, LP T cells and epithelial cells from normal intestinal mucosa . In contrast, although TNF-alpha was found in LP T cells, this cytokine was virtually undetectable in either IELs or epithelial cells . In coeliac disease, intracellular levels of MIF were significantly higher in epithelial cells compared with control subjects (P = 0.005) . Raised levels of TNF-alpha were found in epithelial cells (P = 0.03) as well as IELs (P = 0.045) from coeliac patients compared with controls . The findings from this study show up-regulated expression of MIF and TNF-alpha in IELs and epithelial cells of histologically normal mucosa in patients with coeliac disease . Increased expression of proinflammatory cytokines in cells occupying the epithelial layer could help explain the rapidity with which the coeliac mucosa may respond to gluten challenge. Z Naturforsch {C}, 2001 Jul-Aug, 56(7-8), 559 - 69 Triaziflam and Diaminotriazine derivatives affect enantioselectively multiple herbicide target sites; Grossmann K et al.; Enantiomers of triaziflam and structurally related diaminotriazines were synthesized and their herbicidal mode of action was investigated . The compounds caused light and dark-dependent effects in multiple test systems including heterotrophic cleaver and photoautotrophic algal cell suspensions, the Hill reaction of isolated thylakoids and germinating cress seeds . Dose-response experiments revealed that the (S)-enantiomers of the compounds preferentially inhibited photosystem II electron transport (PET) and algae growth with efficacies similar to that of the herbicide atrazine . In contrast, the (R)-enantiomers of the diaminotriazines were up to 100 times more potent inhibitors of growth in cleaver cell suspensions and cress seedlings in the dark than the (S)-enantiomers . The most active compound, the (R)-enantiomer of triaziflam, inhibited shoot and root elongation of cress and maize seedlings at concentrations below 1 microM . The meristematic root tips swelled into a club shape which is typical for the action of mitotic disrupter herbicides and cellulose biosynthesis inhibitors . Microscopic examination using histochemical techniques revealed that triaziflam (R)-enantiomer blocks cell division in maize root tips 4 h after treatment . The chromosomes proceeded to a condensed state of prometaphase but were unable to progress further in the mitotic cycle . Disruption of mitosis was accompanied by a loss of spindle and phragmoplast micotubule arrays . Concomitantly, cortical microtubules decreased which could lead to isodiametric cell growth and consequently to root swelling . In addition, a decline in cellulose deposition in cell walls was found 24 h after treatment . Compared to the (R)-form, triaziflam (S)-enantiomer was clearly less active . The results suggest that triaziflam and related diaminotriazines affect enantioselectively multiple sites of action which include PET inhibitory activity, mitotic disruption by inhibiting microtubule formation and inhibition of cellulose synthesis. Cesk Fysiol, 2001 Aug, 50(3), 108 - 14 {Precision-cut tissue sections--an important system for study of metabolism in vitro}; Cervenkova K et al.; Tissue slices represent a useful in vitro method for metabolic, pharmacologic and toxicologic studies . To their major advantage belong preserved cell to cell interactions, architecture and complexity of the tissue . They enable to reduce the number of laboratory animals, that are necessary for research purposes . Tissue slices are more similar to in vivo situation than other in vitro models such as microsomes, cell suspensions or cell cultures . Slices can be cultured up to 72 hours or even more . Preparation of the slices is rapid and easy . Slices can be prepared from any organ . There is a possibility of cross-species comparison, co-culturing of slices derived from different organs . Excess of the material can be cryopreserved for later studies. Phytochemistry, 2001 Sep, 58(1), 53 - 8 7-Deoxyloganin 7-hydroxylase in Lonicera japonica cell cultures; Katano N et al.; The activity of 7-deoxyloganin 7-hydroxylase, an enzyme catalyzing the conversion of 7-deoxyloganin into loganin, was detected in a microsomal preparation from the cell suspension cultures of Lonicera japonica . It was dependent on NADPH and molecular oxygen . The enzymatic reaction was inhibited by carbon monoxide as well as by several cytochrome P450 inhibitors, especially ketoconazole, indicating that the reaction was mediated by cytochrome P450 . The enzyme showed substrate specificity for 7-deoxyloganin . The K(m) values for 7-deoxyloganin and NADPH were estimated as 170 and 18 microM, respectively, from Lineweaver-Burk plots. Mol Genet Genomics, 2001 Aug, 265(6), 954 - 63 Ionising radiation induces the expression of PARP-1 and PARP-2 genes in Arabidopsis; Doucet-Chabeaud G et al.; By screening for Arabidopsis genes activated by ionising radiation (IR)-induced DNA damage, we have isolated a cDNA hybridising with a 3.2-kb mRNA that accumulates rapidly and strongly in irradiated cell suspensions or whole plants . The cDNA codes for a 110-kDa protein that is highly homologous to the 116-kDa vertebrate poly(ADP-ribose) polymerase (PARP-1) . It is recognised by a human anti-PARP-1 antibody, binds efficiently to DNA strand interruptions in vitro, and catalyses DNA damage-dependent (ADP-ribose) polymer synthesis . We have named this protein AtPARP-1 . We have also extended our observations to the Arabidopsis app (AtPARP-2) gene, demonstrating for the first time that IR-induced DNA strand interruptions induce rapid and massive accumulation of AtPARP-1 and AtPARP-2 transcripts, whereas dehydration and cadmium preferentially induce the accumulation of AtPARP-2 transcripts . The IR-induced PARP gene expression seen in Arabidopsis is in striking contrast to the post-translational activation of the PARP-1 protein that is associated with genotoxic stress in animal cells . AtPARP-1 transcripts accumulate in all plant organs after exposure to ionising radiation, but this is followed by an increase in AtPARP-1 protein levels only in tissues that contain large amounts of actively dividing cells . This cell-type specific accumulation of AtPARP-1 protein in response to DNA damage is compatible with a role for the AtPARP-1 protein in the maintenance of DNA integrity during replication, similar to the role of "guardian of the genome" attributed to its animal counterpart. Cell Biol Int, 2001, 25(9), 919 - 30 Impact of proliferative activity and tumorigenic conversion on mitochondrial function of fibroblasts in 2D and 3D culture; Kunz-Schughart LA et al.; The purpose of the present study was to examine mitochondrial function in differently transformed cells relative to their tumorigenic state and proliferative activity in vitro . An established two-step carcinogenesis model consisting of immortal and tumorigenic rat embryo fibroblasts that can be cultured as monolayers and multicellular spheroids was investigated . Flow cytometric measurements were carried out using the two mitochondrial-specific fluorochromes rhodamine 123 (Rh123) and 10-N-nonyl acridine orange (NAO), in combination with the DNA dye Hoechst 33342 for simultaneous cell cycle analysis . Since the accumulation of Rh123 depends on mitochondrial membrane potential, Rh123 fluorescence intensity gives an estimate of mitochondrial activity per cell, as determined by both overall mitochondrial function and mass . In contrast, NAO uptake reflects mitochondrial mass only, as it binds to cardiolipin in the inner mitochondrial membrane independently of membrane potential . Aliquots of cell suspensions derived from exponential monolayer, confluent monolayer, and a range of sizes of multicellular spheroids were stained with either Rh123 or NAO and Hoechst 33342, then mitochondrial mass and activity per unit cell volume and cellular DNA content were measured by flow cytometry . Differences in the average mitochondrial activity per cell in different cell lines and culture conditions were primarily due to alterations in cell volume . Importantly, tumorigenic conversion by ras-transfection did not consistently change mitochondrial activity per unit cell volume . The mitochondrial mass per unit cell volume increased for all cells when cellular quiescence was induced, either in monolayers or spheroids . However, mitochondrial function (activity/mass) decreased when cells became quiescent, resulting in a positive correlation between mitochondrial function and S-phase fraction, independent of transformation status or culture condition . We conclude that mitochondrial function reflects proliferative activity rather than tumorigenic conversion . Int Immunopharmacol, 2001 Aug, 1(8), 1571 - 81 Acute morphine treatment alters cellular immune function in the lungs of healthy rats; Coussons-Read ME et al.; Previous work has shown that morphine suppresses the pulmonary immune response to infection and reduces pulmonary inflammation . No published studies have addressed the impact of morphine on lymphocyte function in the lungs without infection . This study addressed this question by assessing the impact of acute morphine treatment on proliferation, cytokine production, and natural killer (NK) cell activity in resident pulmonary lymphocytes from healthy rats . Male Lewis rats received either a single 15 mg/kg morphine sulfate or vehicle injection 1 h prior to sacrifice . Lungs were minced and passed through wire mesh following collagenase digestion . The resulting cell preparations were pooled (2 rats/pool) to yield sufficient cell numbers for the functional assays, and a portion of these suspensions were separated using a density gradient . Crude and purified cell suspensions were used in assays of NK cell activity and mitogen-induced proliferation and cytokine production . Morphine significantly suppressed lymphocyte proliferation and cytokine production in whole cell suspensions, but not in purified cultures . NK activity was enhanced by morphine treatment in purified treated cultures . Studies of nitrate/nitrite levels in crude and purified cultures suggest that macrophage-derived nitric oxide may be a mechanism of the suppression observed in whole cell suspensions following morphine treatment . These data are consistent with previous work showing that morphine suppresses mitogenic responsiveness and NK activity in the spleen and peripheral blood, and may do so through a macrophage-derived nitric oxide mechanism. Indian J Exp Biol, 2001 May, 39(5), 483 - 4 A simple and inexpensive "cell dissociation sieve-tissue grinder" apparatus; Pai K et al.; A simple and inexpensive cell dissociation sieve-tissue grinder apparatus consisting essentially of stainless steel sieve (the one popularly used for sieving tea leaves) and a glass syringe plunger acting as pestle, is described for making single cell suspension. Biophys J, 2001 Sep, 81(3), 1360 - 72 Stochastic simulation of hemagglutinin-mediated fusion pore formation; Schreiber S et al.; Studies on fusion between cell pairs have provided evidence that opening and subsequent dilation of a fusion pore are stochastic events . Therefore, adequate modeling of fusion pore formation requires a stochastic approach . Here we present stochastic simulations of hemagglutinin (HA)-mediated fusion pore formation between HA-expressing cells and erythrocytes based on numerical solutions of a master equation . The following elementary processes are taken into account: 1) lateral diffusion of HA-trimers and receptors, 2) aggregation of HA-trimers to immobilized clusters, 3) reversible formation of HA-receptor contacts, and 4) irreversible conversion of HA-receptor contacts into stable links between HA and the target membrane . The contact sites between fusing cells are modeled as superimposed square lattices . The model simulates well the statistical distribution of time delays measured for the various intermediates of fusion pore formation between cell-cell fusion complexes . In particular, these are the formation of small ion-permissive and subsequent lipid-permissive fusion pores detected experimentally (R . Blumenthal, D . P . Sarkar, S . Durell, D . E . Howard, and S . J., J . Cell Biol . 135:63-71) . Moreover, by averaging the simulated individual stochastic time courses across a larger population of cell-cell-complexes the model also provides a reasonable description of kinetic measurements on lipid mixing in cell suspensions (T . Danieli, S . L . Pelletier, Y.I . Henis, and J . M . White, 1996, J . Cell Biol . 