Cell suspensions

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 TCL produced a wide range of interferon-gamma levels (138 to 15,020 pg/ml) . Clones from 3 patients showed low levels (60 to 1,410 pg/ml), from 2 patients high levels (6,105 to 43,040 pg/ml) and from 1 patient a wide range (405 to 36,010 pg/ml) of interferon-gamma production . Thus epidermal CD8+ Tc1 cells in chronic plaque psoriasis produce highly heterogeneous levels of interferon-gamma, which may reflect clinical diversity.

Exp Hematol, 2001 Jun, 29(6), 720 - 7
Injection of CD4(+) and CD8(+) cells with donor or host accessory cells induces acute graft-vs-host disease in human skin in immunodeficient mice; Matsumoto M et al.; OBJECTIVE: We examined cell subsets with respect to cutaneous graft-vs-host disease by cell sorting selection of subsets of human mononuclear cells and injecting the subsets subcutaneously in a mouse model . MATERIALS AND METHODS: Cell suspensions containing cultured human epidermal cells and dermal fibroblasts from a single donor mixed with lymphoid cell subsets positively selected using the FACSVantage cell sorting instrument and/or MACS cell isolation kits from unrelated individuals were injected into immunodeficient mice . This model is known to generate human skin with histologic findings similar to human graft-vs-host disease . RESULTS: Donor T-cell subsets CD4(+) and CD8(+) plus either host or donor CD14(+) cells were necessary to cause acute cutaneous graft-vs-host disease . Although graft-vs-host disease can result from recognition of class I antigens expressed on human cutaneous cells by donor peripheral blood mononuclear cells, additional recognition of class II antigens expressed on host mononuclear cells resulted in more severe histologic manifestations . Dendritic cells that differentiated from donor and host monocytes also showed competent accessory cell function in this system . CONCLUSIONS: Based on this model, human cutaneous graft-vs-host disease was caused by donor CD4(+) cells and CD8(+) cells activated through recognition of host antigens, including class I and class II antigens presented by either donor or host CD14(+) cells or dendritic cells.

J Nat Prod, 2001 May, 64(5), 630 - 1
Biotransformation of thujopsene by Caragana chamlagu; Sakamaki H et al.; Biotransformation of thujopsene (1) using a cell suspension culture of Caragana chamlagu for 14 days gave mayurone (2, 52%) and two new compounds, 3beta-hydroxy-4-thujopsene (4, 16%) and 3beta-epoxythujopsa-5beta-ol (3, 22%).

Plast Reconstr Surg, 2001 Apr 15, 107(5), 1208 - 15
Aerosolization of epidermal cells with fibrin glue for the epithelialization of porcine wounds with unfavorable topography; Cohen M et al.; Aerosolized epidermal cell suspension was previously found to be effective for the epithelialization of full-thickness wounds . This suspension is less expensive than and requires a shorter preparation time than the currently used cultured epithelial autografts . Still, convex and irregular wounds present unfavorable conditions for homogenous dispersion of the aerosolized cell suspension . The authors hypothesized that the addition of fibrin glue to the aerosol of cells would reduce cell movement and ensure homogenous dispersion of the cells, thereby promoting wound epithelialization . The objectives of the study were to evaluate the healing of wounds with unfavorable topography after autotransplantation of an epidermal cell aerosol with and without fibrin glue.Six Yorkshire piglets were studied . An epidermal suspension was made from full-thickness groin skin . Dispase was used to separate the epidermis from the dermis, and trypsin was used to separate the epidermal cells from one another . Twenty-four hours later, full-thickness wounds with unfavorable topography were created adjacent to the vertebral column of six pigs . Twelve wounds were treated with an aerosol of epidermal cell suspension mixed with fibrin glue (study group), and 12 wounds were treated with the same suspension without the fibrin glue (control group) . The percentages of total wound contraction and the epithelialized and nonepithelialized areas were evaluated 1, 2, 3, and 4 weeks after aerosolization . The histologic characteristics of the newly formed skin were examined by light microscopy using slides stained with hematoxylin and eosin.Study wounds were characterized by central epithelialization, whereas control wounds were characterized by peripheral epithelialization . Study wounds contracted at a slower rate than control wounds, but wound size was the same in both groups after 4 weeks . The addition of fibrin glue facilitated epithelialization: Study wounds showed 75.5 +/- 22.4 percent (mean +/- SD) and 94.2 +/- 8.8 percent epithelialization after 3 and 4 weeks, respectively, compared with 46.3 +/- 9.5 percent and 47.9 +/- 13.1 percent epithelialization of the control wounds at the same times . These differences between the study and control groups were statistically significant (p < 0.001, paired t test).The addition of fibrin glue to an aerosol of epidermal cells significantly enhances the epithelialization of wounds with unfavorable topography in pigs.

Reproduction, 2001 Jun, 121(6), 881 - 7
Studies on the metabolism of essential fatty acids in isolated human testicular cells; Retterstol K et al.; The essential fatty acid 22:6(n-3) is a minor component of the Western diet, but a major fatty acid in human testis and semen . In mature spermatozoa, the physical and fusogenic properties of the plasma membrane are probably influenced by its particular fatty acid composition . In this study, the synthesis of 22:6(n-3) and 22:5(n-6) was investigated in isolated human testicular cells . {1-(14)C}20:4(n-6), {1-(14)C}20:5(n-3), {1-(14)C}22:4(n-6) and {1-(14)C}22:5(n-3) were incubated in a 'crude' cell suspension (consisting of a mixture of the cells in the seminiferous tubule), and in fractionated pachytene spermatocytes and round spermatids . The esterification of fatty acids in lipid and phospholipid classes and the fatty acid chain elongation and desaturation were measured . The crude cell suspension metabolized the fatty acids more actively than did the fractionated germ cell suspension, indicating that types of cell other than the germ cells are important for fatty acid elongation and desaturation and thus the production of 22:6(n-3) . This finding is in agreement with previous results in rats that indicated that the Sertoli cells are the most important type of cell for the metabolism of essential fatty acids in the testis . Some {1-(14)C}20:5(n-3) was elongated to {(14)C}22:5(n-3) in the fractionated germ cells, but very little was elongated further to {(14)C}24:5(n-3),possibly restricting the formation of {(14)C}22:6(n-3) . In the fractionated germ cells, the fatty acid substrates were recovered primarily in the phospholipid fraction, indicating an incorporation in the membranes, whereas in the crude cells, more substrates were esterified in the triacylglycerol fraction . In the phospholipids, more radioactivity was recovered in phosphatidylcholine than in phosphatidylethanolamine and more radioactivity was recovered in phosphatidylethanolamine than in phosphatidylinositol or phosphatidylserine.

Biophys J, 2001 Jun, 80(6), 2493 - 504
Pore-to-pore hopping model for the interpretation of the pulsed gradient spin echo attenuation of water diffusion in cell suspension systems; Jiang PC et al.; A simplified pore-to-pore hopping model for the two-phase diffusion problem is developed for the analysis of the pulsed gradient spin echo (PGSE) attenuation of water diffusion in the condensed cell suspension systems . In this model, the two phases inside and outside the cells are treated as two different kinds of pores, and the spin-bearing molecules perform hopping diffusion between them . The size and the orientations of those two respective pores are considered, and then the diffraction pattern of the PGSE attenuation may be well simulated . Nevertheless, the intensity of the characteristic peak decreases with increasing membrane permeability, from which the exchange time may be estimated . We then analyze the experimental 1H PGSE results of the erythrocytes suspension system . The water-residence lifetime in the erythrocyte is obtained to be 10 ms, which is the same as that estimated from the two-region approximation . Furthermore, the PGSE attenuation curve of addition of p-Chloromercuribenzenesulfonate (p-CMBS) is also discussed . It predicts that the alignment of erythrocytes will become normal to the magnetic field direction after the addition of p-CMBS, and inspection using a light microscope confirms that result.

Z Naturforsch {C}, 2001 Mar-Apr, 56(3-4), 235 - 44
The oxidative processes induced in cell suspensions of Solanum species by culture filtrate of Phytophthora infestans; Polkowska-Kowalczyk L et al.; Solanum genotypes that differ in the level of polygenic resistance to the oomycete plant pathogen Phytophthora infestans were studied for their oxidative response to culture filtrate (CF) of the pathogen . Reactive oxygen species (ROS) production, peroxidase activity and lipid peroxidation have been studied in the CF-treated cell suspensions derived from leaves of the resistant S . nigrum (nonhost) and S . tuberosum cv . Bzura as well as from the susceptible S . tuberosum cv . Tarpan and clone H-8105 . In both the resistant and susceptible cells the CF induced similar processes, but these varied with respect to the kinetics and intensity . In all cells probably the membrane-bound NADPH oxidase, was responsible for the ROS production . This process was more intensive and prolonged in the susceptible cells than in the resistant ones . The CF treatment slightly affected peroxidase activity in all cells studied . Lipid peroxidation that occurred as a consequence of the ROS accumulation was pronounced mainly in the susceptible cells . We suggest that lack of stringent control of the oxidative processes and sensitivity to the pathogen toxins may be decisive for limited polygenic resistance in potato.

Arch Biochem Biophys, 2001 Feb 1, 386(1), 17 - 24
The effects of the site-directed removal of N-glycosylation from cationic peanut peroxidase on its function; Lige B et al.; Peanut peroxidase has been diffracted . The location of its heme and calcium moieties have been shown and their role demonstrated . However, the structure and role of its glycans is only now being elucidated . The role of three N-linked complex glycans on cationic peroxidase (cPrx) of peanut (Arachis hypogaea L cv . Valencia), as expressed by prxPNC1 in transgenic tobacco, was analyzed by site-directed replacement of each of the three glycosylation sites, N-60, N-144, and N-185 with Q, individually . The mutant prxPNC1 cDNAs with a 3' histidine-tag were expressed in transgenic tobacco . The effect on the catalytic ability, thermal stability, and unfolding properties of the mutant peroxidases, isolated from the medium of transgenic tobacco cell suspension cultures were compared with those of the wild cPrx from peanut . It was found that the ablation of the glycans at N-60 and N-144 influences the full expression of the cPrx catalytic ability . The glycan at N-185 is important for the thermostability, as is the removal of the carbohydrate chain at N-185, resulting in rapid enzymatic decrease at temperatures of 50 degrees C . All three glycans appeared to influence the folding of the protein.

Clin Exp Allergy, 2001 Apr, 31(4), 602 - 8
Mast cells and their secretory granules are angiogenic in the chick embryo chorioallantoic membrane; Ribatti D et al.; BACKGROUND: Many data suggest that the density of mast cells is highly correlated with the extent of both normal and pathological angiogenesis . OBJECTIVE: In this study we have compared in an in vivo assay, the chick embryo chorioallantoic membrane, the angiogenic potential of mast cell suspensions isolated from rats, degranulated mast cells and their secretory granules . METHODS: Gelatin sponges adsorbed with cell suspensions of rat mast cells, degranulated mast cells and their secretory granules were implanted on the top of the chorioallantoic membrane at day 8 of incubation . At day 12 the angiogenic response was evaluated macroscopically, microscopically and by a morphometric method of 'point counting' . RESULTS: Isolated mast cells and their secretory granules, but not degranulated mast cells, induced an angiogenic response in the chorioallantoic membrane . The addition of antifibroblast growth factor-2 or antivascular endothelial growth factor antibodies reduced the angiogenic response of both mast cells and their secretory granules by 50% and 30%, respectively . CONCLUSION: These data support the evidence that the angiogenic properties of mast cells depend on the angiogenic molecules contained in their secretory granules and indicate that fibroblast growth factor-2 and vascular endothelial growth factor are the angiogenic cytokines primarily and perhaps synergistically responsible for this vasoproliferative activity.

J Asian Nat Prod Res, 2001, 3(1), 9 - 14
Diglucosylation of salicyl alcohol by cell suspension cultures of Solanum laciniatum; Syahrani A et al.; A new biotransformation product, salicyl alcohol-7-O-beta-D-(beta-1,6-D-glucopyranosyl)-gluco-pyranoside was isolated from cell suspension cultures of Solanum laciniatum, following administration of salicyl alcohol, and its structure was elucidated using a combination of one- and two-dimensional 1H and 13C-NMR data, and positive and negative ion ESMS data.

J Neurosurg, 2001 May, 94(5), 765 - 74
Neuroprotective and behavioral efficacy of nerve growth factor-transfected hippocampal progenitor cell transplants after experimental traumatic brain injury; Philips MF et al.; OBJECT: Immortalized neural progenitor cells derived from embryonic rat hippocampus (HiB5), were transduced ex vivo with the gene for mouse nerve growth factor (NGF) to secrete NGF (NGF-HiB5) at 2 ng/hr/10(5) cells in culture . METHODS: Fifty-nine male Wistar rats weighing 300 to 370 g each were anesthetized with 60 mg/kg sodium pentobarbital and subjected to lateral fluid-percussion brain injury of moderate severity (2.3-2.4 atm, 34 rats) or sham injury (25 rats) . At 24 hours postinjury, 2 microl (150,000 cells/microl) of {3H}thymidine-labeled NGF-HiB5 cells were transplanted stereotactically into three individual sites in the cerebral cortex adjacent to the injury site (14 rats) . Separate groups of brain-injured rats received nontransfected (naive {n})-HiB5 cells (12 animals) or cell suspension vehicle (eight animals) . One week postinjury, animals underwent neurological evaluation for motor function and cognition (Morris water maze) and were killed for histological, autoradiographic, and immunocytochemical analysis . Viable HiB5 cell grafts were identified in all animals, together with reactive microglia and macrophages located throughout the periinjured parenchyma and grafts (OX-42 immunohistochemistry) . Brain-injured animals transplanted with either NGF-HiB5 or n-HiB5 cells displayed significantly improved neuromotor function (p < 0.05) and spatial learning behavior (p < 0.005) compared with brain-injured animals receiving microinjections of vehicle alone . A significant reduction in hippocampal CA3 cell death was observed in brain-injured animals receiving transplants of NGF-HiB5 cells compared with those receiving n-HiB5 cells or vehicle (p < 0.025) . CONCLUSIONS: This study demonstrates that immortalized neural stem cells that have been retrovirally transduced to produce NGF can markedly improve cognitive and neuromotor function and rescue hippocampal CA3 neurons when transplanted into the injured brain during the acute posttraumatic period.

J Asian Nat Prod Res, 2001, 3(1), 9 - 14
Diglucosylation of salicyl alcohol by cell suspension cultures of Solanum laciniatum; Syahrani A et al.; A new biotransformation product, salicyl alcohol-7-O-beta-D-(beta-1,6-D-glucopyranosyl)-gluco-pyranoside was isolated from cell suspension cultures of Solanum laciniatum, following administration of salicyl alcohol, and its structure was elucidated using a combination of one- and two-dimensional 1H and 13C-NMR data, and positive and negative ion ESMS data.

Plant Mol Biol, 2001 Mar, 45(4), 477 - 88
Catharanthus roseus G-box binding factors 1 and 2 act as repressors of strictosidine synthase gene expression in cell cultures; Siberil Y et al.; The enzyme encoded by the strictosidine synthase (Str) gene catalyses a key step in the biosynthesis of therapeutically valuable terpenoid indole alkaloids . In Catharanthus roseus the Str gene was shown to be regulated by a wide variety of signals including auxin, methyl jasmonate and fungal elicitors in cell suspension cultures and by tissue-specific control in plant organs . The Str promoter contains a functional G-box (CACGTG) cis-regulatory sequence . In order to understand better the mechanisms involved in the regulation of Str gene expression, we isolated the C . roseus cDNAs encoding G-box binding factors Crgbf1 and Crgbf2 . The binding specificity of their protein products CrGBF1 and CrGBF2 was analysed by competitive electrophoresis mobility and saturation binding assays . CrGBF1 had a high binding specificity for class I G-boxes including the Str G-box . CrGBF1 showed a lower affinity for class II G-boxes and for the G-box-like element (AACGTG) found in the tryptophan decarboxylase (Tdc) gene which encodes another enzyme involved in TIA biosynthesis . CrGBF2 showed a high affinity for all types of G-boxes tested and to a lesser extent for the Tdc G-box-like element . Transient bombardment experiments demonstrated that both CrGBF1 and CrGBF2 can act in vivo as transcriptional repressors of the Str promoter via direct interaction with the G-box . These data indicate that GBFs may play functional role in the regulation of expression of the terpenoid indole alkaloid biosynthetic gene Str.

Planta, 2001 Apr, 212(5-6), 888 - 95
WY-14,643 and other agonists of the peroxisome proliferator-activated receptor reveal a new mode of action for salicylic acid in soybean disease resistance; Tenhaken R et al.; Inoculation of soybean (Glycine max {L.} Merr.) cell-suspension cultures with avirulent bacteria results in a salicylic acid (SA)-controlled programmed cell death (pcd) . To unravel the nature of the SA-dependent step in pcd, a screening procedure for complementing compounds was performed . Diverse chemicals that are well known as activating ligands for orphan receptors in animals, particularly receptors of the PPAR (peroxisome proliferator-activated receptor) subfamily, were found to be active . These include the compounds WY-14643, flufenamic acid, LY-171883, tolbutamide, indomethacin and clofibrate . A new marker gene (DD-CA9) from soybean that is induced in the hypersensitive reaction by SA and by PPAR ligands was isolated by differential display, and showed homology to antifungal lectins . In plants, SA is also involved in a signal transduction pathway leading to systemic acquired resistance (SAR) . The PPAR ligands which act on the pcd pathway for plant resistance induce a beta-1,3-glucanase gene in soybean at high concentrations but do not induce marker genes of the SAR pathway such as the PR-1 gene in tobacco or Arabidopsis . Thus SA seems to act on two independent plant defence pathways that can now be separately activated by synthetic compounds . We propose a model for the control of pcd by SA in soybean, in which SA induces the transcription of (novel) genes required for the final completion of the cell death program.

Planta, 2001 Apr, 212(5-6), 765 - 73
Regulation of respiration in rotenone-treated tobacco cell suspension cultures; Zhang Q et al.; Cells of Nicotiana tabacum L . suspension cultures were treated with the respiratory inhibitor rotenone, which specifically inhibits complex I activity of mitochondria . Rotenone retarded cell growth, as shown by decreases in fresh weight, dry weight and cell numbers on a suspension-volume basis . However, rates of the coupled respiration were higher in rotenone-treated compared to control cells when expressed on a fresh-weight basis . Rates of the rotenone-insensitive respiration increased substantially on both a fresh-weight and extractable-cellular-protein basis 24 h after rotenone treatment . ATP/ADP ratios were not significantly different between control and rotenone-treated cells . Our results indicated that cells of tobacco suspension cultures were able to maintain a slow rate of growth and adequate ATP/ADP ratios without the operation of complex I.

Eur J Cardiothorac Surg, 2001 May, 19(5), 576 - 9
Iliac crest biopsy versus rib segment resection for the detection of bone marrow isolated tumor cells from lung and esophageal cancer; Mattioli S et al.; OBJECTIVE: The presence of isolated tumor cells in the bone marrow affects the prognosis of both esophageal cancer and non-small cell lung cancer (NSCLC) . Therefore, preoperative assessment of isolated tumor cells may be useful to plan multimodality treatment . Rib segment resection at surgery provides adequate amounts of bone marrow for the detection of isolated tumor cells while bone marrow aspirate from the iliac crest does not . The iliac crest biopsy according to the Jamshidi technique procures a core of tissue apt for histology and not simply for cytology . The aim of this study was to compare the accuracy of iliac crest biopsy versus rib segment resection in the diagnosis of isolated tumor cells in order to obtain a useful preoperative approach . MATERIAL AND METHODS: Twenty-one consecutive patients (18 NSCLC, three esophageal cancer) were evaluated . None had chemotherapy prior to evaluation . Bone marrow was obtained preoperatively by iliac crest biopsy using the Jamshidi needle and at surgery by rib segment resection . Positive cytokeratin neoplastic cells were searched by immunohistochemistry on tissue sections from the iliac crest biopsies and by flow cytometry on cell suspensions from the rib segments . RESULTS: Isolated tumor cells were detected in the rib segments of ten patients . In all cases the Jamshidi needle biopsy was not diagnostic . CONCLUSION: Our results suggest that, if the diagnosis of bone marrow isolated tumor cells has clinical relevance, the preoperative assessment should be performed by rib segment resection or methods other than iliac crest aspirate or biopsy . Further investigation is needed to determine whether isolated tumor cells have a preferential spread to chest bones other than distant bone sites.

Enzyme Microb Technol, 2001 May 7, 28(7-8), 666 - 672
Selection of fungal elicitors to increase indole alkaloid accumulation in catharanthus roseus suspension cell culture; Zhao J et al.; Various fungal elicitors derived from 12 fungi were tested to improve indole alkaloid production in Catharanthus roseus cell suspension cultures . Results show that different fungal mycelium homogenates stimulate different kinds of indole alkaloid (ajmalicine, serpentine and catharanthine) accumulation, which ranged from 2- to 5-fold higher than the control . Some fungal culture filtrates also efficiently elicited the biosynthesis of different indole alkaloids . The optimal elicitor addition and exposure time for the maximal alkaloid production were on day 7 after subculture and for 3 days of treatment but different fungal elicitors showed the different optimal treatment dosages . Additions of elicitor at the doses ranging from 5 mg/l to 30 mg/l of carbon hydrate equivalent resulted in varieous amounts and kinds of indole alkaloid accumulation . Exposed to a same fungal elicitor, several different cell lines generated the different responses regarding as growth rate, culture color and alkaloid production.

Plant Sci, 2001 May, 160(6), 1221 - 1228
Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica; Mizuhiro M et al.; Protoplasts were isolated from cell suspension cultures of Primula malacoides cv . 'Lovely Tokyo' and P . obconica cv . 'Aalsmeer Giant White' . P . obconica protoplasts were embedded in 0.1% (w/v) gellan gum-solidified discs comprising MS medium supplemented with 3 mg/l of 2,4-D or picloram, 0.1 mg/l of zeatin, 0.2 M glucose and 0.2 M mannitol, and surrounded by a liquid medium of the same composition except for the addition of 0.1% (w/v) activated charcoal . The protoplasts formed visible colonies, which were transferred to the regeneration medium containing 30 g/l of sucrose, 0.1 mg/l of picloram and 2 mg/l of zeatin for shoot induction . P . malacoides protoplasts formed visible colonies when cultured in disc culture using 0.1% (w/v) gellan gum-solidified MS medium containing 5 mg/l of 2,4-D, 1 mg/l of NAA, 0.1 mg/l of zeatin and 0.4 M glucose . Small calli were transferred to MS medium supplemented with 5 mg/l of zeatin for shoot regeneration . The shoots of both species readily rooted on plant growth regulator-free 1/2 MS medium and successfully acclimatized to greenhouse conditions . The protoplast-derived plants showed some alterations in morphological characteristics from those of the in-vitro-germinated control plants.

Phytochemistry, 2001 May, 57(1), 33 - 42
Tracer studies with 13C-labeled carbohydrates in cultured plant cells . Retrobiosynthetic analysis of chelidonic acid biosynthesis; Shen Z-W et al.; The biosynthesis of chelidonic acid was studied in cell suspension cultures of Leucojum aestivum . Cell cultures were supplied with {U-13C}glucose, {l-13C}glucose or {U-13Cs}ribose/ribulose in standard medium containing unlabeled glucose . 13C labeling patterns of amino acids obtained by hydrolysis of biomass were determined by NMR spectroscopy and compared to the labeling pattern of chelidonic acid . The data document the incorporation of a contiguous 4-carbon fragment derived from the pentose phosphate pool into chelidonic acid . This suggests a biosynthetic pathway involving the condensation of phosphoenolpyruvate with a pentose phosphate followed by dehydration, dehydrogenation, ring closure and decarboxylation conducive to the loss of C-5 of the pentose precursor.

J Chromatogr B Biomed Sci Appl, 2001 Apr 5, 753(2), 343 - 53
High-performance liquid chromatographic-tandem mass spectrometric method for the determination of ethionamide in human plasma, bronchoalveolar lavage fluid and alveolar cells; Conte JE Jr et al.; We have developed and validated an accurate, sensitive, and rapid high-performance liquid chromatographic-tandem mass spectrometric method (HPLC-MS-MS) for the determination of ethionamide in plasma, bronchoalveolar fluid (BAL) and alveolar cells (AC) . The retention times for ethionamide, clemastine fumarate (internal standard for plasma), promethazine (internal standard for plasma) and propranolol (internal standard for BAL and AC) were approximately 2.62, 1.21, 2.14, and 2.22 min, respectively, with a total run time of 3.2 min . Ethionamide detection for plasma was carried out on a PE Sciex API III (Perkin-Elmer, Foster City, CA, USA) . BAL and cell pellets and some plasma specimens were analyzed on a Micromass Quattro LC (Micromass Co., Manchester, UK) . The detection limits for ethionamide were 0.05 microg/ml for plasma, and 0.005 microg/ml for BAL supernatants and alveolar cell suspensions.

Perfusion, 2001 Mar, 16 Suppl, 61 - 6
Cell salvage and leucodepletion; Cross MH; Cell salvage has been used as a method of blood conservation for more than three decades . Although the principles and development of the Latham bowl had occurred in the 1960s, it was not until the early 1970s that washing of the concentrated red cells was introduced and a product that was universally acceptable was obtained . The last 25 years have seen little in the way of development of cell salvage, although significant refinement has taken place . Although the simple picture of cell salvage involves removal of the buffy coat, including platelets and leucocytes, in practice there are reports of great variation in the removal of these cells . Most recent studies suggest that there is very little removal of leucocytes by cell salvage . The leucocytes that remain in the red cell suspension following cell salvage have undergone significant morphological changes and the surface expression of leucocyte adhesion receptors increases dramatically during the process . There is little evidence that removal of these activated leucocytes has any significant clinical benefit . Although leucofiltration of blood before storage has been shown to be an extremely safe process, 'bedside leucofiltration', including leucofiltration of cell salvage blood, may not be without problems . Reports of hypotensive events while receiving blood products through a bedside leucocyte reduction filter have emerged during the last few years . This may be due to bradykinin production following platelet exposure to negatively charged leucocyte filters.

Perfusion, 2001 Mar, 16 Suppl, 51 - 5
Filtration of malignant cells: tumour cell depletion in an ex vivo model using a leukocyte adhesion filter; Fruhauf NR et al.; The clinical use of leukocyte adhesion filters is based on differentiated depletion of blood cells, although most of the mechanisms of filtration are still unclear . There is also evidence that leukocyte adhesion filters can remove disseminated tumour cells from patients' blood . The following proves this observation and consists of three parts: in the first part we analysed the depletion capacity of the LeucotrapWB filter medium (Pall) in an employed small-scale model for the cell line HT29 (colorectal origin) in phosphate-buffered saline (PBS) solution . A depletion rate of more than 99.6% for HT29 cells was seen . In the second part of the study, samples of whole blood were spiked with HT29 cells in a similar setting, showing comparable results . In the last part, defined cell suspensions of various human primary tumours were admixed to whole blood samples and afterwards filtrated in a scale-up model . Again, a nearly complete tumour cell reduction was seen after filtration . Results show a significant tumour cell reduction and, for most cases, a complete depletion of tumour cells independently from quality and origin of the tumour tissue . These experiments and further investigation are supposed to help to reduce the risk of dissemination of tumour cells in patients undergoing oncological surgery.

J Photochem Photobiol B, 2000 Dec, 59(1-3), 115 - 22
NO-hemoglobin may be a light-sensitive source of nitric oxide both in solution and in red blood cells; Vladimirov Y et al.; Hemoglobin in solution and inside red blood cells forms a complex with nitric oxide exhibiting a specific EPR signal both at room and liquid nitrogen temperatures . In the present paper it was shown that the nitrosyl complex of hemoglobin (NO-Hb) is photochemically sensitive and hence may serve as a source of free NO under He-Cd laser irradiation (441 nm) . It was found that at laser light radiant power of 3.9 mW, room temperature and in the presence of oxygen, 50% decrease of NO-Hb EPR signal occurred at doses of 54, 30, and 18 kJ/m2 for NO-hemoglobin solution, hemolysed and intact erythrocytes, respectively . The detection of free NO produced as a result of NO-Hb photolysis was performed by means of a spin trap, nitronyl nitroxyl radical NNR, which in the presence of NO is transformed into imino nitroxyl radical (INR) showing different EPR signal . In isolated hemoglobin solution, 20 mM INR was accumulated under irradiation with the maximal dose of 700 kJ/m2 . In intact cells the HbFe(2+)-NO photolysis and NO release occur with essentially higher efficacy . To produce 100 mM INR, a dose of 290 kJ/m2 was needed in erythrocyte lysates and 100 kJ/m2 in intact red blood cell suspension . Measurements of absorption spectra showed that in all systems studied (NO-Hb in solution, intact erythrocytes and hemolysed erythrocytes) NO-Hb concentration decreased after irradiation by 14-22% with simultaneous formation of methemoglobin . These observations show that NO-Hb may serve as a store of nitric oxide from which free NO can be released by intensive illumination.

J Clin Pathol, 2001 May, 54(5), 377 - 80
Distribution and viral load of type specific HPVs in different cervical lesions as detected by PCR-ELISA; Zerbini M et al.; AIMS: To investigate the distribution and viral load of the most prevalent high risk human papillomavirus (HPV) types 16, 18, 31, 33, and 45 and low risk HPV types 6 and 11 in a variety of cervical lesions . METHODS: One hundred and seventy six cytological specimens from women with different cervical lesions were investigated . For an accurate standardisation of the sample, cervical cells were counted and a volume of the cell suspension processed by polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) . Semiquantitative determinations were achieved in relation to an external reference titration curve . RESULTS: HPV DNA was detected in 60.2% of the samples . HPV-16 was the prevalent genotype (57.6%), followed by HPV-33, HPV-31, HPV-6, HPV-18, and HPV-45 . HPV-11 was not detected . HPV-16 showed a pronounced increase in prevalence with the evolution of cervical disease . Semiquantitative evaluation of the results showed that only HPV-16 DNA could reach very high values (> 1000 genome copies/cell) and a very high HPV-16 load correlated with the severity of cervical disease . CONCLUSIONS: Only HPV-16 load appears to be associated with the severity of cervical disease.

FEMS Microbiol Lett, 2001 Apr 20, 198(1), 23 - 9
Metabolite and enzyme profiles of glycogen metabolism in Methanococcoides methylutens; Maitra PK et al.; When a buffered anaerobic cell suspension of Methanococcoides methylutens was maintained under methanol-limited conditions, intracellular glycogen and hexose phosphates were consumed rapidly and a very small amount of methane formed at 4 h of a starvation period . When methanol was supplemented after a total of 20 h of starvation, a reverse pattern was observed: the glycogen level and the hexose phosphate pool increased, and formation of methane took place after a lag period of 90 min . A considerable amount of methane was formed in 120 min after its detection with a rate of 0.18 micromol mg(-1) protein min(-1) . When methane formation decreased after 270 min of incubation and finally came to a halt, probably due to complete assimilation of supplemented methanol, the levels of glycogen and hexose monophosphates decreased once again . However fructose 1,6-diphosphate levels showed a continuous increase even after exhaustion of methane formation . In contrast to the hexose phosphate pool, levels of other metabolites showed a small increase after addition of methanol . The enzyme profile of glycogen metabolism showed relatively high levels of triose phosphate isomerase . Glyceraldehyde 3-phosphate dehydrogenase reacted with NADPH with a three-fold higher activity as compared to that with NADH.

