Plant Cell, 2000 Oct, 12(10), 1939 - 50 Glutathione and a UV light-induced glutathione S-transferase are involved in signaling to chalcone synthase in cell cultures; Loyall L et al.; UV irradiation stimulates expression of the gene encoding the key enzyme chalcone synthase (CHS), which leads to the generation of protective flavonoids in parsley cell cultures . CHS transcripts increase after 3 to 4 hr, and early genes are involved in the signal transduction to the CHS promoter . By using the fluorescent differential display technique in a large-scale screening, several early UV light-induced genes were isolated . Of these, a novel glutathione S-transferase (PcGST1) is induced within 2 hr and precedes CHS expression . Overexpression of PcGST1 in transformed cell lines containing a CHS promoter/luciferase reporter (CHS-LUC) affected the onset of LUC transcription . Supplementing these cell lines with glutathione immediately stimulated CHS-LUC expression within 2 hr in dark-incubated cells and resulted in a biphasic induction profile in UV-irradiated cells . Our data indicate the involvement of glutathione and PcGST1 in early events of a UV light-dependent signal transduction pathway to CHS . In this context, the oxidative status of a cell acts as a central regulating element. J Neurosci Methods, 2000 Jul 31, 100(1-2), 157 - 63 A microtiter trypan blue absorbance assay for the quantitative determination of excitotoxic neuronal injury in cell culture; Uliasz TF et al.; An automated method for the determination of neuronal cell death using trypan blue is described . Following various excitotoxic insults, murine mixed cortical cell cultures are stained with trypan blue (0.05%; 15 min), followed by SDS (1%) lysis . The absorbance of the dye is measured spectrophotometrically at 590 nm using a microtiter plate reader . When compared to the biochemical lactate dehydrogenase assay, no statistical difference in the calculated levels of excitotoxic neuronal cell death was noted between the assays in any given paradigm . This method is fast and reliable . It eliminates the need for cell counting, thus allowing for high volume sample analysis with a minimum of sample error . Utility of this trypan blue absorbance spectrophotometric assay is likely to extend beyond the study of excitotoxic neuronal injury and should complement existing methods for measuring neuronal viability and cytotoxicity in cell culture. Mol Cell Probes, 2000 Oct, 14(5), 305 - 10 Sheep poxvirus identification from clinical specimens by PCR, cell culture, immunofluorescence and agar gel immunoprecipitation assay; Mangana-Vougiouka O et al.; Some 40 clinical specimens of skin lesions from sheep pox suspected cases were investigated by four different diagnostic assays: PCR, virus isolation in lamb testis cell cultures, direct immunofluorescent assay (DIFA) and antigen detecting agar gel immune precipitation test (AGIPT) . All the specimens were positive by PCR and virus isolation, 29 were positive by DIFA and 16 by AGIPT . Using virus isolation on cell cultures as the gold standard, the PCR sensitivity was 100%, while that of DIFA and AGIPT was 73% and 40%, respectively . Skin samples with orf lesions or normal skin biopsies were PCR-negative . Cross-reactions with orf virus were observed in three samples only in the AGIPT assay . The PCR described combines high specificity and sensitivity with speed . PCR was therefore shown to be the method of choice for sheep poxvirus diagnosis directly from clinical specimens . Chin J Biotechnol, 1999, 15(4), 231 - 7 Optimization of conditions for safflower cell culture and accumulation of cellicolous product tocopherols; Wang P et al.; The culture of calli, which were induced from cotyledon of Carthamus tinctorius L, was able to synthesize tocopherols . Results determined by NP-HPLC showed that the main component was alpha-tocopherol . When 0.1% casein was added into MS medium, and 2,4-D concentration was adjucted to 0.30 mg/L and 6-BA to 1.80 mg/L, alpha-, beta- and gamma-forms of tocopherol reached 4.1706, 0.3120 and 0.1650 microg/g fresh calli, respectively . The effective tocopherol content was 4.4091 microg/g fresh calli, and was 57 times higher than that in the control. Int J Pharm, 2000 Oct 10, 207(1-2), 21 - 30 A comparison of the permeation enhancement potential of simple bile salt and mixed bile salt:fatty acid micellar systems using the CaCo-2 cell culture model; Meaney CM et al.; The aim of this study was to compare the permeation enhancing potential and toxicity of simple bile salt and bile salt:fatty acid mixed micellar systems using the CaCo-2 cell culture model . The effects of micellar systems of sodium cholate, (NaC), and sodium taurocholate, (NaTC), on the permeability of the hydrophilic markers, mannitol (182) and polyethylene glycols (PEGS) 900 and 4000, were assessed . Simple micelle systems of the unconjugated bile salt, NaC, caused greater enhancement of the hydrophilic markers than the conjugated bile salt, NaTC . In the case of NaC systems the enhancement was coincident with excess membrane disruption and toxicity as indicated by altered TEERs, TEMs, MTT values, and, the lack of recovery following removal of the enhancer . In contrast, the NaTC systems were less toxic, and, in the simple micelle form the likely mechanism of enhancement of the hydrophilic markers is via a transient effect on the tight junctions . Formation of mixed micellar systems with linoleic acid (LA) accentuated the toxic effects of NaC . In comparison, NaTC:LA mixed micelles showed superior permeability enhancement versus simple micelles without increasing membrane toxicity . The mechanism of enhancement of NaTC:LA appears more complex and involves a possible combination effect on both the paracellular and transcellular routes. Pathol Oncol Res, 2000, 6(3), 202 - 9 Tenascin expression in primary and recurrent breast carcinomas and the effect of tenascin on breast tumor cell cultures; Tokes AM et al.; Tenascin is generally classified as an anti-adhesive protein . Many cells do not adhere to tenascin or if they adhere they do not spread . In this study we analysed the stromal expression of tenascin-C in primary, second primary and recurrent breast carcinomas and the ability of tenascin-C to stimulate the focal adhesion plaques in MDA-MB-435 breast carcinoma cell line . To assess the tenascin-C expression formalin-fixed, paraffin-embedded specimens of 20 specially selected breast carcinomas and their recurrences (14) or a second primary breast cancer of the same patient (6) were examined with immunohistochemical methods . We also studied the effect of tenascin-C on focal adhesion plaques added to MDA-MB-435 breast carcinoma cell line . During a median 2,9-year patient follow up 14 local recurrences and 6-second primary breast carcinomas developed in the 20 patients . In 3 cases a second recurrence occurred . The presence of tenascin in tumor cells, in the proliferating and some normal ducts, near to the tumor cell nests, in the stroma and in ductal carcinoma in situ component of the invasive carcinoma may suggest the role of tenascin played in tumor cell migration . Soluble tenascin added to the cell culture had minimal or no effect on focal adhesion plaques . Tenascin only seems not to be of prognostic value in predicting the local recurrence of breast cancer. Eur J Pharm Sci, 2000 Oct, 11 Suppl 2, S51 - 60 Cell cultures as tools in biopharmacy; Braun A et al.; A survey is given on a few selected cell culture models that are used for transport studies . They are characterised for growth, transcellular electrical resistance and cytoarchitecture . The importance of standardisation in view of their use as transport models is documented . Their potential for studies on passive permeation and P-glycoprotein-mediated transport is explored and related to published data . Transport studies are presented that were performed in a two-chamber set-up, the Costar "vertical diffusion system" . A series of non-homologous compounds showed similar permeability data (P(app)) in the different cell cultures . The origin of the cell type had no remarkable influence on passive transcellular permeation . MDCK cells, an epithelial cell line of canine kidney origin, are perfectly suited to screen for passive permeation . They have low expression of transporter proteins and low metabolic activity . In general, they probably represent the best-known epithelial cell line with respect to genetics as well as lipid and protein composition . MDCK cells are easy to handle . Transport experiments can be done between 7 and 14 days after seeding, when the stationary growth phase is reached . To screen for P-glycoprotein substrates, efflux and uptake studies were performed with mdr1-transfected MDCK cells (MDR1-MDCK) in a one-chamber system in the presence or absence of verapamil or cyclosporin A as inhibitor . Evidence is presented why the transfected cells, which express large amounts of P-glycoprotein, are not suitable for two-chamber transport studies. Crit Rev Oncol Hematol, 2000 Nov-Dec, 36(2-3), 141 - 57 The multilayered postconfluent cell culture as a model for drug screening; Padron JM et al.; New drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals . In this paper, we review the characteristics and scope of a relatively simple cell-culture system with a three-dimensional organisation pattern - the multilayered postconfluent cell culture model . Solid tumour cell lines from diverse origins when grown in V-bottomed microtiter plates reach confluence in 3-5 days and then start to form multilayers . The initial exponential growth of the culture is followed by a plateau phase when cells reach confluence . This produces changes in the morphology of the cells . For some cell lines, it is possible to observe cell differentiation . A substantial advantage of the system is the use of the sulforodamine B (SRB) assay to determine relative cell growth or viability, which allows semiautomation of the experiments . Several experiments were performed to assess the differences and similarities between cells cultured as monolayers and multilayers, and eventually, compared with the results for solid tumours and some other models such as spheroids . Cell-cycle analysis for multilayers showed a lower S-phase arrest, which is accompanied by a decrease in the expression of cell-cycle-related proteins and a decrease in cellular nucleotide pools . Gene and protein expression of topoisomerase I, topoisomerase II and thymidylate synthase expression were lower for multilayers, but no substantial changes were observed for the expression of DT-diaphorase . P53 expression increased . Multilayer cultures present distinctive properties for drug transport across the membrane, drug accumulation and retention . In fact, the transport of antifolates across the membrane, accumulation of topotecan and gemcitabine-triphosphate are reduced in multilayers when compared with monolayers, which may be related to a decrease in drug penetration to the inner regions of the multilayers . Alteration of these pharmacodynamic parameters is directly related to a decrease in drug activity . The most powerful application of multilayers is in the assessment of cytotoxicity . Solid tumour cell lines from different origins have been treated with several conventional and investigational anticancer drugs . The data show that multilayers are more resistant to the drugs than the corresponding monolayers, but there are substantial differences between the drugs depending on culture conditions, e.g . the difference was rather small for a drug such as cisplatin, miltefosine and EO9, a drug, which is activated under hypoxic conditions . Gemcitabine was active against ovarian cancer but not against colon cancer, resembling the in vivo situation . This observation was not evident with monolayer experiments . Another interesting application is the possibility to perform drug combination studies . The combination of gemcitabine and cisplatin proved to produce selective cell kill in H322 cells (non-small cell lung cancer cell line) . Neither of the drugs was independently able to produce similar effects . In summary, multilayer cultures are relatively simple three-dimensional systems to study the effect of microenvironmental conditions on anticancer drug activity . The model might serve as a base for a more rigorous secondary in vitro screening. Biotechnol Prog, 2000 Sep-Oct, 16(5), 885 - 92 The lactate issue revisited: novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures; Patel SD et al.; It is well established that cell proliferation in batch (unfed) hematopoietic cell cultures is greatly inhibited relative to that in cultures with feeding . What is not known, however, is the nature of this inhibition . On the basis of our observations in hematopoietic cultures that cell proliferation ceases when the lactate concentration ({lactate}) exceeds 20 mM (accompanied by a decrease in culture pH), we investigated the effect of lactate accumulation on cell proliferation, metabolism, and differentiation . We differ in our approach from previous efforts in that we have tried to more accurately recreate the manner in which lactate accumulates in culture by employing a daily feeding protocol in which {lactate} and/or