133:559-569). Cell Calcium, 2001 Sep, 30(3), 151 - 6 Measurement of stress-induced Ca(2+) pulses in single aequorin-transformed tobacco cells; Cessna SG et al.; Signaling patterns measured in large cell populations are the sum of differing signals from separate cells, and thus, the detailed kinetics of Ca(2+) pulses can often be masked . In an effort to evaluate whether the cytosolic Ca(2+) pulses previously reported in populations of elicitor- and stress-stimulated tobacco cells accurately represent the pulses that occur in individual cells, a study of single cell Ca(2+) fluxes in stress-stimulated tobacco cells was undertaken . Individual aequorin-transformed cells were isolated from a tobacco suspension culture and placed directly on a sensitive photo-multiplier tube mounted in a dark chamber . Ca(2+)-dependent luminescence was then monitored after stimulation with hypo- or hyper-osmotic shock, cold shock, or defense elicitors (oligogalacturonic acid and harpin) . Hypo-osmotic shock induced a biphasic Ca(2+) transient in 67% of the single cells tested that exhibited similar kinetics to the biphasic pulses measured repeatedly in 1ml cell suspensions . In contrast, 33% of the stimulated cells displayed Ca(2+) flux patterns that were not previously seen in cell suspension studies . Additionally, because only 29% of the cells tested responded with measurable Ca(2+) pulses to oligogalacturonic acid and 33% to the harpin protein, we conclude that not all cells in a suspension are simultaneously sensitive to stimulation with defense elicitors . In contrast, all cells tested responded with an immediate Ca(2+) influx after cold or hyperosmotic shock . We conclude that in many cases the Ca(2+) signaling patterns of single cells are accurately represented in the signaling patterns of large populations, but that single cell measurements are still required to characterize the Ca(2+) fluxes of the less prominent cell populations . J Acoust Soc Am, 2001 Jul, 110(1), 588 - 96 Ultrasound-mediated disruption of cell membranes . I . Quantification of molecular uptake and cell viability; Guzman HR et al.; Ultrasound-mediated drug delivery is a nonchemical, nonviral, and noninvasive method for targeted transport of drugs and genes into cells . Molecules can be delivered into cells when ultrasound disrupts the cell membrane by a mechanism believed to involve cavitation . This study examined molecular uptake and cell viability in cell suspensions (DU145 prostate cancer and aortic smooth muscle cells) exposed to varying peak negative acoustic pressures (0.6-3.0 MPa), exposure times (120-2000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent . With increasing pressure and exposure time, molecular uptake of a marker compound, a calcein, increased and approached equilibrium with the extra cellular solution, while cell viability decreased . Varying pulse length produced no significant effect . All viability and molecular uptake measurements collected over the broad range of ultrasound conditions studied correlated with acoustic energy exposure . This suggests that acoustic energy exposure may be predictive of ultrasound's nonthermal bioeffects. Bioelectrochemistry, 2001 Aug, 54(1), 91 - 5 Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses . Part II . Reduced electrolytic contamination; Kotnik T et al.; The paper presents a comparative study of the contamination of a cell suspension by ions released from aluminum cuvettes (Al(3+)) and stainless steel electrodes (Fe(2+)/Fe(3+)) during cell membrane electropermeabilization by unipolar and by symmetrical bipolar rectangular electric pulses . A single pulse and a train of eight pulses were delivered to electrodes at a 2-mm distance, with 100-micros and 1-ms pulse durations, and amplitudes ranging from 0 to 400 V for unipolar, and from 0 to 280 V for bipolar pulses . We found that the released concentrations of Al(3+) and Fe(2+)/Fe(3+) were always more than one order of magnitude lower with bipolar pulses than with unipolar pulses of the same amplitude and duration . We then investigated the viability of DC-3F cells after 1 h of incubation in the medium containing different concentrations of Al(3+) or Fe(2+)/Fe(3+) within the range of measured released concentrations (up to 2.5 mM for both ions), thus separating the effects of electrolytic contamination from the effects of electropermeabilization itself . For Fe(2+)/Fe(3+), loss of cell viability became significant at concentrations above 1.5 mM, while for Al(3+), no effect on cell survival was detected within the investigated range . Still, reports on the biochemical effects of released Al(3+) also suggest that with aluminum cuvettes, electrolytic contamination can be detrimental . Our study shows that electrolytic contamination and its detrimental effects can be largely reduced with no loss in efficiency of electropermeabilization, if bipolar rectangular pulses of the same amplitude and duration are used instead of the commonly applied unipolar pulses. Bioelectrochemistry, 2001 Aug, 54(1), 83 - 90 Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses . Part I . Increased efficiency of permeabilization; Kotnik T et al.; The paper presents a comparative study of electropermeabilization of cells in suspension by unipolar and symmetrical bipolar rectangular electric pulses . While the parameters of electropermeabilization by unipolar pulses have been investigated extensively both in cell suspensions and in tissues, studies using bipolar pulses have been rare, partly due to the lack of commercially available bipolar pulse generators with pulse parameters suitable for electropermeabilization . We have developed a high-frequency amplifier and coupled it to a function generator to deliver high-voltage pulses of programmable shapes . With symmetrical bipolar pulses, the pulse amplitude required for the permeabilization of 50% of the cells was found to be approximately 20% lower than with unipolar pulses, while no statistically significant difference was detected between the pulse amplitudes causing the death of 50% of the cells . Bipolar pulses also led to more than 20% increase in the uptake of lucifer yellow . We show that these results have a theoretical background, because bipolar pulses (i) counterbalance the asymmetry of the permeabilized areas at the poles of the cell which is introduced by the resting transmembrane voltage, and (ii) increase the odds of permeabilization of cells having a nonspherical shape or a nonhomogeneous membrane . If similar results are also obtained in tissues, bipolar pulse generators could in due course gain a wide, or even a predominant use in cell membrane electropermeabilization. Bioelectrochemistry, 2001 Aug, 54(1), 53 - 61 Reduction of the contribution of electrode polarization effects in the radiowave dielectric measurements of highly conductive biological cell suspensions; Bordi F et al.; Electrode polarization effects in dielectric spectra of highly conductive biological cell suspensions cause a severe difficulty in the estimation of dielectric parameters of cells under physiological conditions . This problem becomes particularly serious with the increase of the electrical conductivity of the sample, preventing the use of low frequencies in the characterization of biological systems, especially aqueous biological systems.Although a variety of methods to correct the electrode polarization have been proposed in the past, no simple technique for its correction has been available so far . Since the magnitude of the polarization effect can be time-dependent owing to changes in the conductance of the suspending medium or to possible alteration in the electrode surface structure, it is clear that correction procedure should be based on a kind of "self-correction" method, avoiding the so-called "comparison methods" which, on the contrary, require time-independent effects.This note is aimed to address this problem considering an electrode polarization modelled by a constant phase angle (CPA) element in series with the sample admittance . A scaling-law frequency dependence has found to describe the a.c . response of the interface between the electrode and the bulk electrolyte solution . Although this approach has been extensively proposed in the past in the analysis of dielectric spectra of biological suspensions, we have somewhat modified the way it has been previously applied and have re-examined in detail its effectiveness in typical systems of biological interest . The results give support to the proposed analysis, allowing the complete low-frequency dielectric spectra characterization at frequencies of the order of 1 kHz for samples with a bulk ionic conductivity as large as that of the order of 1 mho/m . Typical examples with different dielectric behaviours are extensively discussed in order to show the applicability of the proposed method to biological samples. Planta, 2001 Jul, 213(3), 435 - 45 Solubilization of rhamnogalacturonan I galactosyltransfrases from membranes of a flax cell suspension; Peugnet I et al.; Galactosyltransferases (GalTs), capable of transferring a galactosyl residue from UDP-galactose (UDP-Gal) to polysaccharide acceptor, were solubilized from flax (Linum usitatissimum L.) membranes using 0.5% CHAPS . The observed requirement for a rhamnogalacturonan I (RG-I) exogenous substrate to stimulate the solubilized GalT activity provided the first evidence for the presence of RG-I GalT activities in flax cells . An assay to measure specifically the products of this RG-I GalT activity was designed, based on size-exclusion chromatography . Labelled products were characterized as an RG-I polymer by using purified RG-I hydrolase or lyase . At pH 8 and in the presence of 5 mM CaCl2, beta-D-galactosyl residues were specifically transferred onto RG-I branches of short beta-(1 --> 4)-D-galactan side chains . These side chains were liable to hydrolysis by beta-galactosidase and endo-beta-(1 --> 4)-D-galactanase . The RG-I GalT had a temperature optimum of 30 degrees C . an apparent Km for UDP-Gal and exogenous RG-I substrate of 460 +/- 40 microM and 1.1 +/- 0.1 mg ml(-1) respectively, and a Vmax of 3.0 +/- 0.5 pkat mg(-1) protein. Biotechnol Bioeng, 2001 Sep, 76(2), 126 - 31 Utilizing genetically engineered bacteria to produce plant-specific glucosides; Arend J et al.; Plant-derived glucosides have attracted much attention due to their widespread applications . This class of products is difficult to isolate or to synthesize in pure form because of the resulting low yields . Thus, simple approaches for the generation of such glucosides would be highly beneficial . We purified and characterized a novel glucosyltransferase from plant cell suspension cultures of Rauvolfia serpentina, which showed rather low substrate specificity . We obtained its cDNA and expressed the active recombinant protein in bacteria (Escherichia coli) with excellent plant-specific glucosylation efficiencies . Compared with the plant system, the bacteria delivered the new enzyme, which was in the form of a soluble or matrix-bound enzyme, approximately 1800 times more efficiently for the synthesis of a wide range of glucosides . More importantly, the engineered E . coli strain allowed for in vivo glucosylation and release of the product into the culture medium, as shown by the formation of arbutin, which is a potent inhibitor of human melanin biosynthesis with commercial value . Arch Environ Contam Toxicol, 2001 Oct, 41(3), 282 - 8 Effects of experimental manipulation of pH and salinity on Cd(2+) uptake by the sponge Microciona prolifera and on sponge cell aggregation induced by Ca(2+) and Cd(2+); Philp RB; Marine sponges (Microciona prolifera) collected in St . Joseph Bay, Florida panhandle, were exposed for 2 h to pH/salinity unit combinations of 7.4/30, 6.3/30, 7.4/11, and 6.3/11 . Cell suspensions from these were aggregated with 24 microM of either CaCl(2) or CdCl(2) . Cells exposed to the low/low (11/6.3) combination aggregated spontaneously (no added stimulus) in 8/11 experiments, suggesting a significant disturbance of normal function, possibly involving disrupted ion uptake . In all other combinations aggregation proceeded normally and there were no statistically significant differences among the groups . CdCl(2) was as effective an aggregation stimulus as CaCl(2) . The calcium channel blocker verapamil (100 microM) reduced calcium-induced aggregation by 15% but had no effect on cadmium (Cd)-induced aggregation, indicating that L-type calcium channels do not play a major role in aggregation induced by these divalent cations . Sponge tissue was exposed for 48 h to the same pH/salinity combinations but containing Cd (15 or 150 microg/ml) and then dried and analyzed for Cd . All sponges concentrated Cd but those exposed to low salinity concentrated it most (in one case x13) . Low pH alone had no appreciable effect but appeared to increase the effect of low salinity . One sponge with a native Cd content of 47.2 microg/g dry weight had the highest acquired Cd content . The results of this study indicate that low levels of salinity and pH, similar to those we recorded in the study area, facilitate the accumulation of Cd, but not via L-type calcium channels, and disrupt normal aggregation responses of the cell . These results may help explain a previous observation that cells from M . prolifera from this area, with high native levels of Cd, failed to aggregate in response to CaCl(2){Philp RB (1999) Comp Biochem Physiol 124C:41-49} and also the frequent die-offs of Microciona that have virtually eliminated this sponge from its local habitat. Plant Physiol, 2001 Aug, 126(4), 1403 - 15 A new C-type cyclin-dependent kinase from tomato expressed in dividing tissues does not interact with mitotic and G1 cyclins; Joubes J et al.; Cyclin-dependent kinases (CDKs) form a conserved superfamily of eukaryotic serine-threonine protein kinases whose activity requires the binding of a cyclin protein . CDKs are involved in many aspects of cell biology and notably in the regulation of the cell cycle . Three cDNAs encoding a C-type CDK, and a member of each B-type CDK subfamily, were isolated from tomato (Lycopsersicon esculentum Mill.) and designated Lyces;CDKC;1 (accession no . AJ294903), Lyces; CDKB1;1 (accession no . AJ297916), and Lyces;CDKB2;1 (accession no . AJ297917) . The predicted amino acid sequences displayed the characteristic PITAIRE (CDKC), PPTALRE (CDKB1), and PPTTLRE (CDKB2) motives in the cyclin-binding domain, clearly identifying the type of CDK . The accumulation of all transcripts was associated preferentially with dividing tissues in developing tomato fruit and vegetative organs . In contrast to that of CDKA and CDKBs, the transcription pattern of Lyces;CDKC;1 was shown to be independent of hormone and sugar supply in tomato cell suspension cultures and excised roots . This observation, together with the absence of a patchy expression profile