Sheng Li Xue Bao, 1998 Feb, 50(1), 28 - 36
{A new way for neural transplantation: the grafted central neurons migration from the subarachnoid space into the spinal cord and cerebral cortex}; Shu J et al.; In the studies of neural transplantation, Chen and his colleagues observed that the grafted central neurons could migrate from the subarachnoid space into the spinal cord and cerebral cortex . This finding promises an attractive possibility that a lot of neurons might be introduced into a long distance of the spinal cord and the extensive superficial layer of the brain which has suffered neuronal loss and thus might reinnervate a wide range of denervated area . Female rats were used in the study . Neural tissues or cell suspension containing fetal monoaminergic or arginine vasopressin (AVP) neurons were implanted into the subarachnoid space of the transected spinal cord or the normal brain and spinal cord . The rats were treated in different ways: some were grafted and the spinal cord was cut at the same time; grafted one month later or before the spinal cord was cut; grafted but the brain and spinal cord remained intact . Immunohistochemical method was used to monitor the grafted neurons after these rats survived for one or nine months . The results showed that there were a few to hundreds of 5-HT, TH or AVP-immunoreactive neurons and fibers found in the spinal cord or cerebral cortex near the grafted region . These neurons grew well and displayed their capacity of adaptability and long-time survival in the host CNS . No neurons, however, were found in the subarachnoid space of the grafted rats which had survived for another month or longer . On the other side, grafted with tissue block, it was found that transplants left in the grafted region grew as a "nodule" attaching to the surface of the spinal cord . The "nodule" was also found occasionally when the cell suspension was centered heavily at one region of the subarachnoid space . In both cases, the pia mater between them disappeared . Immunoreactive neurons were found in the "nodule" and neighboring spinal cord, some fibers from the neurons in the "nodule" extended into the spinal cord . It is supposed that a part of cells in the suspension died after grafted in the subarachnoid space and released hydrolases which injured the near pia mater by hydrolysis . As a result, some neurons in the suspension had a chance to migrate into the spinal cord and cerebral cortex . After the transplant in the subarachnoid space was gradually cleaned out, the enzymatic hydrolysis to the pia mater became weaker and finally stopped, the lesioned pia mater was gradually repaired . If the transplant was not cleared timely, the pia mater could not withstand the persistent hydrolysis and collapsed finally, leading the transplant to be fused with the host CNS.

Eur J Cell Biol, 2001 Mar, 80(3), 222 - 9
Rat germinal cells require PARP for repair of DNA damage induced by gamma-irradiation and H2O2 treatment; Atorino L et al.; The ability of rat germinal cells to recover from genotoxic stress has been investigated using isolated populations of primary spermatocytes and round spermatids . Using a comet assay at pH 10.0 to assess single strand breakage (SSB) in DNA, it was found that a high level of damage was induced by 5 Gy gamma-irradiation and acute exposure to 50 microM H2O2 . This damage was effectively repaired during a subsequent recovery period of 1-3 hours culture in vitro but repair was significantly delayed in the presence of the poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide (3-ABA) . Immunofluorescence detection of PARP with specific antibodies localised the protein to discrete foci within the nucleus of both spermatocytes and spermatids . Poly(ADP-ribose) (pADPR) could also be detected in spermatid nuclei following gamma-irradiation or H2O2 treatment . Moreover, PARP activation occurs both in spermatocytes and spermatids left to recover after both genotoxic stresses . The NO donors, 3-morpholino-sydnonimine (SIN-1) and S-nitrosoglutathione (SNOG), caused significant SSBs in both spermatocytes and spermatids . The effects of SIN-1 could be prevented by exogenous catalase (CAT), but not superoxide dismutase (SOD), in the cell suspensions . SNOG-induced SSBs were insensitive to both CAT and SOD . It is concluded that DNA in spermatocytes and spermatids is sensitive to damage by gamma-irradiation and H2O2 and that efficient repair of SSBs requires PARP activity.

Pharmacol Toxicol, 2001 Apr, 88(4), 192 - 7
Bioactivation and cytotoxicity of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) in isolated rat hepatocytes; Ferrara R et al.; The bioactivation and cytotoxicity of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), a replacement for some ozone-depleting chlorofluorocarbons, were investigated using freshly isolated hepatocytes from non-induced male rats . A time- and concentration-dependent increase in the leakage of lactate dehydrogenase and a concentration-dependent loss of total cellular glutathione were observed in cells incubated with 1, 5 and 10 mM HCFC-123 under normoxic or hypoxic (about 4% O2) conditions . Lactate dehydrogenase leakage was completely prevented by pretreating the cell suspension with the free radical trapper N-t-butyl-alpha-phenylnitrone . The aspecific cytochrome P450 (P450) inhibitor, metyrapone, totally prevented the lactate dehydrogenase leakage from hepatocytes, while two isoform-specific P450 inhibitors, 4-methylpyrazole and troleandomycin (a P450 2E1 and a P450 3A inhibitor, respectively), provided a partial protection against HCFC-123 cytotoxicity . Interestingly, pretreatment of cells with glutathione depletors, such as phorone and diethylmaleate, did not enhance the HCFC-123-dependent lactate dehydrogenase leakage . Two stable metabolites of HCFC-123, 1-chloro-2,2,2-trifluoroethane and 1-chloro-2,2-difluoroethene, were detected by gas chromatography/mass spectrometry analysis of the head space of the hepatocyte incubations carried out under hypoxic and, although at a lower level, also normoxic conditions, indicating that reductive metabolism of HCFC-123 by hepatocytes had occurred . The results overall indicate that HCFC-123 is cytotoxic to rat hepatocytes under both normoxic and hypoxic conditions, due to its bioactivation to reactive metabolites, probably free radicals, and that P450 2E1 and, to a lower extent, P450 3A, are involved in the process.

Klin Monatsbl Augenheilkd, 2001 Mar, 218(3), 192 - 6
{Autologous subretinal transplantation of cultivated porcine iris pigment epithelial cells (IPE)}; Steinhorst UH et al.; BACKGROUND: Subretina transplantation of epithelium may be a therapeutic option for surgical treatment of age-related macular degeration (AMD) . Various experimental data have demonstrated that homologous transplantation of retinal pigment epithelium (RPE) can prevent photoreceptor deterioration . However, most investigators experienced immunogenic graft rejection when using homologous pigmented cells for grafting . Autologous cells were soon considered as an alternative for subretinal grafting . Particularly iris pigment epithelium (IPE) appeared suitable to replace homologous RPE for it embryogenetic similarity and its simple availability . Recent studies have shown, that IPE is capable of taking over functions of RPE in maintaining retinal metabolism . the purpose of this study was to evaluate if autologous IPE cells would survive when being transplanted subretinally . In addition, immunogenic reponses to the presence of "foreign" iris pigment cells needed to be excluded . MATERIALS AND METHODS: Iris tissue was obtained by peripheral iridectomy in the anesthetized pig . Sheets of pigment iris epithelium were separated from the specimens and transferred into tissue culture . After the cells had been grown to confluency, cell suspensions were injected into the subretinal space of the donor animal's fellow eye . After 4 weeks, the grafted eye was enucleated and examined histologically. . RESULTS: The histological exam revealed that the graft cells had survived in the subretinal space . No evidence of immunogenic rejection was observed . CONCLUSION: Autologous IPE-cells can survive in the host's sub-retinal space without creating inflammatory reactions . Transplanted IPE appears to interact with photoreceptor outer segments.

Am J Chin Med, 2001, 29(1), 161 - 72
Effect of norcantharidin on N-acetyltransferase activity in HepG2 cells; Wu LT et al.; The inhibition ofarylamine N-acetyltransferase (NAT) activity by norcantharidin (NCTD), the demethylated form of cantharidin, in human hepatocellular carcinoma HepG2 cells was investigated . By using high performance liquid chromatography, NAT activity on acetylation of 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) were examined . Two assay systems were performed, one with cellular cytosols, the other with intact HepG2 cell suspensions . The NAT activity in HepG2 cell line was inhibited by norcantharidin in a dose-dependent manner in both types of examined systems: i.e . the greater the concentration of norcantharidin in the reaction, the greater the inhibition of NAT activities . This report is the first to show that norcantharidin has an inhibitory effect on NAT activity in HepG2 cell.

Biotechnol Bioeng, 2001 Jun 5, 73(5), 338 - 46
Characterization of monoclonal antibody fragments produced by plant cells; Sharp JM et al.; Production of a murine IgG1 was investigated using hairy roots, shooty teratomas, and suspended cells of transgenic tobacco . In all cases, in addition to complete assembled antibody, two to four major antibody fragments accumulated in the biomass . A range of protease inhibitors, protein-stabilizing agents, inhibitors of N-glycosylation and protein secretion, glycan-reactive agents, and affinity probes was used to characterize these fragments and investigate their sites and mechanisms of formation . The fragments were not experimental artifacts caused by antibody degradation during tissue homogenization and sample preparation, nor did they represent glycosylation variants . All of the molecules were actively secreted into the culture media and some showed evidence of Golgi-associated glycan processing, indicating they were not assembly intermediates . Antibody fragments of 50 and 80 kDa were identified mainly as the products of extracellular degradation in the root and shoot apoplast; the 80-kDa fragment was also present in cell suspension medium, and in suspended cell biomass toward the end of the growth phase . Larger 120- and 135-kDa fragments were most likely produced by proteolytic degradation along the secretory pathway outside of the endoplasmic reticulum (ER) and Golgi apparatus; the carbohydrate residues of the 135-kDa antibody suggest formation between these organelles . Inhibition of protein secretion and retention of antibody in the ER and/or Golgi reduced fragmentation and increased antibody accumulation levels, probably by reducing exposure to the principal sites of protease activity . This work highlights the importance of foreign protein degradation in plant tissues as a mechanism for posttranslational product loss . Identifying the nature of these degradative processes is a first step toward alleviating their effects, improving protein yields, and enhancing the feasibility of plants as a commercial means for large-scale protein production .

Physiol Plant, 2001 May, 112(1), 142 - 148
Increasing ploidy level in cell suspension cultures of Doritaenopsis by exogenous application of 2,4-dichlorophenoxyacetic acid; Mishiba KI et al.; To clarify the causal factors for ploidy variation in plant cell culture, we attempted to alter ploidy distribution in cell cultures of a tetraploid cultivar of Doritaenopsis by changing the plant growth regulators (PGRs) in the culture medium . The original suspension cultured cells, which had been maintained in medium containing 0.1 mg l-1 1-naphthaleneacetic acid and 1 mg l-1 benzyladenine, were transferred onto various gellan gum solidified media with a single application of PGRs, and the ploidy distributions of the cells were examined using flow cytometry analysis during 3 weeks of culture . Among the PGRs tested, 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid caused a drastic reduction in the 4C-cell proportion in cell cultures with an increased cell proportion of 8C or higher C-values . In the case of 2,4-D application, a reduction of cell viability was observed . A decreasing proportion was also observed in the 8C-cell population accumulated by 2,4-D treatment, following transfer back to the medium containing the standard PGR composition . These results suggest that the exogenous application of 2,4-D arrested the cell cycle at G2 phase in the Doritaenopsis cells, and the removal of 2,4-D might induce further endoreduplication or recover the mitotic cycle of the G2-arrested cells.

Appl Biochem Biotechnol, 2001 Mar, 90(3), 261 - 72
Phosphodiesterase production in an aqueous two-phase system by Nicotiana tabacum 1507; Ilieva M et al.; Studies were conducted on the production of phosphodiesterases by Nicotiana tabacum 1507 cell suspension in an aqueous two-phase system formed by adding 4% polyethylene glycol (mol wt 20,000) and 7.5% dextran (mol wt 70,000) to the medium . The time course of growth, biosynthesis, secretion, and partitioning of phosphodiesterases was followed in comparison with N . tabacum 1507 cultivation as a free suspension . Partitioning of phosphodiesterases took place mainly in the bottom dextran phase, and a possibility was revealed for obtaining an enzyme preparation with high phosphodiesterase activity.

Exp Neurol, 2001 May, 169(1), 23 - 9
Diminished survival of mesencephalic dopamine neurons grafted into aged hosts occurs during the immediate postgrafting interval; Sortwell CE et al.; The survival rate of dopamine (DA) neurons in mesencephalic grafts to young adult rats is poor, estimated at 5-20%, and even poorer in grafts to the aged striatum . Grafted cells die in young adult rats during the first 4 days after implantation . The present study was undertaken to determine whether the decreased survival of DA neurons in grafts to aged rats is (1) due to additional cell death during the immediate postgrafting interval or (2) due to protracted cell loss during longer postgrafting intervals . We compared survival rates of tyrosine hydroxylase-immunoreactive (THir) neurons in cell suspension grafts to young adult (3 months) and aged (24 months) male Fischer 344 rats at 4 days and 2 weeks after transplantation . At 4 days after grafting, mesencephalic grafts within the aged rat striatum contain approximately 25% of the number of THir neurons in the same mesencephalic cell suspension grafted to young adult rats . This corroborates the decreased survival of grafted DA neurons we have demonstrated previously at 10 weeks postgrafting . THir neurons in grafts to the intact striatum possessed a significantly shorter "long axis" than their counterparts on the lesioned side . No significant differences in the number of apoptotic nuclear profiles or total alkaline phosphatase staining between mesencephalic grafts to young and aged rats were detectable at 4 days postgrafting . In summary, the present study indicates that the exaggerated cell death of grafted DA neurons that occurs following implantation to the aged striatum occurs during the immediate postgrafting interval, timing identical to that documented for young adult hosts .

Toxicol Lett, 2001 Apr 8, 121(1), 57 - 61
Donors of NO and pulsed radiation at lambda = 820 nm exert effects on cell attachment to extracellular matrices; Karu TI et al.; The adhesion of HeLa cells to a glass matrix was evaluated after the irradiation of the cell suspension with a pulsed near-infrared light-emitting diode (lambda = 820 nm, frequency 10 Hz, dose 8-120 J/m(2)) and treatment with two donors of nitric oxide, sodium nitroprusside (SNP, 5 x 10(-4) M) and NaNO(2) (4 x 10(-4) M) . It was found that in an irradiated cell suspension, the cell-glass adhesion increases in a dose-dependent manner (a bell-shaped curve with a maximum at 60 J/m(2)) . The treatment of cells with SNP or NaNO(2) before the irradiation eliminates the radiation-induced attachment stimulation . Pretreatment of cells with SNP not only eliminates the radiation-induced attachment stimulation but inhibits the attachment of irradiated (but not non-irradiated) cells . It is suggested that a modulation of the activity of respiratory chain (probably the alteration of the activity of cytochrome c oxidase) is involved in radiation-induced increase of cell attachment.

Cytometry, 2001 May 1, 44(1), 45 - 56
Early functional apoptotic responses of thymocytes induced by Tri-n-butyltin; Grundler W et al.; BACKGROUND: Programmed cell death, also termed apoptosis, is the main focus of interest in a variety of scientific and clinical areas . For a better understanding of the mechanisms of apoptosis, from the onset of the cellular death program to the late stages of apoptosis or apoptotic necrosis, very early functional events have to be quantified because they might be involved in temporal and causal relationships between apoptosis-related key processes . METHODS: We have established a flow cytometric technique to quantify time-dependent signals simultaneously with high temporal resolution (Deltat = 1 s) in living cells . With this technique, the response of cells to apoptosis-stimulating agents can be analyzed over 15 min . For this purpose, a thermostatted sample tube holder for repeatable interruption-free injection of substances into the cell suspension was developed . Early detectable fluorescence and scatter parameters were related to intracellular free Ca2+ concentration, {Ca2+}i (Indo-1 fluorometry), membrane permeability (propidium iodide {PI} influx), and cell volume (forward scatter) . RESULTS: A T-cell line (Jurkat) served as a model system . Apoptosis was induced by the biozid Tri-n-butyltin (TBT) . Dependent on the TBT concentration (0.3-10 microM), the mean free {Ca2+}i increased by a factor of 1.2-6 during a short time interval of just 2 min . Especially after low TBT concentrations (< 0.5 microM), this {Ca2+}i increase was nearly transient during the observation time of 15 min . Higher TBT concentrations (0.5-10 microM), however, induced a transient increase of {Ca2+}i (Ca-TR) only in a fraction of the cells; in another subpopulation, a steady-state Ca2+ signal (Ca-SST) was observed . The analysis of the simultaneously registered PI signals of the Ca-SST cells showed a shift to increasing PI fluorescence (by a factor of about 4) with increasing Ca2+ concentrations . In Ca-TR cells, the PI fluorescence remained nearly unchanged . These apoptosis-related changes (increase in {Ca(2+)}i and membrane permeability) could be confirmed by the additional observation of a TBT concentration-dependent decrease in cell volume measured during the same early time period . CONCLUSIONS: The simultaneously analyzed parameters (i.e., {Ca2+}i, membrane permeability, and cell volume) suggested that, in our model system of Jurkat T-cells treated with TBT, an apoptotic cell fate was indicated very early (within 15 min) by the steady-state {Ca2+}i level .

Cytometry, 2001 May 1, 44(1), 30 - 7
Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry; Siiman O et al.; BACKGROUND: Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method . A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous . METHODS: Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors . We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution . Also presented is a new method of obtaining the amount of soluble antigen in a sample . We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand . RESULTS: Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay . These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen . CONCLUSIONS: The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available .

Int Endod J, 2000 Mar, 33(2), 103 - 12
The preparation of periapical lesions for flow cytometry; Fernando K et al.; AIM: To devise an optimal protocol and to analyse the leucocyte composition of periapical (PA) lesions by flow cytometry . METHODOLOGY: PA lesions were mechanically agitated, with and without proteolysis . This was with either 0.2% collagenase alone, or in combination with 0.02% DNA-ase in serial incubations until all tissue was digested . The efficacy of each method was assessed by counting total cell yield and cell viability . Phenotype stability was gauged by the percentage of peripheral blood leucocytes (PBL) which expressed CD45RB, CD3, CD20, CD4 and CD8 before and after mechanical and collagenase treatment . RESULTS: Disaggregation of PA lesions was superior if collagenase was present, but cell clumping was problematic unless the DNA-ase was also added, and serial digestion with this combination produced optimal cell yield and viability . Nevertheless, the total number of cells released rarely exceeded 105, though viability was in excess of 80% . Mechanical agitation and proteolysis adversely affected PBL phenotypes, but collagenase digestion limited to 10 min caused least damage . Flow cytometric analysis of disaggregated PA lesions failed to identify more than 7.9% (mean, range 6-10%) CD45RB + cells . CONCLUSIONS: Because of the necessity for single cell suspensions, flow cytometry is not easily applied to the analysis of leucocytes in PA lesions, and further refinements in tissue disaggregation and cell preparation are required.

Eur J Nucl Med, 2001 Feb, 28(2), 214 - 20
Bone marrow uptake of 99mTc-MIBI in patients with multiple myeloma; Fonti R et al.; In a previous study, we showed the ability of technetium-99m methoxyisobutylisonitrile (99mTc-MIBI) scan to identify active disease in patients with multiple myeloma (Eur J Nucl Med 1998; 25: 714-720) . In particular, a semiquantitative score of the extension and intensity of bone marrow uptake was derived and correlated with both the clinical status of the disease and plasma cell bone marrow infiltration . In order to estimate quantitatively 99mTc-MIBI bone marrow uptake and to verify the intracellular localization of the tracer, bone marrow samples obtained from 24 multiple myeloma patients, three patients with monoclonal gammopathy of undetermined significance (MGUS) and two healthy donors were studied for in vitro uptake . After centrifugation over Ficoll-Hypaque gradient, cell suspensions were incubated with 99mTc-MIBI and the uptake was expressed as the percentage of radioactivity specifically retained within the cells . The cellular localization of the tracer was assessed by micro-autoradiography . Twenty-two out of 27 patients underwent 99mTc-MIBI scan within a week of bone marrow sampling . Whole-body images were obtained 10 min after intravenous injection of 555 MBq of the tracer; the extension and intensity of 99mTc-MIBI uptake were graded using the semiquantitative score . A statistically significant correlation was found between in vitro uptake of 99mTc-MIBI and both plasma cell infiltration (Pearson's coefficient of correlation r=0.69, P<0.0001) and in vivo score (Spearman rank correlation coefficient r=0.60, P<0.01) . No specific tracer uptake was found in bone marrow samples obtained from the two healthy donors . Micro-autoradiography showed localization of 99mTc-MIBI inside the plasma cells infiltrating the bone marrow . Therefore, our findings show that the degree of tracer uptake both in vitro and in vivo is related to the percentage of infiltrating plasma cells which accumulate the tracer in their inner compartments.

Eur J Cell Biol, 2001 Feb, 80(2), 178 - 86
Uptake of endocytic markers by rice cells: variations related to the growth phase; Bahaji A et al.; Endocytosis is now considered a basic cellular process common to plant cells . Although both non-specific and receptor-mediated endocytosis appear to take place in plant cells, the physiological role of the latter remains unclear . We have investigated the endocytic process in rice cell suspensions using two biotinylated proteins, peroxidase and bovine serum albumin (bHRP and bBSA), as markers . First, we show that markers are internalized by rice cells and appear in intracellular membranes . The uptake of the two markers is temperature dependent, saturable with time and markers dose and it is competed by free biotin . Thus, it shows the properties of a receptor-mediated process . We also show that uptake of markers is strongly influenced by growth phase as optimal uptake occurs during the lag phase, but the initiation of the exponential growth phase decreases uptake drastically . Arrest of the cell cycle by starvation of either a nutrient (phosphate) or a growth regulator (2,4-dichlorophenoxyacetic acid), both components of the culture medium, does not modify the rate of bBSA uptake . Subsequent readdition of these components results in growth recovery and a dramatic decrease in bBSA uptake . On the other hand, nocodazole treatment, a method to arrest the cell cycle by microtubule depolymerization, inhibited bBSA uptake . The possible causes for this arrest of endocytosis are discussed.

Planta Med, 2001 Mar, 67(2), 150 - 2
Growth and production of camptothecin by cell suspension cultures of Nothapodytes foetida; Fulzele DP et al.; Callus cultures were initiated from stem parts of Nothapodytes foetida on Murashige and Skoog's medium supplemented with different growth regulators . Suspension cultures were established and the cell biomass was higher in the presence of NAA in comparison with 2,4-D . Culture medium supplemented with NAA (10.74 microM) and BA (2.22 microM) attained 31.3 g/l DW during 20 days of cultivation in shake flasks . In the presence of NAA, maximum concentrations of camptothecin (0.035 mg/ml) and 9-methoxycamptothecin (0.026 mg/ml) were found in the medium . Alkaloid production was reduced in presence of 2,4-D in the culture medium . Cells contained trace amount of alkaloids . Alkaloids were detected and identified by means of TLC and HPLC.

Plant Physiol, 2001 Apr, 125(4), 1831 - 41
A novel dark-inducible protein, LeDI-2, and its involvement in root-specific secondary metabolism in Lithospermum erythrorhizon; Yazaki K et al.; Lithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives . They are accumulated exclusively in the roots of this plant . The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized . Thus, L . erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis . LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems . Its mRNA accumulation showed a similar pattern with that of shikonin . In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin . LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice) . Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L . erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g . p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone . This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities.

Physiol Plant, 2001 Apr, 111(4), 512 - 518
Correlation of resistance and H2O2 production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo-ulmi; De Rafael MA et al.; The Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi Buissm . elicited the production of H2O2 in cell suspension cultures of the resistant species Ulmus pumila L . This response was not observed in suspensions of the susceptible elm U . campestris Mill . H2O2 production started after a lag time of 30-40 min following inoculation, peaked between 4 and 6 h and lasted up to 24 h . Treatment of the suspensions with exogenously added H2O2 did not cause accumulation of the sesquiterpene phytoalexins mansonones nor of the coumarin scopoletin . Spore germination and growth of O . novo-ulmi were significantly delayed with different amounts of H2O2 (0.1-1 mM) . These results suggest that H2O2 production is an inducible defence response which may contribute to DED resistance by delaying the growth of the pathogen at the earliest stages of infection . Whether H2O2 is involved in other elm defence responses to the pathogen is presently unknown, but its production seems to be an independent event from phytoalexin formation.

J Gen Virol, 2001 May, 82(Pt 5), 1169 - 74
Demonstration by single-cell PCR that Reed--Sternberg cells and bystander B lymphocytes are infected by different Epstein--Barr virus strains in Hodgkin's disease; Faumont N et al.; Epstein--Barr virus (EBV) is associated with Hodgkin's disease (HD) . However, EBV-positive Reed--Sternberg (RS) cells and EBV-positive B lymphocytes co-exist in the same EBV-positive lymph node affected by HD . In a previous report, using total lymph node DNA, the presence of two distinct EBV strains was demonstrated, but their cellular localization (i.e . RS cells vs B lymphocytes) could not be determined . To address this question, three patients with EBV-associated HD were selected in the present study and single-cell PCR of the latent membrane protein-1 (LMP-1) gene from isolated RS cells was performed . In one case, it was clear that RS cells and B lymphocytes were infected by different EBV strains . In the two remaining cases, only one band was detected from total lymph node DNA . However, single-cell PCR showed that RS cells in each sample were infected by single EBV strains, which were different from those detected in lymphoblastoid cell lines derived from EBV-positive B lymphocytes of lymph node cell suspensions from these two patients.

Lasers Surg Med, 2001, 28(3), 227 - 36
Cell attachment modulation by radiation from a pulsed light diode (lambda = 820 nm) and various chemicals; Karu TI et al.; BACKGROUND AND OBJECTIVE: Adhesive interactions between cells and extracellular matrices play a regulative role in wound repair processes . The objective of this investigation is to study the mechanisms of light action on cellular adhesion in vitro . The adhesion of HeLa cells to a glass matrix is evaluated after irradiation with a pulsed near-infrared (IR) diode and treatment with various chemicals . STUDY DESIGN/MATERIALS AND METHODS: A semiconductor diode (820 +/- 10 nm, 10Hz, 16--120 J/m(2)) is used for irradiation of the cell suspension . In parallel experiments, various chemicals (mannitol, melatonin, ethanol, ascorbic acid, superoxide dismutase, catalase, rotenone, azide, dinitrophenol (DNP), methylene blue, and hydrogen peroxide) are added to the cell suspension before or after the irradiation procedure . The cell-glass adhesion is studied by using the adhesion assay technique (Lasers Surg . Med . 1996;18:171) . RESULTS: It has been found that cell-glass adhesion increases in a dose-dependent manner after irradiation . The treatment of the cells with antioxidants (free radical scavengers), e.g., mannitol, melatonin, ethanol, and ascorbic acid, as well as with the ionophore DNP, eliminated the light effect . The respiratory chain inhibitors rotenone and azide strongly modified the light effect, depending on the dose . The oxidative agents hydrogen peroxide (in a low concentration) and methylene blue increased the cell adhesion . Superoxide dismutase did not modify the light effect . The effect of the catalase (stimulative or suppressive) was dependent on its concentration and treatment sequence . Preirradiation was found to decrease (or normalize to the control level) the suppressive effects of some chemicals . CONCLUSION: The results obtained are evidence that first, pulsed IR radiation with certain parameters modulates the cell-matrix attachment . second, free radical and redox processes are involved in the cell-matrix interaction, probably at some stage(s) of the photosignal transduction . Third, both types of the primary reactions in the respiratory chain, namely, the increase of the electron flow and production of the reactive oxygen species, cause a transient oxidative stress in the cytoplasm .

Cell Transplant, 2001 Jan-Feb, 10(1), 25 - 30
Neural xenotransplantation: pretreatment of porcine embryonic nigral tissue with anti-Gal antibodies and complement is not toxic for the dopaminergic neurons; Brevig T et al.; The immunogenicity of porcine tissue is a major obstacle to its use as donor material in xenotransplantation for neurodegenerative diseases . We are currently evaluating a novel strategy for reducing the immunogenicity, in which the alpha-galactosyl epitope (Galalpha1,3Galbeta1,4GlcNAc-R) is used as a target for antibody- and complement-mediated removal of microglia . In the present study, our aim was to determine whether a pretreatment with antibodies against the alpha-galactosyl epitope (anti-Gal) and complement would lyse or otherwise damage dopaminergic neurons in porcine embryonic ventral mesencephalon (VM), the donor tissue for treatment of Parkinson's disease by xenotransplantation . Cell suspensions prepared from VM tissue from 27-day-old pig embryos were incubated with anti-Gal, purified from normal human serum by affinity chromatography, or medium only (control), and subsequently with rabbit complement . After these pretreatments, the cell suspensions were transplanted into the right striatum of 14 adult rats (two groups of 7 animals) . The animals were sacrificed 20 days after transplantation, the brains were processed for histology, and the sections were stained for Nissl substance, porcine neurofilament, tyrosine hydroxylase, and rat CD45 to determine graft volume, presence of porcine neurons, content of dopaminergic cells, and leukocyte infiltration, respectively . The VM tissue pretreated with anti-Gal and complement gave rise to dopaminergic grafts that were indistinguishable from those derived from VM tissue given the control pretreatment . In 5 of the 14 animals, the grafts were infiltrated by host leukocytes, but in two of these recipients, the infiltration was only minimal . We conclude that anti-Gal and complement can be applied to porcine embryonic VM tissue without damaging the dopaminergic neurons and their precursors.

Nucleic Acids Res, 2001 Apr 15, 29(8), 1791 - 800
Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro; Jansen J et al.; The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information . In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage . Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR) . In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells . To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene . Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR . In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells . However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity . We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

Int J Cancer, 2001 Apr 15, 92(2), 176 - 80
The pattern of metastasis of human melanoma to the central nervous system is not influenced by integrin alpha(v)beta(3) expression; Kusters B et al.; We investigated the effect of integrin alpha(v)beta(3) expression on the metastatic pattern of human melanoma cells in the central nervous system (CNS) . For this purpose, we developed a hematogenous CNS melanoma metastasis model in nude mice using a modified internal carotid artery infusion technique . This protocol revealed 2 different patterns of CNS metastasis . The integrin alpha(v)beta(3)-expressing melanoma lines Mel57 and Zkr nearly exclusively produced metastases in the brain parenchyma, whereas cells of the BLM and MV3 lines, devoid of integrin alpha(v)beta(3) expression, preferentially metastasized to dura mater and leptomeninges . Treatment with hyaluronidase to obtain single BLM cell suspensions did not influence the metastatic pattern, indicating that this was not simply the result of entrapment of tumor cell aggregates in large-sized leptomeningeal vessels . The role of integrin alpha(v)beta(3) expression in the process of metastasis was tested by transfection of BLM, but did not lead to an altered pattern of metastasis . We did observe, however, slower growth of the transfected tumors, although the in vitro growth rate was unaltered, indicating a reduction in tumorigenicity . We conclude from our findings that CNS metastasis of melanoma cells in the mouse xenograft model occurs in at least 2 different but very reproducible patterns . Although it is predicted that adhesion of tumor cells to endothelial cells plays a role in this phenomenon, tumor cell integrin alpha(v)beta(3) expression per se does not explain the difference in metastatic behavior in the CNS . We assume that other, as yet unknown factors, must be involved .