pH in the fresh medium was adjusted to match the conditions prior to feeding . We conclude that the decrease in pH associated with lactate accumulation significantly inhibits both cell proliferation and metabolism . Although inhibition in cultures with high {lactate} and low pH is similar to that in unfed cultures, pH control in unfed cultures does not alleviate the inhibition, indicating that other inhibitory factors are also present . Thus, pH control is necessary, but not sufficient, to eliminate inhibition of cell growth and metabolism in unfed hematopoietic cell cultures . We also conclude that high {lactate} and low pH have little effect on cell differentiation in fed cultures, although there is evidence to suggest that low pH may play a role in monocyte differentiation in unfed cultures. Biotechnol Prog, 2000 Sep-Oct, 16(5), 829 - 36 A tool for modeling strategic decisions in cell culture manufacturing; Farid SS et al.; The development of a prototype tool for modeling manufacturing in a biopharmaceutical plant is discussed . A hierarchical approach to modeling a manufacturing process has been adopted to confer maximum user flexibility . The use of this framework for assessing the impact of manufacturing decisions on strategic technical and business indicators is demonstrated via a case study . In the case study, which takes the example of a mammalian cell culture process delivering a therapeutic for clinical trials, the dynamic modeling tool indicates how manufacturing options affect the demands on resources and the associated manufacturing costs . The example illustrates how the decision-support software can be used by biopharmaceutical companies to investigate the effects of working toward different strategic goals on the cost-effectiveness of the process, prior to committing to a particular option. Biotechnol Prog, 2000 Sep-Oct, 16(5), 815 - 21 A comparison of different methods to determine the end of exponential growth in CHO cell cultures for optimization of scale-up; Schoenherr I et al.; Maximizing cell growth rate and cell yield are among the most important features of a successful mammalian cell culture production process . To minimize time and resources needed to scale up cell mass it is important to maintain the cultures in exponential growth at every scale . Here we report results comparing viable cell counts, packed cell volume, intracellular nucleotide ratios, cell cycle analysis, and on-line oxygen uptake rates (OUR) and optical density for the determination of the end of exponential growth to optimize transfer times during scale-up of CHO cell cultures . Viable cell concentration, packed cell volume, and relative abundance of cells in S-phase were not very reliable at determining the end of exponential growth during the process . In contrast, on-line determination of OUR and off-line determination of intracellular nucleotide ratios (U-ratio) were very sensitive to changes in growth rate, enabling clear determination of the end of exponential growth within a short time . Although on-line OUR was found to be the most convenient and fastest method, it is restricted to instrumented and continuously monitored cultures . In contrast the nucleotide method can be applied with any culture scale and condition but needs the availability of an operator running an HPLC system and takes about an hour from sampling to result . Optical density showed an inflection along with OUR and U-ratio but was less sensitive in determining the end of exponential growth. Biotechnol Prog, 2000 Sep-Oct, 16(5), 809 - 14 Biomass and aggregation analysis of human embryonic kidney 293 suspension cell cultures by particle size measurement; Tsao YS et al.; A method has been developed to monitor the cell growth of aggregated human embryonic kidney 293 (HEK293) suspension cultures by measuring cumulative particle volume and the particle size distribution . This method employs a particle size analyzer that determines the size of individual particles by detecting their light obscuration (blockage) or scattering . Cell counts derived from the cumulative volume of the cell particle correlate well with manual cell counts from a hemacytometer at different stages of growth . This correlation was further confirmed by quantifying total cellular protein of the samples . Simultaneously, the aggregation state of the samples can also be monitored and mathematically described . Results from this study demonstrate that this simple and reproducible method allows the direct measurement of cumulative cell volume and the degree of cell aggregation, as well as an indirect assessment of cell counts. Biotechnol Prog, 2000 Sep-Oct, 16(5), 803 - 8 On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process; Zeiser A et al.; On-line monitoring of insect cell cultures used for the production of recombinant proteins with the baculovirus expression vector system (BEVS) provides valuable tools for the optimization, operation, and control of the production process . The relative permittivity (epsilon') and CO(2) evolution rates (CER) were measured on-line using the biomass monitor and the infrared CO(2) analyzer, respectively . The growth and infection phases of two different cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni(High-5), were monitored using the above measurements . These in turn were correlated to the progress of the culture by using the off-line measurements of protein produced, virus titer, and biovolume, which is the product of viable cell density and mean cell volume . The epsilon', CER, and the biovolume profiles were closely matched during the growth phase of cells when grown in a batch or fed batch culture . The relationship became more complex when the cultures were either in stationary phase or in the postinfection phase . The epsilon' profile was found to be a good indicator of the process of synchronous baculoviral infection, showing a plateau between 18 and 24 h postinfection (hpi), the period during which budded virus is produced, and a peak at approximately 48 hpi correlated to the onset of accelerated cell lysis . The CER profile continues to increase after the growth period with a peak around the 24 hpi period, after which there is a decline in the profile corresponding to release of virus as seen from virus titer determinations . This was examined for Sf-9 cultures under conditions of cell densities from 3 to 50 x 10(6) cells/mL and MOI values ranging from 0.001 to 1000 . The profiles were found to be similar also in the case of the High-5 cells . Thus both measurements give reliable information regarding the physiological status of the cells as seen from their correlation to virus and protein production . A further combination of these with the off-line measured parameters such as the biovolume and metabolite concentrations can give a more detailed understanding of the process and help in the better design and automation of these processes. Biotechnol Prog, 2000 Sep-Oct, 16(5), 800 - 2 Using the Microcyte flow cytometer to monitor cell number, viability, and apoptosis in mammalian cell culture; Harding CL et al.; The Microcyte is a novel, portable flow cytometer based on diode laser technology whose use has been established for yeast and bacterial analysis . We present data that demonstrate its suitability for routine mammalian cell counting and viability determination . To extend its range of applications in the field of animal cell culture biotechnology, a test to determine the number of apoptotic cells present has been developed for use with the instrument . Apoptosis was induced in hybridoma cell cultures by treatment with camptothecin . Apoptotic cells were labeled with biotinylated Annexin V and then visualized using a streptavidin-allophycocyanin conjugate . Their numbers were counted, and the cell size of the apoptotic cell population was determined using the Microcyte. Biotechnol Prog, 2000 Sep-Oct, 16(5), 795 - 9 Effect of low inoculation density in the scale-up of insect cell cultures; Marteijn RC et al.; The scale-up of insect cell cultures and the production of baculovirus with these cultures is dependent on the inoculation density applied . The effect of applying a low inoculation on the specific growth rate and on the duration of the lag phase was tested . Three different cell lines, HzAm1, Ha2302, and Sf21 were tested in a total of five cell line/medium combinations . Growth in suspension culture was examined, and data obtained were fitted with the Gompertz equation . A significant decline in specific growth rate with decreasing inoculation density was observed in all cell line/medium combinations, except for HzAm1 . No critical inoculation density, below which no growth would occur, was found . In suspension culture in shake flasks, an inoculation density of 5 x 10(4) cells/mL is achievable, without severely influencing the overall growth rate . A lower inoculation density in suspension culture results in less steps in the scale-up process and might be a tool in bypassing the viral passage effect. Biotechnol Prog, 2000 Sep-Oct, 16(5), 775 - 81 Osmoprotective effect of glycine betaine on thrombopoietin production in hyperosmotic Chinese hamster ovary cell culture: clonal variations; Kim TK et al.; When 23 recombinant Chinese hamster ovary (rCHO) cell clones were cultivated in hyperosmolar medium resulting from NaCl addition (533 mOsm/kg), their specific thrombopoietin (TPO) productivity (q(TPO)) was increased . However, due to depressed cell growth at elevated osmolality, no enhancement in the maximum TPO titer was made in batch cultures of all 23 clones . To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved TPO production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures of 23 clones were performed in the presence of 15 mM glycine betaine . Glycine betaine was found to have a strong osmoprotective effect on all 23 clones . Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled 22 clones to grow at 542 mOsm/kg, where most clones could not grow in the absence of glycine betaine, but at a cost of reduced q(TPO) . However, the relative decrease in q(TPO) varied significantly among clones . Thus, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among clones . Six out of 23 clones displayed more than a 40% increase in the maximum TPO titer in the hyperosmolar medium containing glycine betaine, compared with that in the standard medium with a physiological osmolality . Taken together, the results obtained here emphasize the importance of selection of clones for the successful use of hyperosmotic pressure and glycine betaine as an economical means to improve TPO production. Biotechnol Prog, 2000 Sep-Oct, 16(5), 769 - 74 Measurement and control of dissolved carbon dioxide in mammalian cell culture processes using an in situ fiber optic chemical sensor; Pattison RN et al.; At high viable cell concentrations in large-scale mammalian cell culture processes, the accumulation of dissolved carbon dioxide (dCO(2), typically quantified as an equilibrium gas-phase concentration) becomes problematic as a result of low CO(2) removal rates at reduced surface-to-volume ratios . High dCO(2) concentrations have previously been shown to inhibit cell growth and product formation in mammalian cells and to alter the glycosylation pattern of recombinant proteins . Therefore, reliable monitoring and control of dCO(2) are important for successful large-scale operation . Off-line measurements by instruments such as blood gas analyzers (BGA) are constrained by the low frequency of data collection and cannot be used for on-line control . In a preliminary evaluation of the YSI 8500 in situ sensor, a response time (t(90%)) of 6 min, sensitivity of 0.5% CO(2) (3.6 mmHg), and linearity of measurement (R(2) = 0.9997) between the equivalent gas-phase partial pressure of 0-180 mmHg (0% and 25% CO(2)) were established . Measurements were found to be unaffected by culture pH and typical mammalian cell culture concentrations of glucose, glutamine, glutamate, lactate, and ammonium . The sensor withstood repeated sterilization and cleaning cycles . The reliability of this sensor was demonstrated in microcarrier-based Chinese hamster ovary (CHO) cell perfusion cultures at reactor scales of 30, 40, 340, and 2000 L and was successfully implemented in a dCO(2) control strategy using N(2) sparging. Biotechnol Prog, 2000 Sep-Oct, 16(5), 760 - 8 Ammonia removal using hepatoma cells in mammalian cell cultures; Choi YS et al.; It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures . It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia . Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level . However, primary hepatocytes lost the liver function gradually and finally died after 2-3 weeks . Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures . Hep G2 cells, which are immortal, also showed a strong ammonia removal activity . The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes . However, urea could not be detected in the course of ammonia removal by Hep G2 cells . Instead of urea, Hep G2 cells secreted glutamine into the culture medium . The capacity for ammonia removal was higher in the absence than in the presence of glutamine . Thus we checked the activity of glutamine synthetase in the Hep G2 cells . The level of glutamine synthetase activity increased with the addition of ammonium chloride . This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis . Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures . These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected . In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells. Biotechnol Prog, 2000 Sep-Oct, 16(5), 693 - 7 Effects of insulin and LongR(3) on serum-free Chinese hamster ovary cell cultures expressing two recombinant proteins; Morris AE et al.; Insulin is the most commonly used growth factor for sustaining cell growth and viability in serum-free Chinese hamster ovary (CHO) cell cultures . In the present study insulin and IGF-1 analogue (LongR(3)) were compared for their ability to support growth, viability, and production of two serum-free CHO cell lines expressing recombinant protein . The first cell line, VA12, expresses protein B, and the second cell line, CL23, expresses protein C . Both molecules are recombinant cytokine receptors . VA12 will grow in serum-free media lacking growth factor, while CL23 requires either insulin or LongR(3) for cell growth . Both cell lines, however, require a growth factor for optimal performance under production conditions . In this study, LongR(3) was better able to sustain the viability of both cell lines under production conditions than insulin . These data indicate that while insulin and LongR(3) can both serve as growth and viability factors for CHO cells, LongR(3) is the preferred growth factor for cell lines VA12 and CL23. Neurotoxicology, 2000 Aug, 21(4), 599 - 605 Use of aggregating brain cell cultures to study developmental effects of organophosphorus insecticides; Zurich MG et al.; Aggregating brain cell cultures of fetal rat telencephalon can be grown in a chemically defined medium for extended periods of time . After a phase of intense mitotic activity, these three-dimensional cell cultures undergo extensive morphological differentiation, including synaptogenesis and myelination . To study the developmental toxicity of organophosphorus compounds (OP), aggregating brain cell cultures were treated with parathion . Protein content and cell type-specific enzyme activities were not affected up to a concentration of 10(5) M . Gliosis, characterized by an increased staining for glial fibrillary acidic protein (GFAP), was observed in immature and in differentiated cells . In contrast, uridine incorporation and myelin basic protein (MBP) immunoreactivity revealed strong differences in sensitivity between these two developmental stages . These results are in agreement with the view that in vivo the development-dependent toxicity is not only due to changes in hepatic detoxification, but also to age-related modifications in the susceptibility of the different populations of brain cells . Furthermore, they underline the usefulness of histotypic culture systems with a high developmental potential, such as aggregating brain cell cultures, and stress the importance of applying a large range of criteria for testing the developmental toxicity of potential neurotoxicants. Anal Biochem, 2000 Oct 15, 285(2), 211 - 9 Differential use of Alcian blue and toluidine blue dyes for the quantification and isolation of anionic glycoconjugates from cell cultures: application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes; Terry DE et al.; Alcian blue and toluidine blue dyes form complexes with anionic glycoconjugates (AG) such as proteoglycans (PG) and glycosaminoglycans (GAG) . However, the Alcian blue-AG complexes do not readily dissociate, while the toluidine blue-AG complexes do so in salt solutions . This differential dissociation of the dye-AG complexes has been utilized in the analysis and isolation of radiolabeled AG elaborated by articular chondrocyte cultures incubated with the radiolabeled precursors of AG . For the rapid quantification of newly synthesized (35)S-labeled PG, small replicate aliquots of the radiolabeled culture media were applied directly to cellulose acetate strips, stained with Alcian blue and the stained immobilized radiolabeled PG was quantified by liquid scintillation counting . Comparison of anionic glycoconjugates quantified in the culture media employing toluidine blue and Alcian blue staining on cellulose acetate trips gave similar results . Staining on cellulose acetate strips using these two dyes is particularly suited for the simultaneous processing of large numbers of samples, as illustrated by the screening of the effects of biological materials and drugs on AG synthesis, in cultures labeled with {(35)S}-sulfate and {(3)H}-glucosamine . The Alcian blue and toluidine blue precipitation methods yielded similar results for the total AG recovered from the media of TGF-beta-stimulated chondrocytes . Electrophoretic analysis of toluidine blue- and Alcian blue-precipitated AG followed by autoradiography and Alcian blue staining in combination with silver nitrate demonstrated that both dyes yielded similar pattern of bands on gels . However, some AG from Alcian blue precipitate did not enter the gel, suggesting incomplete dissociation of Alcian blue-AG complex . The application of the toluidine blue precipitation method, in combination with enzymatic digestion of the GAG chains of the PGs, is illustrated by the isolation of a non-PG high-molecular-weight AG, as well as the PGs from the media of chondrocyte cultures stimulated by TGF-beta . ASAIO J, 2000 Sep-Oct, 46(5), 522 - 6 New type of matrix support for bone marrow cell cultures: in vitro culture and in vivo transplantation experiments; Tun T et al.; A new type of bone marrow cell culture system was developed by using a highly porous substrate matrix, i.e., porous polyvinyl formal (PVF) resin . Murine bone marrow (BM) cells were cultured without the use of exogenous growth factors in a three-dimensional matrix support made of collagen coated porous PVF resin . To examine the optimal conditions for highest stromal cell density, short-term and long-term in vitro culture experiments using PVF were performed . In the short-term culture experiments, it was found that cubes of PVF (10 x 10 x 2 mm and 130 microm in pore size) coated with type I collagen with a seeding density of 2x10(7) BM cells offered the most appropriate culture conditions . In the long-term cultures, BM cells in PVF maintained their viability for up to 6 weeks . In another series of re-inoculation experiments, freshly isolated BM cells were inoculated onto the already developed stromal layer . In this study, a higher cell density of the stromal layer was obtained in the PVF culture compared with those in the control dish culture . Based upon the results of in vitro experiments, in vivo transplantation studies were also performed . Histologic examinations of the subcutaneously transplanted PVF with stroma revealed host derived hematopoiesis inside the PVF matrix . Moreover, survival of approximately 15% of the transplanted BM cells that were cultured in PVF were confirmed in X-ray irradiated recipients . From these results, it is suggested that PVF resin is a promising three-dimensional substrate for BM cell culture and that it can maintain hematopoietic stem cells or progenitor cells after transplantation. Kidney Int, 2000 Oct, 58(4), 1588 - 602 Elastic fiber proteins in the glomerular mesangium in vivo and in cell culture; Sterzel RB et al.; BACKGROUND: Glomerular capillaries of the mammalian kidney are exposed to high intraluminal hydrostatic pressures and require elastic constraint to maintain size, shape, and integrity . Previous morphological and functional studies indicated that the extracellular matrices of glomeruli, that is, basement membrane and mesangial matrix, contribute to glomerular resilience and mechanical stability . Immunofluorescence microscopy findings demonstrated elastic fiber components to be located in the renal vasculature, including glomeruli . The aim of this study was to clarify the exact glomerular localization, composition, and cellular production of these proteins . METHODS: We examined the renal distribution of the elastic fiber proteins fibrillin-1, emilin, microfibril-associated glycoproteins (MAGPs) 1 and 2, latent transforming growth factor-binding protein-1 (LTBP-1), and elastin using immunohistology and immunoelectron microscopy of human, rat, and mouse kidneys . In mesangial cell cultures, we also studied the expression and extracellular deposition of such proteins by use of Northern blotting and immunocytochemistry . RESULTS: Fibrillin-1, emilin, MAGPs 1 and 2, and LTBP-1 were present in glomeruli of mouse, rat, and human kidney, where they were located predominantly in the mesangial extracellular matrix underlying glomerular endothelium and basement membrane . Several of these proteins, as well as elastin, were also expressed in the renal vasculature . While elastin localized to the glomerular vascular pole in afferent and efferent arterioles extending to Bowman's capsule, it was not found in the glomerular capillary tuft . Cultured mesangial cells of rat, mouse, and human kidneys expressed mRNAs of fibrillin-1, emilin, MAGP-2, and elastin, and the respective proteins localized within and outside of mesangial cells, as shown by immunocytochemistry . mRNA expression of fibrillin-1, emilin, and elastin was strong in quiescent mesangial cells; their gene expression was further up-regulated by transforming growth factor-beta1, while it was transiently reduced when cells were exposed to mitogenic 10% fetal calf serum and platelet-derived growth factor . CONCLUSIONS: These findings demonstrate that specific elastic fiber proteins are produced and secreted by mesangial cells . This process is regulated by growth factors . Their abundance in the extracellular matrix of the mesangium is in keeping with the concept that elastic fiber proteins contribute to the mechanical stability and elastic strength of the glomerular capillary tuft. Stem Cells, 1998, 16 Suppl 1, 51 - 73 Unilineage hematopoietic differentiation in bulk and single cell culture; Ziegler B et al.; The rarity of hematopoietic stem and progenitor cells (HSCs, HPCs) has hampered the analysis of cellular and molecular mechanisms underlying early hematopoiesis . Methodology for HPC purification has partially offset this limitation . A further hurdle has been represented by the heterogeneity of the analyzed HPC/precursor populations: recently, development of unilineage HPC differentiation cultures has provided homogeneous populations of hematopoietic cells, particularly in the early differentiation state, i.e., populations pertaining to a single lineage and a restricted stage of differentiation/maturation, but sufficiently large for cellular/molecular analysis . This report focuses on the development and characterization of the unilineage HPC differentiation culture systems . A section is devoted to selected cellular and molecular mechanisms underlying hematopoiesis, which have been investigated by the HPC unilineage culture approach . Finally, recent advances in the development of HPC unilineage cultures at single cell level are discussed. J Appl Physiol, 2000 Oct, 89(4), 1553 - 60 A novel cell culture model for studying ischemia-reperfusion injury in lung transplantation; Cardella JA et al.; Many cell culture models have been developed to study ischemia-reperfusion injury; however, none is specific to the conditions of lung preservation and transplantation . The objective of this study was to design a cell culture model that mimics clinical lung transplantation, in which preservation is aerobic and hypothermic . A549 cells, a human pulmonary epithelial cell line, were preserved in 100% O(2) at 4 degrees C for varying periods in low-potassium dextran glucose solution, simulating ischemia, followed by the introduction of warm (37 degrees C) DMEM plus 10% fetal bovine serum to simulate reperfusion . Cultures were assayed for cell attachment and viability . Sequential extension of ischemic times to 24 h showed a time-dependent loss of cells . There was a further decrease in cell number after simulated reperfusion . Cell detachment was due mainly to cell death, as determined by cell viability . The effects of chemical components such as dextran 40 and calcium in the preservation solution and various preservation gas mixtures were examined by use of this model system . With its design and validation, this model could be used to study mechanisms related to ischemia-reperfusion injury at the cellular and molecular level. Dig Dis Sci, 2000 Aug, 45(8), 1667 - 9 Method to obtain endoscopic esophageal samples for primary cell culture: focus on infectious contamination; Constant-Neto M et al.; Cell culture techniques hold great importance for the development of molecular biology . However, when used to study oncology, most of the samples come from surgical specimens . Endoscopy is a interesting alternative to get samples for culture . We studied a protocol to allow the control of infectious contamination potentially related to endoscopy, which could preclude it as a method to obtain cells for culture . Esophageal biopsies from 30 patients were taken through upper gastrointestinal endoscopy, using a previously flamed forceps, and were cultivated with and without amphotericin . Our