in in situ hybridization experiments, suggests a non-cell cycle regulation of Lyces;CDKC;1 . Using a two-hybrid assay, we showed that Lyces;CDKC;1 did not interact with mitotic and G1 cyclins . The role of plant CDKCs in the regulation of cell division and differentiation is discussed with regard to the known function of their animal counterparts. Inhal Toxicol, 2001 Sep, 13(9), 773 - 88 The influence of suspension nebulization or instillation on particle uptake by guinea pig alveolar macrophages; Suarez S et al.; Phagocytosis represents a crucial event in the host defense against pathogens . Experimental methods are required that allow a range of particle doses to be delivered . However, it is not clear that these methods result in the same sites of deposition or mechanisms of clearance . The effect of particle administration by nebulization or instillation on the uptake by guinea pig alveolar macrophages (AMs) has been studied . Suspensions of escalating quantities of 1-microm fluorescent polystyrene latex microspheres were delivered by 15 min of nebulization (1.4 x 10(7)-11.1 x 10(7) particles) or instillation (19 x 10(7)-152 x 10(7) particles) into the lungs of guinea pigs . These doses were selected to maximize delivery using each of these methods . Macrophages were collected by alveolar lavage 6 h postadministration . The total number of cells recovered was 3 x 10(6) and the cell viability was >97%, which was measured by trypan blue exclusion . Differential cell counts of lavaged cell suspensions were conducted and results showed no difference for the two methods of administration with various concentrations of latex particles and control samples . The uptake of particles was measured using epifluorescence, confocal microscopy, and flow cytometry . AMs showed a dose-dependent increase in associated particles measured by microscopy and flow cytometry . There was a direct correlation (R(2) =.99) in the phagocytic indices (PIs) measured by flow cytometry and fluorescence microscopy . The PI was 15 times higher after instillation than that obtained after particle nebulization . The percentage of AMs involved in phagocytosis observed after instillation was 55% and after nebulization 23% . The uptake of aerosolized particles by AMs and the number of cells involved in phagocytosis were dependent on the particle dose and the efficiency of aerosol delivery to the lungs. Prostate, 2001 Aug 1, 48(3), 156 - 64 Primary culture of microvascular endothelial cells from human benign prostatic hyperplasia; Stachon A et al.; BACKGROUND: Prostate growth seems to be influenced by paracrine factors like IL-6 originating from the microvascular endothelium . Therefore, our efforts were focused on the primary culture and behavior of microvascular endothelial cells (HPEC) derived from tissue of human benign prostatic hyperplasia (BPH) . Until now, the isolation and culture of HPEC from BPH have not been reported . METHODS: BPH tissue was cut into small cubes and gently squeezed after incubation with dispase . HPEC were cultured from the resulting cell suspension after a stepwise selection by use of superparamagnetic beads coated with antibodies against endothelial specific antigens . HPEC were characterized by flow cytometry and immunohistochemistry . gamma-Glutamyl transpeptidase activity (specific for microvascular endothelium) was measured after dissolution of the HPEC with Triton X-100 . After the incubation of HPEC either with ATP, VEGF, or TNF-alpha, the release of IL-6 was measured by enzyme linked immunosorbent assay (ELISA) . RESULTS: HPEC showed a typical endothelial morphology . They were positive for von Willebrand factor, CD31, CD62E (after stimulation with TNF-alpha), alpha-actin and were negative for fibroblastic antigens and PSA . Proliferation was stimulated by vascular endothelial growth factor (VEGF) . gamma-Glutamyl transpeptidase activity in HPEC was 6.3 microIU/microg protein, whereas in human umbilical vein endothelial cells (HUVEC) no gamma-glutamyl transpeptidase activity was detectable . The IL-6 secretion of HPEC was stimulated by VEGF and TNF-alpha, but not by ATP and bradykinin . CONCLUSIONS: For the first time, the primary culture of microvascular endothelial cells from BPH tissue was successfully performed . Our results suggest that HPEC may be actively involved in prostate growth, due to the secretion of regulatory factors such as IL-6 . Plant J, 2001 Jul, 27(1), 1 - 12 Cold-activation of Brassica napus BN115 promoter is mediated by structural changes in membranes and cytoskeleton, and requires Ca2+ influx; Sangwan V et al.; Previous studies on cold-triggered events leading to Ca2+ influx during cold acclimatization have been conducted on either unicellular cyanobacterium Synechocystis or plant cell suspensions, and used transcript levels of cold-induced genes as end-point markers . Whether the results of these studies are valid for intact plants or their organs is not known . Here we examine cold signaling in transgenic Brassica napus seedlings carrying, in addition to the endogenous cold-inducible BN115 gene, the beta-glucuronidase (GUS) gene placed under control of the BN115 promoter . The activity of BN115 promoter was monitored at the transcriptional and translational levels by determining accumulation of BN115 transcripts and by histochemical assay of GUS activity . Cold-activation of BN115 was strongly inhibited by the membrane fluidizer benzyl alcohol, but mimicked at 25 degrees C by the membrane rigidifier dimethylsulfoxide (DMSO) . The cold induction of BN115 was also inhibited by stabilizers of microtubules and actin microfilaments, taxol and jasplakinolide, respectively, but was mimicked at 25 degrees C by microtubule destabilizer oryzalin or colchicine, or by microfilament destabilizer latrunculin B . Gd3+ or ruthenium red prevented the cold activation of BN115, but Ca2+ ionophore A23187 or cyclic ADP-ribose activated it at 25 degrees C . Inhibitors of tyrosine kinases, protein kinase C and phosphoinositide kinases prevented the cold activation of BN115, but inhibitors of protein phosphatases (PP) 1 and 2 A activated BN115 at 25 degrees C . Constitutively expressed GUS activity in another transgenic line of the same cultivar of B . napus, was not affected by cold or any of the chemical treatments used in the experimentation . Activation of BN115 at 25 degrees C by DMSO, Ca2+ ionophore, cADPR, and by inhibitors of PP1 and 2A was accompanied by an increased freezing tolerance . It was concluded that the cold-activation of BN115 requires membrane rigidification, cytoskeleton reorganization, Ca2+ influx and action of several types of protein kinases. Clin Oral Implants Res, 2001 Aug, 12(4), 332 - 8 Migration of osteoblastic cells on various guided bone regeneration membranes; Takata T et al.; To evaluate the biological effects of guided bone regeneration (GBR) barrier materials on osteoblastic cell migration, migration of mouse osteoprogenitor cells (MC3T3-E1) was examined, in vitro, on various membranes . Eight commercially available GBR membranes - bovine type I collagen (BioMend; BM), porcine type I collagen (BioGide; BG), bovine type I atelocollagen (Tissue Guide; TG), polylactic acid (Epi-Guide; EG), co-polymer of polylactic acid and polyglycolic acid (Resolute; RL, Resolut XT; RL-XT), expanded polytetrafluoroethylene (e-PTFE; Gore Tex; GT) and co-polymer of cellulose acetate and nitrocellulose (Millipore filter; MP) - were tested . A 3x5 mm section of the membrane was fixed to the bottom of a culture dish with double-sided adhesive tape, and half of the membrane was closely covered by PARAFILM (American National Can) to leave an unexposed area for cell migration . The border between exposed and unexposed areas was marked as a baseline of cell migration . Membranes were then plated with 3 ml of cell suspension at an initial density of 1x105 cells/ml in alpha-MEM culture medium with 10% fetal bovine serum and ascorbic acid . After a 5-hour incubation, non-attached cells were completely washed out with phosphate buffered saline and the PARAFILM cover was removed . After 3 days cultivation, specimens were fixed with 10% buffered formalin and stained briefly with hematoxylin . The area of cell migration on a membrane was analyzed using a LA 500 Image Analysis System and migration area per unit length of the baseline (mm2/mm) was compared among membranes . Results demonstrated that cell migration was greater in the order: RL>RL-XT, BM, TG, MP>EG, BG . Membranes except for BG, EG and GT showed the migration rate equal to or higher than a plastic culture cover slip (Celldesk) (P<0.01) on which cells generally grow favorably . Only a small number of the cells attached to GT, and the net cell migration for the membrane could not be determined . These results indicate that GBR barrier materials per se may influence the process of bone regeneration in vivo through the effects of their presence on cell migration. Planta Med, 2001 Jul, 67(5), 475 - 7 Synthesis and biosynthesis of isocordoin; Vitali A et al.; In the search of a convenient synthesis for isocordoin (1), a potential anticancer natural product, 2',4'-dihydroxychalcone was inoculated in cell suspension cultures of Morus nigra, which were expected to contain an active prenyltransferase . After 24 hours the target compound was easily isolated from the metabolite extract . Optimization of the biotransformation resulted in a 85% yield of the prenyl derivative. J Pineal Res, 2001 Aug, 31(1), 39 - 45 Melatonin reduces UV-induced reactive oxygen species in a dose-dependent manner in IL-3-stimulated leukocytes; Fischer TW et al.; Reactive oxygen species (ROS) are presumed to be involved in inflammatory UV reactions of the skin . This in vitro study was performed to investigate the suppressive effect of melatonin in interleukin-3 (IL-3) stimulated leukocytes . Neutrophilic granulocytes were isolated from EDTA-treated whole blood and placed in a phosphate-buffered saline (PBS) containing IL-3 . Cell suspensions were either treated with PBS (control) or with increasing doses of melatonin (0.1, 0.5, 1, 2, 3, 5, 7.5, 10 mmol) . One PBS solution was left unirradiated and the other nine solutions (PBS and melatonin) were irradiated with 750 mJ/cm2 UVB light (280-360 nm, max: 310 nm) . Radical formation was measured by the chemiluminescence technique . UV-irradiated leukocytes showed a 5-fold higher radical formation than unirradiated leukocytes . Melatonin, in increasing doses in powers of ten, led to a maximum suppression of free radicals at 10 nmol (P= 0.01) and 1 mmol melatonin (P= 0.001), showing a biphasic, non-linear, dose response relationship . Melatonin, given in amounts of 0.1-10 mmol, led to a direct dose-dependent suppression of ROS . Radical formation was suppressed significantly in a range from 0.5 to 10 mmol (P= 0.001) . Melatonin is known to function as a radical scavenger and antioxidant; some of these melatonin effects may be receptor independent, while others may be receptor dependent. J Clin Neurosci, 2001 Mar, 8(2), 151 - 6 Medulloblastoma/primitive neuroectodermal tumour studied as a Matrigel enhanced subcutaneous xenograft model; White L et al.; An important role for pre-clinical models of medulloblastoma/primitive neuroectodermal tumour (MB/PNET) is inhibited by the limitations of conventional heterotransplantation . Nine cohorts of MB/PNET were studied for subcutaneous engraftment in nude mice by both conventional and Matrigel supplemented methods . While no subcutaneous tumours resulted from 63 conventional attempts, an aggregate 41 xenografts from 72 injections (57%) were produced when Matrigel was added to the cell suspension . In subsequent passage, engraftment rate approached 100% . To study the response to chemotherapeutic agents in the model, a total of 221 tumours in 3 cohorts were treated using one of the following: cisplatin, carboplatin, vincristine, cyclophosphamide, diaziquone, or saline control . While all agents demonstrated statistically significant activity, cyclophosphamide proved to be particularly effective . The potential applications of this xenograft model in the biologic as well as therapeutic study of MB/PNET deserve continuing investigation. Protein Expr Purif, 2001 Aug, 22(3), 443 - 54 Human aromatase in high yield and purity by perfusion chromatography and its characterization by difference spectroscopy and mass spectrometry; Gartner CA et al.; Expression of human cytochrome P450 aromatase (CYP19A1, aromatase) was accomplished at a high level using a baculovirus expression system in an insect cell suspension culture . Using the relatively new chromatographic technique of perfusion chromatography, a very rapid procedure for purification of the protein from solubilized cells was developed . At extraordinary flow rates of between 3 and 9 column volumes per minute, all chromatographic procedures could be performed, including setup, equilibration, and column regeneration steps, in less than 2 h, not including brief dialysis periods . Total yields were 40-52% and resulted in preparations with specific content values of 17.1 nmol aromatase/mg protein . Final purified preparations showed virtually no typical P450 spectra under standard conditions, but displayed full activity with typical enzyme kinetic parameters . These unusual results suggest that standard methods of P450 measurement are inappropriate when applied to aromatase . The findings are fully consistent with those encountered previously for purified preparations from a human placental source and led us to a new aromatase quantification method based on ligand-induced difference spectroscopy . A new HPLC assay is described which rapidly separates heme and apoprotein while measuring total heme content . Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was employed with both glycosylated and deglycosylated forms of the final purified product to confirm its identity as a glycosylated cytochrome P450 . Indian J Exp Biol, 2001 Feb, 39(2), 111 - 8 Induction of lymphomas on implantation of human oral squamous cell carcinomas in nude mice; Teni TR et al.; Cancer cells from five oral cancer patients and pleomorphic adenoma cells from one individual were inoculated as single cell suspension into subcutis of 30 Swiss nude mice and tail vein of additional 30 mice . Further, tumor tissue pieces from three oral cancer patients were xenografted s.c . in 18 nude mice, and 10 mice were kept as controls . In animals implanted with tumor pieces, 7/18 (39%) mice, developed squamous cell carcinoma at the site of inoculation within 8-15 days, while tumors were not observed in mice inoculated with single cell suspension, up to 60/90 days . In 8/68 (12%) mice, white foci were observed in several tissues, with hepatomegaly and splenomegaly noted in 27/68 (39%) mice . Histopathological examination of various tissues revealed presence of large cell lymphoma in several organs in 14/68 (21%) mice . No regional or distant metastasis of the implanted oral tumor cells was detected . Mice injected with cells from pleomorphic adenoma, also demonstrated large cell lymphoma in 2/10 (20%) mice, whereas none of the 10 control animals showed any gross abnormalities or microscopic abnormalities in several organs . 2/16 (12%) lymphomas exhibited positive reaction with mouse B cell antibodies illustrating the murine origin of the lymphomas, and these were immunophenotyed as B cell lymphomas . The lymphomas were also examined with mouse T cell antibodies and none reacted positively with the mouse T cell antibodies . The lymphomas also failed to react with human T cell, B cell and human Leucocyte common antigen (LCA) antibodies, indicating that the induced lymphomas were not of human origin . The tumor specimens from seven of eight oral cancer patients and the pleomorphic adenoma patient induced lymphomas in nude mice . Thus it appears that xenografting oral tumor cells into nude mice may cause induction of the murine lymphomas, and this needs further investigation. J Exp Bot, 2001 Aug, 52(361), 1625 - 33 Analysis of cell division parameters and cell cycle gene expression during the cultivation of Arabidopsis thaliana cell suspensions; Richard C et al.; Arabidopsis thaliana cell suspension cultures were characterized for the first time in detail in terms of biomass accumulation, cell division rate and cell cycle phase durations . Subsequently, this model system was used to follow the transcription profile of key cell cycle genes during a complete cultivation cycle . According to the calculated changes in the relative division rate over time, the cell cycle genes could be classified into four groups based on their transcriptional expression pattern . These differential patterns of gene expression are discussed with respect to the putative roles of the different cell cycle genes in the division cycle . Analysis of protein levels showed that mRNA levels did not correlate with protein levels in all cases . Results obtained in other systems, such as BY-2 cell suspensions or plants, confirm that cell suspension cultures of A . thaliana are suitable for the analysis of cell cycle regulation. Hum Reprod Update, 2001 Jul-Aug, 7(4), 378 - 83 Cryopreservation of testicular tissue in young cancer patients; Hovatta O; Cryopreservation of testicular tissue might benefit prepubertal boys who must undergo chemotherapy or radiotherapy . Cryopreservation of testicular tissue and testicular cells for intracytoplasmic sperm injection (ICSI) is feasible and widely applied . Testicular tissue from prepubertal boys can also be frozen, by applying techniques used with other tissues and with testicular tissue from adult men before ICSI . Good results have been obtained when propanediol is used as a cryoprotectant, but glycerol has also been used when freezing testicular tissue . Spermatogonia might also be isolated and cryopreserved as a cell suspension, though practical experience in humans is lacking . Transplantation of the frozen-thawed cells back to the testes after cancer treatment might result in restoration of spermatogenesis . Live offspring have been born to mice after transplantation of fresh, but not cryopreserved, testicular cells . Transplantation is technically feasible also in larger species, but to date no offspring have been born . Spermatogenesis in vitro would be an excellent option for boys with haematological malignancies who carry a risk of relapse after transplantation; however, at present the method is feasible only for the late stages of spermatogenesis. Hum Reprod, 2001 Aug, 16(8), 1575 - 82 Live human germ cells in the context of their spermatogenic stages; Johnson L et al.; BACKGROUND: Various types of live, dispersed, human testicular cells in vitro were previously compared with the morphologic characteristics of human spermatogenic germ cells in situ within seminiferous tubules . The current study extends those observations by placing live human germ cells in the context of their developmental steps and stages of the spermatogenic cycle . METHODS: Live human testicular tissue was obtained from an organ-donating, brain-dead person . A cell suspension was obtained by enzymatic digestion, and dispersed cells were observed live with Nomarski optics . Testes from 10 men were obtained at autopsy within ten hours of death, fixed in glutaraldehyde, further fixed in osmium, embedded in Epon, sectioned at 20 microm, and observed unstained by Nomarski optics . RESULTS: In both live and fixed preparations, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells . For germ cells, size, shape, and chromatic pattern of nuclei, the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece are characteristically seen in live dispersed cells and those in the fixed seminiferous tubules . These lead to identification of live germ cells in man and placement of each in the context of their developmental steps of spermatogenesis at corresponding stages of the spermatogenic cycle . CONCLUSIONS: 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 in clinical procedures or by scientists interested in studying live cells at known steps in spermatogenic development characteristic of germ cells in specific stages of the spermatogenic cycle. Curr Issues Mol Biol, 2001 Jan, 3(1), 15 - 26 In situ NMR systems; Shanks JV; In situ NMR is becoming an established technology for applications in bioprocessing and metabolic engineering . The in situ NMR biosensor acts as a noninvasive pH, ion, and concentration meter, with 31P and 13C as the two main isotopes of study . A substantial data base now exists for phosphorus and carbon spectra of bacteria and yeast . In situ NMR can provide many of the state variables needed for modeling glycolytic pathway function . NMR micro-reactor technology has improved significantly in the last decade . Several designs for immobilized cell reactors have been tested, and in particular, considerable gains have been made in the feasibility of studying aerobic, chemostat cultures with in situ NMR . Acquisition of 31P spectra from cell suspensions of 3-5% v/v under controlled conditions can be made in 3-7 minute time resolution in several systems. J Craniomaxillofac Surg, 2000 Oct, 28(5), 300 - 7 Evaluation of the VX2 rabbit auricle carcinoma as a model for head and neck cancer in humans; van Es RJ et al.; INTRODUCTION: This study investigates whether the VX2 carcinoma cell line, transplanted into the rabbit auricle, can be used as a head and neck cancer model . The biologic behaviour of this model is evaluated, comparing tumour transplantation with either tissue pieces or cell suspensions . MATERIAL: Thirty-six adult NZW rabbits received s.c . injections of VX2-suspensions (Group S) and 11 rabbits received solid VX2-pieces (Group P) into both auricles . METHODS: In Group S, 16 rabbits were sacrificed at various days before (S1) and 15 after (S2) the 28th day following transplantation . In the other five rabbits transplantation failed . Animals from Group P were sacrificed every 2 weeks after the 28th day . At autopsy the size of the primary tumours and of lymph node, lung and other metastases were assessed . If transplantation failed, the maximal tumour size and the time at which regression took place were recorded . Exponential trend lines were used to create growth curves of metastases . Differences between groups were evaluated with the chi2 test, correlations between parameters with Kendall's tau . RESULTS: The tumour take-rate in Groups S and P was 78% and 59% respectively . The maximal size and time at which regression occurred was significantly different, amounting to 83 +/- 7 mm2 at 10.4 +/- 1.6 days (Group S) and 243 +/- 30 mm2 at 20.9 +/- 2.0 days (Group P), respectively . Development of lymph node metastases was not different . In Groups P and S2, over 90% of the necks contained lymph node metastases . There was a higher incidence of lung metastases in Group S2 when compared to Group P (47% vs . 14%) but it was not statistically significant . A significant correlation (p<0.05) between weight loss and the size of lung metastases was found . CONCLUSION: Transplantation of the VX2-tumour with cell suspensions produces a useful head and neck cancer model for locoregional disease in which anti-tumour regimens against both the primary and lymph node metastases can be tested . Transplantation with tumour pieces is not advised as the take-rate is low and spontaneous remissions occur at a late stage. Phys Rev E Stat Nonlin Soft Matter Phys . 2001 Jul;64(1 Pt 1):012903 . Epub 2001 Jun 28. First-principle approach to dielectric behavior of nonspherical cell suspensions; Lei J et al.; We present a theoretical study of the dielectric behavior of cell suspensions by employing the Bergman-Milton spectral representation of the effective dielectric constant . By means of the spectral representation, we derive the dielectric dispersion spectrum in terms of the electrical and structure parameters of the cell models . Our results show that a better agreement with the experimental data can be obtained, provided that we introduce a conductivity contrast t=sigma(2)/(sigma(2)-sigma(1)) . We find that the conductivity of the cell cytoplasm sigma(1) can be much larger than that of the suspending medium sigma(2), in contrast to the previous claim that sigma(1) approximately sigma(2). J Natl Cancer Inst, 2001 Jul 18, 93(14), 1075 - 81 Heterogeneity of angiogenic activity in a human liposarcoma: a proposed mechanism for "no take" of human tumors in mice; Achilles EG et al.; BACKGROUND: Tumor cells are known to be heterogeneous with respect to their metastatic activity, proliferation rate, and activity of several enzymes . However, little is known about the heterogeneity of tumor angiogenic activity . We investigated whether heterogeneity of angiogenic activity could be responsible for the well-known observation of "no take" of human tumors transplanted into immunodeficient mice . METHODS: Severe combined immunodeficient (SCID) mice were xenotransplanted subcutaneously with tumor tissue (n = 55) or cell suspension of a human liposarcoma cell line (SW-872) or subclones (n = 28), with varying cell proliferation rates . Xenograft tumor growth was recorded for up to 6 months . Tumor tissues were then removed and analyzed for tumor cell apoptosis, microvessel density, and cell proliferation . All statistical tests were two-sided . RESULTS: Pieces of tumor derived from the parental cell line or its clones gave rise to three kinds of tumors: 1) highly angiogenic and fast-growing (aggressive) tumors, 2) weakly angiogenic and slow-growing tumors, and 3) nonangiogenic and stable tumors . Most tumors retained the original phenotype of their parental tumor . Tumor volume correlated positively with microvessel density (Spearman correlation coefficient {r} =.89; P< or =.0001) and inversely with tumor cell apoptosis (Spearman r = -.68; P =.002) . Tumor volume was less strongly but still positively correlated with tumor cell proliferation in vivo (Spearman r =.55; P =.02) . CONCLUSIONS: Human liposarcoma cells appear to be heterogeneous in their angiogenic activity . When tumor cells with little or no angiogenic activity are transplanted into SCID mice, a microscopic, dormant tumor results that may not grow further . Because such tiny tumors are neither grossly visible nor palpable, they have previously been called "no take." The finding that an angiogenic tumor can contain subpopulations of tumor cells with little or no angiogenic activity may provide a novel mechanism for dormant micrometastases, late recurrence, and changes in rate of tumor progression. Pest Manag Sci, 2001 Jan, 57(1), 46 - 56 Metabolism of the herbicide glufosinate-ammonium in plant cell cultures of transgenic (rhizomania-resistant) and non-transgenic sugarbeet (Beta vulgaris), carrot (Daucus carota), purple foxglove (Digitalis purpurea) and thorn apple (Datura stramonium); Muller BP et al.; The metabolism of the herbicide glufosinate-ammonium was investigated in heterotrophic cell suspension and callus cultures of transgenic (bar-gene) and non-transgenic sugarbeet (Beta vulgaris) . Similar studies were performed with suspensions of carrot (Daucus carota), purple foxglove (Digitalis purpurea) and thorn apple (Datura stramonium) . 