Int J Radiat Oncol Biol Phys, 2001 Apr 1, 49(5), 1361 - 8
Radiobiologic significance of apoptosis and micronucleation in quiescent cells within solid tumors following gamma-ray irradiation; Masunaga S et al.; PURPOSE: To determine the frequency of apoptosis in quiescent (Q) cells within solid tumors following gamma-ray irradiation, using four different tumor cell lines . In addition, to assess the significance of detecting apoptosis in these cell lines . METHODS AND MATERIALS: C3H/He mice bearing SCC VII or FM3A tumors, Balb/c mice bearing EMT6/KU tumors, and C57BL mice bearing EL4 tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells . The mice then received gamma-ray irradiation at a dose of 4--25 Gy while alive or after tumor clamping . Immediately after irradiation, the tumors were excised, minced, and trypsinized . The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU . Meanwhile, 6 hours after irradiation, tumor cell suspensions obtained in the same manner were fixed . The apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU . The MN and apoptosis frequency in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU . RESULTS: In total cells, SCC VII, FM3A, and EMT6/KU cells showed reasonable relationships between MN frequency and surviving fraction (SF) . However, fewer micronuclei were induced in EL4 cells than the other cell lines . In contrast, a comparatively close relationship between apoptosis frequency and SF was found in total cells of EL4 cell line . Less apoptosis was observed in the other cell lines . Quiescent tumor cells exhibited significantly lower values of MN and apoptosis frequency probably due to their large hypoxic fraction, similar to total tumor cells on clamped irradiation . CONCLUSION: gamma-ray irradiation induced MN formation in SCC VII, FM3A, and EMT6/KU tumor cells, and the apoptosis was marked in EL4 cells compared with the other cell lines . Our method for detecting the Q cell response to gamma-ray irradiation using P cell labeling with BrdU and the MN frequency assay was also applicable to apoptosis detection assay.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2001 Mar, 15(2), 118 - 21
{Transplantation of cultured human keratinocyte on collagen sponge}; Xu LH et al.; OBJECTIVE: To investigate the skin regeneration using cultured human keratinocytes with collagen sponge transplanted into thickness wound of nude mice . METHODS: Human foreskin from foreskin ectomy procedures was detached with 0.5% Dispase II . Epidermis sheets were separated from dermis and digested with 0.05% Trypsin into single cell suspension . Keratinocytes were cultured and seeded into collagen sponge during logarithmic growth phase . After 3 days, the keratinocytes-collagen sponge were grafted on full thickness wound of nude mice, compared with simple collagen sponge without keratinocytes . The histological, immunohistochemical examination and electron microscopy were detected . RESULTS: After the epidermal substitute was grafted onto wound, the human keratinocytes were able to further proliferate and differentiate and develop into new epithelia . Compared with the control group, the wound healed earlier and contracted less, epithelia matured earlier, and the collagen fiber was less beneath epithelia . CONCLUSION: Keratinocytes can grow on collagen sponge and migrate onto wound to develop into stratified epithelia and inhibit wound contract . The keratinocyte graft can be used to repair skin defect.

Biochem J, 2001 Apr 15, 355(Pt 2), 389 - 95
Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects; Wieser W et al.; The original aim of the present study was to deal with two problems that had emerged from a study on hierarchies of ATP-consuming processes in cells {Buttgereit and Brand (1995) Biochem . J . 312, 163-167} . Firstly, we wanted to find out whether the results of that study had been influenced by the method used for the determination of process activity and, secondly, we wondered whether and to what extent the structure of the hierarchy established for cell suspensions under energy-limiting conditions might depend on the type of cell or on the lifestyle, ecology and phylogenetic status of the species from which the cells were derived . We confined our study to the two most prominent ATP consumers of cells: protein synthesis and the Na(+)/K(+)-ATPase, measuring their activity directly by {3H}leucine incorporation and Rb(+)-flux respectively . We found large differences in the sensitivity of protein synthesis to energy limitation between hepatocytes from an anoxia-tolerant fish species and an anoxia-sensitive fish species (goldfish and rainbow trout respectively) . On the other hand, Na(+)/K(+)-ATPase activity was hardly affected by energy limitation in the hepatocytes from both fish species . We also studied the response of a human hepatoma cell line, HepG2, to energy limitation and found both protein synthesis and Na(+)/K(+)-ATPase activity to be equally sensitive to energy limitation, but more sensitive than the Na(+)/K(+)-ATPase of the two fish species . A comparison of the indirect and direct methods for measuring protein synthesis revealed the rate of oxygen consumption to be functionally related to the concentration of cycloheximide, the inhibitor used . It was found that at 15 mM cycloheximide {three orders of magnitude higher than the concentration at which the incorporation of free amino acids (FAA) into protein is inhibited} total oxygen consumption was suppressed by 71-75%, whereas the measured rate of {3H}leucine incorporation into protein suggested that the cycloheximide-sensitive fraction should have amounted to not more than approx . 10% of the total oxygen consumption . On the other hand, the amount of oxygen consumption suppressed with the high concentration of cycloheximide corresponded almost exactly to the increase in oxygen consumption of cells incubated in an FAA-enriched medium compared with cells incubated in a standard, FAA-free medium . Our major conclusions are, firstly, that high concentrations of cycloheximide disrupt cellular metabolism, bringing to a standstill all those processes that can be stimulated by incubating starved cells in an FAA-enriched medium, secondly, that the attempt to estimate the metabolic cost of protein synthesis by inhibiting oxygen consumption with cycloheximide leads to spurious results, and, thirdly, that the structure of a 'hierarchy' of ATP-consumers may reflect the lifestyle and physiology of the species studied.

Stroke, 2001 Apr, 32(4), 1012 - 9
Resolution of stroke deficits following contralateral grafts of conditionally immortal neuroepithelial stem cells; Veizovic T et al.; BACKGROUND AND PURPOSE: Grafts of MHP36 cells have previously been shown to reduce dysfunction after global ischemia in rats . To test their efficacy after focal ischemia, MHP36 cells were grafted 2 to 3 weeks after transient intraluminal middle cerebral artery occlusion (tMCAO) in rats . METHODS: MHP36 cells were implanted into the hemisphere contralateral to the lesion, with 8 deposits of 3 microL of cell suspension (25 000 cells per microliter) . Sham grafted rats received equivalent volumes of vehicle . Three groups, sham-operated controls (n=11), MCAO+sham grafts (n=10), and MCAO+MHP36 grafts (n=11), were compared in 3 behavioral tests . RESULTS: In the bilateral asymmetry test, MCAO+MHP36 grafted rats exhibited neglect before grafting but subsequently showed no significant dysfunction, whereas MCAO+sham grafted rats showed stable sensorimotor deficits over 18 weeks relative to controls . MCAO+sham grafted rats demonstrated spontaneous motor asymmetry and increased rotational bias after injection of dopamine agonists . MCAO+MHP36 and control groups exhibited no bias in either spontaneous or drug-induced rotation . In contrast to motor recovery, MCAO+MHP36 grafted rats showed no improvement relative to MCAO+sham grafted rats in spatial learning and memory in the water maze . MCAO produced large striatal and cortical cavitations in both occluded groups . Lesion volume was significantly reduced (P<0.05) in the MCAO+MHP36 grafted group . The majority of MHP36 cells were identified within the intact grafted hemisphere . However, MHP36 cells were also seen in the cortex, striatum, and corpus callosum of the lesioned hemisphere . CONCLUSIONS: MHP36 cells may improve functional outcome after MCAO by assisting spontaneous reorganization in both the damaged and intact hemispheres.

Phytochemistry, 2001 Mar, 56(6), 523 - 7
L-Tyrosine beta-naphthylamide is a potent competitive inhibitor of tyramine N-(hydroxycinnamoyl)transferase in vitro; Negrel J et al.; L-Tyrosine beta-naphthylamide, a synthetic substrate designed to measure tyrosine aminopeptidase activity, is a potent inhibitor of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) purified from elicited tobacco cell-suspension cultures . The inhibition is competitive, with the inhibitor binding reversibly to the tyramine binding site of the enzyme . Similar results were obtained with THT extracted from elicited potato cell-suspension cultures . Ki values were found to be 0.66 microM for the enzyme from tobacco and 0.3 microM for the enzyme from potato . L-Tyrosine 7-amido-4-methylcoumarin, a fluorogenic substrate for tyrosine aminopeptidases, the structure of which is close to that of L-tyrosine beta-naphthylamide . was also a powerful inhibitor, but slightly less effective with Ki values of 0.72 and 0.42 microM for tobacco and potato THT, respectively . L-Tyrosine beta-naphthylamide was rapidly hydrolysed when fed in vivo to tobacco or potato cell cultures or when incubated in crude enzymic extracts prepared from these cultures . This hydrolysis, which is presumably catalysed by aminopeptidases, precludes the use of L-tyrosine amides as inhibitors of THT in vivo.

J Biol Chem, 2001 Jun 15, 276(24), 21083 - 8 Epub 2001 Mar 28.
Virally induced lytic cell death elicits the release of immunogenic GRP94/gp96; Berwin B et al.; Necrotic cell death yields the release of cellular components that can function in the initiation of cellular immune responses . Given the established capacity of the endoplasmic reticulum chaperone GRP94 (gp96) to elicit CD8(+) T cell activation, we have investigated the cellular fate and antigenicity of GRP94 in differing scenarios of cell death . Virally induced cell death or mechanical cell death, elicited by freeze/thaw treatment of cell suspensions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 release . To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVA(Met258-265), a recombinant, ovalbumin epitope-expressing vaccinia virus) or mechanical cell death (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant fractions were pulsed onto antigen-presenting cells, and antigen re-presentation was assayed as activation of an ovalbumin-specific T cell hybridoma . For both cell death scenarios, released GRP94 elicited a dose-dependent, ovalbumin-specific, hybridoma activation . In contrast, calreticulin derived from rVV ES-OVA(Met258-265)-infected cell extracts did not stimulate B3Z activity . These data identify GRP94 as an antigenic component released upon pathological, but not apoptotic, cell death and provide an assay system for the identification of cellular components of related activity.

J Biol Chem, 2001 May 25, 276(21), 18139 - 45 Epub 2001 Mar 05.
A novel tobacco mitogen-activated protein (MAP) kinase kinase, NtMEK1, activates the cell cycle-regulated p43Ntf6 MAP kinase; Calderini O et al.; Two-hybrid screening of a tobacco BY-2 cell suspension cDNA library using the p43(Ntf6) mitogen-activated protein (MAP) kinase as bait resulted in the isolation of a cDNA encoding a protein with features characteristic of a MAP kinase kinase (MEK), which has been called NtMEK1 . Two-hybrid interaction analysis and pull-down experiments showed a physical interaction between NtMEK1 and the tobacco MAP kinases p43(Ntf6) and p45(Ntf4), but not p43(Ntf3) . In kinase assays NtMEK1 preferentially phosphorylated p43(Ntf6) . Functional studies in yeast showed that p43(Ntf6) could complement the yeast MAP kinase mutant mpk1 when co-expressed with NtMEK1, and that this complementation depended on the kinase activity of p43(Ntf6) . Expression analysis showed that the NtMEK1 and ntf6 genes are co-expressed both in plant tissues and following the induction of cell division in leaf pieces . These data suggest that NtMEK1 is an MEK for the p43(Ntf6) MAP kinase.

J Biochem (Tokyo), 2001 Apr, 129(4), 543 - 9
Suppression of the accumulation of triosephosphates and increased formation of methylglyoxal in human red blood cells during hyperglycaemia by thiamine in vitro; Thornalley PJ et al.; The accumulation of triosephosphates and the increased formation of the potent glycating agent methylglyoxal in intracellular hyperglycaemia are implicated in the development of diabetic complications . A strategy to counter this is to stimulate the anaerobic pentosephosphate pathway of glycolysis by maximizing transketolase activity by thiamine supplementation, with the consequent consumption of glyceraldehyde-3-phosphate and increased formation of ribose-5-phosphate . To assess the effect of thiamine supplementation on the accumulation of triosephosphates and methylglyoxal formation in cellular hyperglycaemia, we incubated human red blood cell suspensions (50% v/v) in short-term culture with 5 mM glucose and 50 mM glucose in Krebs-Ringer phosphate buffer at 37 degrees C as models of cellular metabolism under normoglycaemic and hyperglycaemic conditions . In hyperglycaemia, there is a characteristic increase in the concentration of the triosephosphate pool of glycolytic intermediates and a consequent increase in the concentration and metabolic flux of the formation of methylglyoxal . The addition of thiamine (50-500 microM) increased the activity of transketolase, decreased the concentration of the triosephosphate pool, decreased the concentration and metabolic flux of the formation of methylglyoxal, and increased the concentration of total sedoheptulose-7-phosphate and ribose-5-phosphate . Biochemical changes implicated in the development of diabetic complications were thereby prevented . This provides a biochemical basis for high dose thiamine therapy for the prevention of diabetic complications.

Zhongguo Yao Li Xue Bao, 1999 Oct, 20(10), 893 - 6
Inhibitory effects of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on intracellular Ca2+ elevated by neurotransmitters in brain cells; Zhang XQ et al.; AIM: To study the effects of TMB-8 on {Ca2+}i elevation induced by neurotransmitters in dissociated brain cells . METHODS: The brain cell suspension was made using a gentle trituration for 1 min with a polished pipette . The changes of {Ca2+}i were detected by the fluorescent indicator, Fura 2-AM . RESULTS: In the presence of extracellular Ca2+ 1.3 mmol.L-1, sodium glutamate (Glu), histamine (His), and serotonin (5-HT) markedly increased the {Ca2+}i which were reduced by TMB-8 30 mumol.L-1 . TMB-8 3 mumol.L-1 produced inhibitory effects on the increase of {Ca2+}i by His and 5-HT in a Ca(2+)-free Hanks' solution . The increase of {Ca2+}i by His and 5-HT was reduced to control level by TMB-8 10 mumol.L-1 . CONCLUSION: TMB-8 inhibited the {Ca2+}i elevation induced by Glu, 5-HT, and His in brain cells.

J Chromatogr A, 2001 Mar 9, 911(1), 55 - 61
Studies on factors influencing stability and recovery of paclitaxel from suspension media and cultures of Taxus cuspidata cv Densiformis by high-performance liquid chromatography; Nguyen T et al.; An HPLC method was developed for quick scanning of taxanes from large numbers of plant cell suspension samples . The method was optimized for analysis of a range of taxanes of differing polarity . Identification of a standard mixture of paclitaxel and 12 related taxanes was achieved in less than 15 min using a gradient mode and a Microsorb-MV C8 column . The method was used to investigate the influence of several factors on stability and recovery of paclitaxel from suspension media and cultures of Taxus cuspidata cv Densiformis . Incubation time had the most significant influence on stability of paclitaxel, contributing 88% to the total variation . Shaking contributed 6% to the total variation . Light contributed only 0.25% to the total variation . Analysis of test samples of suspension cultures of T . cuspidata cv Densiformis over a 4 week period show paclitaxel, 10-deacetylbaccatin III, and baccatin III levels ranging from 0 to 149 microg/L, from 0 to 1.9 mg/L, and from 0 to 583 microg/L, respectively.

J Neurosci . 2001 Apr 1;21(7):RC138.
The in vitro fate of rabbit fetal brain cells after acute in vivo hypoxia; Derrick M et al.; In the investigation of ischemia-induced brain damage, traditional methods using histopathology estimate brain cell death at a time remote from ischemic insult . These observations fail to take into account endogenous repair processes or ongoing injury cascades like apoptosis . The cells that are injured but not killed initially are the population most amenable to rescue . The hypothesis was that in vivo uterine ischemia-reperfusion would result in more cell death and apoptosis in fetal brain cells cultured in vitro . Near-term, 29 d gestation, pregnant New Zealand White rabbits were subjected to repetitive uterine ischemia for a cumulative time of 40 min ischemia and 20 min reperfusion . Immediately after uterine ischemia, the fetal brains were removed and dissociated into a cell suspension . The ischemic group had more cell death than non-ischemic controls as assessed by Trypan Blue exclusion and propidium iodide (PI) uptake on a flow cytometer . Aliquots of cells were plated and cultured for 24 and 48 hr . The ischemic group had significantly more cell death (propidium iodide) than non-ischemic controls at 24 hr and significantly more apoptosis, as assessed by annexin-V binding in cells at 24 hr and caspase-3 activity at 48 hr . Fewer cells attached to the culture plates at 48 hr in the ischemia group . After uterine ischemia, certain fetal brain cells die immediately, and other cells undergo ongoing damage resulting in necrosis and apoptosis that is manifest later . This method offers insight into the fate of those cells and provides a tool for assessing interventions to decrease cell injury.

J Bone Joint Surg Am, 2001, 83-A Suppl 1(Pt 1), S23 - 30
Growth/differentiation factor-5 (GDF-5) and skeletal development; Buxton P et al.; BACKGROUND: Growth/differentiation factor-5 (GDF-5) has been shown to be essential for normal appendicular skeletal and joint development in humans and mice . In brachypod, a Gdf-5 gene mouse mutant, the defect is first apparent during early chondrogenesis, with the cartilage blastema already reduced in size by E12.5 . This defect is associated with changes in the expression of cell surface molecules . METHODS: To understand further how GDF-5 controls cartilage formation, we first mapped the expression of the Gdf-5 gene during skeletal development (please note that the abbreviation for the gene is given in italics and the abbreviation for the protein expressed by the gene is given in capital letters) . Subsequently, we over-expressed GDF-5 in the developing chick embryo using a replication competent retrovirus, RCAS(BP) . We determined its effects on skeletal development by histological examination and its effects on early growth by autoradiography of proliferating cells . In addition, we examined the effect of GDF-5 on chondrogenic differentiation using micromass and single cell suspension cultures of limb mesenchymal cells, RESULTS: These studies show that the Gdf-5 gene is expressed in the early cartilage condensation, the perichondrium, and the joint interzone . Over-expresSion of GDF-5 in chick limb buds, during the condensation stage or later when the skeletal elements have formed, increased the size of the affected elements . In both cases, the increase in size was associated with an increase in cell number and, at later stages, this was correlated with an increase in S-phase cells . In vitro studies showed that GDF-5 could increase cell adhesiveness, and this may be a mechanism through which GDF-5 initiates condensation formation . CONCLUSION: These studies show that GDF-5 acts at two stages of skeletal development and by two distinct mechanisms . First, GDF-5 promotes the initial stages of chondrogenesis by promoting cell adhesion, which is consistent with the expression of Gdf-5 in the cartilage condensation . Second, GDF-5 can increase the size of the skeletal elements by increasing proliferation within the epiphyseal cartilage adjacent to its expression within the joint interzone.

Zhonghua Xue Ye Xue Za Zhi, 1998 Dec, 19(12), 638 - 41
{Establishment and biological characterization of a transplantable BALB/c mouse erythroblastic leukemia model}; Ding S et al.; OBJECTIVE: Biological characterization of a BALB/c mouse transplantable erythroblastic leukemia model EL9611 . METHODS AND RESULTS: The original erythroblastic leukemia (EL) mouse was obtained by 7,12-DMBA induction . The spleen cell suspension from the original EL mouse was injected into the same inbred BALB/c strain mice . After successive transplantation for 20 generations, the incidence of EL was 100% without spontaneous remission . There was a linear relationship between the survival time of the EL mice and the number of leukemic cells inoculated . When 10(6) leukemic cells were inoculated intravenously, the EL mice survived 10.2 +/- 1.3 days . Invasion of leukemic cells involved mainly in the bone marrow, the spleen and the liver . There was no obvious infiltration of leukemic cells in lymph-nodes and thymus . In peripheral blood, there were a large number of leukemic cells and most of them were basophilic or polychromatophilic erythroblasts . At the advanced stage, the mice developed anemia and thrombocytopenia . The reaction of the leukemic cells for hemoglobin staining was positive or weakly positive . While the peroxydase reaction was negative, Thy-1 antigen and Fc-recepter were not presented . No virus-like particles was found in the cells under electron microscope . EL9611 leukemia model was sensitive to some antitumor drugs used in clinical therapy . CONCLUSION: The establishment of EL9611 leukemia model offered a useful tool for studying proliferation and differentiation of erythroid cells, as well as pathogenesis and experimental treatment of non-virus erythroid malignancy.

J Infect Dis, 2001 Apr 15, 183(8), 1221 - 8 Epub 2001 Mar 16.
Borrelia burgdorferi stimulates in vitro a selective expansion of CD3-CD4-CD8- (triple negative) autoreactive cells in naive rhesus monkey lymphocytes; Ganapamo F et al.; Autoreactive cell lines were generated from cell suspensions of freshly isolated naive monkey lymph node (LN) cells and peripheral blood mononuclear cells by cocultivation with freeze-thawed Borrelia burgdorferi spirochetes (Bb/FT) . These cells produced interleukin (IL)-6 when cocultured with autologous antigen-presenting cells (APCs) alone without Bb/FT . IL-6 production was not observed when control cell lines were stimulated in the same fashion . CD4+-enriched T cell populations obtained from the LN autoreactive cell line also produced IL-6 when cultivated with APCs alone . When these cells were cultivated further in the presence of APCs, a population of cells whose phenotype was CD56+/-CD4-CD8-CD3- was predominantly selected . These cells both proliferated and produced IL-6 when cocultured with APCs alone . The possible relevance of these cells to Lyme disease pathogenesis remains to be determined.

J Asian Nat Prod Res, 1998, 1(2), 111 - 7
Glucosylation of salicyl alcohol by cell suspension cultures of Solanum laciniatum; Syahrani A et al.; Cell suspension cultures of Solanum laciniatum were able to transform exogenously inoculated salicyl alcohol into salicyl alcohol 7-O-beta-D-glucopyranoside (isosalicin) . The highest level of isosalicin (54.6 mg/g dry weight) in the cells was formed within 2 days after inoculation with salicyl alcohol (37.5 mg/flask containing 50 ml of medium) . The biotransformation capacity of the cell suspension cultures was about 31.1%.

Haematologica, 2001 Mar, 86(3), 227 - 36
Mouse plasmacytoma: an experimental model of human multiple myeloma; Gado K et al.; BACKGROUND AND OBJECTIVES: There is no ideal animal model for human multiple myeloma (MM) . All the models resemble the human disease in some respect, but none of them fulfils all the criteria of a perfect animal model . EVIDENCE AND INFORMATION SOURCES: The pristane oil (2,6,10,12-tetramethylpentadecane)-induced mouse plasmacytoma (MPC) model is the most widely used and accepted model and has provided the most data on plasmacytomagenesis so far . This model gives the opportunity to study the role of c-myc dysregulations, the mechanisms leading to cytogenetic changes involving Ig genes, the role of chronic inflammatory factors, the role of interleukin-6 (IL-6), insulin-like growth factor-I, prostaglandins, as well as signal transduction pathways in the neoplastic process . Therapeutic agents have been successfully tested . Although MPC growth is usually restricted to the peritoneal environment, intraperitoneal injection of MPC cell suspensions can reproduce the disseminated characteristics of the human disease in recipients . The IL-6 transgene and knockout models are important tools for clarifying the role of IL-6 in the pathogenesis of MM . Transgenic mice and retroviral gene transfer facilitate the study of oncogenes in neoplastic transformation . Spontaneous development of plasmacytomas in C57BL/ KaLwRij aging mice has several advantages, mainly because the disseminated growth, the typical bone lesions and renal involvement resemble, in part, the human disease . Furthermore, this model has already proved useful in studies on the effect of bisphosphonate in the treatment of bone disease in MM . The severe combined immunodeficiency (SCID) mouse model is also very attractive . A disseminated-like disease can be reproduced in this model . Multiple osteolytic bone lesions and bone marrow involvement are generated, and conventional drugs applied in the treatment of human multiple myeloma have proven to be effective . Nevertheless, the immune system of SCID mice basically differs from that of a MM patient . PERSPECTIVES: Taken together, all these models have contributed to our understanding of MM, but demonstrate the opportuness of developing a more appropriate model of the human disease.

Int J Cancer, 2001 Feb 15, 91(4), 529 - 37
Role of Fas ligand expression in promoting escape from immune rejection in a spontaneous tumor model; Cefai D et al.; Tumors escape immune-mediated rejection by a variety of mechanisms during tumor progression . The elucidation of these mechanisms in vivo suffers from a lack of suitable models of spontaneous tumor formation escaping active specific immunotherapy (ASI) . In a rat neu transgenic (rNeu-TG) mouse model of spontaneous breast tumor formation, we showed that rNeu-TG mice developed late escape tumors despite the presence of a persistent rNeu-specific immune response after ASI . Cell suspensions derived from these escape tumors grew in vaccinated tumor-free mice, whereas injected spontaneous tumor cells were rejected . Escape tumors retained rNeu or MHC class I expression but significantly upregulated Fas (CD95, Apo-1) ligand . We further demonstrated that Fas-L on escape tumor cells correlated with apoptosis of infiltrating T lymphocytes . Thus, our results provide evidence that spontaneous breast tumors upregulate Fas-L expression after vaccination that may promote tumor escape in vivo after ASI .

J Asian Nat Prod Res, 2000, 2(4), 305 - 9
N-acetylation and N-formylation of m-aminobenzoic acid by cell suspension cultures of Solanum laciniatum; Syahrani A et al.; Two new biotransformation products, N-acetyl-m-aminobenzoic acid and N-formyl-m-aminobenzoic acid were isolated from cell suspension cultures of Solanum laciniatum following administration of m-aminobenzoic acid, and their structures were elucidated using one- and two-dimensional 1H- and 13C-NMR data.

J Neurocytol, 1998, 27(7), 541 - 53
The demonstration by transplantation of the very restricted remyelinating potential of post-mitotic oligodendrocytes; Crang AJ et al.; To examine the remyelinating ability of post-mitotic oligodendrocytes, we subjected cell preparations derived from neonatal and adult rats to 40 Grays of X-irradiation to remove mitotically active cells and injected them into areas of demyelination in which the inherent ability to generate remyelinating cells had been inhibited . The extensive remyelination seen following implantation of non-irradiated neonatal and adult cells was almost completely abolished when the transplanted cell suspension was exposed to 40 Grays of X-irradiation, demonstrating that effective remyelination requires the generation of cells by mitosis . Radiation-resistant and therefore non-dividing oligodendrocytes were detected in areas of demyelination following transplantation of neonatal cultures and oligodendrocyte preparations derived from the adult nervous system . However, the pattern of myelin formation associated with the radiation-resistant oligodendrocytes from the two sources was different . Following implantation of X-irradiated neonatal cultures, a small number of oligodendrocytes could be found within the area of demyelination, and although these cells formed sheets of myelin membrane, they did not form myelin sheaths . After implantation of X-irradiated adult cells, in addition to the aberrant myelin formation seen with the neonatal cells, some myelin sheaths were observed . Our findings confirm that effective remyelination requires cell division and suggest that there may be diverse populations of radiation-resistant oligodendrocytes in the adult nervous system, some of which can form myelin sheaths and others of which can only make myelin sheets . Important for the interpretation of our previous studies is the demonstration here that 40 Grays of X-irradiation per se does not inhibit oligodendrocytes from remyelinating axons.

Cancer Res, 2001 Feb 15, 61(4), 1707 - 16
Anoikis and metastatic potential of cloudman S91 melanoma cells; Zhu Z et al.; Anoikis is a form of apoptosis induced in normal cells as a result of loss of their adhesion to substrate . In the present study, we have tested whether tumor cells are also sensitive to anoikis and whether selection of tumor cells for resistance to anoikis could increase their metastatic ability . In vitro cultured Cloudman S91 melanoma cells are strongly adherent to the plastic . Prevention of their adherence by rocking or by covering culture plates with polyhydroxyethylmethacrylate resulted in induction of anoikis and death of almost all cells . Their death was prevented in the presence of caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone . To select anoikis-resistant cells, S91 cells floating in the culture medium were sequentially isolated and transferred for seven generations . As a result, a new subline of S91 cells capable of growing in free cell suspension was selected . These S91 nonadherent (S91Nadh) cells were completely resistant to anoikis and manifested higher metastatic ability than S91Adh cells . Anoikis resistance of S91Nadh cells was not attributable to their resistance to other apoptotic signals in vitro, and they showed no increase in their survival in vivo in the lungs after i.v . inoculation . Increased metastatic potential of the anoikis-resistant S91Nadh cells was associated with various phenotypic changes, including increased proliferation and loss of VLA-4 integrin expression because of down-regulation of the VLA-49alpha (CLD49d) gene . In parallel, they showed a reduction in homotypic aggregation and binding to endothelial cells, increased Matrigel invasiveness, and decreased matrix metalloproteinase-2 and matrix metalloproteinase-9 activity that paralleled up-regulation of the TIMP-1 gene . S91Nadh cells also manifested changes in cell surface carbohydrates, such as appearance of alpha-galactosyl epitopes as a result of up-regulation of the alpha1,3-galactosyltransferase gene and concomitant reduction in cell membrane sialylation . Thus, selection of S91 melanoma cells for anoikis resistance resulted in an increase in their metastatic potential in parallel with multiple alterations in their phenotypic properties.

Leukemia, 2001 Jan, 15(1), 177 - 83
Detection of secondary genetic aberrations in follicle center cell derived lymphomas: assessment of the reliability of comparative genomic hybridization and standard chromosome analysis; Viardot A et al.; Secondary chromosomal aberrations in follicle center cell derived lymphomas (FCDL) usually involve gains and losses of genetic material and may be an important prognostic value . In the present study, we aimed to determine the power of comparative genomic hybridization (CGH) as compared to standard chromosome analysis (CA) to detect such secondary aberrations . The same lymph node cell suspensions prepared from 30 patients with FCDL were analyzed in parallel by CGH and CA based on R banding . In all, 73 discrepancies were found . Sixty-two imbalances were detected only by CA and 11 only by CGH . In cases with completely resolved karyotypes (n= 17), the median number of discrepancies between CGH and CA was one . However, when the karyotype was partially resolved (n = 12), the median was four (P < 0.01) . Discrepant results were further studied by fluorescence in situ hybridization using locus-specific probes . These data confirm, that not only for the detection of balanced aberrations, but also for the detection of unbalanced aberrations in FCDL, standard chromosome analysis is still the 'gold standard' . In contrast, CGH is useful to detect chromosomal imbalances when no metaphases are found or no fresh material is available.

Diagn Cytopathol, 2001 Mar, 24(3), 200 - 5
Application of laser scanning cytometry for evaluation of DNA ploidy in routine cytologic specimens; Wojcik EM et al.; The laser scanning cytometer (LSC) is a relatively new instrument that combines the features of both flow and static image cytometry . The purpose of this study was to examine the application of the LSC for evaluation of DNA ploidy in routine cytologic specimens . The material for this study consisted of 60 routine cytologic specimens obtained from 33 males and 27 females ranging in age from 23-87 yr (mean, 58 yr) . The specimens were simultaneously stained with propidium iodide and FITC-cytokeratin, either on Thin-Prep slide (35 cases) or in a concentrated cell suspension (25 cases) . In each case a minimum of 500 cells was evaluated (range, 527-17,963; mean, 3,889) . All abnormal cell populations were relocated for the presence of malignant cells . The results were defined as diploid and aneuploid/tetraploid . In 10 bladder washes, the results of LSC were compared to results of flow cytometry . Out of 60 specimens, 7 (11%: 6 bladder washes and 1 renal wash) were excluded due to low cellularity . Of the remaining 53 cases, 11 (20%) were aneuploid/tetraploid, and 42 (80%) were diploid . All but one cytologically diagnosed malignancy had abnormal DNA content . Additionally, two bladder washes diagnosed as suspicious and atypical were aneuploid . All abnormal LSC results were confirmed by relocation of the cells . The concordance between flow cytometry and LSC in the 10 control bladder washes was 100% . In conclusion, LSC proved to be a suitable instrument for the evaluation of DNA ploidy in routine cytologic specimens .

J Comp Neurol, 2001 Mar 26, 432(1), 35 - 60
Motor neurons rapidly accumulate DNA single-strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy; Liu Z et al.; The mechanisms of neuronal degeneration in motor neuron disease are not fully understood . We tested the hypothesis that oxidative stress in vitro and axotomy in vivo induce single-strand breaks (SSB) in DNA, a form of early DNA damage, in adult motor neurons early during their degeneration . We developed and characterized a novel cell suspension system enriched in motor neurons from adult rat spinal cord ventral horn . This cell system is approximately 84% neurons, with approximately 86% of these neurons being motor neurons; approximately 72% of these motor neurons are alpha-motor neurons . Motor neuron viability in suspension is approximately 100% immediately after isolation and approximately 61% after 12 hours of incubation . During incubation, isolated motor neurons generate high levels of superoxide . We used single-cell gel electrophoresis (comet assay) to detect DNA-SSB in motor neurons . Exposure of motor neurons to nitric oxide (NO) donors (sodium nitroprusside or NONOate), H2O2, or NO donor plus H2O2 rapidly induces DNA-SSB and causes motor neuron degeneration, the occurrence of which is dose and time related, as represented by comet formation and cell loss . Motor neuron toxicity is potentiated by cotreatment with NO donor and H2O2 (at nontoxic concentrations alone) . Peroxynitrite causes DNA-SSB in motor neurons . The DNA damage profiles (shown by the comet morphology and moment) of NO donors, NO donor plus H2O2, and peroxynitrite are similar . In an in vivo model of motor neuron apoptosis, DNA-SSB accumulate slowly in avulsed motor neurons before apoptotic nuclear features emerge, and the comet fingerprint is similar to NO toxicity . We conclude that motor neurons challenged by oxidative stress and axotomy accumulate DNA-SSB early in their degeneration and that the formation of peroxynitrite is involved in the mechanisms .