results showed contamination in 3.3% of the wells without the antifungal and in 0.8% of those with it . Regarding the 30 cases studied, the described protocol was able to provide samples free of contamination in all of them. J Clin Pathol, 2000 Aug, 53(8), 630 - 3 Toxoplasma dye test using cell culture derived tachyzoites; Ashburn D et al.; AIMS: To assess the diagnostic usefulness of Toxoplasma gondii tachyzoites produced by serial passage in HeLa cell culture . METHODS: Tachyzoites derived from serial passage in cell culture were used in the dye test . Human sera were also examined to determine their suitability for use as accessory factor . Using the optimum conditions, the dye test using cell culture derived tachyzoites was compared with the current method of production (animal culture) on 105 randomly selected sera . Start up and maintenance costs of each system were compared . RESULTS: Tachyzoites in most cell culture harvests (84%) from both early and later passes were useable . Tachyzoite yield and viability were maintained during serial passage in cell culture . Sodium citrate was used to modify accessory factor and improve its suitability . The performance of the accessory factor was improved by the addition of 1% and 3% sodium citrate for the current and cell culture systems, respectively . Under optimum conditions, dye test titres using cell culture and current systems were compared on 105 randomly selected sera . The results from 92 of 105 (87.6%) patients agreed or were within one dilution, but all discrepancies were resolved on re-testing . Start up costs for the current system would be 2.5 times and overall maintenance three times more expensive than cell culture . CONCLUSIONS: Tachyzoites derived from cell culture can be used routinely in the dye test . Production in cell culture is more cost effective than animal culture . It is possible for general hospitals to perform the dye test, thus obtaining faster and more specific results. Biochim Biophys Acta, 2000 Sep 18, 1469(2), 63 - 77 Ganglioside molecular species containing C18- and C20-sphingosine in mammalian nervous tissues and neuronal cell cultures; Sonnino S et al.; Gangliosides exist as a very complex mixture of species differing in both the hydrophilic and hydrophobic moieties . They are particularly abundant in the central nervous system (CNS), where they have been associated with development and maturation of the brain, neuritogenesis, synaptic transmission, memory formation and synaptic aging . Today, many data suggest that some of the effects exerted by gangliosides are due to interactions with proteins that participate in the transduction of signals through the membrane in membrane microdomains . A specific characteristic of CNS gangliosides is the structure of their long-chain base (LCB) . In fact, considering all the mammalian cell sphingolipids, gangliosides, sulphatides, neutral glycosphingolipids, sphingomyelin and ceramides, it would seem that while the LCB with 18 carbons is the main component of all sphingolipids, only CNS gangliosides contain significant amounts of LCB with 20 carbons . C18-Sphingosine is always present in cell gangliosides; the individual ganglioside species containing C18-sphingosine increase during cell differentiation then remain constant during cell aging . Gangliosides containing C20-sphingosine are absent, or present only in traces, in undifferentiated cells but with the onset of cell differentiation they appear, their content slowly but continuously increasing throughout the life span . In this review we discuss the chemistry, physico-chemistry and metabolism of ganglioside species differing in LCB length and introduce the hypothesis that the varying ratio between C18- and C20-gangliosides during CNS development and aging can be instrumental in modulating membrane domain organisation and cell properties. Virology, 2000 Sep 30, 275(2), 306 - 17 Varicella-zoster virus gE escape mutant VZV-MSP exhibits an accelerated cell-to-cell spread phenotype in both infected cell cultures and SCID-hu mice; Santos RA et al.; Varicella-zoster virus is considered to have one of the most stable genomes of all human herpesviruses . In 1998, we reported the unanticipated discovery of a wild-type virus that had lost an immunodominant B-cell epitope on the gE ectodomain (VZV-MSP); the gE escape mutant virus exhibited an unusual pattern of egress . Further studies have now documented a markedly enhanced cell-to-cell spread by the mutant virus in cell culture . This property was investigated by laser scanning confocal microscopy combined with a software program that allows the measurement of pixel intensity of the fluorescent signal . For this new application of imaging technology, the VZV immediate early protein 62 (IE 62) was selected as the fluoresceinated marker . By 48 h postinfection, the number of IE 62-positive pixels in the VZV-MSP-infected culture was nearly fourfold greater than the number of pixels in a culture infected with a low-passage laboratory strain . Titrations by infectious center assays supported the above image analysis data . Confirmatory studies in the SCID-hu mouse documented that VZV-MSP spread more rapidly than other VZV strains in human fetal skin implants . Generally, the cytopathology and vesicle formation produced by other strains at 21 days postinfection were demonstrable with VZV-MSP at 14 days . To assess whether additional genes were contributing to the unusual VZV-MSP phenotype, approximately 20 kb of the VZV-MSP genome was sequenced, including ORFs 31 (gB), 37 (gH), 47, 60 (gL), 61, 62 (IE 62), 66, 67 (gI), and 68 (gE) . Except for a few polymorphisms, as well as the previously discovered mutation within gE, the nucleotide sequences within most open reading frames were identical to the prototype VZV-Dumas strain . In short, VZV-MSP represents a novel variant virus with a distinguishable phenotype demonstrable in both infected cell cultures and SCID-hu mice . Biol Pharm Bull, 2000 Sep, 23(9), 1118 - 21 Hepatoprotective and hepatotoxic activities of sophoradiol analogs on rat primary liver cell cultures; Kinjo J et al.; As a part of our studies of hepatoprotective drugs, we prepared kaikasaponin I (2), sophoradiol monoglucuronide (SoMG, 3) and sophoradiol (4) from kaikasaponin III (1) . We examined the hepatoprotective effects of these analogs, using immunologically-induced liver injury in primary cultured rat hepatocytes and found that compound 1 was more effective than soyasaponin I (1a) while 2 was more effective than 1 . On the other hand, 3 was less effective than 2 at 30-200 microm . Further, compound 3 was strongly cytotoxic at 500 microM while 4 exhibited hepatoprotective activity at the same dose, although less potent . When the cytotoxicity toward hepatocytes of these analogs was tested, only 3 was cytotoxic at doses of 200 and 500 microM . This is the first example of an oleanene glucuronide (OG) which is cytotoxic toward hepatocytes . Compound 3 exhibited hepatoprotective activity at 200 microM, while it was also cytotoxic at the same dose without antiserum . Therefore, the hepatoprotective activity of OG represents a balance between a hepatoprotective action and its cytotoxicity toward hepatocytes. Virus Res, 2000 Aug, 69(1), 31 - 9 The French neurotropic vaccine strain of yellow fever virus accumulates mutations slowly during passage in cell culture; Holbrook MR et al.; This study of the yellow fever French neurotropic vaccine strain from the Institut Pasteur (FNV-IP) demonstrates that this viral genome is not as stable as that of the 17D-204 vaccine virus . FNV-IP was plaque-purified three times and then passaged eight times in Vero cells . Viral populations from the second and eighth passage post purification were sequenced and compared to the published sequences of FNV-IP . The passage-2 viral population had 31 nucleotide and nine amino acid changes compared to the parental virus while the passage-8 virus had six additional nucleotide changes encoding a single amino acid substitution . The plaque-purified virus also had two sequence deletions in the 3'-noncoding region . The plaque purification resulted in selection of a passage-2 virus that had a mouse LD(50) of 20 pfu/ml, 67-fold greater than parental FNV-IP which had an LD(50) of 0.3 pfu/ml . Subsequent passage in Vero cells resulted in a passage-8 virus which had increased neurovirulence with an LD(50) of 3.2 pfu/ml . The only amino acid difference between the passage-2 and passage-8 viruses was at amino acid 638 of NS5 which lies within domain V of the RNA-dependent-RNA polymerase . Overall, these data indicate that FNV-IP virus has an inherently less stable genome than 17D vaccine virus and a variable viral population. Pharmacol Res, 2000 Oct, 42(4), 345 - 53 Long-term effects of parathyroid hormone, 1,25-dihydroxyvitamin d(3), and dexamethasone on the cell growth and functional activity of human osteogenic alveolar bone cell cultures; Adelina Costa M et al.; The proliferation-differentiation behaviour of human alveolar bone cell cultures grown for 32 days in conditions that allowed the complete expression of the osteoblastic phenotype was significantly affected by the continuous presence of parathyroid hormone, 1, 25-dihydroxyvitamin D(3), or dexamethasone . Parathyroid hormone and, in particular, dexamethasone significantly induced the differentiation of osteoblastic cells . Moreover, cultures exposed to these hormones presented an earlier appearance and higher levels of alkaline phosphatase, and an increased ability to form calcium phosphate deposits in the extracellular matrix . Growth Horm IGF Res, 1998 Apr, 8(2), 125 - 32 Insulin-like growth factor: receptor and binding proteins in human retinal endothelial cell cultures of diabetic and non-diabetic origin; Spoerri PE et al.; Human retinal endothelial cell (HREC) cultures of diabetic and non-diabetic origin were examined for the production of insulin-like growth factor I (IGF-I), IGF-I receptor and IGF-binding proteins (IGFBPs) using colloidal gold quantitative immunocytochemistry and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) . The levels of immunoreactivity for IGF-I receptor and for four IGFBPs (IGFBP-1, -2, -3 and -5) were significantly increased in diabetic HREC cultures . Moreover, diabetic HREC cultures showed significantly less immunoreactivity for IGF-I and for IGFBP-4 as compared to non-diabetic HREC cultures . Message levels for IGF-I decreased two-fold in diabetic HREC and correlated with protein levels . Message levels for IGFBP-1, -2 and -5 increased 1.5-, 1.7- and 1.6-fold, respectively, in diabetic HREC and correlated with protein levels . However, the protein levels for IGF-I receptor and IGFBP-3 and -4 did not correlate with mRNA levels . There were no differences in mRNA levels for IGF-I receptor and IGFBP-3 and -4 between diabetic and non-diabetic HREC cultures, suggesting a post-transcriptional regulation of IGF-I receptor and the two IGFBPs . The net effect, however, supports enhanced IGF-I action in HREC cultures of diabetic origin which is an important cellular event in diabetic retinopathy. Blood, 2000 Sep 15, 96(6), 2219 - 25 Soluble CD40 ligand induces selective proliferation of lymphoma cells in primary mantle cell lymphoma cell cultures; Andersen NS et al.; Interaction between CD40 and the CD40 ligand (CD40L) is critical for the survival and proliferation of B cells during immunopoiesis . However, the role of CD40L in the pathogenesis of malignant lymphomas is ambiguous . Primary mantle cell lymphoma (MCL) cells were cultured in the presence of recombinant human CD40L trimer (huCD40LT), and a significant time- and dose-dependent induction of DNA synthesis was observed in thymidine incorporation assays (n = 7, P <.04) . The maximal rate of DNA synthesis was reached at huCD40LT doses of 100 ng/mL and above after 4 days of culture, but a significant increase of DNA synthesis was detected already at doses of 1 ng/mL (P =.03) . HuCD40LT never inhibited the basal level of DNA synthesis . These findings established 400 ng/mL of huCD40LT for 4 days as standard conditions in the system . Under these conditions, huCD40LT significantly increased the proportion of cells in the S/G(2)/M phases of the cell cycle in 4 of 7 studied cases, while the fraction of apoptotic cells remained unchanged (n = 7) . HuCD40LT also induced expression of CD80/B7-1, CD86/B7-2, and CD95/Fas and up-regulated the expression of HLA-DR (n = 6) . With the use of bromodeoxyuridine incorporation in triple-color flow cytometric analysis, it was found that huCD40LT induced cell-cycle progression in light chain-restricted cells only, of which a median of 14% (range, 0.5% to 29%; n = 4) returned to G(0/1) phase DNA content after bromodeoxyuridine incorporation, demonstrating completion of at least one cell cycle in the presence of huCD40LT . Thus, primary clonal MCL cells are activated and can proliferate in the presence of huCD40LT as a single agent. Neurosci Lett, 2000 Sep 15, 291(2), 121 - 5 Neurotrophic factor protection against ethanol toxicity in rat cerebellar granule cell cultures requires phosphatidylinositol 3-kinase activation; Heaton MB et al.; Neonatal rat cerebellar granule cells were used to assess the possible role