14C-labelled chemicals were the (racemic) glufosinate, L-glufosinate, and D-glufosinate, as well as the metabolites N-acetyl L-glufosinate and 3-(hydroxymethylphosphinyl)propionic acid (MPP) . Cellular absorption was generally low, but depended noticeably on plant species, substance and enantiomer . Portions of non-extractable residues ranged from 0.1% to 1.2% of applied 14C . Amounts of soluble metabolites resulting from glufosinate or L-glufosinate were between 0.0% and 26.7% of absorbed 14C in non-transgenic cultures and 28.2% and 59.9% in transgenic sugarbeet . D-Glufosinate, MPP and N-acetyl L-glufosinate proved to be stable . The main metabolite in transgenic sugarbeet was N-acetyl L-glufosinate, besides traces of MPP and 4-(hydroxymethylphosphinyl)butanoic acid (MPB) . In non-transgenic sugarbeet, glufosinate was transformed to a limited extent to MPP and trace amounts of MPB . In carrot, D stramonium and D purpurea, MPP was also the main product; MPB was identified as a further trace metabolite in D stramonium and D purpurea. Haematologica, 2001 Jul, 86(7), 693 - 9 Quantification of human cells in NOD/SCID mice by duplex real-time polymerase-chain reaction; Nitsche A et al.; BACKGROUND AND OBJECTIVES: The aim of this study was the development of a fast and reliable polymerase chain reaction (PCR) assay which quantifies the proportion of human cells in immunodeficient chimeric mice, for example transplanted with human hematopoietic stem cells . DESIGN AND METHODS: We developed a TaqMan chemistry-based, real-time duplex PCR assay to quantify human and murine DNA in a single-tube reaction in parallel (HUmu PCR) . Two independent sets of primers and exonuclease probes, located in the tumor necrosis factor-a gene of both species, were selected to amplify specifically human and murine genomic DNA . Serial dilutions of defined numbers of human cells in mouse cells served to construct calibration curves . The test was applied to NOD/SCID mice transplanted with CD34(+) cells isolated from human cord blood and compared to FACS analysis . RESULTS: Analysis of DNA from human cells diluted stepwise into a fixed number of murine cells - and vice versa - led to calibration curves with good correlation for human and murine cells (r(2)>0.99) with a detection limit of 2% human cells . Results obtained with the HUmu PCR paralleled those of FACS analysis . However, in contrast to FACS analysis, which requires fresh single cell suspensions, the HUmu PCR can be carried out on already stored samples, even from solid organs and, moreover, the quantity of material required for analysis is very low . INTERPRETATION AND CONCLUSIONS: The HUmu PCR presented here is the first real-time PCR assay for simultaneous quantification of human and murine cells . It is extremely fast, accurate and is an interesting alternative method for quantifying the proportion of human DNA in organs of chimeric mice. Fitoterapia, 2000 Sep, 71(5), 501 - 6 Factors affecting volatile terpene and non-terpene biotransformation products in plant cell cultures; Zhu W et al.; Suspension cultures from Peganum harmala were shown to carry out biotransformations of a number of terpenes and non-terpenes . The rate of biotransformation was dependent upon substrate concentration, density of cell suspensions, and the structure and isomeric form of the substrates. Plant Sci, 2001 Jul, 161(2), 273 - 278 Involvement of GTP-binding protein in the induction of phytoalexin biosynthesis in cultured carrot cells; Kurosaki F et al.; Biosynthetic activity of carrot phytoalexin 6-methoxymellen was induced in cell suspension culture by the treatment with oligogalacturonide elicitor; however, the elicitor-induced activity appreciably reduced in the presence of suramin, a potent inhibitor of GTP-binding proteins . In contrast, addition of G-protein activators, such as mastoparan or GTP-gamma-S, to carrot cell culture triggered 6-methoxymellein production even in the absence of uronide elicitor . An appreciable GTPase activity was found in purified plasma membrane of cultured carrot cells, and the hydrolytic activity was significantly increased by the addition of elicitor . Carrot plasma membrane was capable of associating with GTP-gamma-S, and the binding ability was markedly increased in the presence of elicitor . However, the binding activity markedly decreased when the membrane preparation was pre-incubated with GTP but not with ATP . These observations strongly suggest that a certain GTP-binding protein located at plasma membrane of cultured carrot cells plays an important role in the oligogalacturonide elicitor-induced 6-methoxymellein production. Plant J, 2001 May, 26(3), 237 - 47 Characterization of five tomato phospholipase D cDNAs: rapid and specific expression of LePLDbeta1 on elicitation with xylanase; Laxalt AM et al.; Phospholipase D (PLD, EC 3.1.4.4.) has been implicated in a variety of plant processes, including signalling . In Arabidopsis thaliana a PLD gene family has been described and individual members classified into alpha-, beta- and gamma-classes . Here we describe a second PLD gene family in tomato (Lycopersicon esculentum) that includes three alpha- and two beta-classes . Different expression patterns in plant organs were observed for each PLD . In testing a variety of stress treatments on tomato cell suspensions, PLDbeta1 mRNA was found to rapidly and specifically accumulate in response to the fungal elicitor xylanase . The greatest increase was found 2 h after treatment with 100 microg m1(-1) xylanase (ninefold) . In vivo PLD activity increased nearly threefold over a 1.5 h period of treatment . When the elicitor was injected into tomato leaves, PLDbeta1 mRNA accumulation peaked at 2 h (threefold increase), before decreasing to background levels within 72 h . Mutant, non-active xylanase was as effective as the active enzyme in eliciting a response, suggesting that xylanase itself, and not the products resulting from its activity, functioned as an elicitor . When chitotetraose was used as elicitor, no PLDbeta1 mRNA accumulation was observed, thus it is not a general response to elicitation . Together these data show that PLD genes are differentially regulated, reflecting potential differences in cellular function . The possibility that PLDbeta1 is a signalling enzyme is discussed. Anal Biochem, 2001 Jul 15, 294(2), 161 - 8 A high-performance liquid chromatography radio method for determination of L-ascorbic acid and guanosine 5'-diphosphate-l-galactose, key metabolites of the plant vitamin C pathway; Wolucka BA et al.; A simple, rapid, and quantitative high-pressure liquid chromatography radio method is described for the determination of in vivo (14)C-labeled l-ascorbate, dehydro-l-ascorbate, and total l-ascorbate of Arabidopsis thaliana cell suspensions upon incubation of cultures with exogenous d-{(14)C}mannose . The same radio-HPLC conditions can be used to follow the products of in vitro enzymatic conversions of GDP-d-mannose by enzyme extracts of A . thaliana, namely GDP-l-galactose, GDP-4"-keto,6"-deoxy-d-mannose, and GDP-l-fucose . In particular, an accurate assay for GDP-d-mannose 3",5"-epimerase, a key enzyme of the plant vitamin C pathway, is presented . Surg Endosc, 2001 Aug, 15(8), 833 - 6 Epub 2001 May 07. Validation of a new experimental model of colon cancer; Balague C et al.; BACKGROUND: Most of the published animal studies that have evaluated tumor growth and port site metastases in laparoscopy have utilized a cell suspension model and thus cannot be compared to the clinical situation . Although solid tumor models have been developed, there has been no experimental model that establishes an orthotopic tumor in the rectum, reflecting the clinical situation of a solid colonic cancer . METHODS: Tumor cells (colon adenocarcinoma DHD/K1/TRb) were administered intraperitoneally in rats, which were used as solid tumor donors . A 20-mg piece of solid tumor from the donor was placed in a submucosal blister created in the rectum wall of the study rats . The approach to the submucosal blister was made through the mucosa after contralateral enterotomy . In order to validate the model, this intervention was performed in 10 cases (group A) . After 10 days of intervention, the rats were submitted to resection of the rectum and histological examination of the specimen . In another 10 rats (group B), manipulation of the tumor was performed after 10 days to cause tumor cell spillage . The likelihood of tumor dissemination was investigated in this group 20 days after this intervention . RESULTS: Group A developed solid tumors in seven of 10 cases (70%) . All of the tumors were localized between the muscular and the mucosal layer, with preservation of the serosa and without affecting the enterotomy . In all of the rats in group B, macroscopic tumor was observed in the upper rectum (100%) 10 days after its induction . Twenty days after tumor manipulation, nine rats had local tumor dissemination; two of them also had general tumor dissemination in the abdominal cavity . CONCLUSIONS: We established a novel solid colonic tumor model in rats for the investigation of intraoperative tumor cell spillage during resection of the colon and the development of port site metastases. Placenta, 2001 Jul, 22(6), 550 - 9 Flow cytometric measurement of intracellular Th1 and Th2 cytokine production by human villous and extravillous cytotrophoblast; Sacks GP et al.; A wide variety of cytokines are present at the maternal-fetal interface, but the extreme cellular complexity of the placenta has made it difficult to determine which cytokines are produced by which cells . Hence novel flow cytometric methods have been applied to determine intracellular cytokine production by specific cell-types in placental cell suspensions . Cell suspensions were prepared from first and third trimester chorionic villi and third trimester amniochorion by enzymatic digestion and Percoll density gradient centrifugation . After overnight incubation in the presence of monensin, cells were fixed, permeabilized and labelled with antibodies for villous cytotrophoblast (cytokeratin+, MHC class I-), extravillous cytotrophoblast (cytokeratin+, MHC class 1+) and leucocytes (CD45+) . These cell types were further characterized by their expression of EGFR (proliferative cytotrophoblast) and c-erbB2 (invasive cytotrophoblast) . Production of IL-4, IL-10, TNF-alpha, IFN-gamma and IL-12 was determined by simultaneous labelling with the appropriate monoclonal antibodies . Only IL-4 was detected consistently in all samples of cytotrophoblast . IL-10 was not detected but IL-10 mRNA was demonstrated in third trimester chorionic villus digests by RT-PCR . Although IL-4 secretion has not been demonstrated, these data suggest that, in vivo there may be a "Th2 type cytokine bias" orchestrated by the trophoblast . It is proposed that other cytokines (including IL-10 and TNF-alpha) are produced by decidual leukocytes, and not cytotrophoblast, at the maternal-fetal interface . Acta Biochim Pol, 2001, 48(1), 157 - 61 Mitochondrial uncoupling does not influence the stability of the intracellular signal activating plasma membrane calcium channels; Zablocki K et al.; The effects of various concentrations of thapsigargin, a specific inhibitor of Ca2+-ATPase in the endoplasmic reticulum (ER) membrane, on calcium homeostasis in lymphoidal T cells (Jurkat) were investigated . Preincubation of these cells suspended in nominally calcium-free medium with 0.1 microM thapsigargin resulted in a complete release of Ca2+ from intracellular calcium stores . When the medium was supplemented with 3 mM CaCl2 the cells maintained constantly elevated level of cytosolic Ca2+ . However, thapsigargin applied at lower concentration produced only a partial depletion of the stores . For example, in the cells pretreated with 1 nM thapsigargin and suspended in calcium-free medium approximately 75% of the calcium content was released from the intracellular stores . The addition of 3 mM CaCl2 to such cell suspension led to a transient increase in cytosolic calcium concentration, followed by a return to a lower steady-state . This phenomenon, related to the refilling of the ER by Ca2+, allowed to estimate the half-time for the process of cell recovery after activation of store-operated calcium channels . By this approach we have found that carbonyl cyanide m-chlorophenylhydrazone, which has been documented to inhibit calcium entry into Jurkat cells, does not influence the stability of the intracellular signal involved in the activation of store-operated calcium channels. Plant Mol Biol, 2001 May, 46(1), 1 - 15 Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus; Zheng D et al.; Three polyketide synthase genes (PKS1, PKS2, PKS3) from cell suspension cultures of raspberry (Rubus idaeus L . cv . Royalty) were characterized . They showed high similarity in both their nucleotide and deduced amino acid sequences . All three proteins contain the amino acid residues identified in previous work as essential for chalcone synthase (CHS) function . Enzyme activities were investigated after heterologous expression in Escherichia coli . RiPKS1 is a typical naringenin CHS that synthesizes the chalcone as the main reaction product, and p-coumaryltriacetic acid lactone (CTAL) as a minor by-product . RiPKS3 differed from RiPKS1 in four positions (K49R, M64R, P120L, V188A), and the products in vitro were predominantly CTAL and low levels of chalcone . RiPKS2 had the same four differences from RiPKS1 as RiPKS3, but in addition two further exchanges (R259H, F344L), and the protein had no detectable enzyme activity . Experiments with RiPKS1 containing either 259H or 344L showed that each of the exchanges was sufficient to completely eliminate enzyme activity . These experiments identify amino acid residues in CHS which are important for folding of the tetraketide intermediate to the chalcone (PKS3) and which are in general