Anat Rec, 2001 Mar 1, 262(3), 279 - 92
Development of the follicle-associated epithelium and the secretory dendritic cell in the bursa of fabricius of the guinea fowl (Numida meleagris) studied by novel monoclonal antibodies; Nagy N et al.; Two stromal elements, follicle-associated epithelium and secretory dendritic cells of the bursa of Fabricius were studied by light microscopy and two novel MAbs, that were produced against splenic cell suspensions of guinea fowls . Both antigens recognized by these MAbs, designated GIIF3 and NIC2, are localized in the cytoplasm of the stromal cells, and their molecular weights are 50 and 30 kD, respectively . During embryogenesis the GIIF3 and NIC2 cells emerge in the mesenchyme of the folds before follicle formation . The GIIF3 and the NIC2-positive cells accumulate under the surface epithelium of the plicae and migrate into the epithelium, that precedes the bud-formation . From the bud, the GIIF3-positive cells migrate up to the luminal surface, and they transform to distinct, highly polarized follicle-associated epithelial cells . Single GIIF3-positive cells are also present in the interfollicular epithelium . The NIC2 MAb recognized mesenchymal cells harbor in the lymphoepithelial compartment of the folliculus, and they elaborate cytoplasmic granules . Around Day 20 of embryogenesis large amount of NIC2-positive substance appear extracellularly in the medulla and around it . This period well correlates with the starting up of the bursal functions; clonal expansion of B cells, and generation of immune repertoire . After hatching the NIC2 stainability diminishes, and it is restricted to the medullary bursal secretory dendritic cells . The NIC2-positive, possibly elderly bursal secretory dendritic cells, are capable for migration into the follicle-associated epithelium . In eight-day old birds some cells of the follicle-associated epithelium reveals temporary NIC2 positivity, that may prove the transport of the follicle-associated epithelial cells into luminal direction . By 12 weeks of age the presence of NIC2-positive substance in the intercellular space of the FAE, rather than in the cells of FAE may indicate the termination of the transport of secretory substance . In conclusion, two types of mesenchymal cells enter the surface epithelium of the bursal folds . The GIIF3-positive cells appear on the luminal surface of the follicles and occupy the place of the follicle-associated epithelial cells . The NIC2-positive cells become secretory in nature and differentiate to bursal secretory dendritic cells . The follicle formation possibly, requires the joint presence of both GIIF3 and NIC2 cells in the epithelium .

Mol Ther, 2001 Feb, 3(2), 128 - 38
Rhesus monkey model for fetal gene transfer: studies with retroviral- based vector systems; Tarantal AF et al.; Many life-threatening conditions that can be diagnosed early in gestation may be treatable in utero using gene therapy . In order to determine in utero gene transfer efficiency and safety, studies were conducted with fetal rhesus monkeys as a model for the human . Included in these studies were Moloney murine leukemia virus (MLV)-based amphotropic retrovirus, vesicular stomatitis virus-G (VSV-G) pseudotyped MLV, and a VSV-G pseudotyped HIV-1-based vector, all expressing the enhanced green fluorescent protein (EGFP) as a reporter gene and driven by a cytomegalovirus-immediate early promoter (N = 16) . Rhesus monkey fetuses were administered viral vector supernatant preparations by the intraperitoneal (ip) (N = 14) or intrahepatic (ih) (N = 2) routes via ultrasound guidance at 55 +/- 5 days gestation (late first trimester; term 165 +/- 10 days) . Fetuses were monitored sonographically, specimens were collected prenatally and postnatally, and tissue harvests were performed at birth or 3 or 6 months postnatal age (3-10 months post-gene transfer) . PCR analyses demonstrated that transduced cells were present at approximately 1.2% in peripheral blood mononuclear cells from fetuses administered amphotropic MLV, <0.5% in fetuses receiving MLV/VSV-G, and approximately 4.2% for the lentiviral vector, which decreased to 2% at birth . Hematopoietic progenitors showed that overall (mean of all time points assessed), approximately 25% of the collected colonies were positive for the EGFP transgene with the lentiviral vector, which was significantly greater than results achieved with the MLV-based vector systems (4-9%; P < or = 0.001-0.016) . At necropsy, 0.001-10% of the total genomic DNA was positive for EGFP in most tissues for all groups . EGFP-positive fluorescent cells were found in cell suspensions of thymus, liver, spleen, lymph nodes, cerebral cortex, and bone marrow (0.5-6%) . Overall, the results of these studies have shown: (1) healthy infants expressing vector sequences up to 10 months post-gene transfer, (2) fetal primate administration of retroviral vectors results in gene transfer to multiple organ systems, (3) the highest level of gene transfer to hematopoietic progenitors was observed with the lentiviral vector system, and (4) there was no evidence of transplacental transfer of vector sequences into the dams . The rhesus monkey is an important preclinical primate model system for exploring gene transfer approaches for future applications in humans.

Can J Physiol Pharmacol, 2001 Feb, 79(2), 109 - 13
{Induction of apoptosis of splenic lymphocytes in mice by accelerated carbon ions}; Holl V et al.; To assess the capacity of heavy ions to induce apoptosis in lymphocytes, mice have been irradiated with accelerated carbon ions (95 MeV/nucleon) at doses ranging from 0.1 to 4 Gy . Their spleens were removed 24 h later and gently dissociated to prepare a single cell suspension . Mononuclear cells were then maintained in culture at 37 degrees C, and the occurrence of apoptosis in these cells was analysed 24 h later . Lymphocytes were also irradiated in vitro, in the presence of Ac-DEVD-CHO, a potent caspase-3 and -7 inhibitor . Results from three experiments performed at the Grand Accelerateur National d'Ions Lourds (GANIL, Caen, France) are reported here . They indicate that carbon ions induce a marked, dose-dependent, reduction of the spleen weight and cellularity . However, in sharp contrast with spleen cells prepared from X-ray irradiated mice, only a slight increase of apoptosis is evidenced in cultured lymphocytes from mice irradiated with heavy ions . The significance of such results is discussed . So far, few data exist concerning the biological effects of heavy ions, in particular their capacity to induce apoptosis in lymphocytes; the present study provides useful clues for further investigations.

J Photochem Photobiol B, 2000 Nov, 58(2-3), 123 - 9
On recording the true absorption spectrum and the scattering spectrum of a turbid sample: application to cell suspensions of the cyanobacterium Anabaena variabilis; Merzlyak MN et al.; An integrating sphere is often used for recording the absorption spectrum of a turbid sample . If the sample is placed inside the sphere, scattering losses are eliminated, but the recorded spectrum suffers from other distortions . These distortions can be avoided by positioning the sample outside the sphere; but, since some of the scattered light escapes the detector, the recorded spectrum suffers from residual scattering losses . A method proposed by Latimer and Eubanks more than 30 years ago (Arch . Biochem . Biophys . 98 (1962) 274), is put to a quantitative examination, which has shown that one can obtain, by recording two spectra at different distances from the sphere, not only the true absorption spectrum but also the scattering spectra of the sample . Conditions for the validity of the basic assumption underlying the method are investigated by examining suspensions containing various concentrations of cells of the cyanobacterium Anabaena variabilis, and it is shown that the calculated absorbance is proportional to the number density of the cells . The application of the method for quantitative spectrophotometric analysis of pigments in cell suspensions is discussed.

Arch Pathol Lab Med, 2001 Mar, 125(3), 364 - 74
Two-color, cytokeratin-labeled dna flow cytometric analysis of 332 breast cancers: lack of prognostic value with 12-year follow-up; Prasad AR et al.; CONTEXT: DNA flow cytometry of breast cancer is a proposed tumor marker of prognostic significance that is of controversial clinical utility because of lack of standardization and confirmatory studies . OBJECTIVE: To evaluate the prognostic significance of the more informative technique of multiparametric 2-color DNA flow cytometry as recommended by the 1992 DNA Cytometry Consensus Conference . DESIGN: Three hundred thirty-two breast carcinomas with 7 to 12 years of follow-up were prospectively analyzed as fresh tumors that were mechanically dissociated into whole cell suspensions . These suspensions were dual fluorescence-labeled with propidium iodide (DNA) and antibodies to cytokeratin (epithelium) and leukocyte common antigen (internal leukocyte control) for gated analysis of subpopulations . Multicycle software with histogram-dependent algorithms employing background, aggregate, and debris correction were used in DNA and cell-cycle quantitation . Data were analyzed according to the DNA Flow Cytometry Consensus Conference recommendations . RESULTS: DNA ploidy and proliferation stratified into 3 categories were not predictive of overall or disease-free survival . Sixty-five percent of tumors were nondiploid, and 35.4% were diploid . Two hundred six tumors were able to be evaluated for synthesis-phase fraction (SPF) analysis, with 74 of 206 cases in the low range (<13.4%), 36.4% in the intermediate range (>13.5 to <25.4%), and 27.6% in the high SPF (>25.5%) category . Aneuploid tumors tended to have a higher SPF . Univariate survival analysis showed prognostic significance of the following: tumor size, stage, TNM components, vascular invasion, nuclear grade, and histologic grade . Only T classification, presence of positive axillary lymph nodes, and distant metastases were significant independent predictors of survival in multivariate Cox regression models . Age and hormone receptor status showed no prognostic significance . Synthesis-phase fraction was significantly correlated with tumor size, stage, T classification, nuclear and histologic grade, presence of estrogen or progesterone receptors, and axillary lymph node status . None of the histologic parameters showed any significant association with DNA aneuploidy, except for high nuclear and histologic grade and the absence of estrogen receptors . CONCLUSIONS: Despite the use of state-of-the-art processing and flow cytometry analytic techniques, DNA ploidy and proliferation measurements were not predictive of survival in any stage of breast cancer . However, select histopathologic parameters and TNM stage were significant predictors of survival in univariate and multivariate analyses . We conclude that DNA ploidy and proliferation measurements do not provide significant prognostic information for clinicians to integrate into therapeutic decision making for patients with breast cancer.

J Burn Care Rehabil, 2001 Jan-Feb, 22(1), 41 - 6
Melanocyte repopulation in full-thickness wounds using a cell spray apparatus; Navarro FA et al.; Melanocyte restoration is critical in reconstituting skin color . We developed a spotted (piebald) pig wound model to study methods of restoring melanocytes to the epidermis . Paired, full-thickness, porcine wounds were covered with nonpigmented, fully expanded, 3:1 meshed, split-thickness skin grafts and were sprayed with an epidermal cell suspension . The suspensions were highly pigmented skin (HPS) cell isolates for half of the wounds (n = 16) and nonpigmented skin (NPS) cell isolates for the remaining wounds (n = 16) . Histologic sections showed 6.0 +/- 3.0 and 15 +/- 4.0 pigmented melanocytes per high-power field on days 8 and 20 in HPS-treated wounds and no pigmented melanocytes in NPS-treated wounds . Melanin pigment was dispersed in all layers of the epithelium for the HPS group on day 20 compared with a lack of melanin pigment observed in the NPS group . Cell spraying may provide a clinical method to restore color to skin; further work is needed to control the expression of melanin.

Acad Radiol, 2001 Feb, 8(2), 121 - 7
Sonographic evaluation of orthotopic bladder tumors in mice treated with TNP-470, an angiogenic inhibitor; Rooks V et al.; RATIONALE AND OBJECTIVES: The purpose of this study was to determine the feasibility of using transabdominal ultrasonography (US) to monitor tumor growth and response to therapy in a mouse model of orthotopic bladder carcinoma . MATERIALS AND METHODS: Human bladder carcinoma cell suspensions were injected into the bladders of 18 SCID mice, allowed to grow for 3 weeks, and monitored weekly with gray-scale US . After 23 days, five animals were treated with TNP-470, an angiogenic inhibitor, and five control animals were treated with saline solution . US images were evaluated for tumor location, size, and neovascularity . All untreated animals (n = 8) were imaged and sacrificed at 25 days . Eight of the treated animals were imaged and sacrificed after 14 days of treatment . US findings for both groups were compared with autopsy findings . RESULTS: While saline-treated tumors continued to grow, the growth of TNP-470-treated tumors was arrested within 7 days of therapy (P < .02) . Tumors as small as 1.5 mm were identified prospectively with US . US volume estimates correlated well with autopsy volume measurements (r2 = 1.0, P < .0001) . Although tumor neovascularity was identified in every animal, the pattern of neovascularity did not correlate with tumor volume or therapy . CONCLUSION: US can provide accurate intermediate end points for monitoring experimental intraabdominal tumor growth and response to therapy in the mouse model.

Neuroscience, 2001, 102(3), 581 - 92
No functional effects of embryonic neuronal grafts on motor deficits in a 3-nitropropionic acid rat model of advanced striatonigral degeneration (multiple system atrophy); Waldner R et al.; Intrastriatal injection of 3-nitropropionic acid results in secondary excitotoxic local damage and retrograde neuronal cell loss in substantia nigra pars compacta, thus mimicking salient features of striatonigral degeneration, the core pathology underlying Parkinsonism associated with multiple system atrophy . We used 3-nitropropionic acid to create a rat model of advanced striatonigral degeneration in order to assess the effects of embryonic allografts upon rotational and complex-motor behavioural abnormalities . Following stereotaxic intrastriatal administration of 500nmol 3-nitropropionic acid in male Wistar rats we observed consistent amphetamine- and apomorphine-induced ipsiversive rotation . Furthermore, there were marked deficits of contralateral paw reaching . Subsequently, animals received intrastriatal implantations of either E14 mesencephalic or striatal or mixed embryonic cell suspensions . In addition, one group received sham injections . Grafted rats were followed for up to 21 weeks and repeated behavioural tests were obtained during this period . Drug-induced rotation asymmetries and complex motor deficits measured by paw reaching tests were not compensated by embryonic grafts . Persistence of drug-induced rotations and of paw reaching deficits following transplantation probably reflects severe atrophy of adult striatum, additional nigral degeneration as well as glial demarcation of embryonic grafts . We suggest that dopamine rich embryonic grafts fail to induce functional recovery in a novel 3-nitropropionic acid rat model of advanced striatonigral degeneration (multiple system atrophy).

Urol Res, 2000 Dec, 28(6), 391 - 7
Potentiation of effects of anticancer agents by local electric pulses in murine bladder cancer; Ogihara M et al.; Electrochemotherapy is a novel cancer treatment in which electric pulses (EP) are used as a means of delivering anticancer agents to the cytoplasm of cancer cells (electroporation) . The present study evaluates whether electrochemotherapy has in vitro and in vivo anticancer effects in murine bladder cancer . Using mouse bladder tumor cells (MBT-2 cells), in vitro electrochemotherapy was performed by applying EP to the cell suspension immediately after the addition of anticancer agents . The cytotoxicity of adriamycin (ADM), bleomycin (BLM) and cis-diamminedichloroplatinum(II) (CDDP) was determined by measuring succinate dehydrogenase (SD) activity in both electroporated and non-electroporated cells . In addition, intracellular concentrations of these anticancer agents were also measured . In the in vivo study, tumor-bearing C3H/He mice were treated with an intraperitoneal injection of anticancer agents followed by a local delivery of EP at the tumor site . Then, tumor growth rate (TGR) was determined and compared to that in the sham-treated control group, the EP-only group and the drug-only group . The in vitro study showed that, with electroporation, the cytotoxicity of BLM in electroporated cells was increased by as much as 95.7-fold compared to that of non-electroporated MBT-2 cells; CDDP showed only an increase of 1.8-fold and ADM showed no increase . After electroporation, the intracellular concentration of BLM, CDDP and ADM showed an increase of 120-, 1.7- and 0.8-fold, respectively . In electrochemotherapy for in vivo growing tumors, the potentiation of the antitumor effect was most prominent when combined with BLM, only slightly with CDDP, and totally absent with ADM . It is clear from in vitro and in vivo studies that, in a murine bladder tumor, the anticancer effect of BLM can be considerably potentiated by applying EP . Thus, BLM seems to be the most suitable anticancer agent for electrochemotherapy of bladder cancer.

Acta Cient Venez, 2000, 51(2), 90 - 5
{Massive multiplication of coffee (Coffee arabica L . cv . Catimor) through embryogenic cell suspension culture}; Flermoso-Gallardo L et al.; Cell suspensions offer several advantages as a system for massive propagation because of the high rates of multiplication, the higher homogeneity in the culture conditions and the possibility of automatization . In this study, different experimental conditions were analyzed to establish embryogenic cell suspension cultures of coffee . The best conditions to establish the embryogenic cell suspension cultures of coffee were as follows: coffee leaf sections were cultivated during 12 weeks (Stage I) in a solid medium with the Murashige and Skoog salts, 2 mg/l kinetin and 0.5 mg/l 2,4-dichlorophenoxiacetic acid (medium 1) . Under these conditions the explants formed a callus tissue that was transferred to a liquid medium containing 5 mg/l of 6-benzylamlno-purine (medium 2) . After 12 days in a shaking liquid medium (Stage II), the cultures were sieved and were maintained In the same media, which was renewed every eight days (Stage III) . This method yielded 1884 embryos in 50 ml; placing the embryos under conditions for germination yielded plantlets of normal appearance.

Tissue Eng, 2001 Feb, 7(1), 81 - 8
The effect of fibroblast growth factor and transforming growth factor-beta on porcine chondrocytes and tissue-engineered autologous elastic cartilage; Arevalo-Silva CA et al.; Elastic cartilage responds mitogenically in vitro to transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (basic FGF) . We studied the effects of these growth factors separately or in a combination on porcine auricular chondrocytes in vitro and on the autologous elastic cartilage produced . Cells were harvested from the elastic auricular cartilage of 16- to 18-kg Yorkshire swine . Viability and quantification of the cells was determined . Cells were plated at equal concentration and studied in vitro in one of four identical media environments except for the growth factors: Group I contained Ham's F-12 with supplements but no growth factors, Group II also contained basic-FGF, Group III also contained TGF-beta, and Group IV also contained a combination of both growth factors . After 3 weeks in vitro, the cells were chemically dissociated with 0.25% trypsin . Cell suspensions composed of 3 x 10(7) cells/cc in 30% Pluronic F-127/Ham's F-12 were injected subcutaneously . Implants were harvested at 6, 8, 10, and 12 weeks of in vivo culture and then were examined with histologic stains . After 3 weeks of in vitro culture the total number of cells was as follows: Group I, 1.8 x 10(8); Group II, 3.5 x 10(8); Group III, 1.3 x 10(8); Group IV, 2.5 x 10(8) . After 8 weeks of in vivo autologous implantation, the average weight (g) and volume (cm3) of each group was as follows: Group I, 0.7 g/0.15 cm3; Group II, 1.5 g/0.8 cm3; Group III, 0.6 g/0.1 cm3; Group IV, 1.2 g/0.3 cm3 . Histologically, Groups I, II, and IV generated cartilage similar to native elastic cartilage, but Group III specimens demonstrated fibrous tissue ingrowth . Basic FGF produced the most positive enhancement on the quantity and quality of autologous tissue engineered elastic cartilage produced in this porcine model both in vitro and in vivo.

Clin Hemorheol Microcirc, 2000, 23(1), 31 - 42
Validation of a test of the red cell membrane osmotic resistance; Didelon J et al.; The aim of the present work was to validate a new technique for the measurement of resistance of the red blood cell membrane using an automated apparatus called a Fragilimeter . Its principle lies in the measurement of the extinction of a laser beam projected through a red blood cell suspension subjected, by diffusion, to a variation of salinity from an isotonic equilibrium (154 mM NaCl) to, a hypotonic one, 25 mM NaCl . The variation of osmotic pressure induces on the cells a progressive lysis and a modification of the extinction of the transmitted light . The validation of the method was based on the comparison between results obtained with the Fragilimeter and those obtained using the reference DACIE technique . Analyses were based on blood samples from healthy donors . The determination of the initial, the 50% and the full haemolysis thresholds allowed observation of the fragility of the cell, through its membrane resistance . The physical phenomenon measured in these cells when subjected to various ionic strengths is discussed on the basis of observations realised by means of an optical microscope.

Inflamm Res, 2000 Dec, 49(12), 666 - 72
Studies on the effects of adenosine A3 receptor stimulation on human eosinophils isolated from non-asthmatic or asthmatic donors; Reeves JJ et al.; OBJECTIVE: This study was designed to demonstrate the presence of adenosine A3 receptors on human peripheral blood eosinophils, and to investigate the effect of A3 receptor stimulation on eosinophil function . MATERIAL: Eosinophils from either non-asthmatic or asthmatic donors . METHODS: Eosinophils were isolated from peripheral venous blood by discontinuous gradient centrifugation and negative immunoselection . Receptor localisation was investigated by immunoblotting and by immunocytochemistry using a novel antibody specific for the human A3 receptor . Two pharmacological responses were studied: elevation of intracellular calcium in single eosinophils, measured by microfluorimetry, and hydrogen peroxide generation in cell suspensions . RESULTS: The expression of A3 receptors by eosinophils was confirmed using the selective antibody . Addition of the A3 receptor selective agonist, IB-MECA (100 nM), produced increases in intracellular calcium in less than 10% of the eosinophils isolated from non-asthmatic donors . These responses were only partially attenuated with the A3 receptor antagonist, I-ABOPX . IB-MECA (0.001-1000 nM) did not stimulate hydrogen peroxide (H2O2) generation, nor did it enhance fMLP- or C5a-stimulated generation of H2O2 . In fact high concentrations of IB-MECA inhibited the generation of H2O2 (when stimulated by fMLP or C5a), an effect probably mediated by A2 receptors . Similar results were obtained using eosinophils from asthmatic donors . CONCLUSIONS: Stimulation of adenosine A3 receptors does not appear to be a prime mechanism for free radical generation by human peripheral blood eosinophils.

Life Sci, 2000 Mar 3, 66(15), 1451 - 9
Supraphysiological level of estrogen exposure in vivo increases lymphoid cell death in mice; Zajchowski S et al.; Estrogen can enhance or reduce lymphocyte functions in vitro depending on dose and exposure duration . The purpose of this study was to determine the effect of in vivo 17 beta-estradiol (E2) on apoptosis and necrosis in lymphoid tissue of female C567BL/6 mice . Animals were ovariectomized (OVX), ovariectomized and 17 beta-estradiol supplemented (OVX + E2; 71 micrograms E2 per day for 14 days), sham ovariectomized (SHAM), or unhandled controls (CONTROL) . Thymus and spleen were removed aseptically, cells dispersed into single cell suspensions in RPMI-1640, and measures of cell damage performed: an annexin V flow cytometric assay for markers of apoptosis and an enzyme-linked immunoassay for measures of DNA fragmentation and necrosis . OVX + E2 mice had 620 +/- 72 pg/ml 17 beta-estradiol in serum in contrast to OVX mice which had 7.6 +/- 5 pg/ml, the SHAM mice which had 2.8 +/- 1 pg/ml of serum E2, and the CONTROL mice which had 3.9 +/- 0.8 pg/ml of serum E2 (p < 0.001) . There was a significantly lower percentage of viable thymocytes in OVX + E2 mice compared to the other treatment conditions (p < 0.001, respectively) . There was also a significantly higher percentage of annexin V positive thymocytes in OVX + E2 mice (p < 0.005) . Measures of DNA fragmentation by ELISA were higher in splenocytes from OVX + E2 mice than in the OVX, SHAM or CONTROL mice (p < 0.005) . These results suggest that supraphysiological levels of estrogen in vivo induce damage in lymphoid cells; however, the impact of estrogen associated lymphoid tissue damage on specific immune functions remains to be determined.

Clin Exp Metastasis, 2000, 18(1), 57 - 60
A highly metastatic Lewis lung carcinoma orthotopic green fluorescent protein model; Rashidi B et al.; The Lewis lung carcinoma has been widely used for many important studies . However, the subcutaneous transplant or orthotopic cell-suspension injection models have not allowed the expression of its full metastatic potential . A powerful new highly metastatic model of the widely-used Lewis lung carcinoma is reported here using surgical orthotopic implantation (SOI) of tumor fragments and enhanced green fluorescent protein (GFP) transduction of the tumor cells . To achieve this goal, we first developed in vitro a stable high-expression GFP transductant of the Lewis lung carcinoma with the pLEIN retroviral expression vector containing the enhanced Aequorea victoria GFP gene . Stable high-level expression of GFP was found maintained in vivo in subcutaneously-growing Lewis lung tumors . The in vivo GFP-expressing tumors were harvested and implanted as tissue fragments by SOI in the right lung of additional nude mice . This model resulted in rapid orthotopic growth and extensive metastasis visualized by GFP-expression . 100% of the animals had metastases on the ipsilateral diaphragmatic surface, contralateral diaphragmatic surface, contralateral lung parenchima, and in mediastinal lymph nodes . Heart metastases were visualized in 40%, and brain metastases were visualized in 30% of the SOI animals . Mice developed signs of respiratory distress between 10-15 days post-tumor implantation and were sacrificed . The use of GFP-transduced Lewis lung carcinoma transplanted by SOI reveals for the first time the high malignancy of this tumor and provides an important useful model for metastasis, angiogenesis and therapeutic studies.

Pharm Res, 2000 Nov, 17(11), 1402 - 7
Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: immunosuppressive effects after in vivo administration to rats; Mehvar R et al.; PURPOSE: To study the immunosuppressive activities of a macromolecular prodrug of methylprednisolone (MP), dextran-methylprednisolone succinate (DEX-MPS), in rats . METHODS: Single 5 mg/kg (MP equivalent) doses of MP or DEX-MPS were administered intravenously to rats, and blood and spleen samples were collected over 96 h . The immunosuppressive activity was determined by the effects of the free or dextran-conjugated drug on the mitogen-stimulated spleen lymphocyte proliferation . Additionally, the number of lymphocytes in the spleen cell suspensions was estimated . Further, the plasma and spleen concentrations of the conjugated and free MP were determined using size-exclusion and reversed-phase chromatographic methods, respectively . RESULTS: Both MP and DEX-MPS injections resulted in the inhibition of the spleen lymphocyte proliferation . However, the maximal effect of DEX-MPS was significantly (P < 0.003) more intense (approximately 100% inhibition) and delayed (24 h) relative to that of MP (approximately 50% inhibition at 2 h) . The DEX-MPS injection also resulted in a significantly (P < 0.0001) higher decline in the estimated number of spleen lymphocytes (approximately 80% at 24 h), compared with the MP injection (approximately 30% at 2 hr) . Whereas the plasma and spleen concentrations of MP could not be measured at > or = 2 h after the drug injection, relatively high concentrations of DEX-MPS persisted in plasma and spleen for 24 h and 96 h, respectively . CONCLUSION: Dextran-methylprednisolone conjugate can effectively deliver the corticosteroid to its site of action for immunosuppression, resulting in more intense and sustained effects when compared with the free drug administration.

Z Naturforsch {C}, 2000 Nov-Dec, 55(11-12), 957 - 64
The role of lipid peroxidation in aluminium toxicity in soybean cell suspension cultures; Rath I et al.; The primary reactions leading to Al toxicity in plant cells have not yet been elucidated . We used soybean (Glycine max {L.} Merr.) cell suspension cultures to address the question whether lipid peroxidation plays an important role in Al toxicity . Upon transfer to an Al-containing culture medium with a calculated Al3+ activity of 15 microM soybean cells showed a distinct and longtime increase in lipid peroxidation within 4 h . At the same time a drastic loss of cell viability was observed . Butylated hydroxyanisole (BHA) and N,N'-diphenyl-p-phenylenediamine (DPPD), two lipophilic antioxidants, were able to almost completely suppress lipid peroxidation in Al-treated cells at a concentration of 20 microM . This effect was dose-dependent for DPPD and was observed at minimum concentrations of 1-2 microM . When lipid peroxidation was suppressed by DPPD or BHA cell viability remained high even in the presence of toxic Al concentrations . These results suggest that Al-induced enhancement of lipid peroxidation is a decisive factor for Al toxicity in suspension cultured soybean cells.

Endod Dent Traumatol, 2000 Dec, 16(6), 287 - 90
Cytotoxic effect of four root filling materials; Miletic I et al.; The aim of this study was to evaluate the cytotoxic effect of four root canal sealers: AH26, AH Plus, Diaket and Apexit . In the experiment two cell lines, human cervical carcinoma (HeLa) cells and mouse skin fibroblasts (L929), were used . Under aseptic conditions, the sealers were prepared according to the manufacturers' directions, and 0.01 mL of each material was placed in a 24-well plate . The sealers were covered with cell suspension . The cytotoxicity was estimated by determining the number of viable cells by a light microscope, as well as the total number of cells 24 h, 48 h and 120 h after the treatment with mentioned materials . The results obtained in this study showed the high cytotoxcity of the new AH Plus root canal sealer, which was shown to be equally or more toxic to the standard AH26 and Diaket materials . Apexit was the least toxic sealer.

Plant Mol Biol, 2000 Dec, 44(6), 733 - 45
Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.); Chiron H et al.; Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation . A SAM-dependent PMT protein was purified and partially characterised . A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library . Southern blot analysis of the genomic DNA suggested the presence of a gene family . The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs . PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures . The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin . Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated . Recombinant PMT thus had a relatively broad substrate specificity . Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level . Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

Asian J Androl, 2000 Dec, 2(4), 271 - 5
Production of chicken chimeras by fusing blastodermal cells with electroporation; Aritomi S et al.; AIM: To establish techniques for producing somatic and germline chimeric chicken by transferring blastodermal cells fused with electroporation . METHODS: Stage-X blastodermal cells isolated from freshly laid fertile unincubated white Leghorn and Rhode Island red chicken eggs were fused with electroporation . The treated cell suspension was transferred to the recovery medium (DMEM containing 10% FBS) and was injected into the subgerminal cavity of recipient unincubated embryos (stage X) . RESULTS: Of 177 recipient embryos injected with the fusing blastodermal cells, 6 (3.4%) survived to hatching . Somatic chimerism was examined in the melanocyte of the feather . The presence of feathers originating from the donor cell was observed in 1 bird (16.7%) out of the 6 hatched birds . After 21 days of incubation two birds out of five embryos were subjected to polymerase chain reaction (PCR) analysis for W-chromosome-specific DNA for each tissue . One bird possessed W-chromosome-specific DNA in the stomach, and the other exhibited the same DNA in the left and right gonads and other tissues, but not the stomach . CONCLUSION: Recipient embryo having electrofused blastodermal cells yields somatic and germline chimeric chickens more successfully.

Planta Med, 2000 Dec, 66(8), 773 - 5
Isolation of taxoids from cell suspension cultures of Taxus wallichiana; Agrawal S et al.; Cell suspension cultures of stem-derived callus of Taxus wallichiana in MS-F culture media were found to produce three C-14 oxygenated taxoids and one regular taxoid . The taxoids were identified as yunnanxane (1), 2 alpha, 5 alpha, 10 beta, 14 beta-tetraacetoxy-4(20),11-taxadiene (2), 2 alpha, 5 alpha, 10 beta-triacetoxy-14 beta-(2-methyl)-butyryloxy-4(20),11-taxadiene (3) and 1 beta-hydroxybaccatin I (4).