of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway in the neuroprotective effects of neurotrophic factors against ethanol toxicity . Culture conditions included medium with ethanol (400 and 600 mg/dl), nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), ethanol+NGF or BDNF, the PI3-K inhibitor wortmannin (10 or 100 microM), and wortmannin+ethanol+NGF or BDNF . Neuronal survival was determined via the MTT assay . The results indicated that both NGF and BDNF ameliorate ethanol neurotoxicity, and wortmannin abolished this effect, except at the higher ethanol concentration combined with the lower wortmannin level . These data strongly implicate the PI3-K pathway in growth factor protection against ethanol neurotoxicity. J Nat Prod, 2000 Aug, 63(8), 1077 - 81 Application of cell culture for the production of bioactive compounds from sponges: synthesis of avarol by primmorphs from Dysidea avara; Muller WE et al.; Among all metazoan phyla, sponges are known to produce the largest number of bioactive compounds . However, until now, only one compound, arabinofuranosyladenine, has been approved for application in humans . One major obstacle is the limited availability of larger quantities of defined sponge starting material . Recently, we introduced the in vitro culture of primmorphs from Suberites domuncula, which contain proliferating cells . Now we have established the primmorph culture also from the marine sponge Dysidea avara and demonstrate that this special form of sponge cell aggregates produces avarol, a sesquiterpenoid hydroquinone, known to display strong cytostatic activity especially against mammalian cells . If dissociated sponge cells are transferred into Ca(2+)- and Mg(2+)-containing seawater, they form after a period of two to three days round-shaped primmorphs (size of 1 to 3 mm) . After longer incubation, the globular primmorphs fuse and form meshes of primmorphs that adhere to the bottom of the incubation chamber . Later, during incubation, freely floating mesh-primmorphs are formed . No bacterial rRNA could be detected in the primmorphs . We were able to prove that the primmorphs produce avarol . Levels (1.4 microg of avarol/100 microg of protein) close to those identified in specimens from the field (1.8 microg/100 microg) are reached . Avarol was extracted from the cells with EtOAc and subsequently purified by HPLC . The identification was performed spectrophotometrically and by thin-layer chromatography . Single cells apparently do not have the potency to produce this secondary metabolite . It is concluded that the primmorph model is a suitable system for the synthesis of bioactive compounds in vitro. J Chromatogr A, 2000 Aug 18, 890(1), 15 - 23 Use of spray-dried zirconia microspheres in the separation of immunoglobulins from cell culture supernatant; Subramanian A et al.; A method suitable for the isolation of monoclonal antibodies (MAbs) on novel zirconia microspheres (20-30 microm) is described . Zirconia microspheres were generated by spray drying colloidal zirconia . Spray-dried zirconia microspheres were further classified and characterized by X-ray diffraction, BET porosimetry and scanning electron microscopy . Spray-dried zirconia microspheres were modified with ethylenediamine-N,N'-tetra(methylenephosphonic) acid (EDTPA) to create a cation-exchange chromatographic support . The chromatographic behavior of a semi-preparative column packed with EDTPA-modified zirconia microspheres was evaluated and implications for scale-up are provided . EDTPA-modified zirconia microspheres were further used to purify MAbs from cell culture supernatant . Analysis by enzyme linked immunosorbent assay and gel electrophoresis demonstrate that MAbs can be recovered from a cell culture supernatant at high yield (92-98%) and high purity (>95%) in a single chromatographic step. Sheng Wu Gong Cheng Xue Bao, 2000 Mar, 16(2), 221 - 4 {Multiplication of the shrimp baculovirus HHNBV with primary cell cultures from lymphoid organ of Penaeus chinensis}; Miao HZ et al.; The hypodermal and hematopoietic necrosis baculovirus HHNBV has been confirmed to be the causative agent for the explosive epidermic disease of farmed shrimp Penaeus chinensis since 1993 . The virus was isolated and multiplied successfully in the primary cell cultures from the lymphoid organ of the shrimp . A cell monolayer was formed in three days in the medium MPS and could be maintained for 1-3 months as the medium replaced every 4-5 days . The cytopathic effect occurred in 5 days after inoculation of the tissue extract from the diseased shrimp . The transmission electron microscopy showed many rod-shaped virions of HHNBV in the nuclei of infected cells. Cancer Detect Prev, 2000, 24(3), 234 - 43 Interleukin 18 induces a synergistic enhancement of interferon gamma production in mixed murine spleen cell-tumor cell cultures: role of endogenous interleukin 12; Micallef MJ et al.; ABSTRACT: Interleukin 18 (IL-18) reportedly synergizes with IL-12 and IL-10 for interferon gamma (IFN-gamma) synthesis and natural killer (NK) cell activity, respectively . Here we show that IL-18 alone induces low level IFN-gamma production by unstimulated Balb/c mouse spleen cells, but production is enhanced synergistically in cocultures of spleen cells and allogeneic living or fixed Yac-1 cells . Spleen cells could be primed with IL-18 prior to coculture with Yac-1 cells for IFN-gamma production, which also was observed in cocultures containing either syngeneic or xenogeneic tumor cells . IFN-gamma production in stimulated cocultures was abrogated almost completely by anti-IL-12 antibody and was unrelated to spleen cell lytic activity . IL-10 moderately inhibited IFN-gamma production induced by IL-18 . Therefore, in spleen cell and tumor cell cocultures exposed to IL-18, high levels of IFN-gamma are produced by the spleen cells arising from a synergistic interaction between the exogenous IL-18 and endogenous IL-12; however, this activity is unrelated to the spleen cell lytic activity. J Endocrinol, 2000 Sep, 166(3), 553 - 63 Transfection of human insulin-like growth factor-binding protein 3 gene inhibits cell growth and tumorigenicity: a cell culture model for lung cancer; Hochscheid R et al.; IGF-I and IGF-II are potent mitogens, postulated to exert autocrine/paracrine effects on growth regulation in human lung cancer . Their proliferative effects are modulated by IGF-binding proteins (IGFBPs), which are found in conditioned medium (CM) of lung cancer cell lines . The biological role of the IGFBPs, which are ontogenetically and hormonally regulated, is not fully understood . Both inhibitory and stimulatory effects on cell growth have been demonstrated . Exogenous IGFBP-3 has been consistently shown to block IGF action, inhibiting cell growth in vitro . In order to evaluate the action of endogenously produced IGFBP-3 on cell growth in lung cancer, we stably transfected the non-small cell lung cancer cell line NCI-H23 with human IGFBP-3 cDNA (resulting in NCI-H23 pOPI3/BP-3) or with the vector pOPI3CAT as control (resulting in NCI-H23 pOPI3CAT) . RT-PCR confirmed expression of IGFBP-3-specific mRNA in NCI-H23 pOPI3/BP-3, but not in N! CI-H23 or NCI-H23 pOPI3CAT . Western ligand blot and Western immunoblot analysis of CMs yielded strong signals of the characteristic 40-44 kDa human IGFBP-3 protein in NCI-H23 pOPI3/BP-3 . An IGFBP-3 ELISA demonstrated a 20-fold increase in IGFBP-3 protein expression in NCI-H23 pOPI3/BP-3 as compared with NCI-H23 . The growth of NCI-H23 pOPI3/BP-3 in serum-containing medium was significantly slower (1.7-fold) than that of NCI-H23 or the vector-transfected control NCI-H23 pOPI3CAT . While the proliferation rate of parental and vector-transfected cells could be stimulated by IGF-I, IGF-II, IGF-I analog Long R(3) IGF-I or insulin, that of NCI-H23 pOPI3/BP-3 could not . Xenotransplantation in nude mice resulted in marked tumor growth after the injection of NCI-H23 or NCI-H23 pOPI3CAT, but absent or minimal growth for the IGFBP-3-transfected cell line . These data suggest that IGFBP-3 is a potent inhibitor of cell growth in human lung cancer c! ell lines and may impair tumorigenicity in vivo. Biotechnol Bioeng, 2000 Oct 20, 70(2), 167 - 75 Osmoprotective effect of glycine betaine on foreign protein production in hyperosmotic recombinant chinese hamster ovary cell cultures differs among cell lines; Ryu JS et al.; When three recombinant Chinese hamster ovary (rCHO) cell lines, CHO/dhfr-B22-4, CS13-1.00*, and CS13-0.02*, were cultivated in hyperosmolar media resulting from NaCl addition, their specific foreign protein productivity increased with medium osmolality . However, due to a simultaneous suppression of cell growth at elevated osmolality, no enhancement in the maximum foreign protein titer was made in batch cultures . To test the feasibility of using glycine betaine, known as a strong osmoprotective compound, for improved foreign protein production in hyperosmotic rCHO cell cultures, hyperosmotic batch cultures were carried out in the presence of 15 mM glycine betaine . Glycine betaine was found to have a strong osmoprotective effect on all three rCHO cell lines . Inclusion of 15 mM glycine betaine in hyperosmolar medium enabled rCHO cell lines to grow at 557 to 573 mOsm/kg, whereas they could not grow in the absence of glycine betaine . However, effect of glycine betaine inclusion in hyperosmolar medium on foreign protein production differed among rCHO cell lines . CHO/dhfr-B22-4 cells retained enhanced specific human thrombopoietin (hTPO) productivity in the presence of glycine betaine, and thereby the maximum hTPO titer obtained at 573 mOsm/kg was increased by 72% over that obtained in the control culture with physiological osmolality (292 mOsm/kg) . On the other hand, enhanced specific antibody productivity of CS13-1.00* and CS13-0.02* at elevated osmolality was decreased significantly in the presence of glycine betaine . As a result, the maximum antibody titer at 557 mOsm/kg was similar to that obtained in the control culture with physiological osmolality . The mRNA contents per cell determined by northern blot hybridization correlated with q in all three rCHO cell lines, indicating that transcriptional regulation is responsible in part for q enhancement at hyperosmolality in the absence as well as the presence of glycine betaine . Taken together, efficacy of the simultaneous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among different rCHO cell lines . Br J Dermatol, 2000 Sep, 143(3), 557 - 63 Phenotypic characteristics of Kaposi's sarcoma tumour cells derived from patch-, plaque- and nodular-stage lesions: analysis of cell cultures isolated from AIDS and non-AIDS patients and review of the literature; Simonart T et al.; BACKGROUND: Kaposi's sarcoma (KS) is commonly thought to be derived from endothelial cells because of the predominant expression of endothelial markers in KS lesions . However, the heterogeneity of the spindle-cell compartment makes the precise lineage relationship of KS tumour cells unclear . Cultured KS-derived spindle cells constitutively overexpress antiapoptotic proteins and exhibit invasive properties, which suggests that they may adequately represent the tumour cells of KS . OBJECTIVES: We aimed to investigate the expression of a wide variety of immunohistochemical markers by spindle cells derived from patch-, plaque- and nodular-stage lesions from patients with iatrogenic, sporadic and acquired immune deficiency syndrome-related KS, and to review the data reported by other laboratories . METHODS: Cells from six KS cell cultures derived from four subjects were examined by immunostaining . RESULTS: Comparison of these data indicates that KS-derived spindle cells generally express myofibroblast antigens but lack endothelial and/or leucocyte markers . CONCLUSIONS: As the myofibroblast phenotype is not the predominant feature of KS tissues, our findings further substantiate the view that the in vivo dominant endothelial population represents a reactive hyperplasia rather than the true KS tumour process. J Clin Microbiol, 2000 Sep, 38(9), 3502 - 4 Comparison of a ligase chain reaction-based assay and cell culture for detection of pharyngeal carriage of Chlamydia trachomatis; Winter AJ et al.; In 264 genitourinary medicine clinic attenders reporting recent fellatio, the prevalence of pharyngeal Chlamydia trachomatis determined by an expanded standard including cell culture and two in-house PCR tests was 1.5% in 194 women and zero in 70 men . The ligase chain reaction (Abbott LCx) had a specificity of 99.2% and a positive predictive value of 60%. Folia Histochem Cytobiol, 2000, 38(3), 119 - 27 Effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) release and cell morphology in human pituitary adenoma cell cultures; Fazekas I et al.; Six GH adenomas and three prolactinomas were investigated by light- and electron-microscopic morphological and immunocytochemical methods and the effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) secretion was tested in vitro . The tumour cells of the acromegalic patients revealed both GH and PRL immunoreactivity while prolactinomas showed only PRL activity . All the adenomas stained immunocytochemically also for VIP . By electron microscopy, the tumours included two densely and two