essential for CHS activity (PKS2) . The possible functions of these residues are discussed. Cell Transplant, 2001, 10(3), 295 - 304 Enhanced survival of porcine neural xenografts in mice lacking CD1d1, but no effect of NK1.1 depletion; Larsson LC et al.; Transplantation of embryonic porcine neurons may restore neurological function in patients with Parkinson's disease, if immunological rejection could be prevented . This study was performed to investigate the role of natural killer cells (NK cells) and NK1.1+ T cells (NK T cells) in the rejection of neural xenografts . A cell suspension was prepared from the ventral mesencephalon of 26-27-day-old pig embryos, and 2 microl was implanted in the right striata of mutant CD1d1 null (CD1.1-/-) mice, NK1.1-depleted mice, and controls . The CD1.1-/- mice are deficient in NK T cells and the antigen-presenting molecule CD1d1 . Graft survival and host responses were determined immunohistochemically using markers for dopamine neurons, CD4-, CD8- cells, microglia, and macrophages . At 2 weeks, the grafts were significantly larger in CD1.1-/- mice, 0.09 +/- 0.02 microl (mean +/- SEM), compared with controls, 0.05 +/- 0.01 microl . There was no significant difference between NK1.1-depleted mice, 0.02 +/- 0.01 microl, and controls . At 5 weeks, two grafts were still present in the CD1-/- mice, whereas only scars remained in the controls and in the NK1.1-depleted mice . Immune reactions were strong at 2 weeks and less pronounced at 5 weeks in all groups . Microglial activation was lower in NK-depleted mice than in the controls at 2 weeks . In contrast to organ xenografting, NK1.1+ cells do not seem to be important mediators of the rejection of discordant cellular neural xenografts . However, our results suggest that the antigen-presenting molecule CD1d1 may be involved in the rejection process. Carbohydr Res, 2001 May 18, 332(2), 175 - 82 Density-labelling of cell wall polysaccharides in cultured rose cells: comparison of incorporation of 2H and 13C from exogenous glucose; Thompson JE et al.; Labelling with stable isotopes has under-exploited potential for studies of polysaccharide endotransglycosylation in vivo . Ideally, the labelled polysaccharides should have the highest possible buoyant density . Although {13C6}glucose has previously been used as a precursor, it was unclear whether 2H would be efficiently incorporated from {2H}glucose or lost as D2O . Rose (Rosa sp.) cell-suspension cultures efficiently incorporated 13C from D-{13C6,2H7}glucose into wall polysaccharides with negligible dilution from atmospheric 12CO2 . Also, approximately 70% of the 2H atoms in D-{13C6,2H7}glucose were retained during polysaccharide biosynthesis . This shows that relatively few cycles of intermediary metabolism leading to the release of D2O occurred before sugar residues were incorporated into wall polysaccharides . In agreement with these observations, isopycnic centrifugation in caesium trifluoroacetate gradients showed that the hydrated buoyant density of xyloglucan synthesised by rose cells growing on {13C6,2H7}glucose and {13C6}glucose was 3.7 and 2.6% higher, respectively, than in isotopically non-labelled cultures . Thus, {13C,2H}glucose-feeding enabled a 42% better resolution of 'heavy' from 'light' xyloglucan than {13C}glucose-feeding. J Exp Bot, 2001 Jun, 52(359), 1381 - 2 Identification of novel cyclin-dependent kinases interacting with the CKS1 protein of Arabidopsis; Boudolf V et al.; The SUC1/CKS1 proteins interact with cyclin-dependent kinases (CDKs) and play an essential, but yet not entirely resolved, role in the regulation of the cell cycle . With the Arabidopsis thaliana CKS1At protein as bait in a two-hybrid screen, two novel Arabidopsis CDKs, Arath;CDKB1;2 and Arath;CDKB2;1, were isolated . A closely related homologue of Arath;CDKB2;1 was discovered in the databases and was nominated Arath;CDKB2;2 . Transcript analysis of the five known Arath;CDKA and Arath;CDKB genes revealed that they all had the highest expression in flowers and cell suspensions . Differences in the expression patterns in roots, leaves and stems suggest unique roles for each CDK. J Exp Bot, 2001 Jun, 52(359), 1219 - 26 Ca2+-dependent and Ca2+-independent excretion modes of salicylic acid in tobacco cell suspension culture; Chen HJ et al.; 14C-salicylic acid (SA) was used to monitor SA metabolism and its regulation in tobacco cell suspension culture . Two SA concentrations (20 microM and 200 microM) were used for comparison . SA was quickly taken up in both treatments, and the 200 microM-treated cells absorbed approximately 15 times that of 20 microM-treated cells within 5 min . More than 85% and 50% of the absorbed SA were excreted in free form to the culture medium within 5 h from cells treated with 200 microM and 20 microM SA, respectively . SA excretion was significantly inhibited by EGTA and the inhibition could be reversed by the addition of exogenous Ca2+ to the culture medium in the 200 microM SA treatment . However, EGTA had little or no effect on SA excretion in the 20 microM SA treatment . The data suggest that tobacco suspension-cultured cells may contain both Ca2+-dependent and Ca2+-independent pathways for SA excretion . Reduced glutathione (an active oxygen species scavenger), staurosporine (a protein kinase inhibitor), and cycloheximide (an inhibitor of de novo protein synthesis) also blocked intracellular SA excretion to the culture medium in the 200 microM but not in the 20 microM SA treatment . These data support the existence of alternative SA excretion pathways in tobacco suspension-cultured cells . Tobacco cells may use both Ca2+-dependent and Ca2+-independent excretion pathways to cope with different intracellular SA status, and the pathway influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by SA at 200 microM, but not at 20 microM. Plant Mol Biol, 2001 Apr, 45(6), 641 - 54 Changes in gene expression during programmed cell death in tomato cell suspensions; Hoeberichts FA et al.; To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied . In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase 1 . Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals . Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated . During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold . A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD . This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones . CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified . CTD1 is highly similar to Aux/IAA early-auxin-responsive genes . CTD2 corresponds to the tomato RSI-I gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize . Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death . The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes . The possible role of the various predicted gene products in plant PCD is discussed. J Appl Toxicol, 2000 Dec, 20 Suppl 1, S63 - 72 Response of normal human keratinocytes to sulfur mustard: cytokine release; Arroyo CM et al.; Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (2,2'-dichlorodiethyl sulfide, HD) . This study describes responses of normal human epidermal keratinocytes (NHEK) to HD, defined by interleukin-1beta (IL-1beta), IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) release . Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD . Exposure to 100 microM HD increased the release of cytokines . The amounts of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and 4-fold, respectively, above control levels when NHEK were exposed to 300 microM HD . Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and at other times it decreased in cell suspensions . Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM HD and significantly increased levels of IL-6 were observed . Interleukin-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant . These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury . The present findings suggest that the cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury. Bull Exp Biol Med, 2001 Mar, 131(3), 267 - 8 Induction of blebbing in Ehrlich ascitic adenocarcinoma cells during in vitro hyperthermia; Razin BV et al.; We studied the effect of in vitro hyperthermia on induction of blebbing in cultured Ehrlich ascitic adenocarcinoma cells . Hyperthermia (42-48 degrees C) promotes induction of blebbing in cell culture, but this induction is reversible, and cessation of hyperthermia leads to almost complete recovery of the morphological composition of cell suspension. Appl Environ Microbiol, 2001 Jul, 67(7), 3134 - 9 Nuclear magnetic resonance analysis of {1-13C}dimethylsulfoniopropionate (DMSP) and {1-13C}acrylate metabolism by a DMSP lyase-producing marine isolate of the alpha-subclass of Proteobacteria; Ansede JH et al.; The prominence of the alpha-subclass of Proteobacteria in the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of dimethylsulfoniopropionate (DMSP) metabolism in a representative isolate of this phylotype, strain LFR . {1-(13)C}DMSP was synthesized, and its metabolism and that of its cleavage product, {1-(13)C}acrylate, were studied using nuclear magnetic resonance (NMR) spectroscopy . {1-(13)C}DMSP additions resulted in the intracellular accumulation and then disappearance of both {1-(13)C}DMSP and {1-(13)C}beta-hydroxypropionate ({1-(13)C}beta-HP), a degradation product . Acrylate, the immediate product of DMSP cleavage, apparently did not accumulate to high enough levels to be detected, suggesting that it was rapidly beta-hydroxylated upon formation . When {1-(13)C}acrylate was added to cell suspensions of strain LFR it was metabolized to {1-(13)C}beta-HP extracellularly, where it first accumulated and was then taken up in the cytosol where it subsequently disappeared, indicating that it was directly decarboxylated . These results were interpreted to mean that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase, while added acrylate was beta-hydroxylated on (or near) the cell surface to beta-HP, which accumulated briefly and was then taken up by cells . Growth on acrylate (versus that on glucose) stimulated the rate of acrylate metabolism eightfold, indicating that it acted as an inducer of acrylase activity . DMSP, acrylate, and beta-HP all induced DMSP lyase activity . A putative model is presented that best fits the experimental data regarding the pathway of DMSP and acrylate metabolism in the alpha-proteobacterium, strain LFR. Bone, 2001 Jun, 28(6), 595 - 602 Endothelin- and sarafotoxin-induced receptor-mediated calcium mobilization in a clonal murine osteoblast-like cell line, MC3T3-E1/B; Zach D et al.; Previous studies have demonstrated that, in osteoblast-like MC3T3-E1 cells, various endothelin peptides and their homologous sarafotoxins generate prostaglandin E(2) (PGE(2)) release through an ET(A) receptor subtype . In this study, biphasic Ca(2+) signals elicited with endothelin (ET)-1, ET-2, ET-3, beta-ET, S6a1, and S6b (ET/S6) were measured by microspectrofluorimetric methods in cell suspensions loaded with Fura-2 acetoxymethylester (Fura-2 AM) . Phospholipase C (PLC)-dependent calcium activation mechanisms seem to be involved . We found evidence of Ca(2+) release from thapsigargin-sensitive and non-thapsigargin-sensitive intracellular Ca(2+) stores as well as Ca(2+) transmembrane inflow through multiple voltage-independent and Ni(2+)-sensitive cation channels . Using an ET(A) receptor antagonist, BQ-123, we showed that this receptor was coupled to Ca(2+) mobilization . All agonists tested, except S6c (an ET(B)-receptor-specific agonist) induced receptor desensitization . Our results demonstrate that the ET/S6-induced Ca(2+) signaling pathway is mediated via an ET(A)-receptor subtype in MC3T3-E1/B cells. J Anim Sci, 2001 Jun, 79(6), 1549 - 56 Inflammatory response, growth, and thyroid hormone concentrations are affected by long-term boron supplementation in gilts; Armstrong TA et al.; An experiment was conducted to determine the long-term effects of dietary boron (B) on growth performance, immune function, and plasma and serum characteristics in gilts . Fifty weanling gilts were allotted to 10 pens based on weaning weight and litter origin . Pens were randomly assigned to receive one of two dietary treatments . Treatments consisted of a basal diet low in B (control) and the basal diet supplemented with 5 mg B/kg diet as sodium borate . Gilts remained on their respective experimental diets and with their penmates throughout the nursery, growing, and finishing phases . The B concentration of the basal diet was 0.98, 2.1, and 2.2 mg/kg diet during the nursery, growing, and finishing phases, respectively . At the end of each production phase, animals were weighed and feed consumption was determined to assess growth performance variables . In addition, blood samples were obtained from three randomly selected gilts per pen at the completion of each phase . Boron had no affect (P > 0.58) on growth performance during the nursery phase, but gilts receiving supplemental B had increased (P < 0.05) ADG at the end of the finishing phase and over the entire growing-finishing period . Serum concentrations of triiodothyronine (T3) tended (P < 0.07) to be reduced by dietary B at the end of the nursery phase, but serum thyroxine (T4) was not affected (P = 0.46) by B . At the completion of the growing phase, supplemental B decreased (P < 0.05) the concentrations of T3 and T4 in the serum . In addition, serum concentrations of total cholesterol and the activity of alkaline phosphatase were increased (P < 0.05) by dietary B at the end of the growing phase . Serum concentrations of urea N tended (P < 0.09) to be increased by B at the end of the growing phase . Beginning at d 95 of the experimental period, measures of immune function were assessed in randomly selected gilts . Boron decreased (P < 0.05) the inflammatory response to an intradermal injection of phytohemagglutinin . Boron did not affect (P > 0.30) the blastogenic response of isolated lymphocytes to mitogen stimulation or the humoral immune response against a sheep red blood cell suspension . Results indicate that B may affect serum thyroid hormone concentrations, the inflammatory response, and growth in pigs. Am J Hematol, 2001 Feb, 66(2), 105 - 15 Enhanced low shear stress induced platelet aggregation by Shiga-like toxin 1 purified from Escherichia coli O157; Yagi H et al.; The effect of Shiga-like toxin 1 (Stx1) produced by Escherichia coli O157 on platelets was studied with an argon laser light-assisted shear-induced platelet aggregometer and with binding assays . Stx1 markedly enhanced the platelet aggregation under low shear stress but did not affect it under high shear stress . Minimal concentration of Stx1 required for the enhancement was 0.25 ng/ml, and almost maximal enhancement was observed at a final concentration of > or =2.5 ng/ml . This enhanced platelet aggregation disappeared after leukocyte depletion from normal platelet-rich plasma with a specific filter . In contrast, a standard platelet aggregometer was unable to detect this enhanced platelet aggregation in either the presence or the absence of ADP . 