Cancer Res, 2001 Jan 1, 61(1), 243 - 8
The chemotherapeutic drug 5-fluorouracil induces apoptosis in mouse thymocytes in vivo via activation of the CD95(APO-1/Fas) system; Eichhorst ST et al.; The CD95/CD95 ligand (CD95L) system has been shown to mediate chemotherapeutic drug-induced apoptosis in vitro . However, the contribution of the CD95 pathway to drug-induced apoptosis is controversial . We have shown previously that 5-fluorouracil (5-FU) induces apoptosis in vitro via the activation of the CD95/CD95L system . To study the effects of the chemotherapeutic drug 5-FU and the contribution of the CD95 system to 5-FU-induced apoptosis in vivo, we gave mice an i.p . injection of 5-FU . Apoptotic cell death peaked in thymocytes at 18 h after administration of 5-FU . Total organ weight and cell number in the thymus were reduced by approximately 40% . This cell loss was due to apoptosis, as measured in cell suspensions by measuring hypodiploid DNA content and by terminal deoxynucleotidyl transferase-mediated nick end labeling staining of tissue sections . The number of apoptotic cells correlated with the extent of weight loss and cell attrition of the organs . Furthermore, in the thymi of 5-FU-treated animals, CD95L was strongly up-regulated . Apoptosis of thymocytes was blocked in vivo with neutralizing anti-CD95L antibodies . In addition, cell loss in the thymus was negligible in lpr mice in comparison with wild-type mice . Thus, a significant portion of apoptosis of thymocytes in vivo on treatment with 5-FU is mediated via the CD95/CD95L pathway . Our findings therefore contribute to the understanding how chemotherapeutic drugs exert their effects in vivo.

J Burn Care Rehabil, 2000 Nov-Dec, 21(6), 513 - 8
Sprayed keratinocyte suspensions accelerate epidermal coverage in a porcine microwound model; Navarro FA et al.; Keratinocyte suspensions can potentially treat a variety of epidermal defects, but the mechanism of action has not been fully determined . We developed a porcine model to study the effect of sprayed cell suspensions delivered on small wounds within a meshed autograft . Paired full-thickness surgically excised wounds were covered with a fully expanded 3:1 meshed split-thickness autograft . A keratinocyte cell suspension was sprayed onto half of the wounds at a seeding density of 2.8 x 10(3) cells/cm2; the remaining wounds were sprayed with cell culture medium alone . Histologic analysis at days 5 and 8 showed an increase in average epidermal thickness, confluence, keratin cysts, and blood vessels in the keratinocyte cell suspension group compared with the cell culture medium control group . Wounds sprayed with the cell suspension showed faster and better quality of epithelialization than wounds sprayed with cell culture medium alone.

Trop Gastroenterol, 2000 Oct-Dec, 21(4), 194 - 6
Hepatocyte isolation and transplantation in syngenic rats; Dave S et al.; Refinement of techniques to isolate viable hepatocytes began in the late 1960's . It was established that perfusion of the intact liver as opposed to incubation of liver slices or chopped tissue increased the yield of cells . The present study aims to establish a simple, two-step, collagenase digestion method for hepatocyte isolation . A single inbred Fisher rat was used for hepatocyte isolation . The liver was perfused in-situ with perfusion buffer containing ethylene glycol bis N, N1, tetra acetic acid (EGTA), followed by the collagenase buffer . The liver was excised and gently minced . The tissue was resuspended in the collagenase buffer to complete dissociation . The cell suspension obtained was washed, centrifuged and filtered to complete the isolation procedure . The trypan blue exclusion test showed 80-85% cell viability . The isolated cells were transplanted into the splenic parenchyma of syngenic rats . Survival of the transplanted hepatocytes was confirmed by histological examination at the end of 90 days . This two step technique of in-situ liver perfusion gives a high yield of viable hepatocytes which show long term survival after transplantation.

J Orthop Res, 2000 Nov, 18(6), 909 - 11
Delamination rates of tissue flaps used in articular cartilage repair; Driesang IM et al.; A popular strategy in the treatment of articular cartilage lesions involves the introduction of cell suspensions into the defect void and its closure with a periosteal flap (autologous chondrocyte transplantation technique) . We applied this methodology in goats and discovered that all sutured flaps (n = 6 animals) became detached from nonimmobilized joints during the recovery period . The purpose of this study was to ascertain whether postoperative restriction of joint movement could prevent the delamination of flaps . Partial-thickness defects were created in the knee joint cartilage of 27 goats . These defects were then filled with a fibrin matrix and covered with periosteal (n = 6) or fascial (n = 21) flaps, which were sutured with simple, interrupted stitches to the surrounding tissue . The joints were immobilized by means of a modified Robert Jones bandage for periods of 2-6 weeks, after which time they were inspected for the absence or presence of flaps . In four animals, joint immobilization for 3 weeks was followed by free movement for a similar period . Four of the six periosteal flaps and two of the 21 fascial ones became delaminated after the period of immobilization . In the four goats permitted 3 weeks of free joint movement following a similar period of joint immobilization, all flaps (which had been retained up to the end of the immobilization period) became detached . These findings indicate that joint immobilization hinders the delamination of flaps but that this restriction of movement must be sustained for an undefined period of time . The nature of the tissue used for flaps also influences their rate of retention by immobilized joints.

Environ Microbiol, 1999 Dec, 1(6), 489 - 94
Aerotaxis in Desulfovibrio; Eschemann A et al.; Aerotaxis of two sulphate-reducing bacteria, the freshwater strain Desulfovibrio desulfuricans CSN (DSM 9104) and the marine strain Desulfovibrio oxyclinae N13 (DSM 11498), was studied using capillary microslides, microscopy and oxygen microsensors . The bacteria formed ring-shaped bands in oxygen diffusion gradients surrounding O2 bubbles, which were placed into anoxic sulphate-free cell suspensions in capillary microslides . The radial expansion of the oxic volume by diffusion was stopped by aerobic respiration . Bands were formed by cells avoiding high O2 levels near the O2 bubble, as well as by cells entering from the surrounding anoxic zone . At the inner edge of the bands, O2 levels of up to 20% air saturation (50 microM O2) were found, while the outer edge always coincided with the oxic-anoxic interface . Ring diameters and O2 concentrations at the inner edge of the band depended on the cell density and the strain used in the suspension . Band formation did not occur in the absence of an electron donor (5mM lactate) or when N2 gas bubbles were used . Both strains were highly motile with velocities of approximately equals 32 microm s(-1) during forward runs, and 7 microm s(-1) during backward runs respectively . Within the bands, cells moved in circles of about 20 microm diameter, while cells outside the band exhibited straighter or only slightly bent traces . It is concluded that the capacity of respiration at high rates and the positive and negative aerotactical responses of Desulfovibrio provide an efficient strategy for removing O2 from the habitat in situations where sufficient electron donors and high cell densities are present.

Vet Microbiol, 2001 Feb 26, 78(4), 343 - 51
Rapid screening of H(2)O(2) production by Mycoplasma mycoides and differentiation of European subsp . mycoides SC (small colony) isolates; Rice P et al.; Mycoplasma mycoides strains were screened for the ability to produce H(2)O(2) from glucose and glycerol metabolism using rapid and simple colorimetric assays . In quantitative assays, H(2)O(2) production by washed cell suspensions was detected by the oxidation of o-dianisidine in the presence of peroxidase . In qualitative assays, a 3,3'-diaminobenzidine-peroxidase reagent was applied to colonies on agar plates . Both methods enabled differentiation of European subsp . mycoides SC (small colony) isolates from other M . mycoides strains by their inability to produce H(2)O(2) from glycerol metabolism . In addition, two strains of subsp . capri were identified which produced large amounts of H(2)O(2) from glucose oxidation . In lysed cells of these strains, NADH oxidation gave approximately 1 mol H(2)O(2) per mol NADH oxidised whereas in 36 subsp . mycoides and 10 other subsp . capri strains, the quantity produced was 0.01-0.20mol H(2)O(2) per mol NADH oxidised.

Bone, 2001 Feb, 28(2), 187 - 94
Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclasts cultured on collagen-hydroxyapatite films; Lees RL et al.; Diseases exhibiting excessive bone loss are often characterized by an increase in the size and number of osteoclasts in affected areas, suggesting that osteoclast size is associated with increased resorptive activity or efficiency . Because osteoclastic bone resorption depends on proton extrusion via a bafilomycin A1-sensitive vacuolar type H+ ATPase (V-ATPase), we investigated the relationship between osteoclast size and state of activity on the one hand, and proton-extruding mechanisms (bafilomycin A1-sensitive V-ATPase and amiloride-sensitive Na+/H+ exchange) on the other . In determining resorptive activities of individual osteoclasts, osteoclast-containing cell suspensions obtained from newborn rabbit long bones were cultured on apatite-collagen complex (ACC)-coated coverslips . Large osteoclasts resorbed 2.5 times more per cell than small osteoclasts, but the amount resorbed per nucleus was the same for the two categories . However, a much larger percentage of large osteoclasts was resorbing compared with small osteoclasts . To study pH regulatory mechanisms in individual large and small osteoclasts, the cells were loaded with the pH-sensitive indicator BCECF and analyzed by single-cell fluorescence . Small and large resorbing osteoclasts had significantly higher basal pH(i) than their nonresorbing counterparts . Also, small nonresorbing osteoclasts were insensitive to bafilomycin A1 addition or Na+ removal from the medium, large nonresorbing osteoclasts responded slightly, and all resorbing osteoclasts (small and large) responded strongly . Differences were also seen in the recovery from an acid load: both small and large nonresorbing osteoclasts were more sensitive to amiloride inhibition, while large resorbing cells were more sensitive to bafilomycin A1 inhibition . Small resorbing cells were inhibited equally by bafilomycin A1 and amiloride . These results clearly show that a greater proportion of large osteoclasts are active in resorption and that pH(i) regulation is associated with enhanced proton pump activity in actively resorbing osteoclasts . Thus, large and small osteoclasts differ in the proportion of cells that are resorbing, while pH regulatory mechanisms differ mainly between resorbing and nonresorbing cells.

Ultrasound Med Biol, 2000 Nov, 26(9), 1489 - 501
Assessment of arterial stenosis in a flow model with power Doppler angiography: accuracy and observations on blood echogenicity; Cloutier G et al.; The objective of the project was to study the influence of various hemodynamic and rheologic factors on the accuracy of 3-D power Doppler angiography (PDA) for quantifying the percentage of area reduction of a stenotic artery along its longitudinal axis . The study was performed with a 3-D power Doppler ultrasound (US) imaging system and an in vitro mock flow model containing a simulated artery with a stenosis of 80% area reduction . Measurements were performed under steady and pulsatile flow conditions by circulating, at different flow rates, four types of fluid (porcine whole blood, porcine whole blood with a US contrast agent, porcine blood cell suspension and porcine blood cell suspension with a US contrast agent) . A total of 120 measurements were performed . Computational simulations of the fluid dynamics in the vicinity of the axisymmetrical stenosis were performed with finite-element modeling (FEM) to locate and identify the PDA signal loss due to the wall filter of the US instrument . The performance of three segmentation algorithms used to delineate the vessel lumen on the PDA images was assessed and compared . It is shown that the type of fluid flowing in the phantom affects the echoicity of PDA images and the accuracy of the segmentation algorithms . The type of flow (steady or pulsatile) and the flow rate can also influence the PDA image accuracy, whereas the use of US contrast agent has no significant effect . For the conditions that would correspond to a US scan of a common femoral artery (whole blood flowing at a mean pulsatile flow rate of 450 mL min(-1)), the errors in the percentages of area reduction were 4.3 +/- 1.2% before the stenosis, -2.0 +/- 1.0% in the stenosis, 11.5 +/- 3.1% in the recirculation zone, and 2.8 +/- 1.7% after the stenosis, respectively . Based on the simulated blood flow patterns obtained with FEM, the lower accuracy in the recirculation zone can be attributed to the effect of the wall filter that removes low flow velocities . In conclusion, the small errors reported in vitro may support the clinical use of this technique.

Plast Reconstr Surg, 2001 Jan, 107(1), 97 - 104
Basement membrane formation during wound healing is dependent on epidermal transplants; Andree C et al.; The purpose of the study was to compare directly the effect of healing and the formation of the basement membrane during wound healing from two autologous primary keratinocyte cultures in the liquid environment in full-thickness wounds in pigs . Wounds were either transplanted with cultured epidermal autografts (n = 26) or autologous keratinocyte suspensions (n = 24) or treated with saline alone (n = 40) and covered with a chamber . All wounds transplanted with cultured epidermal autografts and keratinocyte cell suspensions had positive "take" after transplantation . Healing times were significantly shorter for wounds treated with either cultured epidermal autografts or keratinocyte suspensions (p = 0.0001) compared with saline-treated wounds but were not different from each other (p = 0.1835) . There were no differences in cytokeratin and laminin expression; however, staining with monoclonal antibody against collagen type VII showed a lower signal for cultured epidermal autografts only on days 8 and 16 compared with keratinocyte suspensions . Electron microscope evaluation showed a higher incidence of anchoring fibrils and a more mature dermal-epidermal junction in wounds treated with keratinocyte cell suspensions at day 8 . These findings may be due to the single, noncontact-inhibited cells and the early formation of an in vivo neodermis to the wet wound environment . These data suggest that wounds transplanted with autologous keratinocyte suspensions in a wet environment may be an alternative method in the treatment of wounds.

J Biotechnol, 2001 Feb 23, 85(3), 247 - 57
Optimizing production of polyunsaturated fatty acids in Marchantia polymorpha cell suspension culture; Chiou SY et al.; Chlorophyll containing callus cells of Marchantia polymorpha are able to grow under dim illumination in the presence of an organic carbon source and retain the ability to produce polyunsaturated fatty acids (PUFA), including C(20) fatty acids . Highest PUFA production was achieved using 2,4-dichlorophenoxyacetic acid as growth regulator . Inoculum size, illumination intensity, organic carbon source, and ferrous ion are the major factors affecting PUFA productivity . Maximum PUFA productivity is attained under low light intensity, with a photon flux density ca . 20 micromol m(-2) s(-1) . Optimal inoculum size and glucose concentration for PUFA production are 8-12% and 20-30 g l(-1), respectively . Ferrous ion can promote PUFA productivity by increasing the intracellular lipid content . Highest productivities for PUFA, arachidonic acid (ARA), and eicosapentaenoic acid (EPA) were 35.0+/-2.1, 6.7+/-0.4 and 6.6+/-0.4 mg l(-1) day(-1), respectively . PUFA production in the M . polymorpha culture is shown to be strongly growth-associated . Environmental stress (osmotic pressure) is ineffective in promoting PUFA productivity . Chitosan, an elicitor, also has no effect on intracellular PUFA content in cultured M . polymorpha cells.

Biochem Soc Trans, 2000 Dec, 28(6), 827 - 9
Lipoxygenase isoforms in elicitor-treated parsley cell suspension cultures; Noehringer C et al.; Treatment of parsley cell cultures with a fungal elicitor triggered the induction of a lipoxygenase isoform which may be involved in the de novo synthesis of defence-response inducers, such as jasmonic acid or 12-oxo-phytodienoic acid.

Biochem Soc Trans, 2000 Dec, 28(6), 684 - 6
Expression and properties of diacylglycerol acyltransferase from cell-suspension cultures of oilseed rape; Weselake RJ et al.; The expression of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) with predicted molecular mass of 56.9 . kDa (BnDGAT1) was examined using microspore-derived cell suspension cultures of oilseed rape (Brassica napus L . cv Jet Neuf) . As well, a recombinant histidine-tagged N-terminal fragment of BnDGAT1 {BnDGAT1((1-116))His(6)}, which was relatively hydrophilic, was partially characterized . A temporal increase in DGAT activity occurred within a 24 h period following transfer of cells from 6% (w/v) sucrose to 14% (w/v) sucrose . Western blotting indicated that the abundance of BnDGAT1 protein was closely correlated with DGAT activity . BnDGAT1 mRNA also exhibited a temporal increase within the 24 h period following transfer of cells into higher sucrose concentrations, but the transcript level was not closely associated with DGAT activity as BnDGAT1 protein . The fragment BnDGAT1(1-116)His(6) interacted with {1-(14)C}oleoyl-CoA, suggesting that the N-terminal region of BnDGAT1 may have a role in binding cellular acyl-CoA.

J Agric Food Chem, 2001 Jan, 49(1), 146 - 55
Comparative degradation of {14C}-2,4-dichlorophenoxyacetic acid in wheat and potato after Foliar application and in wheat, radish, lettuce, and apple after soil application; Hamburg A et al.; The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) applied foliarly as the 2-ethylhexyl ester (EHE) to wheat and potatoes, to the soil as the dimethylamine (DMA) salt under apple tree canopies, and preplant as the free acid for wheat, lettuce, and radish was studied to evaluate metabolic pathways . Crop fractions analyzed for (14)C residues included wheat forage, straw, and grain; potato vine and tubers; and apple fruit . The primary metabolic pathway for foliar application in wheat is ester hydrolysis followed by the formation of base-labile 2,4-D conjugates . A less significant pathway for 2,4-D in wheat was ring hydroxylation to give NIH-shift products 2,5-dichloro-4-hydroxyphenoxyacetic acid (4-OH-2,5-D), 4-OH-2,3-D, and 5-OH-2,4-D both free and as acid-labile conjugates . The primary metabolic pathway in potato was again ester hydrolysis . 2,4-D acid was further transformed to 4-chlorophenoxyacetic acid and 4-OH-2,5-D . For the soil applications, (14)C residues in the crops were low, and characterization of the (14)C residues indicated association with or incorporation into the biochemical matrix of the tissue . The degradative pathways observed in wheat are similar to those characterized in other intact plant studies but differ from those in studies in wheat cell suspension culture in that no amino acid conjugates were observed.

Biotechnol Prog, 2001 Jan-Feb, 17(1), 89 - 94
Enhanced taxol production and release in Taxus chinensis cell suspension cultures with selected organic solvents and sucrose feeding; Wang C et al.; Suspension culture of Taxus chinensis cells was carried out in aqueous-organic two-phase systems for the production and in situ solvent extraction of taxol (paclitaxel) . Three organic solvents, hexadecane, decanol, and dibutylphthalate, were tested at 5-20% (v/v) in the culture liquid . All of these solvents stimulated taxol release and the yield per cell, though decanol and higher concentrations of the other two solvents depressed biomass growth significantly . Ten percent dibutylphthalate was the optimal solvent for improving taxol production and release with minimal cell growth inhibition . The time of solvent addition to the culture also affected taxol production, with the addition during the late-log growth phase being most favorable . By feeding sucrose to the culture near the stationary growth phase, the cell growth and taxol production period was extended from 27 to 42 days . The combining of the two-phase culture and sucrose feeding increased the taxol yield by about 6-fold compared with the single-phase batch culture, to 36.0 +/- 3.5 mg/L, with up to 63% taxol released . This study shows that in situ solvent extraction combined with nutrient feeding is an effective process strategy for production and recovery of secondary metabolites in plant cell suspension culture.

Cytometry, 2001 Feb 1, 43(2), 101 - 9
Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing; Henegariu O et al.; BACKGROUND: Metaphase spreading is an essential technique for clinical and molecular cytogenetics . Results of classical banding techniques as well as complex fluorescent in situ hybridization (FISH) applications, such as comparative genomic hybridization (CGH) or multiplex FISH (M-FISH), are greatly influenced by the quality of chromosome spreading and pretreatment of the slide prior to hybridization . Materials and Methods Using hot steam and a metal plate with a temperature gradient across its surface, a reproducible protocol for slide preparation, aging, and hybridization was developed . RESULTS: This protocol yields good chromosome spreads from even the most difficult cell suspensions and is unaffected by the environmental conditions . Chromosome spreads were suitable for both banding and FISH techniques common to the cytogenetic laboratory . Chemical aging is a rapid slide pretreatment procedure for FISH applications, which allows freshly prepared cytogenetic slides to be used for in situ hybridization within 30 min, thus increasing analytical throughput and reducing benchwork . Furthermore, the gradually denaturing process described allows the use of fresh biologic material with optimal FISH results while protecting chromosomal integrity during denaturing . CONCLUSION: The slide preparation and slide pretreatment protocols can be performed in any laboratory, do not require specialized equipment, and provide robust results .

Clin Exp Immunol, 2001 Jan, 123(1), 28 - 35
Preliminary studies on the effect of dehydroepiandrosterone (DHEA) on both constitutive and phytohaemagglutinin (PHA)-inducible IL-6 and IL-2 mRNA expression and cytokine production in human spleen mononuclear cell suspensions in vitro; Young DG et al.; In order to gain further insight into the potential immunological benefits of oral administration of DHEA we have examined its effects on the constitutive and PHA-inducible expression by human spleen cell suspensions in vitro of IL-6 and IL-2 . This was studied at both the mRNA and protein levels . The quantification of specific mRNA was undertaken using commercially available quantitative polymerase chain reaction kits . These studies, which were performed on suspensions from six individual spleens, revealed that 10(-5) M DHEA did not impair the expression of IL-6 at either the mRNA or protein level, but may have slightly enhanced the latter . In contrast, IL-2 mRNA levels were increased on most occasions, whilst IL-2 secretion was decreased, albeit slightly . Additional studies revealed that cyclosporin (approx . 10(-5) M) and dexamethasone (10(-7) M) readily inhibited these responses and the production of other cytokines, including interferon-gamma and tumour necrosis factor-alpha . These preliminary studies suggest that high doses of DHEA do not readily inhibit the production of IL-6, and indeed other cytokines, by PHA-stimulated secondary human lymphoid tissue suspensions in vitro . They may also partially explain the meagre immunomodulatory effects noted in some DHEA replacement studies in humans.

Br J Haematol, 2001 Feb, 112(2), 483 - 7
Detection of haemoglobin variants and inference of their functional properties using complete oxygen dissociation curve measurements; Imai K et al.; Complete oxygen dissociation curves for red cell suspensions of three haemoglobinopathies, namely haemoglobin (Hb) H, Hb Koln and Hb Tak/beta thalassaemia diseases, were measured using automatic recording methods . These curves were left-shifted compared with the normal red cell curve and showed a biphasic shape as a result of co-existence of the high and normal affinity haemoglobin components . Computer-assisted simulation of these biphasic curves enabled us to infer the curves for the pure abnormal haemoglobins and their fraction in the total haemoglobin of the red cell . The inferred values of fraction agreed with those determined by haemoglobin type analysis or the literature values . The curve for Hb Koln red cells deviated from the normal red cell curve in the whole range of oxygen saturation, whereas the curve for Hb H was close to the normal curve at the middle and upper portions . This difference in deviation was ascribed to a possible interaction between Hb Koln and Hb A through subunit exchange, and its absence between Hb H and Hb A . The present results indicate that measurement of the complete oxygen dissociation curve is important for the detection of non-interacting variants such as Hb H and is useful for inferring the functional properties of haemoglobin components that are not easily isolated.

Leuk Res, 2001 Feb, 25(2), 115 - 23
Cyclin D1 by flow cytometry as a useful tool in the diagnosis of B-cell malignancies; Elnenaei MO et al.; The translocation (11;14)(q13;q32) and its molecular counterpart the BCL-1 rearrangement are features observed in mantle cell lymphoma (MCL) and less commonly in other B-cell disorders . This rearrangement leads to cyclin D1 overexpression, which may be the main pathogenic event in these tumours and is therefore recognised as a diagnostic marker . We developed a flow cytometry method to detect cyclin D1 overexpression using the monoclonal antibody (MoAb) 5D4, and characterised its frequency in 93 B-cell malignancies . The competitive reverse transcriptase polymerase chain reaction (RT-PCR) for cyclin D1, D2 and D3 was then performed on 40 of these cases to assess the validity of the flow cytometry method . Fluorescence in situ hybridisation (FISH) to detect t(11;14)(q13;q32) was carried out on 31 cases and results were compared with cyclin D1 expression by flow cytometry . Twenty five cases showed cyclin D1 expression using 5D4, including MCL (12/13, 92%), chronic lymphocytic leukaemia (CLL) (4/30), B-prolymphocytic leukaemia (B-PLL) (1/4), splenic lymphoma with villous lymphocytes (SLVL) (4/13), hairy cell leukaemia (HCL) (1/7) and other B-non Hodgkins Lymphoma (B-NHL) (3/15) . There was a good correlation between flow cytometry results and RT-PCR in 36/40 cases (90%), and with FISH for t(11;14) in 25/31 cases (80%) . We concluded that the detection of cyclin D1 expression by flow cytometry in cell suspensions could be applied routinely to the study of B-lymphoproliferative disorders and may be of value for their diagnosis and management.

Plant Sci, 2001 Jan 5, 160(2), 355 - 360
Effects of conditioned medium on activities of PAL, CHS, DAHP synthase (DS-Co and DS-Mn) and anthocyanin production in suspension cultures of Fragaria ananassa; Mori T et al.; A conditioned medium (CM) prepared from cell suspension cultures of strawberry stimulated anthocyanin synthesis . The effect was significantly (P<0.05) greater than that of synthetic medium (SM), with macronutrient concentrations, carbohydrate concentrations and pH adjusted to those of CM . The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) (DS-Mn, DS-Co), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and chalcone synthase (CHS, EC 2.3.1.74) were monitored in the CM- and SM-cultured cells . PAL and CHS activities were found to increase significantly (P<0.05) in the CM-cultured cells . CHS transcript levels were higher in the CM-cultured cells compared to transcript abundance in SM-cultured cells . There was no significant difference in the DS-Mn and DS-Co activities of cells grown in conditioned or synthetic media.

J Maxillofac Surg, 2000 Oct, 28(5), 300 - 307
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 chi(2)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 mm(2)at 10.4+/-1.6 days (Group S) and 243+/-30 mm(2)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 loco-regional 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 .

Cryobiology, 2000 Nov, 41(3), 178 - 94
Red blood cell stabilization reduces the effect of cell density on recovery following cryopreservation; Wagner CT et al.; The relationship between red blood cell hematocrit and hemolysis during cryopreservation has been examined . Cells were frozen with glycerol, thawed, and deglycerolized in a model system based on the protocols used in transfusion medicine . Analysis included determination of hemolysis following thaw (Thaw) and deglycerolization (Overall) and osmotic fragility of the final cell suspensions . Results demonstrate that thaw hemolysis decreased with increasing hematocrit at all glycerol levels tested . Overall hemolysis increased with increasing hematocrit at low (15% w/v) glycerol and decreased with increasing hematocrit at high (40% w/v) glycerol levels . These results were paralleled by changes in the fragility index . Furthermore, these results indicate a distinction between freeze/thaw lysis and damage which leads to lysis during postthaw processing . To examine this further, a biochemical stabilizing solution, having no cryoprotective effects itself, was added to suboptimal glycerol concentrations . This addition resulted in hemolysis levels and fragility indices comparable to those using high (40% w/v) glycerol levels . Thus, the damage observed with increasing hematocrit is not necessarily a function of the packing on the volume of the ice-free zone, but rather an expression of cell damage . Furthermore, this damage is, in part, biochemical in nature and may be protected against through specific cellular stabilization prior to cryopreservation .

J Gen Virol, 2001 Feb, 82(Pt 2), 459 - 64
Banana bunchy top nanovirus DNA-1 encodes the 'master' replication initiation protein; Horser C et al.; Banana bunchy top nanovirus has a multicomponent, circular single-stranded DNA genome comprising at least six integral components, BBTV DNA-1 to -6, which have been consistently associated with bunchy top disease worldwide . At least three other components, BBTV S1, S2 and Y, which have been isolated from Taiwanese BBTV isolates, do not appear to be integral components . We show here that both BBTV DNA-1 and S1, which encode replication initiation (Rep) proteins, were capable of self-replication when bombarded into banana embryogenic cell suspensions . However, only BBTV DNA-1 was capable of directing the replication of two other BBTV genomic components, namely BBTV DNA-3 which encodes the coat protein, and DNA-5 which encodes a retinoblastoma binding-like protein . These results indicate that (i) BBTV DNA-1 is the minimal replicative unit of BBTV and encodes the 'master' viral Rep and (ii) BBTV S1 is possibly a satellite DNA which is unable to replicate integral BBTV components.

Am J Pathol, 2001 Feb, 158(2), 571 - 9
Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells; Wang X et al.; The emergence of cells with hepatocellular properties in the adult pancreas has been described in several experimental models . To determine whether adult pancreas contains cells that can give rise to therapeutically useful and biochemically normal hepatocytes, we transplanted suspensions of wild-type mouse pancreatic cells into syngeneic recipients deficient in fumarylacetoacetate hydrolase and manifesting tyrosinemia . Four of 34 (12%) mutant mice analyzed were fully rescued by donor-derived cells and had normal liver function . Ten additional mice (29%) showed histological evidence of donor-derived hepatocytes in the liver . Previous work has suggested that pancreatic liver precursors reside within or close to pancreatic ducts . We therefore performed additional transplantations using either primary cell suspensions enriched for ducts or cultured ducts . Forty-four mutant mice were transplanted with cells enriched for pancreatic duct cells, but only three of the 34 (9%) recipients analyzed displayed donor-derived hepatocytes . In addition, 28 of the fumarylacetoacetate hydrolase-deficient mice were transplanted with cultured pancreatic duct cells, but no donor-derived hepatocytes were observed . Our results demonstrate for the first time that adult mouse pancreas contains hepatocyte progenitor cells capable of significant therapeutic liver reconstitution . However, contrary to previous reports, we were unable to detect these cells within the duct compartment.

Mol Cell Endocrinol, 2000 Nov 27, 169(1-2), 113 - 5
Cryopreservation of testicular tissue; Hovatta O; Cryopreservation of testicular tissue might benefit prepubertal boys who have to undergo chemotherapy or radiotherapy . Cryopreservation of testicular tissue is feasible . Live offspring have been born to mice, but not other species after transplantation of testicular cells . Spermatogenesis in vitro would be an excellent option for boys with leukaemia, but the method is not feasible for the time being . Cryopreservation of biopsied testicular tissue for intracytoplasmic sperm injection (ICSI) is a feasible option for infertility treatment of azoospermic men . Testicular sperm can be frozen as cell suspension, or within a piece of testicular tissue . Both methods result in pregnancies . Testicular needle biopsy is a simple method to obtain tissue for histological diagnosis, for ICSI and for cryopreservation for use in the future . Testicular sperm should be cryopreserved whenever a testicular biopsy is carried out.

Plant Physiol, 2001 Jan, 125(1), 318 - 28
Free and conjugated benzoic acid in tobacco plants and cell cultures . Induced accumulation upon elicitation of defense responses and role as salicylic acid precursors; Chong J et al.; Salicylic acid (SA) is a key endogenous component of local and systemic disease resistance in plants . In this study, we investigated the role of benzoic acid (BA) as precursor of SA biosynthesis in tobacco (Nicotiana tabacum cv Samsun NN) plants undergoing a hypersensitive response following infection with tobacco mosaic virus or in tobacco cell suspensions elicited with beta-megaspermin, an elicitor from Phytophthora megasperma . We found a small pool of conjugated BA in healthy leaves and untreated cell suspensions of tobacco, whereas free BA levels were barely detectable . Infection of plants with tobacco mosaic virus or elicitation of cells led to a rapid de novo synthesis and accumulation of conjugated BA, whereas free BA was weakly induced . In presence of diphenylene iodonium, an inhibitor of superoxide anion formation, SA accumulation was abolished in elicited cells and much higher BA levels were concomitantly induced, mainly as a conjugated form . Furthermore, piperonylic acid, an inhibitor of cinnamate-4-hydroxylase was used as a powerful tool to redirect the metabolic flow from the main phenylpropanoid pathway into the SA biosynthetic branch . Under these conditions, in vivo labeling and radioisotope dilution experiments with {(14)C}trans-cinnamic acid as precursor clearly indicated that the free form of BA produced in elicited tobacco cells is not the major precursor of SA biosynthesis . The main conjugated form of BA accumulating after elicitation of tobacco cells was identified for the first time as benzoyl-glucose . Our data point to the likely role of conjugated forms of BA in SA biosynthesis.