sparsely granulated GH, two mixed GH/PRL, and three sparsely granulated PRL adenomas . The dissociated cells were explanted, and cultured in vitro . The cultures in micro test plates were treated with VIP at different concentrations between 10(-5)-10(-12) M . GH and PRL contents in the culture media were measured by radioimmunoassay . GH release was significantly stimulated by VIP in a dose-dependent manner over the whole concentration range, while VIP was effective on the PRL release only at 10(-6)-10(-7) M concentration . The cells of a mixed adenoma were grown in Petri dishes and used for ultrastructural and immunocytochemical studies . The cytoplasmic structure of the cells treated with VIP corresponded to that of active hormone-secreting cells with large ergastoplasmic fields and Golgi zones containing secretory granules . Massive exocytotic events were encountered mainly in the GH-type cells . GH and PRL double immunocytochemistry showed the predominance of GH cells, many of them containing low amounts of PRL as well . Cells predominantly containing PRL were spread among them, they also might contain GH as well . Some of the cells contained only a single immunoreactive hormone . The intensity of gold labelling of the secretory granules appeared higher in the VIP-treated cells than in the untreated control ones which showed a cytoplasmic structure characteristic of glandular cells with low secretory activity . As all the adenoma cells both contained and reacted to VIP, our results are in agreement with an autocrine or paracrine effect of this peptide . The fine structure of the cells in the cultures treated with VIP supply an additional argument to the assumption that VIP may serve as a growth factor for these cell types. Med J Malaysia, 1998 Sep, 53(3), 293 - 5 Detection of apoptotic cells in Vero cell cultures inoculated with samples derived from fatal cases of Sarawak acute childhood viral infection; AbuBakar S et al.; Infectious agent(s) causing the fatal Sarawak acute childhood viral infection (SACVI) has not been identified . In the present study, results indicating that inocula prepared from the fatal cases of SACVI induced apoptosis in Vero cell cultures are presented . These findings suggest the possible involvement of apoptotic cellular responses in SACVI. Biologicals, 2000 Sep, 28(3), 137 - 48 Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture; de Wit C et al.; Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses . Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM) . To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide . EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method . The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability . In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles . Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule . Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e . every two copies of retroviral pRNA is equivalent to one retrovirus-like particle . The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe . The TaqMan quantification technique is highly comparable to the EM analysis . In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost . Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell-based production system . Toxicol In Vitro, 2000 Oct, 14(5), 435 - 45 Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes; Follmann W et al.; Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals . After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks . By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented . The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps . A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve . During the culture period the cells were characterized morphologically and enzymatically . By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin . Antibodies against vimentin served as negative control . Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations . The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining . A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions . Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture . Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells . Then activity decreased completely . This culture system with primary epithelial cells from the colon will be used further as a model for the colon epithelium in toxicological studies in vitro. Arch Androl, 2000 Jul-Aug, 45(1), 35 - 42 Secretion of glycosidases in human epididymal cell cultures; Castellon EA et al.; The dynamics of glycosidase secretion was evaluated in human epididymal cell culture . Epithelial cells from caput, corpus, and cauda epididymis were isolated from tissue obtained from patients undergoing therapeutic orchidectomy due to prostatic carcinoma . The activities of alpha-glucosidase, N-acetylglucosaminidase, beta-glucuronidase, and alpha-mannosidase were analyzed in conditioned culture media . Glycosidase activity was significantly higher in corpus and/or cauda than in caput epididymis . There was a time-dependent increase in enzyme activities that was maximal between 10 and 14 days of culture in all epididymal regions . Epididymal glycosidases are secreted by cultured epithelial cell from human epididymis with an increase toward the distal regions of this organ, which may be related to the dynamics of sperm maturation . Cultures from different epididymal regions may represent a valuable tool to study of human epididymal function. J Virol, 2000 Sep, 74(18), 8390 - 401 Mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors demonstrate altered rates of RNase H cleavage that correlate with HIV-1 replication fitness in cell culture; Archer RH et al.; Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA- and DNA-dependent DNA polymerization and RNase H cleavage . All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting . The V106A reverse transcriptase demonstrated a three- to fourfold slowing of both DNA 3'-end-directed and RNA 5'-end-directed RNase H cleavage relative to both wild-type and V179D enzymes, similar to what was observed for P236L in a previously published study (P . Gerondelis et al., J . Virol . 73:5803-5813, 1999) . In contrast, the Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage . The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments . Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage . These findings, in combination with results from previous work, suggest that abnormalities in RNase H cleavage are a common characteristic of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3'-end- and RNA 5'-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1. J Muscle Res Cell Motil, 2000 Apr, 21(3), 223 - 33 Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures; Lewis MP et al.; The remodelling of connective tissue components is a fundamental requirement for a number of pivotal processes in cell biology . These may include myoblast migration and fusion during development and regeneration . In other systems, similar biological processes are facilitated by secretion of the matrix metalloproteinases (MMPs), especially the gelatinases . This study investigated the activity of the gelatinases MMP-2 and 9 by zymography on cell conditioned media in cultures of cells derived from explants of the human masseter muscle and in the murine myoblast cell-line C2C12 . Expression of MMP-9 by western blotting and TIMP-1, the major inhibitor of MMPs, by northern blotting, during all phases of myoblast proliferation, migration, alignment and fusion, was also measured . Irrespective of the origin of the cultures, MMP-9 activity was secreted only by single cell and pre-fusion cultures whilst MMP-2 activity was secreted at all stages as well as by myotubes . The loss of MMP-9 activity was due to the loss of MMP-9 protein expression . TIMP-1 mRNA was not detectable at the single cell stage but its expression increased as cells progressed through the pre-fusion and post-fusion stages to reach a maximal in myotube containing cultures . Migration of cells derived from human masseter muscle was inhibited, using a specific anti-MMP-9 blocking monoclonal antibody (6-6B) . These data are consistent with the concept that regulation of matrix turnover via MMP-9 may be involved in the events leading to myotube formation, including migration . Loss of expression of this enzyme and expression of TIMP-1 mRNA is associated with myotube containing cultures . Consequently, the ratio between MMPs and TIMPs maybe important in determining myoblast migration and differentiation. Virus Genes, 2000, 20(3), 209 - 16 n-Butyrate mediated inhibition of papovavirus DNA replication in vivo and in cell culture: a mechanistic approach; Shadan FF et al.; n-Butyrate, an inhibitor of G1-to-S transition inhibits papovavirus DNA replication in cell culture . To explore the efficacy of n-butyrate in vivo and to better understand its mechanism, we studied the effect of n-butyrate on viral DNA replication in mice acutely infected with polyomavirus and in the papovavirus-infected cells in culture . Newborn mice treated with n-butyrate stop growing and become runted . When infected with polyomavirus, these mice show a strong overall inhibition of viral DNA . However, a notable exception to this was the continued viral DNA replication in the differentiated mouse keratinocytes and renal epithelial cells as determined by in situ hybridization . n-Butyrate significantly inhibited viral DNA replication in the cultured IDL cells, and in polyomavirus-infected C2C12 myoblasts based on Southern blot analysis and in situ hybridization . DNA polymerase alpha (but not DNA polymerase beta) and the characteristic nuclear expression of PCNA were both inhibited in the n-butyrate treated IDL and C2C12 cells . n-Butyrate, therefore, inhibited host and viral DNA synthesis in the undifferentiated cells. Kurume Med J, 2000, 47(2), 115 - 24 Establishment of an osteoblastic osteosarcoma cell line and effects of cell culture conditions on secretion of matrix metalloproteinases from the cultured osteosarcoma cells; Minamitani K; Osteosarcoma cells are useful for investigating bone metabolism as malignant counterpart of osteoblasts . In hematogenous metastases of osteosarcoma cells, the cells need to adjust to various changes in pericellular environment . The changes in pericellular environment may change intracellular environment and consequently the secretion of matrix metalloproteinases (MMPs) which destroy extracellular matrices . In this report, a new cell line, KOS-1, derived from human osteoblastic osteosarcoma was established, and we assumed various culture conditions containing ingredients of the extracellular matrix to make a comparative study on MMPs detected from the culture supernatants . A wide spectrum of MMPs, including MMP-1 and -3 which were increased in the presence of interleukin 1 beta, was detected in this cell line . Production of MMP-1, the enzyme which decomposes types I, II, III and X collagen, by the cells, was increased in the presence of type I collagen . MMP-3 (stromelysin-1) which degrades types III and IV collagen, laminin, fibronectin, proteoglycan, etc . was produced more abundantly in the presence of type IV collagen . MMP-2 (72-kd type IV collagenase/gelatinase A) activity was found to be increased in the presence of gelatin and type IV collagen . The MMPs production in cultured osteosarcoma cells was changed depending on the culture conditions . This indicates that the same osteosarocma cells produce different amounts and kinds of enzymes involved in local infiltration and remote metastases and increase the production of the enzymes most required under a specific environment. Vet Parasitol, 2000 Sep 20, 92(2), 165 - 9 Determination of the activity of ponazuril against Sarcocystis neurona in cell cultures; Lindsay DS et al.; The present study examined the efficacy of ponazuril in inhibiting merozoite production of Sarcocystis neurona in cell cultures . Ponazuril inhibited merozoite production by more that 90% in cultures of S . neurona treated with 1.0 microg/ml ponazuril and greater than 95% inhibition of merozoite production was observed when infected cultures were treated with 5.0 microg/ml ponazuril . Ponazuril may have promise as a therapeutic agent in the treatment of S . neurona induced equine protozoal myeloencephalitis (EPM) in horses. J Appl Microbiol, 2000 Jul, 89(1), 11 - 5 The detection of astrovirus in sludge biosolids using an integrated cell culture nested PCR technique; Chapron CD et al.; The work presented here demonstrates the utility of the integrated cell culture-reverse transcriptase-polymerase chain reaction (ICC-RT-PCR) coupled with nested PCR to detect human astroviruses and enteroviruses in sludge biosolids . Viruses were concentrated by beef extract elution and organic flocculation prior to analysis by a plaque assay and ICC-RT-PCR . Astroviruses were detected in all but one sample and all of the samples were positive for enteroviruses . We have demonstrated the prevalence and frequency ofastrovirus in sludge and validated the ICC-RT-PCR/nested PCR technique as a useful tool to detect viruses in sludge. Histochem J, 2000 Jun, 32(6), 337 - 43 Long-term effect of vital labelling on mixed Schwann cell cultures; Mosahebi A et al.; Schwann cell transplantation