125I-labeled purified Stx1 did not specifically bind to normal washed platelets depleted of leukocytes, and thin-layer chromatographic analysis of glycolipids extracted from normal platelet lysates also confirmed that leukocyte-depleted normal platelets lack Stx1-specific receptor globotriaosylceramide (Gb3) . Supernatant from the monocyte suspension stimulated with Stx1 exhibited the enhanced low shear stress induced platelet aggregation, but that from the polymorphonuclear cell suspension did not . Several cytokines produced from monocytes reproduced this event in vitro . Further, plasmas from six out of seven patients with hemolytic uremic syndrome (HUS) had activity similar to the purified Stx1 . This activity was almost totally impaired after treatment of HUS plasmas with Gb3 in accord with reduction of plasma Stx1 levels . Taken together, these results indicate that platelets lack Gb3, and Stx1 appears to modulate platelet aggregation in an indirect fashion, presumably by the release of cytokines or chemical compounds from the target tissues. Am J Physiol Heart Circ Physiol, 2001 Jul, 281(1), H448 - 56 O(2) release from erythrocytes flowing in a narrow O(2)-permeable tube: effects of erythrocyte aggregation; Tateishi N et al.; The effects of erythrocyte aggregation on O(2) release were examined using O(2)-permeable fluorinated ethylenepropylene copolymer tubes (inner diameter, 25 microm; outer diameter, 100 microm) . Measurements were performed using an apparatus built on an inverted microscope that contained a scanning-grating spectrophotometer with a photon count detector connected to two photomultipliers and an image processor through a video camera . The rate of O(2) release from the cells flowing in the narrow tube was determined based on the visible absorption spectrum and the flow velocity of the cells as well as the tube size . When the tube was exposed to nitrogen-saturated deoxygenated saline containing 10 mM sodium dithionite, the flowing erythrocytes were deoxygenated in proportion to the traveling distance, and the deoxygenation at a given distance increased with decreasing flow velocity and cell concentration (hematocrit) . Adding Dextran T-70 to the cell suspension increased erythrocyte aggregation in the tube, which resulted in suppressed cell deoxygenation and increased marginal cell-free-layer thickness . The deoxygenation was inversely proportional to the cell-free-layer thickness . The relation was not essentially altered even when the medium viscosity was adjusted with Dextran T-40 to remain constant . The rate of O(2) release from erythrocytes in the tube was discussed in relation to the O(2) diffusion process . We conclude that the diffusion of O(2) from erythrocytes flowing in narrow tubes is inhibited primarily by erythrocyte aggregation itself and partly by thickening of the cell-free layer. J Biol Chem, 2001 Aug 17, 276(33), 30717 - 23 Epub 2001 Jun 12. Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum; Grothe T et al.; Salutaridinol 7-O-acetyltransferase (EC ) catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-O-acetate, the immediate precursor of thebaine along the morphine biosynthetic pathway . We have isolated a cDNA clone that corresponds to the internal amino acid sequences of the native enzyme purified from a cell suspension culture of opium poppy Papaver somniferum . The recombinant enzyme acetylated the 7-hydroxyl moiety of salutaridinol in the presence of acetyl-CoA . The apparent K(m) value for salutaridinol was determined to be 9 microm and 54 microm for acetyl-CoA . The gene transcript was detected in extracts from Papaver orientale and Papaver bracteatum in addition to P . somniferum . Genomic DNA gel blot analysis indicated that there is likely a single copy of this gene in the P . somniferum genome . The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of Catharanthus roseus . Salutaridinol 7-O-acetyltransferase is the second enzyme specific to morphine biosynthesis for which we have isolated a cDNA . Taken together with the other cDNAs cloned encoding norcoclaurine 6-O-methyltransferase, (S)-N-methylcoclaurine 3'-hydroxylase, the cytochrome P-450 reductase, and codeinone reductase, significant progress has been made toward accumulating genes of this pathway to enable the end goal of a biotechnological production of morphinan alkaloids. Am J Physiol Regul Integr Comp Physiol, 2001 Jul, 281(1), R66 - 75 Intracellular monocyte and serum cytokine expression is modulated by exhausting exercise and cold exposure; Rhind SG et al.; This study tested the hypothesis that exercise elicits monocytic cytokine expression and that prolonged cold exposure modulates such responses . Nine men (age, 24.6 +/- 3.8 y; VO(2 peak), 56.8 +/- 5.6 ml . kg(-1) . min(-1)) completed 7 days of exhausting exercise (aerobic, anaerobic, resistive) and underwent three cold, wet exposures (CW) . CW trials comprised </=6 h (six 1-h rest-work cycles) exposure to cold (5 degrees C, 20 km/h wind) and wet (5 cm/h rain) conditions . Blood samples for the determination of intracellular and serum cytokine levels and circulating hormone concentrations were drawn at rest (0700), after exercise (approximately 1130), and after CW (~2000) . Whole blood was incubated with (stimulated) or without (spontaneous) lipopolysaccharide (LPS; 1 microgram/ml) and stained for CD14 monocyte surface antigens . Cell suspensions were stained for intracellular cytokine expression and analyzed by flow cytometry . The proportion of CD14(+) monocytes exhibiting spontaneous and stimulated intracellular expression of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha increased after exercise, but these cells produced less IL-1beta and TNF-alpha after CW when CW was preceded by exhausting exercise . Serum cytokine concentrations followed a parallel trend . These findings suggest that blood monocytes contribute to exercise-induced cytokinemia and that cold exposure can differentially modulate cytokine production, upregulating expression of IL-6 and IL-1 receptor antagonist but downregulating IL-1beta and TNF-alpha . The cold-induced changes in cytokine expression appear to be linked to enhanced catecholamine secretion associated with cold exposure. Neurochem Int, 2001 Aug, 39(2), 161 - 7 The development of dopamine overflow from foetal nigral grafts in the intact rat striatum and their influence on contralateral striatal dopamine overflow; Earl CD et al.; In this study, cell suspensions of foetal rat ventral mesencephalic dopaminergic tissue were grafted to the intact (non-lesioned) striatum of adult rats . Differential pulse voltammetry at carbon-fibre micro electrodes (12 microm diameter) was employed to first, monitor the development of dopamine overflow over a 20 week period within the grafts and secondly, their influence on contralateral striatal dopamine overflow . At 8 and 20 weeks, animals were pre-treated with pargyline and both striata were monitored for dopamine overflow for 90 min following d-amphetamine administration . Amphetamine led to a significant increase in dopamine overflow in both the grafted striatum and the contralateral striatum . The time course of dopamine overflow in both the grafted striatum and the striatum contralateral to the graft was similar in all groups of animals . Although the actual concentration of dopamine measured in 20 week old grafts was more (approximately 21%) than that measured in 8 week old grafts, there was no significant difference between the two time points . The concentration of dopamine measured in the striatum contralateral to 8 week old grafts was significantly lower (approximately 43%) than that measured in the striatum of a normal control rats . There was no significant difference between the concentration of dopamine measured in the striatum contralateral to 20 week old grafts and normal control rats . In conclusion, dopamine overflow from a ventral mesencephalic graft does not change significantly between 8 and 20 weeks following grafting . However, the grafted tissue causes a decrease of d-amphetamine-induced dopamine overflow in the contralateral side 8 weeks following grafting, which is restored 12 weeks later. J Asian Nat Prod Res, 2001, 3(2), 161 - 8 C-27 and C-3 glucosylation of diosgenin by cell suspension cultures of Costus speciosus; Indrayanto G et al.; 3-O-{beta-D-glucopyranosyl-(1"--> 2')-beta-D-glucopyranosyl}, 27-O-beta-D-glucopyranosyl-(25R)-spirost-5-ene-3beta,27-diol was isolated from cell suspension cultures of Costus speciosus, following incubation with diosgenin, and its structure was elucidated using a combination of one- and two-dimensional 1H and 13C NMR spectral data, and positive and negative ion ESMS spectral data. Cancer Immunol Immunother, 2001 Apr, 50(2), 87 - 92 Active idiotypic vaccination in a patient with biclonal follicular lymphoma; Barrios Y et al.; Specific immunological responses to the idiotypic epitopes present in the surface immunoglobulin (Ig) of the clonal tumour population can be induced for active immunotherapy in patients with B-cell non-Hodgkin lymphoma (NHL) . The clonality of the tumour cells should have important implications for the success of the implemented therapy . Here we report on the case of a patient enrolled in a protocol of active idiotypic immunotherapy in which previous cytofluorometric analysis showed a major IgM+, kappap+ population in the tumoral cell suspensions . However, sequence analysis of both tumour sample and tumour-derived hybrids revealed the presence of two unrelated clones that used different VH and VK gene segments . It was possible to obtain hybridomas secreting these two different IgM, kappap idiotypic proteins . The patient was immunised with a mixture of these two idiotypic Igs conjugated to keyhole limpet haemocyanin . Anti-idiotypic antibodies directed against both tumour-associated proteins were detected . This is the first case of anti-idiotypic therapy in a patient with a biclonal NHL . Our work calls attention to the question of clonality in the context of idiotypic vaccination in NHL patients. Osteoarthritis Cartilage, 2001 May, 9(4), 341 - 50 Biological freezing of human articular chondrocytes; Almqvist KF et al.; AIM: To preserve viable, metabolically active chondrocytes cultured in alginate beads at -196 degrees C for further use in in vitro and in vivo studies . METHODS: Human articular chondrocytes were isolated from femoral condyles within 24 h post mortem . To optimize the biological freezing procedure, the chondrocytes were control-rate frozen in different concentrations of dimethyl sulfoxide (DMSO) in Dulbecco's MEM supplemented with 10% FCS before being thawed and the cell viability was determined by Trypan Blue exclusion test . To investigate the effect of control-rate freezing on chondrocyte metabolism, control-rate frozen chondrocytes in 5% DMSO were thawed and cultured in gelled agarose for 2 weeks . Non-frozen chondrocytes cultured in agarose served as controls . Furthermore, human articular chondrocytes were cultured in 2% alginate beads for 2 weeks after which the beads were incubated with 5% DMSO for 0 h, 2.5 h, 5 h and 10 h and frozen at -196 degrees C . Non-frozen alginate beads containing chondrocytes and incubated with 5% DMSO served as a control . After 2 weeks in culture, chondrocytes in agarose or in alginate were sulfated with 10 microCi(35)SO(4)/ml for 48 h . The total production of aggrecans, and the aggrecan subtypes, were subsequently determined . RESULTS: Five percent DMSO in the culture medium was the optimal condition to control-rate freeze and recover viable and functional isolated chondrocytes . Total aggrecan synthesis of control-rate frozen chondrocytes cultured in gelled agarose was not significantly reduced when compared with control cells . The proportion of aggrecan in the aggregate form of control-rate frozen chondrocytes kept in agarose remained unaltered . Chondrocytes, control-rate frozen in the alginate matrix, showed a 0-30% decrease in total aggrecan synthesis rates in culture when compared with the non-frozen chondrocytes . The optimal pre-incubation time of the alginate beads with 5% DMSO was 5 h, without any change in aggrecan synthesis rates when compared with the control situation . Shorter pre-incubation times resulted in an insufficient diffusion of DMSO into the beads and in cell death . There was no difference in the synthesis of the different aggrecan subtypes between frozen and non-frozen chondrocytes in alginate . CONCLUSION: Human articular chondrocytes can be stored at -196 degrees C for 24 h without important decreases in their aggrecan synthesis rates when control-rate frozen as a cell suspension in 5% DMSO . Proportions of the aggrecan subtypes (monomers, aggregates) synthesized by chondrocytes cultured in agarose remained unchanged . The control-rate freezing procedure in the alginate beads pre-incubated with 5% DMSO for 5 h produced no decrease in total aggrecan synthesis rates and no change in the synthesized aggrecan subtypes . Further experiments have to confirm the suitability of this freezing method for long-term storage of chondrocytes allowing us to set up a 'chondrocyte' bank for further use in in vitro and in vivo manipulations . Appl Microbiol Biotechnol, 2001 May, 55(4), 404 - 10 Enhancement of Taxol production and excretion in Taxus chinensis cell culture by fungal elicitation and medium renewal; Wang C et al.; An endophytic fungus, Aspergillus niger, isolated from the inner bark of a Taxus chinensis tree, was used as an elicitor to stimulate the Taxol (paclitaxel) production in a Taxus chinensis cell suspension culture . Different elicitor doses and elicitation times were tested in a batch culture; and the highest volumetric Taxol yield was achieved when 40 mg of the fungal elicitor (carbohydrate equivalent) l(-1) was added to the culture during the late exponential-growth phase . The elicitation resulted in a more than two-fold increase in the Taxol yield and about a six-fold increase in total secretion . The Taxol yield was further improved substantially by applying medium renewal and re-elicitation to the culture . In particular, with repeated medium renewal (in a way similar to medium perfusion) and a second elicitation of the culture, the volumetric Taxol yield was increased to 67.1+/-7.5 mg l(-1), which was about seven times the amount obtained in the non-elicited batch culture . The Taxol productivity of the perfusion-like culture with repeated fungal elicitation was 1.5 mg l(-1) day(-1), which was about 40% higher than that of the elicitor-treated batch culture and three times the productivity of the non-elicited batch culture. Int J Pharm, 2001 Jun 19, 221(1-2), 1 - 22 Biomedical applications of collagen; Lee CH et al.; Collagen is regarded as one of the most useful biomaterials . The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenecity, made collagen the primary resource in medical applications . The main applications of collagen as drug delivery systems are collagen shields in ophthalmology, sponges for burns/wounds, mini-pellets and tablets for protein delivery, gel formulation in combination with liposomes for sustained drug delivery, as controlling material for transdermal delivery, and nanoparticles for gene delivery and basic matrices for cell culture systems . It was also used for tissue engineering including skin replacement, bone substitutes, and artificial blood vessels and valves . This article reviews biomedical applications of collagen including the collagen film, which we have developed as a matrix system for evaluation of tissue calcification and for the embedding of a single cell suspension for tumorigenic study . The advantages and disadvantages of each system are also discussed. Enzyme Microb Technol, 2001 Jun 7, 28(9-10), 796 - 805 The application of continuous culture for plant cell suspensions; van Gulik WM et al.; Continuous culture of plant cell suspensions has been developed during the last 35 years . Starting from rather imperfect set-ups, nowadays much better equipment is used for studies on growth and production kinetics or cell physiology . In this review the development of equipment and theory, as well as the applications are discussed. Biomaterials, 2001 Jul, 22(13), 1911 - 7 Cytocompatibility and response of osteoblastic-like cells to starch-based polymers: effect of several additives and processing conditions; Gomes ME et al.; This work reports on the biocompatibility evaluation of new biodegradable starch-based polymers that are under consideration for use in orthopaedic temporary applications and as tissue engineering scaffolds . It has been shown in previous works that by using these polymers it is both possible to produce polymer/hydroxyapatite (HA) composites (with or without the use of coupling agents) with mechanical properties matching those of the human bone, and to obtain 3D structures generated by solid blowing agents, that are suitable for tissue engineering applications . This study was focused on establishing the influence of several additives (ceramic fillers, blowing agents and coupling agents) and processing methods/conditions on the biocompatibility of the materials described above . The cytotoxicity of the materials was evaluated using cell culture methods, according to ISO/EN 109935 guidelines . A cell suspension of human osteosarcoma cells (HOS) was also seeded on a blend of corn starch with ethylene vinyl alcohol (SEVA-C) and on SEVA-C/HA composites, in order to have a preliminary indication on cell adhesion and proliferation on the materials surface . In general, the obtained results show that all the different materials based on SEVA-C, (which are being investigated for use in several biomedical applications), as well as all the additives (including the novel coupling agents) and different processing methods required to obtain the different properties/products, can be used without inducing a cytotoxic behaviour to the developed biomaterials. Bull Exp Biol Med, 2001 Feb, 131(2), 153 - 5 Allotransplantation of pituitary cells to rat testicle; Dendeberov ES; Fifty-three transplantations of pituitary tissue (intact and minced with scissors) under the tunica albuginea testis were carried out . In both series pituitary cells retained viability for up to 6 months . Transplantation of cell suspension was not accompanied by the formation of necrotic zone in the transplant; the adjacent testicular tubules and the entire testicular structure remained unchanged, while transplantation of large fragments of the pituitary tissue impaired of spermatogenesis in the adjacent testicular tubules, although most of them remained unchanged . Hence, cell suspension is preferable for transplantation of pituitary tissue into the testicle. Biosci Biotechnol Biochem, 2001 Apr, 65(4), 962 - 5 Effect of zinc deficiency on betacyanin production in a cell suspension culture of table beet (Beta vulgaris L.); Akita T et al.; The effect of microelements in the Linsmaier-Skoog (LS) medium on betacyanin production was investigated in suspension cultures of table beet (Beta vulgaris L.) . Removing zinc from the medium resulted in a high betacyanin content of the cells, the betacyanin content of the cells decreasing with increasing zinc concentration in the medium . The betacyanin content of cells cultured in the medium without zinc was twice as high as that in the medium containing 0.03 mM of zinc . In the revised LS medium without zinc, the maximum betacyanin yield was obtained of 590 mg/l from a 21-day culture. C R Acad Sci III, 2001 Apr, 324(4), 335 - 43 Purification of several pectin methyltransferases from cell suspension cultures of flax (Linum usitatissimum L.); Bourlard T et al.; Three pectin methyltransferases (PMT5, PMT7, PMT18; EC 2.1.1.6.x) were solubilized from the endo-membrane complex of flax cells, with 0.05% Triton X-100 . After a 3 step-chromatography procedure, PMT7 and PMT5 were purified to apparent homogeneity . PMT5 and PMT7 differed regarding their optimum pH (5 or 7), the methyl acceptor (low or highly methylesterified pectin), their focusing pH range (6-7 or 8-9) and relative molecular mass (40 +/- 5 or 110 +/- 10 kDa) . SDS-PAGE of PMT5 and PMT7 did not reveal bands at 40 or 110 kDa but only a silver stained band of about 18 kDa . Two independent methods (photo labelling and enzymatic activity) showed that this silverstained band corresponded to a methyltransferase with affinity for pectins . This polypeptide was of the same size as the enzyme designed PMT18 (18 +/- 3 kDa; pl 4-4.5) recovered during size exclusion chromatography of either PMT7 or PMT5, suggesting that PMT18 bears the catalytic site of PMT5 and PMT7. J Steroid Biochem Mol Biol, 2001 Jan-Mar, 76(1-5), 203 - 11 MHC class II expression and antigen presentation by human endometrial cells; Wallace PK et al.; It has been demonstrated previously that mixed cell suspensions from the female reproductive tract consisting of human epithelial and stromal cells were capable of presenting foreign antigen to autologous T cells . There have been, however, no reported studies examining antigen presentation by isolated epithelial cells from the human female reproductive tract . It is now shown that freshly isolated epithelial cells from the uterine endometrium constitutively express MHC class II antigen and that class II was upregulated on cultured epithelium by interferon gamma (IFNgamma) . Using a highly purified preparation, it was demonstrated that these epithelial cells were able to process and present tetanus toxoid recall antigen driving autologous T cell proliferation . Cells isolated from the basolateral sub-epithelium stroma were also potent antigen presenting cells in this model system . Thus, isolated endometrial epithelial cells were able to directly process and present antigen to T cells and may be responsible for the transcytosis and delivery of antigen to professional antigen presenting cells found in the sub-epithelial stroma. Biorheology, 2001, 38(2-3), 249 - 62 Theoretical and experimental analysis of the sedimentation kinetics of concentrated red cell suspensions in a centrifugal field: determination of the aggregation and deformation of RBC by flux density and viscosity functions; Lerche D et al.; The flow properties of blood are mostly determined using various viscometric approaches, and described in terms of a shear rate or shear stress dependent apparent viscosity . The interpretation of results are rather difficult, especially at low shear rates when particle sedimentation and migration within the viscometer gap are significant . By contrast, analysing the separation process in concentrated RBC suspensions in a centrifugal field also yields information about the viscosity function, including particle-particle interaction and deformation parameters . In this paper, the sedimentation process is approached by means of the theory of kinematic waves and theoretically described by solving the corresponding one-dimensional quasi-linear partial differential equation based on viscosity/flow function as a function of volume concentration . The sedimentation kinetics of rigid spherical RBC suspended in saline and normal RBC suspended in Dx-saline solutions were investigated by means of a separation analyser (LUMiFuge 114) . The instrument detects the light transmission over the total length of the cell containing the suspension . During centrifugation the analyser automatically determines the position of the particle free fluid/suspension interface or the sediment by means of a special algorithm . The data obtained with sedimentation of rigid spherical RBC at different volume concentrations demonstrate that, in the case of suspensions rotated in containers of constant cross section, there is good agreement between the theory of kinematic waves developed by Anestis and Schneider (1983) and the results of the experiments . Such good agreement was obtained even though a restrictive one-dimensional model was used to obtain the theoretically derived sedimentation time course . In addition, we describe an algorithm enabling the experimental determination of the viscosity and related flux density function to be made for any suspension . Through this approach, we investigated in detail the rheological behavior of suspended rigid spheres at low Reynolds numbers ranging from 10(-6) to 10(-3) . The method here introduced also enabled us to investigate RBC suspensions with respect to the deformability and interactions of the cells by means of the separation analysis . Normal, rigid as well as aggregating RBC exhibited marked differences in the sedimentation kinetics, which were quantified by means of the flux and viscosity functions based on the theory of kinematic waves. Exp Dermatol, 2001 Jun, 10(3), 168 - 74 Epidermal CD8+ T cells in chronic plaque psoriasis are Tc1 cells producing heterogeneous levels of interferon-gamma; Ovigne JM et al.; The majority of epidermal CD8+ T cells in chronic plaque psoriasis are activated Tc1 cells producing interferon-gamma and no interleukin-4, a small proportion of which express NK-T receptors . To quantitate their level of cytokine production and characterize them further, CD8+ T cells were isolated from epidermal cell suspensions of lesional biopsies from 24 patients with chronic plaque psoriasis . T-cell lines (TCL) were established by culture of CD8+ T cells with feeders and IL-2 for 11 days and expansion with PHA . Ten TCL were stained for surface markers; 6 were cloned with PHA by limiting dilution . Interferon-gamma, interleukin-4 and interleukin-10 production was measured by ELISA after PMA/anti-CD3 activation of 15 TCL and 39 CD8+ T-cell clones . The 10 TCL stained were CD8alphabeta+ (93.3%), T-cell receptor-alphabeta+ (99.5%), costimulatory molecule CD28+ (90.1%), with a small CD8alphaalpha+ population (2.3%) . No NK-T-cell receptor CD158a or CD158b expression was detected, whilst CD94 was expressed on 6.2% of cells in 6/9 TCL . All the TCL and 37/39 CD8+ T-cell clones produced interferon-gamma but no or minimal interleukin-4 or interleukin-10 . The