J Cancer Res Clin Oncol, 2000 Dec, 126(12), 699 - 706
Tumor cell adhesion of human colon carcinoma cells with different metastatic properties to extracellular matrix under dynamic conditions of laminar flow; Haier J et al.; PURPOSE: Shear forces have an important influence on cell adhesion and other cellular functions, and malignant cell lines appear to possess different adhesive properties under static and dynamic conditions . Thus, we analyzed human colon carcinoma cell adhesion under dynamic conditions and examined the interactions of HT-29 colon carcinoma cells of different metastatic properties with various immobilized ECM components . METHODS: Wall shear adhesion threshold (WSAT), dynamic adhesion rate (DAR), and adhesion stabilization rate (ASR) were compared between the cell lines using dynamic conditions in a laminar flow chamber by decreasing the flow (wall shear stress) of cell suspensions . Patterns of cell adhesion under dynamic conditions were compared to adhesive interactions in static microtiterplate assays . RESULTS: Poorly metastatic HT-29P cells adhered six times more than highly metastatic cells to type I collagen under laminar fluid flow, whereas only highly metastatic HT-29LMM showed adhesive interactions with fibronectin under static and dynamic conditions . High rates of cell adhesion to collagen IV were found under static, but not under dynamic, conditions . CONCLUSIONS: Although poorly and highly metastatic HT-29 cells express similar patterns of integrins, they differ in their adhesive properties to ECM components under static and dynamic conditions . Hydrodynamic shear forces appear to influence adhesive properties of HT-29 cells, and differences between dynamic and static cell adhesion were found.

Biochem, Eng . J. . 2001 Jan, 7(1), 69 - 78
Perfluorocarbon-mediated aeration applied to recombinant protein production by virus-infected insect cells; Gotoh T et al.; Perfluorocarbon (PFC) was used as an oxygen carrier in the cultures of insect cells and virus-infected insect cells . The cell suspensions were placed on a planar layer of PFC, which was re-oxygenated in an outer aeration unit and continuously recirculated, and were agitated by two sets of impeller blades, lower one of which was set in such a way that the ridge of the blade touched the PFC layer . The maximum cell density attained in the PFC-mediated aeration culture was higher than that in surface aeration culture . On viral infection, a recombinant protein yield was significantly high in the PFC-mediated aeration culture as compared with that in the surface aeration culture, though the production was largely decreased by setting apart the lower set of the blade from the PFC-medium interface . These results showed that the PFC-mediated aeration would be a useful technique for insect cell/baculovirus expression system . Overall mass-transfer coefficient K(L) for oxygen was examined in both the PFC-mediated aeration and surface aeration systems, by using a flask whose dimensions were identical to those of spinner flasks used for the cultures . The K(L) value in the PFC-mediated system was 2.60x10(-3)cms(-1), 1.6 times higher than that in the surface aeration system, when impeller blades were positioned at PFC-medium and medium-air interfaces, respectively . However, the K(L) values in both the PFC-mediated and surface aeration systems were decreased and their differences were brought so close, as the blade was set apart from the interfaces . DO behavior in the cultures was well explained by the model calculation using the determined K(L) values and oxygen-consumption rates of viable cells . This calculation further suggested that crucial DO, under which recombinant protein productions were unsuccessful, was 0.24-0.5ppm (3-7%) in the insect cell/baculovirus expression system.

Cell Stress Chaperones, 2000 Apr, 5(2), 113 - 20
Cellular and biochemical responses to environmental and experimentally induced stress in sea urchin coelomocytes; Matranga V et al.; Coelomocytes are considered to be immune effectors of sea urchins . Subpopulations of coelomocytes can be purified from a total cell suspension . The proportion of each cell type can vary not only among species, but also between individuals of the same species, according to their size and physiological conditions . We tested the hypothesis that coelomocytes play a role in defense mechanisms activated by adverse external conditions . Total coelomocytes from control and stressed (temperature, pollution, and injuries) sea urchins were analyzed for their expression of the 70 kDa heat shock protein (hsp70), a well recognized stress marker . Further analysis was performed by separation of coelomocytes into subpopulations by step gradients . We demonstrated that sea urchin coelomocytes respond to temperature shock and to polluted seawater by the upregulation of hsp70 . Among coelomocytes certain cells, known as red spherula cells, showed a great increase in number in animals collected from polluted seawaters or subjected to "accidental" injury . The present study confirms the immunological function of sea urchin coelomocytes, as indicated by the upregulation of the hsp70 molecular marker, and suggests that sea urchin coelomocytes can be utilized as sensitive bio-indicators of environmental stress.

Cell Transplant, 2000 Sep-Oct, 9(5), 577 - 84
Grafting of nigral tissue hibernated with tirilazad mesylate and glial cell line-derived neurotrophic factor; Petersen A et al.; Transplantation of embryonic ventral mesencephalon is a potential therapy for patients with Parkinson's disease . As only around 5-10% of embryonic dopaminergic neurons survive grafting into the adult striatum, it is considered necessary to use multiple donor embryos . To increase the survival of the grafted dopaminergic neurons, the clinical transplantation program in Lund currently employs the lipid peroxidation inhibitor, tirilazad mesylate, in all solutions used during tissue storage, preparation, and transplantation . However, the difficulty in obtaining a sufficient number of donor embryos still remains an important limiting factor for the clinical application of neural transplantation . In many clinical transplantation programs, it would be a great advantage if human nigral donor tissue could be stored for at least 1 week . This study was performed in order to investigate whether storage of embryonic tissue at 4 degrees C for 8 days can be applied clinically without creating a need to increase the number of donors . We compared the survival of freshly grafted rat nigral tissue, prepared according to the clinical protocol, with tissue transplanted after hibernation . Thus, in all groups tirilazad mesylate was omnipresent . One group of rats was implanted with fresh tissue and three groups with hibernated tissue with or without addition of glial cell line-derived neurotrophic factor (GDNF) in the hibernation medium and/or the final cell suspension . Earlier studies have suggested that GDNF improves the survival of hibernated nigral transplants . We found no statistically significant difference between the groups regarding graft survival after 3 weeks . However, there was a nonsignificant trend for fewer surviving dopaminergic neurons in grafts from hibernated tissue compared to fresh controls . Furthermore, we show that the addition of GDNF to the hibernation medium and/or to the final cell suspension does not significantly increase the survival of the dopaminergic neurons.

Amino Acids, 2000, 19(3-4), 687 - 703
Modulation of taurine uptake in the goldfish retina and axonal transport to the tectum . Effect of crushing the optic nerve or axotomy; Guerra A et al.; Although there are a great number of studies concerning the uptake of taurine in several tissues, the regulation of taurine transport has not been studied in the retina after lesioning the optic nerve . In the present study, isolated retinal cells of the goldfish retina were used either immediatly after cell suspension or in culture . The high-affinity transport system of {3H}taurine in these cells was sodium-, temperature- and energy-dependent, and was inhibited by hypotaurine and beta-alanine, but not by gamma-aminobutyric acid . There was a decrease in the maximal velocity (Vmax) without modifications in the substrate affinity (Km) after optic axotomy . These changes were mantained for up to 15 days after the lesion . The results might be the summation of mechanisms for providing extracellular taurine to be taken up by other retinal cells or eye structures, or regulation by the substrate taurine, which increases after lesioning the optic nerve . The in vivo accumulation of {3H}taurine in the retina after intraocular injection of {3H}taurine was affected by crushing the optic nerve or by axotomy . A progressive retinal decrease in taurine transport was observed after crushing the optic nerve, starting at 7 hours after surgery on the nerve . The uptake of {3H}taurine by the tectum was compensated in the animals that were subjected to crushing of the optic nerve, since the concentration of {3H}taurine was only different from the control value 24 hours after the lesion, indicating an efficient transport by the remaining axons . On the contrary, the low levels of {3H}taurine in the tectum after axotomy might be an index of the non-axonal origin of taurine in the tectum . Axonal transport was illustrated by the differential presence of {3H}taurine in the intact or crushed optic nerve . The uptake of {3H}taurine into retinal cells in culture in the absence or in the presence of taurine might indicate the existence of an adaptive regulation of taurine transport in this tissue, however taurine transport probably differentially occurs in specific populations of retinal cells . The use of a purified preparation of cells might be useful for future studies on the modulation of taurine transport by taurine in the retina and its role during regeneration.

Photochem Photobiol, 2000 Dec, 72(6), 780 - 7
Eradication of multiple myeloma and breast cancer cells by TH9402-mediated photodynamic therapy: implication for clinical ex vivo purging of autologous stem cell transplants; Brasseur N et al.; High-dose chemotherapy combined with autologous transplantation using bone marrow or peripheral blood-derived stem cells (PBSC) is now widely used in the treatment of hematologic malignancies as well as some solid tumors like breast cancer (BC) . However, some controversial results were recently obtained in the latter case . The presence of malignant cells in the autograft has been associated with the recurrence of the disease, and purging procedures are needed to eliminate this risk . The aim of this study was to evaluate the potential of the photosensitizer 4,5-dibromorhodamine methyl ester (TH9402), a dibrominated rhodamine derivative, to eradicate multiple myeloma (MM) and BC cell lines, while sparing more than 50% of normal pluripotential blood stem cells from healthy volunteers . The human BC MCF-7 and T-47D and MM RPMI 8226 and NCI-H929 cell lines were used to optimize the photodynamic purging process . Cell concentration and the cell suspension thickness as well as the dye and light doses were varied in order to eventually treat 1-2 L of apheresis . The light source consisted of two fluorescent scanning tubes emitting green light centered about 515 nm . The cellular uptake of TH9402 was measured during the incubation and washout periods and after photodynamic treatment (PDT) using spectrofluorometric analysis . The limiting dilution assay showed that an eradication rate of more than 5 logs is obtained when using a 40 min incubation with 5-10 microM dye followed by a 90 min washout period and a light dose of 5-10 J/cm2 (2.8 mW/cm2) in all cell lines . Agitating the 2 cm thick cell suspension containing 20 x 10(6) cells/mL during PDT was essential for maximal photoinactivation . Experiments on mobilized PBSC obtained from healthy volunteers showed that even more drastic purging conditions than those found optimal for maximal eradication of the malignant cell lines were compatible with a good recovery of hematopoietic progenitors cells . The absence of significant toxicity towards normal hematopoietic stem cells, combined with the 5 logs eradication of cancer cell lines induced by this procedure suggests that TH9402 offers an excellent potential as an ex vivo photodynamic purging agent for autologous transplantation in MM and BC treatment.

Environ, Toxicol . Pharmacol. . 2000 Dec, 9(1-2), 49 - 55
Toxicity of methylmercury conjugated with L-cysteine on rat thymocytes and human leukemia K562 cells in comparison with that of methylmercury chloride; Oyama Y et al.; In order to reveal the implication of use of methylmercury chloride (MeHgCl) in in vitro study, the effects of 10 microM MeHgCl on rat thymocytes and human leukemia K562 cells were compared with those of methylmercury conjugated with L-cysteine (10 microM MeHg-Cys) using a flow cytometer and fluorescent probes to monitor cellular physiological and pathological parameters . MeHgCl hyperpolarized membranes of thymocytes, followed by depolarization within a few minutes after the application, while MeHg-Cys persistently hyperpolarized them . MeHgCl increased intracellular concentration of Ca(2+), decreased cellular content of glutathione and increased generation of superoxide anion in the cells . The effects of MeHg-Cys were much less than those of MeHgCl . MeHgCl greatly increased both numbers of the cells undergoing apoptosis and dead cells in cell suspension containing thymocytes, while this was not the case for MeHg-Cys . MeHgCl reduced the cell viability of human leukemia K562 cells and completely inhibited the cell growth . The effects of MeHg-Cys on K562 cells were less than those of MeHgCl . It can be concluded that the effects of MeHgCl on rat thymocytes and K562 cells are different from those of MeHg-Cys . The results obtained from the in vitro studies using MeHgCl may be less implicit to elucidate the mechanism of MeHg intoxication in humans and experimental animals because MeHg are present in forms of MeHg-Cys and/or MeHg-S conjugate under the in vivo conditions.

Parasite Immunol, 2001 Jan, 23(1), 19 - 25
Dissemination of Encephalitozoon intestinalis, a causative agent of human microsporidiosis, in IFN-gamma receptor knockout mice; El Fakhry Y et al.; The dissemination of Encephalitozoon intestinalis, a microsporidium causing intestinal diseases and systemic infection in humans, was investigated in IFN-gamma Ro/o mice . Although lesions were seen in organs of autopsied animals, the parasites were rarely detected using histological examination . Nevertheless, infection of the duodenum, liver, kidneys and lungs was demonstrated by polymerase chain reaction . This method also enabled the detection of the parasite in the brain and the heart . The development of E . intestinalis in RK13 cell cultures to which cell suspensions from liver, kidney, lung or brain of infected IFN-gamma Ro/o mice were added, confirmed the spread of intestinal microsporidiosis to these organs . No dissemination was observed in wild-type mice . These results confirm those of previous studies and emphasize the low morbidity of the infection in IFN-gamma Ro/o mice and confirm the role of IFN-gamma in the control of E . intestinalis infection . These mice infected with E . intestinalis offer important information about this interesting and important parasitic disease of man and animals.

J Clin Endocrinol Metab, 2000 Dec, 85(12), 4728 - 33
Development-related increase in cortisol biosynthesis by human granulosa cells; Yong PY et al.; Antiinflammatory mechanisms are important in ovulation and may be regulated by cortisol (F) . We previously showed that after administration of human (h)CG for ovulation induction, luteinized granulosa cells (LGC) abundantly express 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) messenger RNA but not 11betaHSD type 2 (11betaHSD2) messenger RNA . 11ssHSD1 is responsible for the reversible formation of antiinflammatory F from its inactive precursor cortisone (E), whereas 11betaHSD2 unidirectionally converts F to E through 11-oxidation . This pattern of gene expression predicts that LGC from periovulatory follicles would show increased activation of E to F, compared with granulosa cells from immature follicles (IGC), and that follicular fluid concentrations of E and F would alter accordingly . To test this hypothesis, we isolated IGC, thecal cells (TC), and follicular fluid, from ovaries of cyclic women, removed during surgery for benign gynecological disease . LGC and follicular fluid were aspirated from periovulatory follicles, 35 h after hCG injection, in patients undergoing in vitro fertilization treatment . In an 11betaHSD assay based on interconversion of tritiated E and F by cell suspensions in vitro, IGC (% conversion, 0.6 +/- 0.4, mean +/- SEM) and collagenase-dispersed TC (0.2 +/- 0.1%) were unable to convert E to F, whereas LGC (36.3 +/- 3.7%) were highly efficient at this reaction . Immature granulosa cells, LGC, and (to a lesser extent) TC were all able to convert F to E . Correspondingly, follicular fluid concentrations of total F and F:E ratios were significantly higher in periovulatory follicles, compared with immature follicles . Culturing IGC for 48 h in the presence of hFSH resulted in increased 11betaHSD1 reductase activity, paralleling stimulation of estrogen (aromatase activity) and progesterone biosynthesis . Similar treatment with hLH did not influence 11betaHSD1 reductase activity, except in a patient with more mature IGC, which also showed a significant increase in E-to-F conversion, as well as progesterone synthesis in response to hLH . These data confirm that 11betaHSD activity in the human ovary is developmentally regulated and gonadotropin responsive, favoring metabolism of F to E in immature follicles and E to F in periovulatory follicles . Increased formation of F by LGC in periovulatory follicles is consistent with an antiinflammatory function for this glucocorticoid at ovulation.

Br Poult Sci, 2000 Sep, 41(4), 494 - 501
Effect of luteinising-hormone in vivo on ovarian cell subpopulations of newly-hatched chicks; Gonzalez-Moran G et al.; 1 . We analysed the number and size of different ovarian cell subpopulations of newly-hatched chicks by ovarian cell suspension count and morphometric/stereological methods as well as delta5-3beta-hydroxysteroid dehydrogenase (delta5-3beta HSD) activity in these cells treated in vivo with LH during embryonic development . 2 . Fertile White Leghorn eggs received 1 microg LH applied to the chorioallantoic membrane on days 13, 15, and 17 of incubation . All animals were killed within 24 h after hatching, the left ovary was dissected and processed . 3 . The results indicate that the number of germ, pregranulosa, interstitial and undifferentiated cells was not affected by LH treatment . However, we observed an increase in the size of individual interstitial cells of the ovarian medulla . In these cells, delta5-3beta HSD activity was increased in response to LH . 4 . These findings suggest that LH does not exert a proliferative effect on the cells of the prefollicular ovary of the chick and that interstitial cells can be target cells for LH, increasing their steroidogenic activity due to LH treatment.

Histochem J, 2000 Sep, 32(9), 535 - 43
Efficiency of doxorubicin handling by isolated hepatocytes is a valuable indicator for restored cell function; Crivellato E et al.; Pig liver is a possible source of hepatocytes for extracorporeal bio-artificial liver devices . In order to evaluate recovered hepatocyte function following enzymatic isolation, we developed a cytochemical method that is based on the capacity of hepatocytes to sequester the anthracycline antitumour drug doxorubicin within intracellular acidic compartments . Doxorubicin is a naturally fluorescent molecule . Thus, the process of drug concentration within hepatocytes can be visualized in living conditions by fluorescence microscopy . Porcine hepatocytes harvested from heart-beating donors were grown either as isolated cell suspensions or as tissue monolayers . Immediately after isolation and at fixed culture times, cells were incubated with 0.1 mM doxorubicin in Hanks' balanced salt solution for 10 min at 37 degrees C in 5% CO2-humidified atmosphere and observed by fluorescence microscopy . Parallel electron microscopy was performed to compare fluorescence data with general cell morphology . To monitor lysosomal acidification capacity, the fluorescent pH-sensitive vital dye LysoSensor-Blue was used . Doxorubicin fluorescence showed different patterns of nuclear and cytoplasmic staining, according to the time allowed for cell recovery and the culture method . In particular, cytoplasmic fluorescence changed from a diffuse staining, that could be observed after cell isolation and in hepatocyte suspensions, to a punctate perinuclear and pericanalicular fluorescence detectable in fully recovered hepatocyte monolayers . This study indicates that the 'doxorubicin-fluorescence test' may be considered a simple and rapid procedure for assessing hepatocyte functional condition . It may provide valuable and 'real time' guidelines for judging the correct way these cells are to be collected, preserved and utilized for clinical purposes.

Mol Pharmacol, 2001 Jan, 59(1), 54 - 61
"cAMP-specific" phosphodiesterase contributes to cGMP degradation in cerebellar cells exposed to nitric oxide; Bellamy TC et al.; Nitric oxide (NO) functions as a diffusible messenger in the central nervous system and elsewhere, exerting many of it physiological effects by activating soluble guanylyl cyclase, so increasing cellular cGMP levels . Hydrolysis of cyclic nucleotides is achieved by phosphodiesterases (PDEs) but the enzyme isoforms responsible for degrading cGMP in most cells have not been identified . We have devised a method for quantitatively monitoring the rate of breakdown of cGMP within intact cells and have applied it to rat cerebellar cell suspensions previously stimulated with NO . In contrast to previous findings in cultured cerebellar cells, there was no evidence from the use of selective inhibitors that PDE 1 participated importantly in cGMP hydrolysis . Moreover, procedures expected to increase PDE 1 activity by raising cytosolic Ca2+ concentrations (neurotransmitter agonists, Ca2+ ionophore) failed to influence cGMP breakdown . Instead, through the use of inhibitors selective for different PDE families, two isoforms were implicated: a "cGMP-specific" PDE (PDE 5), inhibited by sildenafil and zaprinast, and a "cAMP-specific" PDE (PDE 4), inhibited by low concentrations of rolipram and Ro-20-1724 and by milrinone . An explanation is offered for a participation of PDE 4 based on the high estimated intracellular cGMP concentration (approximately 800 microM) and the low affinity of the enzyme for cGMP . In accordance with predictions, recombinant PDE 4 was shown to hydrolyze high cGMP concentrations in a rolipram-sensitive manner . The widespread use of rolipram to test for a specific involvement of cAMP in cellular phenomena must therefore be questioned.

Am J Physiol Heart Circ Physiol, 2001 Jan, 280(1), H222 - 36
Effect of erythrocyte aggregation on velocity profiles in venules; Bishop JJ et al.; A recent whole organ study in cat skeletal muscle showed that the increase in venous resistance seen at reduced arterial pressures is nearly abolished when the muscle is perfused with a nonaggregating red blood cell suspension . To explore a possible underlying mechanism, we tested the hypothesis that red blood cell aggregation alters flow patterns in vivo and leads to blunted red blood cell velocity profiles at reduced shear rates . With the use of fluorescently labeled red blood cells in tracer quantities and a video system equipped with a gated image intensifier, we obtained velocity profiles in venous microvessels (45-75 microm) of rat spinotrapezius muscle at centerline velocities between 0.3 and 14 mm/s (pseudoshear rates 3-120 s(-1)) under normal (nonaggregating) conditions and after induction of red blood cell aggregation with Dextran 500 . Profiles are nearly parabolic (Poiseuille flow) over this flow rate range in the absence of aggregation . When aggregation is present, profiles are parabolic at high shear rates and become significantly blunted at pseudoshear rates of 40 s(-1) and below . These results indicate a possible mechanism for increased venous resistance at reduced flows.

J Immunol, 2000 Dec 15, 165(12), 7308 - 15
Adoptive T cell immunotherapy of human uveal melanoma targeting gp100; Sutmuller RP et al.; HLA-A*0201-restricted CTL against human gp100 were isolated from HLA-A*0201/K(b) (A2/K(b))-transgenic mice immunized with recombinant canarypox virus (ALVAC-gp100) . These CTL strongly responded to the gp100(154-162) epitope, in the context of both the chimeric A2/K(b) and the wild-type HLA-A*0201- molecule, and efficiently lysed human HLA-A*0201(+), gp100(+) melanoma cells in vitro . The capacity of the CTL to eradicate these tumors in vivo was analyzed in A2/K(b)-transgenic transgenic mice that had received a tumorigenic dose of human uveal melanoma cells in the anterior chamber of the eye . This immune-privileged site offered the unique opportunity to graft xenogeneic tumors into immunocompetent A2/K(b)-transgenic mice, a host in which they otherwise would not grow . Importantly, systemic (i.v.) administration of the A2/K(b)-transgenic gp100(154-162)-specific CTL resulted in rapid elimination of the intraocular uveal melanomas, indicating that anti-tumor CTL are capable of homing to the eye and exerting their tumoricidal effector function . Flow cytometry analysis of ocular cell suspensions with HLA-A*0201-gp100(154-162) tetrameric complexes confirmed the homing of adoptively transferred CTL . Therefore, the immune-privileged state of the eye permitted the outgrowth of xenogeneic uveal melanoma cells, but did not protect these tumors against adoptive immunotherapy with highly potent anti-tumor CTL . These data constitute the first direct indication that immunotherapy of human uveal melanoma may be feasible.

J Anat, 2000 Oct, 197 Pt 3, 495 - 502
Mouse granulated metrial gland cells require contact with stromal cells to maintain viability; Stewart IJ; Granulated metrial gland (GMG) cells differentiate in the uterine wall in pregnancy in mice but the mechanisms which control their differentiation and maintenance are unknown . In vivo, GMG cells share an intimate association with fibroblast-like stromal cells . The importance of this association has been assessed by examining the effects of withdrawal of stromal cell contact on GMG cell maintenance in vitro . When single cell suspensions of cells were prepared from mouse metrial glands there was a steady decline in numbers with days of culture but usually some remained at 7 d of culture . The ability of metrial gland cells to kill Wehi 164 target cells in 51Cr-release cytotoxicity assays was retained by cells cultured for at least 3 d . When explants of metrial gland were maintained in culture to allow GMG cell migration onto the culture flask, the attached GMG cells were lost by 1 d later . Overall, these results suggest that a juxtacrine regulatory mechanism maintains GMG cells . The rapid loss of unsupported GMG cells in culture has major implications in the design of assays to examine GMG cell function.

Plant Mol Biol, 2000 Sep, 44(2), 231 - 43
Arabidopsis ATP A2 peroxidase . Expression and high-resolution structure of a plant peroxidase with implications for lignification; Ostergaard L et al.; Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack . Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown to be involved in lignin biosynthesis . Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA . Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants . Moreover, an Arabidopsis mutant with increased lignin levels compared to wild type shows increased levels of ATP A2 mRNA and of a mRNA encoding an enzyme upstream in the lignin biosynthetic pathway . The substrate specificity of ATP A2 was analysed by X-ray crystallography and docking of lignin precursors . The structure of ATP A2 was solved to 1.45 A resolution at 100 K . Docking of p-coumaryl, coniferyl and sinapyl alcohol in the substrate binding site of ATP A2 were analysed on the basis of the crystal structure of a horseradish peroxidase C-CN-ferulic acid complex . The analysis indicates that the precursors p-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139 . We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant cell wall.

J Exp Bot, 2000 Nov, 51(352), 1799 - 811
The fungal elicitor cryptogein induces cell wall modifications on tobacco cell suspension; Kieffer F et al.; Upon addition of the fungal elicitor cryptogein, suspension cells of tobacco (Nicotiana tabacum cv . Xanthi) aggregated in clusters . Cytochemical experiments indicated that elicited cells displayed fibrillar expansions of pectin along the primary cell wall . Immunocytochemical detection of pectin epitopes indicated that the fibrillar material surrounding the treated cells was mostly composed of low methylated galacturonan sequences, but the use of the cationic probe did not reveal the presence of negatively charged carboxyl groups: the presence of important amounts of calcium ions in these pectic fibrillar expansions accounts for these observations . These data indicate that tobacco cells treated with cryptogein show a cell wall altered by the presence of a calcium pectate gel, resulting from the reorganization of pectin in the middle lamellae . These results are consistent with a drastic reduction in wall digestibility, partially reversed by increasing the pectolyase concentration in the hydrolytic solution . Diphenylene iodonium, an inhibitor of the oxidative burst triggered by cryptogein on tobacco cells, partially prevents elicited cell walls from this loss of digestibility, suggesting a possible role of active oxygen species in the cell wall strengthening . This work represents a new element of the signal transduction cascade triggered on tobacco cells by cryptogein.

Mutat Res, 2001 Jan 5, 461(4), 301 - 9
Endonuclease V of Escherichia coli prevents mutations from nitrosative deamination during nitrate/nitrite respiration; Weiss B; Endonuclease V (Endo V) of Escherichia coli participates in the excision repair of hypoxanthine and xanthine (deaminated adenine and guanine) in DNA . It thereby reduces the mutagenic effects of nitrous acid by attacking lesions caused by nitrosative deamination . Nitrosating agents may be produced endogenously when E . coli is grown in oxygen-poor cultures, during which nitrate and nitrite replace oxygen as preferred electron acceptors . In this study, the protective effect of Endo V was observed under such conditions . During micro-aerobic growth, an nfi (Endo V) mutation enhanced the frequency of nitrate- and nitrite-induced A:T-->G:C and G:C-->A:T transition mutations, which are consistent with a defect in the removal of DNA hypoxanthine and xanthine, respectively . Similar effects were observed in saturated, aerobic cultures but not in well-aerated, logarithmically growing ones . A narG (nitrate reductase) mutation blocked the mutagenesis of the nfi mutant by nitrate but not by nitrite . These results differed from those of previous studies in which cell suspensions generated an exogenous nitrosating agent from nitrite, but not from nitrate, in a reaction that was narG-dependent . Nitrate/nitrite metabolism is also known to generate endogenous alkylating agents through N-nitrosation . However, an nfi mutation did not appreciably enhance mutagenesis by N-methyl-N-nitrosourea, suggesting that the mutator effect of nfi is not due to a defect in alkylation repair . The overall results indicate that Endo V functions during normal growth by helping to repair nitrosatively deaminated bases in DNA, which are by-products of anaerobic nitrate/nitrite respiration.

Microbiology, 2000 Dec, 146 Pt 12, 3109 - 18
The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity; Lloyd D et al.; Trophozoites of the microaerophilic flagellate parasitic protozoon Giardia intestinalis have only a limited capacity to detoxify O(2) . Thus, when exposed to controlled concentrations of dissolved O(2) >8 microM, they gradually lose their ability to scavenge O(2) . In a washed cell suspension stirred under 10% air in N(2) (equivalent to 25 microM O(2)), inactivation of the O(2)-consuming system was complete after 3.5 h; during this period accumulation of H(2)O(2) (3 micromol per 10(6) organisms) and oxidation of cellular thiols to 16% of their initial level occurred . Under 20% air (50 microM O(2)), respiratory inactivation was complete after 1.5 h, and under air (258 microM O(2)), after 50 min . Loss of O(2)-consuming capacity was accompanied by loss of motility . Use of the fluorogen 2, 7-dichlorodihydrofluorescein acetate indicated that intracellular H(2)O(2) is produced at extranuclear sites . Flow cytometric estimation of the plasma membrane electrochemical potentials using bis(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC(4)(3), showed that values declined from -134 mV to -20 mV after 4.5 h aeration . Incubation of organisms with 60 microM H(2)O(2) for 10 min gave partial collapse of plasma membrane potential and complete loss of O(2) uptake capacity; motility and viability as assessed by DiBAC(4)(3) exclusion were completely lost after 1 h . Inactivation of the O(2)-consuming system and loss of viability were also observed on exposure to singlet oxygen photochemically generated from rose bengal or toluidine blue.

Neuroscience, 2000, 100(3), 521 - 30
Enhancement of nigral graft survival in rat brain with the systemic administration of synthetic fibronectin peptide V; Duan WM et al.; A major obstacle in neural transplantation is a severe loss of neurons in grafts soon after implantation . In the present study, we have investigated whether the systemic administration of synthetic fibronectin peptide V can increase the survival of neural grafts . Synthetic fibronectin peptide V is derived from the 33,000 mol . wt carboxyl-terminal heparin-binding domain of fibronectin . Previous studies have shown that these polypeptides possess anti-inflammatory properties . However, it is currently unknown whether this peptide has anti-apoptotic properties . Dissociated neural grafts were prepared from the ventral mesencephalon of pregnant Sprague-Dawley rats and were stereotaxically injected as a cell suspension into the striatum of adult Sprague-Dawley rats . A group of recipient rats received i.v . injections of peptide V (5mg/kg, dissolved in saline) at 24 and 4h prior to transplantation, at the time of transplantation, and 24, 48 and 72h post-transplantation . Saline-treated rats served as controls . The rats were killed at two, four and 42 days post-grafting and the brain tissue was immunologically processed for tyrosine-hydroxylase, major histocompatibility complex class I and class II antigens, complement receptor type 3 and leukocyte common antigen immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay . We found a significant increase (approximately twofold) in the number of dopamine neurons in the grafts for the peptide-treated group at four and 42 days compared with the controls . In contrast, there was no significant difference in the patterns of inflammation using different immunocytochemical markers in the two different groups . The levels of expression for these markers, however, were reduced over time . Interestingly, the number of apoptotic cells in the graft areas was significantly smaller in the peptide-treated group than in the control group two days after grafting.The results demonstrate that the systemic administration of synthetic fibronectin peptide V can dramatically increase the survival of nigral grafts in the brain and substantially reduce the number of apoptotic cells in the graft site, suggesting that this peptide may exert a beneficial effect on survival of nigral grafts through an anti-apoptotic mechanism.

Appl Environ Microbiol, 2000 Dec, 66(12), 5329 - 33
Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture; Annweiler E et al.; Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture . Metabolite analyses revealed two groups of degradation products . The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds . Naphthyl-2-methyl-succinic acid accumulated to 0.5 microM in culture supernatants . Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions . The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 +/- 0.003 nmol min(-1) mg of protein(-1) only in the presence of cells and fumarate . We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation . The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid . These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture . A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.