following neuronal injury could encourage regeneration of spinal cord as well as improving peripheral nerve gap repair . In order to gain a better understanding of the role of transplanted Schwann cells in vivo, it is essential to be able to follow their behaviour after transplantation . Our aim was to evaluate the suitability of two vital fluorescent labels on the proliferation rate and phenotypic stability of Schwann cells, in either pure culture or mixed co-culture . Primary cultures of Schwann cells were obtained from Dark Agouti and Lewis neonatal rats and labelled with H33342 and PKH26, respectively . In mixed cultures, a 50: 50 mixture of Dark Agouti and Lewis Schwann cells was present . Labelled cultured cells were examined at 1, 2 and 4 weeks for viability and phenotypic marker expression of S100, GFAP, p75, MHC I, MHC II and compared with corresponding unlabelled cells . The results showed that although there was no deleterious interaction in the mixed cultures, the viability was reduced by the labelling after 2 weeks . Labelled cells could be distinguished up to 4 weeks, but there was leakage of H33342 label after 2 weeks . Labelled Schwann cells showed reduced expression of phenotypic markers, especially p75 when labelled with H33342 . In conclusion, H33342 and PKH26 can be used as fluorescent markers of Schwann cells for short-term studies, for a maximum of 2 weeks, but different markers may be needed for longer experiments. Endocr J, 2000 Apr, 47(2), 177 - 83 Role of reconstituted basement membrane in human granulosa cell culture; Hwang DH et al.; The effects of Matrigel, a reconstituted basement membrane, on human granulosa cells were investigated . Cells were obtained from follicular aspirate in the course of oocyte retrieval for in vitro fertilization and were cultured on either a surface coated with Matrigel or uncoated plastic . Light and electron microscopy showed that granulosa cells cultured on Matrigel demonstrated three-dimensional aggregated cells with well differentiated morphology: numerous lipid droplets, microvilli, junctional complexes and lumen-like structures were seen . In contrast, cells cultured on plastic were flattened, poorly differentiated and showed apoptotic cells . Immunocytochemistry showed that the proportion of immunopositive cells for 3beta-hydroxysteroid dehydrogenase was increased in cultures on Matrigel . The results of the present study suggest that culture on Matrigel promotes the differentiation of human granulosa cells and provides a useful tool which may improve the efficiency of in vitro fertilization. Cent Eur J Public Health, 2000 Jul, 8 Suppl, 35 - 6 Primary mono-layer cell cultures as model system for studying of environmental toxic agents: organochlorine compounds; Soos K et al.; Organic pollution of water and soil has various harmful effects on biological systems (1) . Chlorine substituted benzol compounds are one these xenobiotic substances, which are toxic to the environment (2) . They can also accumulate in plant and animal tissues (3), which provides ample reason to study the effects of sublethal doses of chloro-benzols on various cell cultures . In this study the toxic effects of chloro-benzols were investigated on avian fibroblast and mammalian hepatocyte cultures . The fibroblast cultures were prepared from eggs preadapted to chloro-benzol during a fourteen-day-long incubation period . The Wistar rat hepatocyte monolayer cultures were exposed to a direct treatment of 1,2,4-tri-chloro-benzol (0.01 microgram/ml-1 microgram/ml) for 3 hours . Following the treatment with chloro-benzol, the viability of the cells was measured, together with lactic dehydrogenase activity, in both kinds of cultures . The effect of tri-chloro-benzol treatment on chicken eggs was not significant . The cells of chicken embryos were not damaged after the 1,2,4-tri-chloro-benzol treatment . The hepatocyte cultures showed the toxic effects of 1,2,4-tri-chloro-benzol after the direct and acute treatment . The cell viability decreased and the LDH activity increased significantly . These results show that the primary cell cultures are suitable for studying the effects of organochlorine compounds. Biotechnol Bioeng, 2000 Oct 5, 70(1), 25 - 31 Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator; Andersen DC et al.; Human tissue-type plasminogen activator (t-PA) contains a variably occupied glycosylation site at Asn-184 in naturally produced t-PA and in t-PA produced in recombinant Chinese hamster ovary (CHO) cells . The presence of an oligosaccharide at this site has previously been shown to reduce specific activity and fibrin binding . In this report, the site occupancy of t-PA is shown to increase gradually over the course of batch and fed-batch CHO cultures . Additional cell culture factors, including butyrate and temperature, are also shown to influence the degree of glycosylation . In each of these cases, conditions with decreased growth rate correlate with increased site occupancy . Investigations using quinidine and thymidine to manipulate the cell cycle distribution of cultures further support this correlation between site occupancy and growth state . Comparison of the cell cycle distribution across the range of cell culture factors investigated shows a consistent relationship between site occupancy and the fraction of cells in the G(0)/G(1) phase of the cell cycle . These results support a correlation between growth state and site occupancy, which fundamentally differs from site occupancy trends previously observed and illustrates the importance of the growth profile of CHO cultures in producing consistently glycosylated recombinant glycoproteins. Tumour Biol, 2000 Sep-Oct, 21(5), 267 - 77 Isolation and properties of human alpha-fetoprotein from HepG2 cell cultures; Deutsch HF et al.; A relatively rapid 3-step fractionation method has been developed for the isolation of human alpha-fetoprotein from culture fluids of HepG2 cells applicable to large volumes . The protein exists as a complex with lipids or lipoproteins but an ethanol precipitation step is effective in separating it . Yields of 50-60% can be obtained from culture fluid containing 30-40 microg/ml . A minor fraction that appears to be a proteolytic product of the AFP is present in the final product . Platelets, 2000 Jun, 11(4), 204 - 14 The mitosis of fibroblasts in cell culture is enhanced by binding GP IIb-IIIa of activated platelets on fibrinogen; Gfatter R et al.; A fibroblast cell culture model enables us to measure the mitogenic ability mediated by growth factors released from stimulated platelets under different conditions . Simultaneously the growth factors secreted in the culture medium were determined . Cell mitotic rate was measured by incorporation of 3H-thymidine on days 3, 5 and 7 of culture . PDGF, TGF-beta, EGF and IGF-I were determined by Western blot . When fibroblasts were grown on surfaces precoated with a mixture of fibrinogen and thrombin-stimulated platelets, the 3H-thymidine uptake (196,645 +/- 56,864 cpm/ml) was increased, in comparison to fibroblasts grown on uncoated surfaces, in medium supplemented with FBS (28,855 +/- 7329 cpm/ml) . Neither thrombin-stimulated platelets without fibrinogen nor fibrinogen alone had positive effects on the mitogenic activity of fibroblast . Growth factors were identified only in a culture medium in which the cells were grown on surfaces precoated with fibrinogen and thrombin-stimulated platelets . Blocking the platelet integrin GP IIb-IIIa inhibited the release of growth factors from thrombin-stimulated platelets and consecutively the stimulation of mitosis by fibrinogen and activated platelets was absent . Antibodies against the growth factors added to the medium suppressed the stimulation of cell mitosis . These results show that delivery of growth factors from platelets' secretory granules is dependent on binding of fibrinogen to GP IIb-IIIa. Invest Ophthalmol Vis Sci, 2000 Aug, 41(9), 2658 - 64 Molecular cloning of ovine connexin44 and temporal expression of gap junction proteins in a lens cell culture; Yang DI et al.; PURPOSE: The lens plasma membranes of several mammalian species have been shown to contain three different connexin proteins . The goal of this study was to clone the sheep homologue of rat connexin46 identified as sheep connexin44 and to determine the temporal changes in the expression of the three sheep connexin proteins in a lens primary cell culture system . METHODS: A sheep genomic library was screened with a rat lens connexin46 cDNA probe . Lens junctional protein and mRNA levels were determined in a sheep primary cell culture system by Western and Northern blot analyses, respectively . RESULTS: Sheep connexin44, the homologue of rat lens connexin46, was identified as a single-copy gene with a predicted molecular weight of 43,989 Daltons that is contained within a single exon . Northern blot analysis detected a 2.2-kb connexin44 transcript in RNA isolated from lens but not that isolated from heart, kidney, liver, or lung . During the in vitro differentiation of lens epithelial cells from 5 to 20 days in culture, connexin43 mRNA levels declined approximately 75%, whereas connexin49 RNA levels increased approximately 24 fold . The 40% decrease in the level of connexin43 protein and the 21-fold increase in the level of connexin49 protein did not directly correlate with the changes in mRNA levels encoding these proteins during this same period . Although detectable, the amount of connexin44 mRNA and protein remained low throughout the 20-day period during which lens cells were grown in culture . Neither mRNA nor protein encoding MP20 or MP26 transcripts could be detected in even the oldest 20-day lens cultures . CONCLUSIONS: Steady state mRNA levels of sheep connexin43 and connexin49 do not appear to be the only factor regulating the expression of these genes during in vitro differentiation of lens cells in culture . Although a decreased level of expression of connexin43 was accompanied by an increased level of expression of connexin49 over the 20-day period in culture, connexin44 mRNA and protein levels remained low throughout this 20-day period . Overall, these results suggest that these junctional proteins have a unique temporal pattern of expression during differentiation, and this lens primary cell culture system provides a valuable tool to better understand this process. Mol Biotechnol, 1999 Nov, 13(1), 45 - 55 Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems . An update; Caughey B et al.; Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res . Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells . Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease-sensitive precursor, PrP-sen, after the latter reaches the cell surface . Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner . These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation. Exp Mol Med, 2000 Jun 30, 32(2), 88 - 92 Dose-dependent effect of resveratrol on proliferation and apoptosis in endothelial and tumor cell cultures; Szende B et al.; Experimental data suggest that Resveratrol, a compound found in grapes and other fruits may influence cell proliferation and apoptosis . The aim of our experiments was to study the effect of Resveratrol on tumor cell cultures and an endothelial cell culture in order to examine the effect of various doses of this compound on active cell death and cell proliferation . Human tumor (HT-29, SW-620, HT-1080) and endothelial (HUV-EC-C) cells were treated with various doses of (0.1 to 100.0 microg/ml) Resveratrol in vitro . Cell number, apoptotic and mitotic index was measured 24, 48 and 72 h after treatment . Low doses (0.1-1.0 microg/ml) of Resveratrol enhance cell proliferation, higher doses (10.0-100.0 microg/ml) induce apoptosis and decrease mitotic activity, which is reflected in changes of cell number . Resveratrol influences dose dependently the proliferative and apoptotic activity of human tumor and endothelial cells . The possible role of formaldehyde in the mechanism of action of Resveratrol is discussed. Dis Aquat Organ, 2000 Jun 19, 41(2), 91 - 104 Ultrastructure of white spot syndrome virus development in primary lymphoid organ cell cultures; Wang CH et al.; Primary cell cultures from the lymphoid organ of Penaeus monodon were used to investigate in vitro propagation and morphogenesis of white spot syndrome virus (WSSV) . Double-strength Leibovitz's L15 supplemented with 20% fetal bovine serum, pH 7.5, with a final osmolarity of 530 +/- 5 mOsm kg-1 was identified as the most suitable culture medium . In this medium, the lymphoid cells remained viable for more than 1 wk . Migrating cells were inoculated with WSSV, and the consequent cytopathic effects documented by light and electron microscopy . WSSV appears capable of following 2 alternative assembly sequences, one similar to the morphogenesis of the Oryctes rhinocerus virus and another which is more typical of baculoviral assembly . Possible relationships between WSSV, Oryctes virus, and baculoviruses are discussed. J Surg Oncol, 2000 Jun, 74(2), 141 - 7 A feasibility study of chemosensitivity assay by adhesive tumor cell culture system using biopsy specimens for gastric cancer; Suzuki T et al.; BACKGROUND AND OBJECTIVES: The