Invest Ophthalmol Vis Sci, 2000 Dec, 41(13), 4268 - 74
Incorporation and differentiation of hippocampus-derived neural stem cells transplanted in injured adult rat retina; Nishida A et al.; PURPOSE: In a previous study it has been shown that adult rat hippocampus-derived neural stem cells can be successfully transplanted into neonatal retinas, where they differentiate into neurons and glia, but they cannot be transplanted into adult retinas . In the current study, the effect of mechanical injury to the adult retina on the survival and differentiation of the grafted hippocampal stem cells was determined . METHODS: Mechanical injury was induced in the adult rat retina by a hooked needle . A cell suspension (containing 90,000 neural stem cells) was slowly injected into the vitreous space . The specimens were processed for immunohistochemical studies at 1, 2, and 4 weeks after the transplantation . RESULTS: In the best case, incorporation of grafted stem cells was seen in 50% of the injured retinas . Most of these cells located from the ganglion cell layer through the inner nuclear layer close to the injury site . Immunohistochemically, at 1 week, more than half of the grafted cells expressed nestin . At 4 weeks, some grafted cells showed immunoreactivity for microtubule-associated protein (MAP) 2ab, MAP5, and glial fibrillary acidic protein (GFAP), suggesting progress in differentiation into cells of neuronal and astroglial lineages . However, they showed no immunoreactivity for HPC-1, calbindin, and rhodopsin, which suggests that they did not differentiate into mature retinal neurons . Immunoelectron microscopy revealed the formation of synapse-like structures between graft and host cells . CONCLUSIONS: By the manipulation of mechanical injury, the incorporation and subsequent differentiation of the grafted stem cells into neuronal and glial lineage, including the formation of synapse-like structures, can be achieved, even in the adult rat retina.

Melanoma Res, 2000 Oct, 10(5), 445 - 50
Establishment of IPC 227 cells as human xenografts in rabbits: a model of uveal melanoma; Bonicel P et al.; This study was designed in order to evaluate the feasibility of establishing an animal model of human uveal melanoma . IPC227, a cell line established from the biopsy of a patient with a spindle cell ciliary body melanoma, was transplanted into the anterior chamber of the eye in immunosuppressed New Zealand rabbits . In a second step, a tumour fragment from the anterior chamber was implanted transclerally into the posterior choroid . Complete ophthalmological examinations were then performed on the animals . Characteristic growth patterns were noted depending on the location of implantation . In the anterior chamber, diffuse, flat, heavily pigmented tumours appeared 8 days after the injection of the cell suspension that covered the iris and the angle by day 25, with a success rate of 100% . Nodular, lightly pigmented tumours were obtained 6-7 weeks after subchoroidal implantation, with a 25% success rate . Clinical examination, including fundus photography, ultrasound and magnetic resonance imaging, demonstrated the same characteristics as those of human uveal melanoma, confirming the value of this model for the evaluation of new therapeutic and diagnostic methods in the management of uveal melanoma.

Biosci Rep, 2000 Jun, 20(3), 167 - 76
Culture and function of electrofusion-derived clonal insulin-secreting cells immobilized on solid and macroporous microcarrier beads; Hamid M et al.; In view of the advantages of the bulk production of clonal pancreatic beta cells, an investigation was made of the growth and insulin secretory functions of an electrofusion-derived cell line (BRIN-BD11) immobilized on a solid microcarrier, cytodex-1 or a macroporous microcarrier, cultispher-G . For comparison, similar tests were performed using BRIN-BD11 cells present in single cell suspensions or allowed to form pseudoislets . Similar growth profiles were recorded for each microcarrier with densities of 4.4 x 10(5) +/- 0.3 cells/ml and 4.2 x 10(5) +/- 0.2 cells/ml achieved using cytodex-1 and cultispher-G, respectively . Cell viability began to decline on day 5 of culture . Insulin concentration in the culture medium reached a peak of 26 +/- 2.0 ng/ml and 24 +/- 2.2 ng/ml for cells grown on cytodex-1 and cultispher-G, respectively . Cells grown on both types of microcarrier showed a significant 1.5-1.8-fold acute insulin-secretory response to 16.7 mmol/l glucose . L-alanine (10 mmol/l) and L-arginine (10 mmol/l) also induced significant 3 4 fold increases of insulin release . BRIN-BD11 cells immobilized on cytodex-1 or cultispher-G out-performed single cell suspensions and pseudoislets in terms of insulin-secretory responses to glucose and amino acids . A 1.3-fold, 2.2-fold and 1.7-fold stimulation of insulin secretion was observed for glucose, L-alanine and L-arginine respectively in single cell suspensions . Corresponding increases for pseudoislets were 1.6-1.8-fold for L-alanine and L-arginine, with no significant response to glucose alone . These data indicate the utility of micro-carriers for the production of functioning clonal beta cells.

Int J Cancer, 2000 Dec 15, 88(6), 956 - 61
T cell responses in colorectal cancer patients: evidence for class II HLA-restricted recognition of shared tumor-associated antigens; Bremers AJ et al.; Few cases of anti-colon cancer specific T lymphocytes have been described so far . Moreover, the majority of these effectors were generated in vitro by stimulating PBMC from patients or healthy donors with peptides that were derived from proteins expressed and/or secreted by colon cancer tissue such as CEA, Mucin or Her-2/neu . The aim of our study was to evaluate the immunogenicity of colorectal carcinomas in an autologous setting . We exploited the antigen processing and presentation capacity of dendritic cells (DC) to establish an in vitro autologous system that can bypass the need of obtaining cultured tumor cells . DC were generated from the adherent monocyte fraction of PBMC taken from stage II/III colorectal cancer patients . A single cell suspension was prepared by mechanical and enzymatic disruption of the surgical specimens immediately after resection . DC were loaded with autologous tumor lysate, obtained by repeated freezing and thawing, before being used as stimulators for autologous PBL . HLA-class II restricted T cells that recognize the autologous tumor could be generated in a proportion of patients . The fine specificity of the anti-tumor T cells indicates that differentiation as well as tumor restricted antigens are expressed in colon cancer and that these antigens can evoke a class II HLA-restricted response in an autologous setting . Altogether these findings may open a new perspective for a DC based vaccination of colon cancer patients .

Plant Cell Physiol, 2000 Nov, 41(11), 1259 - 66
Phenylethylamine-induced generation of reactive oxygen species and ascorbate free radicals in tobacco suspension culture: mechanism for oxidative burst mediating Ca2+ influx; Kawano T et al.; In the previous paper {Kawano et al . (2000a) Plant Cell Physiol . 41: 1251}, we demonstrated that addition of phenylethylamine (PEA) and benzylamine can induce an immediate and transient burst of active oxygen species (AOS) in tobacco suspension culture . Detected AOS include H2O2, superoxide anion and hydroxyl radicals . Use of several inhibitors suggested the presence of monoamine oxidase-like H2O2-generating activity in the cellular soluble fraction . It was also suggested that peroxidase(s) or copper amine oxidase(s) are involved in the extracellular superoxide production as a consequence of H2O2 production . Since more than 85% of the PEA-dependent AOS generating activity was localized in the extracellular space (extracellular fluid + cell wall), extracellularly secreted enzymes, probably peroxidases, may largely contribute to the oxidative burst induced by PEA . The PEA-induced AOS generation was also observed in the horseradish peroxidase (HRP) reaction mixture, supporting the hypothesis that peroxidases catalyze the oxidation of PEA leading to AOS generation . In addition to AOS production, we observed that PEA induced an increase in monodehydroascorbate radicals (MDA) in the cell suspension culture and in HRP reaction mixture using electron spin resonance spectroscopy and the newly invented MDA reductase-coupled method . Here we report that MDA production is an indicator of peroxidase-mediated generation of PEA radical species in tobacco suspension culture.

Lab Invest, 2000 Nov, 80(11), 1617 - 28
Conversion of human colonic adenoma cells to adenocarcinoma cells through inflammation in nude mice; Okada F et al.; The roles of inflammation in the malignant progression of tumors during multistep carcinogenesis have been much discussed but remain to be elucidated . To determine the direct contribution of inflammation to colon carcinogenesis, we established a new model of progression of human colonic adenoma cells using a nude mouse; the progression is accelerated by coimplantation of a plastic plate . The FPCK-1-1 cell line, derived from a colonic polyp in a patient with familial adenomatous polyposis, is nontumorigenic when injected subcutaneously into nude mice in a cell suspension of up to 5 x 106 cells per mouse . However implantation of 1 x 10(5) FPCK-1-1 cells attached to a plastic plate induced first acute and then chronic inflammation, and formed progressively growing tumors that were histologically determined as moderately differentiated adenocarcinoma in 65% of mice . Moreover cell lines established from the growing tumors were found to be tumorigenic when injected into mice even without a plastic plate . The tumor arising from the adenoma cells implanted attached to a plastic plate was surrounded by highly proliferating fibrous stroma . This fibrous tissue was considered essential for malignant progression, rather than for attachment to the plastic plate substrate, because the tumors were formed after injection of FPCK-1-1 cells into the fibrous tissue from which the plastic plate had been removed before the cell injection . The conditioned medium (CM) obtained from the fibroblasts derived from a plastic plate-associated stromal tissue was found to contain factors that stimulated growth of FPCK-1-1 cells, but not of the derivative progressor cell lines . The factor was stable to heating and neuraminidase treatment, but labile to trypsin treatment . The main growth-potentiating activity was contained in the fraction larger than 100 kDa . In contrast, the activity to promote FPCK-1-1 cell growth was not present in the CM of subcutaneous fibroblasts from untreated nude mice or the fibroblast cell lines C3H10T 1/2 and NIH3T3 . These results demonstrated that inflammation-associated stroma promoted the conversion of colonic adenoma cells to adenocarcinoma cells.

Blood, 2000 Dec 1, 96(12), 3971 - 8
Donor stromal cells from human blood engraft in NOD/SCID mice; Goan SR et al.; Little is known about the presence, frequency, and in vivo proliferative potential of stromal cells within blood-derived hematopoietic transplants . In this study, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were injected with human CD34(+) peripheral blood cells (PBCs) or cord blood cells (CBCs, either enriched for CD34 or density-gradient separated mononuclear cells) . Flow cytometric analysis 5 to 11 weeks after transplantation revealed the presence of a human lymphomyeloid hematopoiesis within the murine bone marrow . Immunohistochemical staining of bone marrow cell suspensions using human-specific antibodies showed human cells staining positive for human fibroblast markers, human von Willebrand factor (vWF) and human KDR (vascular endothelial growth factor receptor-2) in mice transplanted with CD34(+) PBCs or CBCs, with mean frequencies between 0.6% and 2.4% . In stromal layers of bone marrow cultures established from the mice, immunohistochemical staining using human-specific antibodies revealed flattened reticular cells or spindle-shaped cells staining positive with human-specific antifibroblast antibodies (mean frequency, 2.2%) . Cell populations of more rounded cells stained positive with human-specific antibodies recognizing CD34 (1.5%), vWF (2.2%), and KDR (1.6%) . Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and subsequent complementary DNA sequencing detected transcripts of human KDR (endothelial specific) and human proline hydroxylase-alpha (fibroblast specific) within the bone marrow and spleen of transplanted mice . Analysis of nontransplanted control mice yielded negative results in immunocytochemistry and RT-PCR . Cells expressing endothelial and fibroblast markers were also detected in the grafts before transplantation, and their numbers increased up to 3 log in vivo after transplantation . These results indicate that stromal progenitor cells are present in human cytokine-mobilized peripheral blood or cord blood that engraft in NOD/SCID mice . (Blood . 2000;96:3971-3978)

Clin Exp Metastasis, 1999, 17(10), 849 - 55
Highly metastatic variant of a mouse colon carcinoma cell line, LM17 and its response to GM-CSF gene therapy; Ikubo A et al.; In order to establish a highly metastatic variant of a mouse colon carcinoma cell line (CT26), BALB/c mice were first subcutaneously injected with CT26 cells . Several weeks later, metastatic tumors in lungs were resected, mechanically dispersed into a single cell suspension and cultured in vitro until cells reached confluency . These tumor cells were then subcutaneously injected into new mice . After repeating this procedure five times, a highly lung metastatic cell line, denoted as LM17, has been established . The LM17 cells grow in vitro with or without serum, whereas parental CT26 cells require serum for their growth . The LM17 cells adhere to type I collagen or fibronectin stronger than CT26 cells do . The LM17 cells invade through Matrigel-coated basement membrane in greater number than CT26 cells . By gelatin zymography, LM17 cells showed higher proteinase activity than CT26 . Furthermore, subcutaneous injection of irradiated LM17 cells infected with adenovirus harboring mouse GM-CSF gene prevents the growth and lung metastasis of pre-existing subcutaneous tumor . The injection of irradiated GM-CSF-producing LM17 cells after the surgical removal of pre-existing tumor also protected the occurrence of lung metastasis . These results suggest that this highly metastatic LM17 cell line could be useful for analysis of the lung metastatic mechanism and as the mouse GM-CSF gene therapy model.

Planta, 2000 Oct, 211(5), 679 - 92
Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures; Fry SC et al.; Maize (Zea mays L.) cell cultures incorporated radioactivity from {14C}cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues . Within 5-20 min, the CoA pool had lost its 14C by turnover and little or no further incorporation into polysaccharides then occurred . The system was thus effectively a pulse-chase experiment . Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age . In young (1-3 d) cultures, polysaccharide-bound {14C}feruloyl- and {14C}diferuloyl residues were both detectable within 1 min of {14C}cinnamate feeding . Thus, feruloyl residues were dimerised < 1 min after their attachment to polysaccharides . For at least the first 2.3 h after {14C}cinnamate feeding, polysaccharide-bound {14C}diferuloyl residues remained almost constant at approximately 7% of the total polysaccharide-bound {14C}ferulate derivatives . Since feruloyl residues are attached to polysaccharides < 1 min after the biosynthesis of the latter, and > 10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically . Exogenous H2O2 (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited . In older (e.g . 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound {14C}diferuloyl residues were a steady 1.4% of the total polysaccharide-bound {14C}ferulate derivatives . In contrast to the situation in younger cultures, exogenous H2O2 induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H2O2-limited . In both 2- and 4-d-old cultures, polysaccharide-bound 14C-trimers and larger coupling products exceeded {14C}diferulates 3- to 4-fold, but followed similar kinetics . Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells . We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively . The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed.

Planta, 2000 Oct, 211(5), 623 - 31
Developmental expression of the Arabidopsis thaliana CycA2;1 gene; Burssens S et al.; The associations of cyclins with highly conserved cyclin-dependent kinases are key events in the regulation of cell cycle progression . The spatio-temporal expression of an Arabidopsis thaliana (L.) Heynh . mitotic cyclin, Arath;CycA2;1, was studied by histochemical beta-glucuronidase (GUS) analysis and in-situ hybridizations . The CycA2,1} promoter was active in the egg apparatus before fertilization . During embryogenesis, CycA2;1:gus expression was found in the embryo and the developing endosperm . Throughout plant development, CycA2;1 transcripts were found in both dividing and non-dividing cells, indicating that the expression of this cyclin is not a limiting factor for cell division . In the pericycle and stelar parenchyma, CycA2;1 transcripts were located at the xylem poles, a position that can be correlated with competence for lateral root formation . In addition, CycA2;1:gus expression was upregulated in roots by auxins and in the shoot apex by cytokinins . Transcription of CycA2;1 was shown by reverse transcription-polymerase chain reaction to be strongly induced by sucrose in A . thaliana cell suspensions.

J Thorac Cardiovasc Surg, 2000 Dec, 120(6), 1158 - 67; discussion 1168
Patch augmentation of the pulmonary artery with bioabsorbable polymers and autologous cell seeding; Stock UA et al.; OBJECTIVE: In recent years bioabsorbable synthetic or biologic materials have been used to augment the pulmonary artery or the right ventricular outflow tract . However, each of these polymers has one or more shortcomings . None of these patch materials has been seeded with cells . Thus, we have tested a fast-absorbing biopolymer, poly-4-hydroxybutyric acid, with autologous cell seeding for patch augmentation of the pulmonary artery in a juvenile sheep model . METHODS: Vascular cells were isolated from ovine peripheral veins (n = 6) . Bioabsorbable porous poly-4-hydroxybutyric acid patches (porosity > 95%) were seeded on 3 consecutive days with a mixed vascular cell suspension (21.3 +/- 1.3 x 10(6) cells) . Forty-five (+/- 2) days after the vessel harvest, 1 unseeded and 6 autologously seeded control patches were implanted into the proximal pulmonary artery . The animals received no postoperative anticoagulation . Follow-up was performed with echocardiography after 1 week and before explantation after 1, 7, and 24 weeks (2 animals each) for the seeded control patches and after 20 weeks for the nonseeded control patch . RESULTS: All animals survived the procedure . Postoperative echocardiography of the seeded patches demonstrated a smooth surface without dilatation or stenosis . Macroscopic appearance showed a smooth internal surface with increasing tissue formation . Histology at 169 days demonstrated a near-complete resorption of the polymer and formation of organized and functional tissue . Biochemical assays revealed increasing cellular and extracellular matrix contents . The control patch showed a slight bulging, indicating a beginning dilatation . CONCLUSION: This experiment showed that poly-4-hydroxybutyric acid is a feasible patch material in the pulmonary circulation.

Arch Virol, 2000, 145(10), 2211 - 6
A convenient semi-quantitative method for the diagnosis of Epstein-Barr virus reactivation; Venard V et al.; A semi-quantitative determination of Epstein-Barr virus (EBV) viremia has been devised . Peripheral blood mononuclear cells are recovered by Ficoll gradient and numerated . Five microl aliquots of recovered cell suspension and 5 microl of two standard dilutions (containing 500 and 100 cells, respectively) are subjected to a nested polymerase chain reaction (PCR) . This technique has been evaluated over 3 years for the follow-up of 45 patients attending the Bone Marrow Transplantation Unit of the "Centre Hospitalier et Universitaire de Nancy" . EBV reactivation was diagnosed in 13 patients (28%) . Positivity of PCR for 100 cells was found in 9 patients of whom 6 developed lymphoma or lymphoproliferative disorder . This technique is easy to perform and doesn't necessitate any specific material besides the one necessary for routine genic amplification.

Liver Transpl, 2000 Nov, 6(6), 686 - 702
Donor and recipient leukocytes in organ allografts of recipients with variable donor-specific tolerance: with particular reference to chronic rejection; Ichikawa N et al.; We have attributed organ engraftment to clonal exhaustion-deletion of host-versus-graft and graft-versus-host reactions that are reciprocally induced and governed by migratory donor and recipient leukocytes . The so-called donor passenger leukocytes that migrate from the allograft into the recipients have been thoroughly studied (chimerism), but not the donor leukocytes that remain in, or return to, the transplanted organ . Therefore, using flow cytometry we determined the percentage and lineages of donor leukocytes in cell suspensions prepared from Lewis (LEW) cardiac allografts to 100 days posttransplantation . The LEW hearts were transplanted to naive untreated Brown Norway (BN) recipients (group 2), to naive BN recipients treated with a 28-day or continuous course of tacrolimus (TAC) (groups 3 and 4), and to drug-free BN recipients pretolerized by earlier bone marrow cell (BMC) or orthotopic LEW liver transplantation (groups 5 and 6) . The findings in the heart cell suspensions were correlated with the results from parallel histopathologic-immunocytochemical studies and other studies of the grafts and of host tissues . Although the LEW heart allografts were rejected in 9.6 days by the unmodified recipients of group 2, all beat for 100 days in the recipients of groups 3 through 6 . Nevertheless, all of the long-surviving cardiac allografts (but not the isografts in group 1) were the targets of an immune reaction at 5 days, reflected by dramatic increases in the ratio of leukocytes to nonleukocyte nucleated cells from normal values of 1:5-1:6 to 1:1-5:1 and by manifold other evidence of a major inflammatory event . The acute changes returned to baseline by 100 days in the chronic rejection (CR) free hearts of groups 4 and 6, but not in the CR-afflicted hearts of short-course TAC group 3 or the less-severely damaged hearts of the BMC-prime group 5 . The freedom from CR in groups 4 and 6 was associated with a large donor contribution to the intracardiac leukocyte population at 5 days (28.6% and 22% in the respective groups) and at 100 days (30.5% in group 4 and 8.4% in group 6) compared with 2% and 1.2% at 100 days in the CR-blighted allografts of the partially tolerant animals of groups 3 and 5 . Whether large or small, the donor leukocyte fraction always included a subset of class II leukocytes that had histopathologic features of dendritic cells . These class II(+) cells were of mixed myeloid (CD11b/c(+)) and lymphoid lineages; their migration was markedly inhibited by TAC and accelerated by donor-specific priming and TAC discontinuance . Although a large donor leukocyte population and a normal leukocyte/nonleukocyte cell ratio were associated with freedom from CR, these findings and the lineage profile of the intracardiac leukocytes were not associated with tolerance in the animals of groups 3 and 4 under active TAC treatment . The findings in this study, singly and in their entirety, are compatible with our previously proposed leukocyte migration-localization paradigm of organ allograft acceptance and tolerance.

Eur J Biochem, 2000 Dec, 267(23), 6775 - 83
Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana; Eeckhout D et al.; In order to obtain recombinant antibody fragments that bind the cell-cycle protein CDC2a from Arabidopsis thaliana (CDC2aAt), two phage display libraries of single-chain variable (scFv) fragments were constructed . One library was derived from mice immunized with recombinant CDC2aAt N-terminally fused to a His6-tag (His-CDC2aAt) and the other was made out of an anti-PSTAIRE hybridoma cell line . Six specific His-CDC2aAt-binding phage clones (3D1, 3D2, 3D10, 3D25, 4D21 and 4D47) were isolated by panning . The isolated monoclonal phage clones, as well as the soluble scFv fragments produced in the periplasm of Escherichia coli, bind His-CDC2aAt in ELISA and on Western blots . Moreover, four clones (3D1, 3D2, 3D10 and 4D21) detect specifically CDC2aAt from Arabidopsis cell suspensions on Western blots . Clone 4D21 binds the PSTAIRE epitope, whereas the 3D1, 3D2 and 3D10 clones bind, as yet unidentified, epitopes of CDC2aAt . Furthermore, the accumulation and antigen-binding activity of these scFv fragments in a reducing environment were assessed . No interaction could be shown between the scFv fragments and CDC2aAt in a yeast two-hybrid assay . However, after transient expression of the scFv fragments in the cytosol of tobacco leaves, three of six scFv fragments (3D1, 3D2 and 3D10) accumulated in the plant cytosol and ELISA results indicate that these scFv fragments retained antigen-binding activity.

Plant Physiol, 2000 Nov, 124(3), 1437 - 48
Auxin-induced ethylene triggers abscisic acid biosynthesis and growth inhibition; Hansen H et al.; The growth-inhibiting effects of indole-3-acetic acid (IAA) at high concentration and the synthetic auxins 7-chloro-3-methyl-8-quinolinecarboxylic acid (quinmerac), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), 4-amino-3,6, 6-trichloropicolinic acid (picloram), and naphthalene acetic acid, were investigated in cleavers (Galium aparine) . When plants were root treated with 0.5 mM IAA, shoot epinasty and inhibition of root and shoot growth developed during 24 h . Concomitantly, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, and ACC and ethylene production were transiently stimulated in the shoot tissue within 2 h, followed by increases in immunoreactive (+)-abscisic acid (ABA) and its precursor xanthoxal (xanthoxin) after 5 h . After 24 h of treatment, levels of xanthoxal and ABA were elevated up to 2- and 24-fold, relative to control, respectively . In plants treated with IAA, 7-chloro-3-methyl-8-quinolinecarboxylic acid, naphthalene acetic acid, 2-methoxy-3,6-dichlorobenzoic acid, and 4-amino-3,6,6-trichloropicolinic acid, levels of ethylene, ACC, and ABA increased in close correlation with inhibition of shoot growth . Aminoethoxyvinyl-glycine and cobalt ions, which inhibit ethylene synthesis, decreased ABA accumulation and growth inhibition, whereas the ethylene-releasing ethephon promoted ABA levels and growth inhibition . In accordance, tomato mutants defective in ethylene perception (never ripe) did not produce the xanthoxal and ABA increases and growth inhibition induced by auxins in wild-type plants . This suggests that auxin-stimulated ethylene triggers ABA accumulation and the consequent growth inhibition . Reduced catabolism most probably did not contribute to ABA increase, as indicated by immunoanalyses of ABA degradation and conjugation products in shoot tissue and by pulse experiments with {(3)H}-ABA in cell suspensions of G . aparine . In contrast, studies using inhibitors of ABA biosynthesis (fluridone, naproxen, and tungstate), ABA-deficient tomato mutants (notabilis, flacca, and sitiens), and quantification of xanthophylls indicate that ABA biosynthesis is influenced, probably through stimulated cleavage of xanthophylls to xanthoxal in shoot tissue.

Biochem, Eng . J. . 2000 Dec 1, 6(3), 185 - 191
Glucocorticoid-induced expression of a foreign gene by the GVG system in transformed tobacco BY-2 cells; Nara Y et al.; A glucocorticoid-induced target gene expression system was used to control the expression of the uidA gene, whose product was beta-glucuronidase (GUS), in tobacco BY-2 cell suspension culture . This targeting system showed quick, sensitive, and reversible response to dexamethazone (DEX), an artificial glucocorticoid hormone . Addition of DEX greatly and quickly enhanced uidA gene expression, whose level was as high as that under the control of the CaMV 35S promoter whereas in the absence of DEX, the GUS specific activity was suppressed to be as low as that of nontransformed BY-2 cells . The dilution of DEX decreased GUS specific activity showing that the concentration of DEX plays a major role in controlling the expression level of the target . The use of the glucocorticoid-induced system in plant cell suspension culture was demonstrated to precisely control target gene expression.

Br J Cancer, 2000 Dec, 83(11), 1525 - 31
Comparison between the comet assay and pimonidazole binding for measuring tumour hypoxia; Olive PL et al.; Pimonidazole is finding increasing use in histochemical analyses of hypoxia in tumours . Whether it can identify every hypoxic cell in a tumour, and whether the usual subjective criteria used to define 'positive' cells are optimal, are less certain . Therefore, our aim was to develop an objective flow cytometry procedure for quantifying pimonidazole binding in tumours, and to validate this method by using a more direct indicator of radiobiologic hypoxia, the comet assay . SCCVII tumours in C3H mice were analysed for pimonidazole binding using flow cytometry and an iterative curve-fitting procedure, and the results were compared to the comet assay for the same cell suspensions . On average, cells defined as anoxic by flow analysis (n = 43 tumours) bound 10.8 +/- 0.95 times more antibody than aerobic cells . In samples containing known mixtures of aerobic and anoxic cells, hypoxic fractions as low as 0.5% could easily be detected . To assess the flow cytometry assay under a wider range of tumour oxygen contents, mice were injected with hydralazine to reduce tumour blood flow, or allowed to breathe various gas mixtures during the 90 min exposure to pimonidazole . Hypoxic fraction estimated by the pimonidazole binding method agreed well with the hypoxic fraction measured using the comet assay in SCCVII tumours (r2 = 0.87, slope = 0.98), with similar results in human U87 glioma cells and SiHa cervical carcinoma xenografts . We therefore conclude that this objective analysis of pimonidazole labelling by flow cytometry gives a convenient and accurate estimate of radiobiological hypoxia . Preliminary analyses of biopsies from 3 patients given 0.5 g m-2 pimonidazole also suggest the suitability of this approach for human tumours .

Plant J, 2000 Nov, 24(3), 317 - 26
Alternative splicing of a novel diacylglycerol kinase in tomato leads to a calmodulin-binding isoform; Snedden WA et al.; Calmodulin is a regulatory protein activated during Ca2+ signalling . We have isolated a cDNA, designated LeCBDGK (Lycopersicon esculentum calmodulin-binding diacylglycerol kinase) encoding a novel calmodulin-binding protein with sequence similarity to diacylglycerol kinases from animals . Diacylglycerol kinases convert diacylglycerol to phosphatidic acid . We delineated the calmodulin-binding domain to approximately 25 residues near the C-terminus of LeCBDGK . We have also isolated a second diacylglycerol kinase cDNA, designated LeDGK1, identical to LeCBDGK, except that it lacks the calmodulin-binding domain . Both recombinant LeCBDGK and LeDGK1 were catalytically active in vitro . Anti-DGK antiserum detected two immunoreactive proteins associated with microsomal and plasma membrane fractions from cell suspensions . The higher molecular weight immunoreactive protein was also present in soluble extracts and bound to calmodulin-agarose in the presence of calcium, demonstrating that native LeCBDGK is a calmodulin-binding protein . In the presence of calcium, LeCBDGK associated with membrane cell fractions in vitro, but calmodulin antagonists disrupted this association, suggesting a possible role of calcium in the recruitment of LeCBDGK from soluble to membrane cell fractions . Native LeCBDGK and calmodulin co-immunoprecipitated from tomato soluble cell extracts, suggesting their interaction in vivo . The same gene encodes both LeCBDGK and LeDGK1 and the calmodulin-binding domain of LeCBDGK is encoded by a separate exon . Thus, alternative transcript splicing leads to calmodulin-binding and non-binding forms of diacylglycerol kinases in tomato . Possible roles of LeCBDGK and LeDGK1 in calcium and lipid signalling are discussed.

Biochim Biophys Acta, 2000 Nov 15, 1502(3), 471 - 80
Acute changes of myocardial creatine kinase gene expression under beta-adrenergic stimulation; Hammerschmidt S et al.; Creatine kinase (CK) plays a crucial role in myocardial energy metabolism . Alterations in CK gene expression are found in hypertrophied and failing heart, but the mechanisms behind these changes are unclear . This study tests the hypothesis that increased adrenergic stimulation, which is observed in heart failure, induces changes of myocardial CK-activity, -isoenzyme distribution and -gene expression that are characteristic of the failing and hypertrophied heart . Isolated rat hearts were perfused (constant pressure of 80 mmHg) with red cell suspensions . Following a 20-min warm-up period, perfusion for 3 h with 10(-8) M (iso 3 h) or without (control 3 h) isoproterenol was started or experiments were immediately terminated (control 0 h) . Left ventricular tissue was analyzed for total CK-activity, CK-isoenzyme distribution and, by use of quantitative RT-PCR, for B-CK, M-CK, mito-CK and GAPDH- (as internal standard) mRNA . After beta-adrenergic stimulation (iso 3 h) but not after control perfusion (control 3 h) a roughly threefold increase in B-CK mRNA levels and a decrease in M-CK mRNA levels by 18% was found . There were no significant differences among the three groups in total CK-activity and in distribution of CK-MM, CK-BB, CK-MB and mito-CK . Thus, beta-adrenergic stimulation induces a switch in CK gene expression from M-CK to B-CK, which is characteristic for the hypertrophied and failing heart . This may be interpreted as an adaptive mechanism making energy transduction via CK more efficient at times of increased metabolic demand.

Vet Immunol Immunopathol, 2000 Nov 23, 77(1-2), 93 - 102
Beta cell and insulin antibodies in treated and untreated diabetic cats; Hoenig M et al.; Beta cell and insulin antibodies are involved in the pathogenesis of diabetes in human patients . Beta cell antibodies have also been found in about 50% of newly diagnosed diabetic dogs . This study's objective was to examine these antibodies' role in feline diabetes . The serum of 26 newly diagnosed untreated diabetic cats, 29 cats on insulin therapy, 30 cats with diseases other than diabetes, and 30 healthy cats was examined for beta cell and insulin antibodies . For beta cell antibody testing, purified beta cells from a radiation-induced transplantable rat insulinoma were used . Serum from cats in which anti-beta cell antibodies were induced by injecting a purified beta cell suspension subcutaneously was used as a positive control . Following incubation with test sera, fluorescein-labeled anti-cat immunoglobulins were used to visualize binding between the beta cells and cat gamma globulins . Each serum was tested on two different tumor preparations . For the detection of insulin antibodies, a charcoal separation method was used . It was found that none of the healthy cats, none of the newly diagnosed, untreated diabetic cats and none of the cats with diseases other than diabetes had antibodies against beta cells or against endogenous insulin . Four diabetic cats (14%) that had been treated with different insulin preparations had insulin antibodies.It is concluded that immune-mediated processes are not causing diabetes in the cat . Further studies are needed to evaluate if antibodies directed against exogenous insulin alter the response of diabetic cats to insulin.

Anal Quant Cytol Histol, 2000 Oct, 22(5), 393 - 7
Proliferation and apoptosis in solid tumors . Analysis by laser scanning cytometry; Abdel-Moneim I et al.; OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC) . STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide . Proliferation was detected by Ki-67 antibody . Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides . RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0) . Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0) . Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination . The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5) . Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%) . CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation . However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells . Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices . Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data . Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.

Br J Radiol, 2000 Sep, 73(873), 978 - 86
Change in oxygenation status in intratumour total and quiescent cells following gamma-ray irradiation, tirapazamine administration, cisplatin injection and bleomycin treatment; Masunaga S et al.; C3H/He mice bearing SCC VII tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells . The mice then received gamma-ray irradiation, or administration of tirapazamine (TPZ), cisplatin or bleomycin . At various time points after each treatment, tumour-bearing mice were irradiated with a series of test doses of gamma-rays, while alive or after being killed, to obtain hypoxic fractions (HFs) in the tumours . Immediately after gamma-ray test irradiation, the tumours were excised, minced and trypsinized . Tumour cell suspensions obtained were incubated with cytochalasin-B, a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling (i.e . quiescent (Q) cells) was determined using immunofluorescence staining for BrdU . MN frequency in the total (P + Q) tumour cells was determined from the tumours that were not pre-treated with BrdU . MN frequency of BrdU-unlabelled cells was then used to calculate the surviving fraction of the unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumour cells . TPZ and cisplatin reduced the HF after treatment, especially in Q cells, and this tendency was particularly marked with TPZ . In contrast, bleomycin increased the HF after treatment . Both reoxygenation following gamma-ray irradiation or bleomycin treatment and a subsequent return to pre-treatment levels of HF following TPZ or cisplatin treatment (rehypoxiation) occurred more rapidly in total (P + Q) cells than in Q cells . Based on our previous report that total (P + Q) and Q cells within this tumour have large acutely and chronically HFs, respectively, we conclude that acute hypoxic cells play a major role in reoxygenation and rehypoxiation in SCC VII tumours.

Connect Tissue Res, 1998, 38(1-4), 9 - 15; discussion 35-41
Isolation of amelogenin-positive ameloblasts from rat mandibular incisor enamel organs by flow cytometry and fluorescence activated cell sorting; Chen WY et al.; The purpose of this study was to use amelogenin as a marker to examine the feasibility of isolating ameloblasts from enamel organ cell populations by fluorescence activated cell sorting . After treating dissected rat enamel organs with proteolytic enzymes to loosen cell attachments and labial connective tissues, dissociated cell suspensions were fixed, then immunostained with rabbit anti-rM179 recombinant amelogenin antibody and FITC-conjugated goat anti-rabbit Ig G antibody . Flow cytometry indicated that about 70% of the total cell sample and virtually all the larger cells therein were amelogenin-positive . Fluorescence activated cell sorting yielded a sample of amelogenin-positive cells at 97% purity . Immunofluorescence microscopy indicated that these isolated amelogenin-positive cells varied widely in size and morphology . This was attributed to loss of intercellular support for ameloblasts once they were dissociated from each other, and to some fragmentation caused when the cells were initially physically removed from the teeth . The results demonstrate that viable ameloblast cell fractions, especially representing cells at the secretory stage, can be purified from enzymic digests of rat enamel organ by sorting on the basis of cell size alone . From these fractions, subpopulations of ameloblasts may be identified when differentiation specific cell surface markers become available.

Connect Tissue Res, 1998, 38(1-4), 3 - 8; discussion 35-41
Primary culture and characterization of enamel organ epithelial cells; Den Besten PK et al.; The cells of the enamel organ are programmed by signals such as growth factors and extracellular matrix components to differentiate and form dental enamel . To study how the enamel organ epithelial cells control enamel development, we have begun to characterize a primary porcine enamel organ epithelial cell culture system . The unerupted molars of 3 month old pigs were isolated, the cells were digested into a single cell suspension and grown in media either with or without serum . Expression of amelogenin and ameloblastin mRNA was monitored by RT PCR, and protein secretion was identified by immunohistochemistry . Cells grown in MEM formed a mixed cell population of epithelial- and fibroblast-like cells which grew past confluence, formed nodules, mineralized, and expressed low levels of amelogenin and ameloblastin protein . In LHC-9 media, which is selective for epithelial cells, the cells did not grow past confluence but secreted amelogenin and ameloblastin proteins more strongly . Cell viability was maintained in both serum-free and serum-containing media . However, in the serum-free media, cell proliferation proceeded slowly . Although cells grown in MEM mineralized, the mixed cell population may make studies of specific ameloblast-like cells more difficult . However, cells grown in a culture media selective for epithelial cells will require modifications such as cell immortalization to allow long term studies of cell regulation and interaction . In summary, we have established an enamel organ epithelial cell culture system which will enable us to study the role of ameloblasts in enamel matrix formation, ameloblast regulation, as well as cell-matrix interactions . Selection of specific culture conditions will depend on the questions being addressed in individual studies.

Anticancer Res, 2000 Sep-Oct, 20(5A), 2939 - 44
Increase of tumour infiltrating lymphocytes in mice treated with antimetastatic doses of NAMI-A; Magnarin M et al.; NAMI-A is a novel antitumour agent, based on ruthenium, which has proved effectiveness against lung metastases of solid mouse tumours . The study focuses on the effects of NAMI-A on leukocyte infiltration into the primary tumour of MCa mammary carcinoma, implanted subcutaneously (s.c.) or intramuscularly (i.m.) into CBA mice . NAMI-A, given with a cycle of daily treatments for six consecutive days on advanced tumours at 35 mg/kg/day, markedly reduces lung metastasis independently of the tumour type (Lewis lung carcinoma, MCa mammary carcinoma or TS/A adenocarcinoma) being treated and of the site of tumour implantation (s.c . or i.m.) . The analysis of leukocyte infiltration of the primary tumour, performed on a single cell suspension of cells isolated from a Ficoll gradient on which a raw suspension of primary tumour cells was layered, showed NAMI-A to significantly increase tumour infiltrating lymphocytes . These lymphocytes are almost all CD3+ cells with a significant increase of the CD8+ over the CD4+ subpopulation that reduces the helper/suppressor ratio from 2.8 to 2.1 . These data indicated the absence of toxicity of NAMI-A for tumour infiltrating lymphocytes and suggested that this compound might even synergize in combined treatments with cancer immunotherapy.

J Urol, 2000 Dec, 164(6), 2173 - 7
Adenovirus mediated cytosine deaminase gene transduction and 5-fluorocytosine therapy sensitizes mouse prostate cancer cells to irradiation; Anello R et al.; PURPOSE: We assess the ability of adenovirus mediated expression of the Escherichia coli cytosine deaminase gene in conjunction with the prodrug 5-fluorocytosine to result in radiation sensitization in the mouse prostate cancer cell line RM-1 in vitro . MATERIALS AND METHODS: To document cytotoxicity of gene therapy, RM-1 cells were exposed to escalating doses of adenovirus mediated cytosine deaminase and a fixed dose of 5-fluorocytosine or phosphate buffered saline . Viable cells as determined by exclusion of trypan blue were counted the following day . Cytosine deaminase expressing RM-1 cells were then irradiated as single cell suspensions at various doses of radiation in a cesium source (4.4 Gy . per minute) and randomized to receive 5-fluorocytosine therapy at different times in relation to the external radiation therapy . End points were determined in a clonogenic assay by counting colonies with greater than 50 cells 7 days after replating . RESULTS: Use of adenovirus mediated cytosine deaminase plus 5-fluorocytosine demonstrated viral dose dependent killing of RM-1 cells to a maximum of 85%, while either therapy alone was nontoxic . Neither adenovirus mediated cytosine deaminase infection nor 5-fluorocytosine alone influenced external radiation therapy killing . However, after controlling for death due to gene therapy alone, the combination of adenovirus mediated cytosine deaminase plus 5-fluorocytosine and external radiation therapy resulted in synergistic activity to approximately 2 logs of cell kill at low doses of radiation (p = 0.001) . While altering the chronology of prodrug exposure in relation to external radiation therapy maintained synergy in all scenarios tested, starting 5-fluorocytosine 24 hours before external radiation therapy resulted in the most profound killing (p = 0.04), which indicates the importance of maintaining prodrug therapy during external radiation therapy . CONCLUSIONS: The combination of adenovirus mediated cytosine deaminase plus 5-fluorocytosine and radiation therapy resulted in radiation sensitization with clinically relevant doses of radiation suggesting a potential usefulness of this treatment in patients with prostate cancer.

J Clin Microbiol, 2000 Nov, 38(11), 4042 - 8
Real-time quantitative PCR for human herpesvirus 6 DNA; Locatelli G et al.; The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection . We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids . The test uses a 5' nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system . The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 10(6) viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule . The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions . Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys.

Fertil Steril, 2000 Nov, 74(5), 916 - 9
A simple and reliable method for meiotic studies on testicular samples used for intracytoplasmic sperm injection; Metzler-Guillemain C et al.; OBJECTIVE: To develop a reliable and simple method allowing meiotic studies to be performed on testicular samples used for ICSI . DESIGN: Evaluation of meiotic abnormalities in patients with severe spermatogenic impairment . SETTING: Centre de Medecine de la Reproduction, Marseille . PATIENT(S): Two azoospermic men undergoing testicular biopsy for ICSI and one control individual with normal testicular histology . INTERVENTION(S): The immature germ cells from the patients came from testicular biopsy used for ICSI, after dispersal into a thin cell suspension . Cells were cytocentrifuged to obtain well-spread spermatocytes and then immunocytochemical techniques were performed . We used rabbit polyclonal antibodies against the specific meiotic proteins Cor1 and Syn1 and a human CREST anti-kinetochore antibody . MAIN OUTCOME MEASURE(S): Synapsis abnormalities in patients with severe spermatogenesis impairment . RESULT(S): Pachytene spermatocytes are easily analyzed with this technique, without damage of the axial core and synaptonemal complex . The loss of germ cells is limited . CONCLUSION(S): The cytocentrifugation method is the most suitable technique for meiotic studies in patients with severe spermatogenic failure, because it can be used on the testicular cell suspension remaining after ICSI with testicular spermatozoa.

Appl Environ Microbiol, 2000 Nov, 66(11), 4679 - 87
Attachment of Escherichia coli O157:H7 to the surfaces and internal structures of apples as detected by confocal scanning laser microscopy; Burnett SL et al.; Confocal scanning laser microscopy (CSLM) was used to demonstrate the attachment of Escherichia coli O157:H7 transformed with a plasmid encoding for green fluorescent protein (GFP) to the surface and within the internal structures of nonwaxed Red Delicious cv . apples . Apples at 2 or 25 degrees C were inoculated with an E . coli O157:H7 cell suspension at 2 or 25 degrees C . The effect of a negative temperature differential (cold inoculum, warm apple), a positive differential (warm inoculum, cold apple), and no differential (warm inoculum, warm apple), in combination with a pressure differential (atmospheric versus 10,130 Pa), on the attachment and infiltration of cells was determined . CSLM stereo images of external surfaces of apples subjected to all combinations of test parameters showed preferential cellular attachment to discontinuities in the waxy cuticle on the surface and to damaged tissue surrounding puncture wounds, where the pathogen was observed at depths up to 70 microm below the skin surface . Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 microm . Infiltration through the floral tube and attachment to seeds, cartilaginous pericarp, and internal trichomes were observed in all apples examined, regardless of temperature differential during inoculation . The pressure differential had no effect on infiltration or attachment of E . coli O157:H7 . Image analysis to count cells at various depths within tissues was used to quantitatively compare the extent of infiltration into various apple structures as well as the effects of the temperature differential . Puncture wounds harbored greater numbers of the pathogen at greater depths than did other sites examined . Attachment or infiltration of cells was greater on the intact skin and in lenticels, russet areas, and the floral tube of apples inoculated under a negative temperature differential compared to those inoculated under no temperature differential . The results suggest that E . coli O157:H7 attached to internal core structures or within tissues of apples may evade decontamination treatments . Interventions designed to deliver disinfectants to these locations or to remove viable cells of E . coli O157:H7 and other pathogens from apples by other means need to be developed and validated.

Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1807 - 12
Production of betacyanins by a cell suspension culture of table beet (Beta vulgaris L.); Akita T et al.; A cell suspension culture of table beet (Beta vulgaris L.) was established for efficient betacyanin production from violet callus induced from the hypocotyls of aseptic seedlings . This suspension culture produced large amounts of betacyanins . The betacyanin content increased with increasing cell growth during the log phase . Reducing the total nitrogen concentration (30 mM) and modifying the ratio of ammonium to nitrate (1:14) resulted in an increased betacyanin content . Supplementation of Fe2+ to the LS medium also promoted betacyanin production . The maximal betacyanin yield was achieved with a 2 mM Fe2+ concentration . Combining these conditions, we established a revised LS medium to improve betacyanin productivity (250 mg/l for a 14-day culture).

Jpn J Cancer Res, 2000 Oct, 91(10), 1065 - 72
Mechanism of anti-tumor effect of combination of bleomycin and shock waves; Kato M et al.; We have previously reported marked enhancement of the cytocidal effect of bleomycin (BLM) on cancer cell suspensions in vitro by the combination with shock waves . In this study, we evaluated the synergistic effects on cancer cell proliferation and apoptosis in solid tumors . A spherical piezo-ceramic element was used as the shock wave source, with a pressure peak of 40 MPa . A human colon cancer cell line, SW480 was implanted onto the back of nude mice . Two thousand shock waves were administered to the tumor immediately following an intravenous injection of BLM at a dose of one-tenth of the LD(50) . The tumor was extirpated at 3, 6, 12, 24, 72 h and 1 week following shock exposure . Cell proliferation and apoptosis were detected by Ki-67 using antibody MIB-1 and by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method . The lowest percentage (35.7%) of Ki-67-positive cells appeared 24 h following the treatment . The maximum apoptotic index was detected within 6 h following the treatment . Moreover, numerous large cells with enlarged nuclei were detected histologically . These results suggest that shock waves may enhance chemotherapeutic effects by increasing apoptosis and decreasing cell proliferation in the tumor tissue.

Neurol Res, 2000 Sep, 22(6), 565 - 6
Numbers of neurons and non-neuronal cells in human trigeminal ganglia; LaGuardia JJ et al.; We analyzed both trigeminal ganglia of eight post-mortem human subjects for their content of neurons and non-neuronal cells using dissociated cell suspension techniques . Neuronal counts in each of the 16 ganglia ranged from 20,000 to 35,400, with an average of 27,400 +/- 4800, and the estimated ratio of non-neuronal to neuronal cells was 100 to 1 . Our numbers are comparable to the lower ranges obtained using different techniques in previous studies.

Physiol Res, 2000, 49(3), 355 - 62
Effects of treatment with interleukin-1 receptor antagonist on endogenous interleukin-1 levels in normal and irradiated mice; Bugarski D et al.; The in vivo effects of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) administration on endogenous IL-1 levels in the circulation and conditioned media (CM) from different immunohematopoietic organ/tissues were studied in CBA mice under steady state and postirradiation conditions . In normal mice, constitutive IL-1 levels were demonstrated in the plasma, CM of peritoneal exudate cells and full-thickness skin explants with low or undetectable levels in CM of splenic and bone marrow cell suspensions . In irradiated mice (2 Gy, X rays) on day 3 post exposure a significant increase of IL-1 levels was seen in the circulation and CM of peritoneal exudate cells, with no significantly different levels in postirradiation bone marrow, spleen and skin . After rhIL-1Ra treatment of the animals (2 x 50 microg/mouse, i.p.), significantly elevated IL-1 levels were observed in the skin and CM of peritoneal exudate cells in normal mice, whereas slightly increased levels were detected in CM of splenic cells . The rhIL-1Ra administration in irradiated mice led to decreased IL-1 concentrations in the circulation, and CM of peritoneal exudate cells and skin . The results pointed out the importance of IL-1 secretion and receptor expression in the maintenance of homeostasis in steady state, as well as during recovery after irradiation . Modulatory effects of IL-1Ra on IL-1 production were dependent on basic endogenous IL-1 concentration.

Neuroreport, 2000 Sep 28, 11(14), 3063 - 5
Serotonergic modulation of hippocampal acetylcholine release after long-term neuronal grafting; Hilgert M et al.; Adult female rats sustained aspirative fimbria-fornix lesions and, 2 weeks later, received intrahippocampal grafts of fetal septal or mixed septal-raphe cell suspensions . Twenty-four months later, the extracellular concentration of hippocampal acetylcholine (ACh) was determined by microdialysis . Basal ACh levels (5-65 fmol/5 microl sham-operated rats) were strongly reduced after lesioning (3-7 fmol/5 microl) . In septally transplanted and septal-raphe co-transplanted rats, hippocampal ACh concentrations were restored to near-normal levels (15-25 fmol/5 microl), indicating long-term functional survival of hippocampal transplants . After administration of citalopram (100 microM by infusion) and fenfluramine (20 mg/kg i.p.), the hippocampal ACh efflux was increased by 2- to 3-fold in all groups of rats . The relative increase of ACh was highest in co-transplanted rats, an effect which was possibly due to functional interactions between grafted raphe and septal neurons.

J Biol Chem, 2001 Jan 26, 276(4), 2644 - 51 Epub 2000 Oct 17.
Sequence-specific and methylation-dependent and -independent binding of rice nuclear proteins to a rice tungro bacilliform virus vascular bundle expression element; He X et al.; Nuclear proteins from rice (Oryza sativa) were identified that bind specifically to a rice tungro bacilliform virus promoter region containing a vascular bundle expression element (VBE) . One set of proteins of 29, 33, and 37 kDa, present in shoot and cell suspension extracts but hardly detectable in root extracts, bound to a site containing the sequence AGAAGGACCAGA within the VBE, which also contains two CpG and one CpNpG potential methylation motifs . Binding by these proteins was determined to be cytosine methylation-independent . However, a novel protein present in all analyzed extracts bound specifically to the methylated VBE . A region of at least 49 nucleotides overlapping the VBE and complete cytosine methylation of the three Cp(Np)G motifs was required for efficient binding of this methylated VBE-binding protein (MVBP).

Bone Marrow Transplant, 2000 Sep, 26(6), 667 - 71
Preparation and analysis of fetal liver extracts; Zwicky C et al.; The aim of this work is to describe the techniques that have been used for preparation and analysis of whole fetal liver extracts destined for in utero transplantation . Nine fetal livers between 12 and 17 weeks of gestation were prepared: cell counts and assessment of the hematopoietic cell viability were performed on cell suspensions . Hepatocytes represented 40 to 80% of the whole cell population . The remaining cells were constituted by hematopoietic cells (mainly erythroblasts), as well as by endothelial cells . The latter expressed CD34 on their surface, interfering with the assessment of CD34+ hematopoietic cells by flow cytometry . Direct visual morphologic control using alkaline phosphatase anti-alkaline phosphatase techniques was needed to differentiate hematopoietic from extra-hematopoietic CD34+ cells . Between 3.0 and 34.6 x 10(6) CD34+ viable hematopoietic cells were collected per fetal liver . Adequate differentiation of these cells into burst-forming units erythroid (BFU-E), colony-forming units granulocyte-macrophage (CFU-GM), and colony-forming units granulocyte erythroid macrophage megakaryocyte (CFU-GEMM) has been shown for each sample in clonogeneic cultures . In conclusion, fetal liver is a potential source of hematopoietic stem cells . Their numeration, based on the presence of CD34, is hampered by the expression of this antigen on other cells contained in the liver cell extract, in particular endothelial cells.

Arch Biochem Biophys, 2000 Sep 15, 381(2), 285 - 94
Differential induction of sesquiterpene metabolism in tobacco cell suspension cultures by methyl jasmonate and fungal elicitor; Mandujano-Chavez A et al.; Jasmonates are well documented for their ability to modulate the expression of plant genes and to influence specific aspects of disease/pest resistance traits . We and others have been studying the synthesis of sesquiterpene phytoalexins in elicitor/pathogen-challenged plants and have sought to determine if methyl jasmonate (MeJA) could substitute for fungal elicitors in the induction of capsidiol accumulation by tobacco cell cultures . The current results demonstrate that MeJA does in fact induce phytoalexin accumulation, but with a much more delayed induction time course than elicitor . While elicitor treatment induced strong but transient changes in key enzymes of sesquiterpene biosynthesis, sesquiterpene cyclase, and aristolochene/deoxy-capsidiol hydroxylase, MeJA did not . Instead, MeJA caused a protracted induction of cyclase activity and only a low level of hydroxylase activity . MeJA induced the expression of at least two sesquiterpene cyclase genes, including one that had not been observed previously in elicitor-induced mRNA populations . Only a small portion of the total sesquiterpene cyclase mRNA induced by MeJA was associated with polysomal RNA, suggesting that the MeJA treatment imposed both transcriptional and posttranscriptional regulation in tobacco cells . These results are not consistent with MeJA playing a role in orchestrating defense responses in elicitor-treated tobacco cells, but do provide evidence that MeJA induces a subset of genes coding for the biosynthesis of sesquiterpene phytoalexins.

Biol Chem, 2000 Aug, 381(8), 741 - 8
Structure-activity relationships of synthetic analogs of jasmonic acid and coronatine on induction of benzo{c}phenanthridine alkaloid accumulation in Eschscholzia californica cell cultures; Haider G et al.; A facile test system based on the accumulation of benzo{c}phenanthridine alkaloids in Eschscholzia californica cell suspension culture (an indicator of defense gene activation) has been used to analyze a series of synthetic compounds for elicitor-like activity . Of the 200 jasmonic acid and coronatine analogs tested with this system, representative results obtained with 49 of them are presented here . The following can be summarized concerning structure-activity relationships: there is a large degree of plasticity allowed at the C-3 of jasmonic acid in the activation of defense genes . The carbonyl moiety is not strictly required, but exocyclic double bond character appears necessary . The pentenyl side chain at C-2 cannot tolerate bulky groups at the terminal carbon and still be biologically active . Substitutions to the C-1' position are tolerated if they can potentially undergo beta-oxidation . Either an alkanoic acid or methyl ester is required at C-1, or a side chain that can be shortened by beta-oxidation or by peptidase hydrolysis . Coronatine and various derivatives thereof are not as effective as jasmonic acid, and derivatives in inducing benzo{c}phenanthridine alkaloid accumulation . Jasmonic acid rather than the octadecanoic precursors is therefore considered to be a likely signal transducer of defense gene activation in planta.

Clin Immunol, 2000 Nov, 97(2), 171 - 81
On the origin of surface proteinase 3 of nonmyeloid cells: evidence favoring an exogenous source; Zhou Z et al.; In Wegener's granulomatosis (WG), when the endogenous Proteinase 3 (PR3) of myeloid cells is translocated to the cell surface, a pathologically consequent interaction is believed to occur with classic anti-neutrophil cytoplasmic antibody (cANCA) . In contrast, the exact origin of surface PR3 on cells of nonmyeloid origin is still debated . By various methods, PR3 mRNA and protein are easily demonstrated in myeloid cells but not in nonmyeloid cells . Exceptionally, the endothelial ECV304 cell line spontaneously produced PR3 mRNA but no PR3 protein . In the other nonmyeloid cells, we could not show cell surface PR3 either spontaneously or after TNFalpha stimulation . On the other hand, under serum-free conditions and using {(3)H}DFP-labeled HL-60 extract, a rapid, dose-dependent, saturable binding was demonstrated to both myeloid and nonmyeloid cells . That was reproduced with purified {(3)H}DFP-PR3 . While we could not demonstrate cell surface PR3 on nonmyeloid cells after incubation with serum-containing supernatants of HL-60 cell cultures, we could do so after an overnight coculture period with HL-60 cell suspensions under the usual serum-containing culture conditions . Overall, our data would suggest that in vivo, the surface PR3 found on nonmyeloid cells is not endogenous but results from adsorption of PR3 extruded in their microenvironment by neighboring myeloid cells coming in close contact with them .

Biorheology, 2000, 37(3), 225 - 37
Detection of red cell aggregation by low shear rate viscometry in whole blood with elevated plasma viscosity; Janzen J et al.; The viscosity of whole blood measured at low shear rates is determined partly by shear resistance of the red cell aggregates present, stronger aggregation increasing the viscosity in the absence of other changes . Effects of cell deformability can confound interpretation and comparison in terms of aggregation, however, particularly when the plasma viscosity is high . We illustrate the problem with a comparison of hematocrit-adjusted blood from type 1 diabetes patients and controls in which it is found the apparent and relative viscosities at a true shear rate of 0.20 s-1 are lower in the patient samples than age matched controls, in spite of reports that aggregation is increased in such populations . Because the plasma viscosities of the patients were higher on average than controls, we performed a series of experiments to examine the effect of plasma protein concentration and viscosity on normal blood viscosity . Dilution or concentration by ultrafiltration of autologous plasma and viscosity measurements at low shear on constant hematocrit red cell suspensions showed (a) suspension viscosity at 0.25 and 3 s-1 increased monotonically with plasma protein concentration and viscosity but (b) the relative viscosity increased, in concert with the microscopic aggregation grade, up to a viscosity of approximately 1.25 mPa-s but above this the value the relative viscosity no longer increased as the degree of aggregation increased in concentrated plasmas . It is suggested that in order to reduce cell deformation effects in hyperviscous pathological plasmas, patient and control plasmas should be systematically diluted before hematocrit is adjusted and rheological measurements are made . True shear rates should be calculated . Comparison of relative viscosities at low true shear rates appears to allow the effects of red cell aggregation to be distinguished by variable shear rate viscometry in clinical blood samples.

J Urol, 2000 Nov, 164(5), 1834 - 7
Immunomagnetic cell enrichment detects more disseminated cancer cells than immunocytochemistry in vitro; Zigeuner RE et al.; PURPOSE: We describe a method to improve tumor cell detection compared to currently available immunocytochemical methods by using immunomagnetic cell enrichment . MATERIALS AND METHODS: Two different methods of immunomagnetic cell enrichment using antibody coated magnetic beads were tested and compared with unenriched immunocytochemistry . One method was positive selection of epithelial cells from mononuclear cells with the antiepithelial antibody BER-EP4 and the other was depletion of mononuclear cells with the antileukocyte antibody CD45 . Mononuclear cells were isolated from peripheral blood by density centrifugation and various numbers of tumor cells were added . The 5 different cell lines from urological malignancies used in the study were DU-145, RT-4, CAKI-2, KTCTL-2 and KTCTL-30 . Following incubation of cell suspensions with the beads, cell separation was performed in a magnetic field . After centrifugation on glass slides immunocytochemical staining for cytokeratin was performed . A total of 112 experiments were completed and negative controls were obtained . RESULTS: The number of tumor cells detected by positive selection and depletion was significantly higher than by immunocytochemistry (p <0.001) . The median enrichment factor and tumor cell recovery rate for positive selection and depletion were 15.3 and 61.2%, and 13.0 and 57.3%, respectively (not significant) . With less than 1 tumor cell suspended in 106 mononuclear cells, the probability of tumor cell detection was 23% for immunocytochemistry alone and 93.3% for both enrichment methods (p <0.01) . No false-positive results were observed . CONCLUSIONS: Compared to immunocytochemistry, immunomagnetic cell enrichment significantly improves the sensitivity of detection of epithelial cells added to mononuclear cells . Both methods of enrichment were equally effective and may be important for clinical practice in the future.

Cytometry, 2000 Oct 15, 42(5), 296 - 306
Contribution of flow cytometric immunophenotyping to the evaluation of tissues with suspected lymphoma?
Dunphy CH.
BACKGROUND: A critical analysis of the contribution of flow cytometric immunophenotyping (FCI) to the evaluation of lymph nodes and extranodal tissues with suspected lymphoma by a large, retrospective approach has not been reported previously and represents the purpose of this study . METHODS: A total of 278 lymph nodes and 95 extranodal tissue specimens submitted over a 2-year period with complete histologic, FCI, and immunohistochemical (IH) data formed the basis of the study . RESULTS: The FCI data contributed significantly to or was consistent with the final tissue diagnosis in the majority (94%) of the tissue samples . There is no well-described utility of flow cytometry markers for Hodgkin's lymphoma (HL) due to the usual scarcity of tumor cells in the final cell suspensions obtained from these tumors . However, the FCI data excluded non-Hodgkin's lymphoma (NHL) and suggested the possible usefulness of CD15 and CD30 by FCI in HL . In addition, immunophenotypic data by FCI in combination with touch imprint cytomorphology was useful in excluding a diagnosis of NHL in cases of nonhematopoietic malignancies and was particularly useful in defining the following hematopoietic tumors and malignancies: thymoma, T-cell lymphoblastic lymphoma, leukemia cutis, and plasma cell dyscrasia . Thus, IH was not essential for the diagnosis in these latter cases and was performed in only two cases (one thymoma and one plasma cell dyscrasia) . Of interest, FCI supported the diagnosis in 3 cases of Ewing's sarcoma/primitive neuroectodermal tumor by detection of CD56 on the surface of the malignant cell . Only 11% of NHL were "negative" by FCI (i.e., an aberrant T-cell or monoclonal B-cell population was not identified) . Reasons for these discrepancies included partial tissue involvement by the NHL with sampling differences, T-cell rich or lymphohistiocytic-rich variants with a small population of monoclonal B cells, marked tumoral sclerosis, poor tumor preservation, and T-cell NHL without an aberrant immunophenotype . Only 60% of CD30+ anaplastic large cell lymphomas (ALCL) were CD30+ by FCI . CONCLUSIONS: FCI data should always be correlated with light microscopy if no FCI abnormalities are detected; IH may need to be performed in selected cases . It is less necessary to perform microscopic examination of tissues when the FCI data are positive and indisputable . However, in selected cases in which FCI data is diagnostic, microscopic observations may provide additional information due to sampling .

Radiat Res, 2000 Oct, 154(4), 439 - 46
Measurement of hypoxia using the comet assay correlates with preirradiation microelectrode pO2 histography in R3327-AT rodent tumors; Sauer G et al.; Polarographic determination of tumor oxygenation by Eppendorf histography is currently under investigation as a possible predictor of radiotherapy outcome . Alternatively, the alkaline comet assay has been proposed as a radiobiological approach for the detection of hypoxia in clinical tumor samples . Direct comparisons of these methods are scarce . One earlier study with different murine tumors could not establish a correlation, whereas a weak correlation was reported for a variety of human tumors . Considering the different end points and spatial resolution of the two methods, a direct comparison for a single tumor entity appeared desirable . Anaplastic R3327-AT Dunning prostate tumors were grown on Copenhagen rats to volumes of 1-6 cm(3) . Eppendorf histography (100-200 readings in 5 parallel tracks) for 8 different tumors revealed various degrees of oxygenation, with median pO(2) values ranging from 1.1 to 23 mmHg . Within 5 min after an acute exposure to 8 Gy (60)Co gamma rays, tumors were excised from killed animals and rapidly cooled to limit repair, and a single cell suspension was prepared for use with the comet assay . The resulting comet moment distributions did not exhibit two subpopulations (one hypoxic and the other aerobic), and a hypoxic fraction could not be calculated . Instead, the average comet moment distribution was taken as a parameter of overall strand break induction . Corresponding experiments with tumor cells grown in vitro allowed us to derive the relationship between the oxygen enhancement ratio (OER) for the average comet moment and oxygen partial pressure (Howard-Flanders and Alper formula) . The validity of this relationship was inferred for cells exposed in situ, and the convolution of a pO(2) distribution with the formula of Howard-Flanders and Alper yielded an array of expected OER values for each tumor . The average expected OER correlated well with the average comet moment (r = 0.89, P < 0.01), and the in situ comet moment distributions could be predicted from the Eppendorf data when 50% repair was taken into account, assuming a 5-min damage half-life . The findings confirm the potential of interstitial polarography to reflect radiobiologically relevant intracellular oxygenation, but also underscore the confounding influence of differences in repair that may occur when cells are prepared from irradiated tissues for use with the comet assay.

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