adhesive tumor cell culture system (ATCCS) is known to produce high colony-forming efficiency . We, therefore, studied the feasibility of ATCCS for gastric cancer by use of biopsy specimens and the relationship between the results of ATCCS and histological effects of anticancer drugs . METHODS: Tumor specimens extracted by gastroendoscopic biopsy were sufficient for obtaining the result of sensitivity to at least one drug in 24 out of 30 (80%) patients . Twenty patients were administered 5-fluorouracil (5-FU) for 14 days prior to surgery, and the results of ATCCS were compared with histological changes of the resected specimens . RESULTS: The histological response rate was found to be 100% (4/4) when the 90% inhibition concentration (IC90) of 5-FU was less than 0.24 microg/ml (sensitive) and was 0% (0/3) when IC90 was greater than 0.40 microg/ml (resistant) . CONCLUSIONS: Although lacking in statistical significance, the results suggest that the drug to which the tumor revealed sensitivity in the ATCCS would produce histological effects and the drug to which the tumor was resistant would have no histological effect. Cancer Genet Cytogenet, 2000 Jul 1, 120(1), 50 - 7 Genetic characterization of immortalized human prostate epithelial cell cultures . Evidence for structural rearrangements of chromosome 8 and i(8q) chromosome formation in primary tumor-derived cells; Macoska JA et al.; We have utilized a combination of conventional and spectral karyotyping (SKY) techniques and allelotype analysis to assess numerical and structural chromosome alterations in two cell lines derived from normal human prostatic epithelium, and three cell lines derived from human prostate primary tumor epithelium, immortalized with the E6 and E7 transforming genes of human papilloma virus (HPV) 16 or the large T-antigen gene of simian virus 40 (SV40) . These studies revealed trisomy for chromosome 20 and rearrangements involving chromosomes 3, 4, 8, 9, 10, 16, 17, 18, 19, 21, or 22 . In addition, the four HPV-immortalized cell lines exhibited extensive duplications or translocations involving the 11q chromosomal region . Interestingly, allelotyping data disclosed loss of 8p sequences in two of the three primary tumor-derived cell lines, and SKY data revealed that the loss of 8p sequences was directly due to i(8q) chromosome formation and/or other structural alterations of chromosome 8 . This provides intriguing evidence that 8p loss in primary human prostate tumors may, in some cases, result from complex structural rearrangements involving chromosome 8 . Moreover, the data reported here provide direct evidence that such complex structural rearrangements sometimes include i(8q) chromosome formation. Mol Biotechnol, 2000 May, 15(1), 69 - 75 Detecting and minimizing glycosidase activities that can hydrolyze sugars from cell culture-produced glycoproteins; Gramer MJ; Heterogeneity of cell culture-produced glycoproteins often results from the presence or absence of a few sugars found on the terminus of glycoprotein oligosaccharides . Variability in bioprocess factors can potentially lead to variability in this oligosaccharide heterogeneity (1) . Although stochastic events in the intracellular biosynthetic process have long been recognized as a cause of oligosaccharide heterogeneity (2), more recent data has demonstrated that extracellular degradation by glycosidases can also contribute to oligosaccharide heterogeneity (3,4) . The purpose of this chapter is to introduce the concept and consequence of glycosidase degradation, to discuss methods for evaluating whether glycosidase degradation is significant for a particular process, and to provide some potential remedies to alleviate undesirable degradation. Dev Growth Differ, 2000 Jun, 42(3), 219 - 27 Synergistic effects of FGF and non-ridge ectoderm on gene expression involved in the formation of the anteroposterior axis of the chick limb bud in cell culture; Kimura J et al.; Skeletal patterning of the vertebrate limb is controlled by the zone of polarizing activity (ZPA), apical ectodermal ridge (AER) and dorsal ectoderm . In the present study, to understand the involvement of fibroblast growth factor (FGF) and non-ridge ectoderm in anteroposterior (AP) axis formation, gene expression in chick limb bud mesenchymal cells in culture was investigated by reverse transcription-polymerase chain reaction and in situ hybridization . It was found that Shh expression was locally maintained in the mesenchymal cells underneath and near non-ridge ectoderm in coculture with the posterior mesenchymal cells and non-ridge ectoderm in the presence of FGF-4 by in situ hybridization . In Shh-expressing anterior limb bud mesenchymal cells cultured with non-ridge ectoderm, it was also discovered that Bmp-2 was activated in the presence of FGF-2, -4 and -8, while Hoxd-13 was activated in the presence of FGF-4 and that FGF-2 had a similar effect but FGF-8 did not . This result indicates that Hoxd-13 activation by SHH depends on non-ridge ectoderm and FGF-2 or FGF-4, and that there may be a difference in the effect on AP axis formation of the limb bud between FGF-2, -4 and -8 . Possible roles of these genes and signal molecules in AP pattern formation are discussed. Ann Oncol, 2000 May, 11(5), 587 - 94 Combined 4-hydroxy-ifosfamide and vinorelbine treatment in established and primary human breast cell cultures; Ricotti L et al.; BACKGROUND: Vinorelbine and ifosfamide are active drugs against breast cancer, but the best treatment schedule has yet to be defined by preclinical or clinical studies . The antitumor activity of 4-hydroxy-ifosfamide (4-OH-IF), the active form of ifosfamide, and vinorelbine (VNB) and their interaction were investigated in two established breast cancer cell lines (MCF-7 and BRC-230) and in 10 primary breast cancer cultures . MATERIALS AND METHODS: Cytotoxic activity was evaluated by a highly efficient clonogenic assay (HECA) . The median-effect principle was applied to evaluate synergistic and antagonistic interactions and the corresponding combination index values were calculated . Cell cycle perturbations were analysed by flow cytometry . RESULTS: In MCF-7 and BRC-230 cell lines the sequence VNB for 4 hours followed by 4-OH-IF for 24 hours produced an antagonistic effect . Conversely, the inverse sequential scheme, 4-OH-IF-->VNB provided synergistic effects on both cell lines . The synergism was associated with a strong block in the G2-M phase . Synergistic activity of 4-OH-IF-->VNB sequence was confirmed in 7 of 10 primary breast cancer cultures . CONCLUSIONS: In conclusion, the sequence 4-OH-IF-->VNB appeared to be the most effective scheme both in established cell lines and in primary breast cancer cultures. J Cell Biochem, 2000 Jul 19, 79(1), 89 - 102 Type I collagen influences cartilage calcification: an immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures; Boskey AL et al.; Chick limb-bud mesenchymal cells, plated in high-density micro-mass culture, differentiate and form a matrix resembling chick epiphyseal cartilage . In the presence of 4 mM inorganic phosphate or 2.5 mM beta-glycerophosphate mineral deposits upon this matrix forming a mineralized tissue that, based on electron microscopy, x-ray diffraction and Fourier Transform Infrared microspectoscopy, is like that of chick calcified cartilage . In this culture system the initial mineral deposits are found on the periphery of the chondrocyte nodules . During differentiation of the cells in the high-density micro-mass cultures there is a switch from expression of type I collagen to type II, and then to type X collagen . However, type I collagen persists in the matrix . Because there is some debate about whether type I collagen influences cartilage calcification, an immunoblocking technique was used to determine the importance of type I collagen on the mineralization process in this system . Studies using nonspecific goat anti-chick IgG demonstrated that 1-100 ng/ml antibody added with the media after the cartilage nodules had developed (day 7) had no effect on the accumulation of mineral in the cultures . Nonspecific antibody added before day 7 blocked development of the cultures . Parallel solution based cell-free studies showed that IgG did not have a strong affinity for apatite crystals, and had no significant effect on apatite crystal growth . Type I collagen antibodies (1-200 ng/ml) added to cultures one time on day 9 (before mineralization started), or on day 11 (at the start of mineralization), slightly inhibited the accumulation of mineral . There was a statistically significant decrease in mineral accretion with 100 or 200 ng/ml collagen antibody addition continuously after these times . Fab' fragments of nonspecific and type I collagen antibodies had effects parallel to those of the intact antibodies, indicating that the decreased mineralization was not attributable to the presence of the larger, bulkier antibodies . The altered accumulation of mineral was not associated with cell death in the presence of antibody (demonstrated by fluorescent labeling of DNA) or with increased apoptosis (TUNEL-stain) . In the immunoblocked cultures, EM analysis demonstrated that mineral continued to deposit on collagen fibrils, but there appeared to be fewer deposits . The data demonstrate that type I collagen is important for the mineralization of these cultures . J Biomed Mater Res, 2000 Oct, 52(1), 142 - 7 Alveolar mononuclear cells can develop into multinucleated osteoclasts: an in vitro cell culture model; Sun JS et al.; Previous studies have shown that osteoclasts are derived from mononuclear cells of hemopoietic bone marrow and peripheral blood . The purpose of this study was to demonstrate the presence of multinucleated osteoclasts after adding alveolar mononuclear cells to new-born rat calvaria osteoblasts in vitro . To utilize osteoclast-free bone, fetal calvariae were obtained from newborn Wistar-rats and cultured in DMEM medium for 14 days . On the day of osteoblast culture, alveolar mononuclear cells were isolated from newborn Wistar rats with a serial washing method and then co-cultured with the calvarial osteoblasts . Bone resorption characteristics were observed both with light and scanning electron microscopy . When alveolar mononuclear cells were cultured for 14 days on the calvarial osteoblasts in response to 1 alpha, 25-dihydroxyvitamin D3, they formed tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells . Resorption pits were seen in the 7-14 days long-term cultures . These results indicate that osteoclasts can be derived from alveolar mononuclear cells in vitro when a suitable microenvironment is provided by calvarial osteoblasts and vitamin D(3) . J Virol, 2000 Aug, 74(16), 7619 - 27 Infectious hematopoietic necrosis virus matrix protein inhibits host-directed gene expression and induces morphological changes of apoptosis in cell cultures; Chiou PP et al.; Infectious hematopoietic necrosis virus (IHNV) infection in tissue culture cells has previously been shown to result in the shutdown of host protein synthesis, cell rounding, and cell death . We report here an investigation of the cytopathogenicity of the viral phosphoprotein (P or M1), matrix (M or M2), and nonvirion (NV) proteins in cultured fish cells . The expression of M alone potently inhibited reporter gene expression from a viral and an interferon (IFN)-inducible promoter, whereas P and NV did not produce a similar effect . Northern blot analysis further revealed a reduction in the steady-state level of reporter mRNA when the M gene was cotransfected into cells; conversely, M mRNA was not drastically reduced in the same cells . By immunofluorescence confocal microscopy, fragmented nuclei were found in some cells expressing M protein but not in cells expressing P, NV, or beta-galactosidase protein . Electron microscopy revealed the morphological changes associated with apoptosis in the M-transfected cells . Furthermore, IHNV infection was shown to produce DNA "laddering" in cultured cells . Taken together, these data suggested at least two functions for M protein in an IHNV infection: down regulation of host transcription and the induction of programmed cell death . In the course of these experiments, we also discovered that NV expression was associated with cell rounding, the first biological effect on cells to be attributed to the NV gene. Pediatr Surg Int, 2000, 16(4), 262 - 6 The effect of phospholipase A2 on bacterial translocation in a cell culture model; Sawai T et al.; The activity of phospholipase (PL)A2 is elevated in the intestinal epithelia of patients with inflammatory bowel disease (IBD) . Recently, we reported that lysophosphatidylcholine (L-PC), the PLA2 hydrolysis product of phosphatidylcholine (PC), stimulates bacterial translocation (BT) in an enterocyte cell-culture model . These two observations stimulated us to examine the effects of extracellular PLA2 on intestinal epithelial permeability . Human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell-culture system . Monolayer integrity and tight-junction permeability were measured by dextran blue (DB) permeability and transepithelial electric resistance (TEER) . Monolayers were treated with PC, L-PC, or PLA2 with and without PC . The magnitude of BT was determined 2 h