Genetika, 2001 Jun, 37(6), 779 - 83 {Inhibition of gene expression by administration of homologous double-stranded RNA in Drosophila melanogaster cell culture}; Aravin AA et al.; Specific inhibition of gene expression by exogenous homologous double-stranded RNA (dsRNA) in invertebrates and in the early development of vertebrates is termed RNA interference . Cultured cells were cotransfected with reporter plasmids and dsRNA . The inhibitor effect on reporter gene expression depended on the extent of homology between dsRNA and the target gene . RNA interference was also studied in cells cotransfected with plasmids directing synthesis of sense and antisense RNAs . Production of antisense RNA only slightly inhibited expression of the reporter gene . Simultaneous expression of both sense and antisense RNAs from a special plasmid did not inhibit expression of the reporter construct. Biomaterials, 2001 Sep, 22(18), 2465 - 73 Grooves affect primary bone marrow but not osteoblastic MC3T3-E1 cell cultures; Bruinink A et al.; To elucidate the influence of microtextures on bone cell performance, primary adult rat bone marrow cells (RBMC) and osteoblastic MC3T3-E1 cells were cultured on tissue culture pretreated plates to which grooves at different density were applied . RBMC cells were found to be significantly affected by grooves in the substratum in contrast to osteoblastic MC3T3-E1 cells, taking culture morphology, total cell number, cell mass, and cell activity (MTT-dehydrogenase), parameter for differentiation of osteoblast progenitor cells into (pre-)osteoblasts (alkalinephosphatase activity, ALP) and tartrate-resistant acid phosphatase (TRAP) activity as indices . TRAP is located in lysosomes and secretory granules mainly although not solely in osteoclasts . By applying grooves to and/or by chemical treatment of unpretreated pure polysterene plates it could be concluded that the effects on RBMC cells were evoked not only by the presence of grooves but also by the surface chemistry of the grooved and ungrooved surface areas. In Vitro Cell Dev Biol Anim, 2001 Jun, 37(6), 353 - 9 Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera: Noctuidae); Garcia JJ et al.; Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium . Long-term continuous culture and maintenance of midgut cells were achieved with P . unipuncta armyworm intestinal cells . Several cells lines were obtained from these P . unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo . The three major midgut cell types were present in the cultures, including stem (regenerative), columnar, and goblet cells . In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed . There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7-8 wk . After approximately six passages, the cell density in T-flasks appeared to be somewhat constant, reaching 10(3)-10(4) cells per milliliter of medium . The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity . Peritrophic membrane-like secretions were observed in all the culture flasks . Infection of these cells with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection. J Biol Chem, 2001 Oct 19, 276(42), 38680 - 4 Epub 2001 Aug 20. ACTH decreases the expression and secretion of apolipoprotein B in HepG2 cell cultures; Xu N et al.; Administration of adrenocorticotropic hormone (ACTH) has been shown to decrease plasma concentrations of apolipoprotein B (apoB) containing lipoproteins, including lipoprotein(a), in man . However, the mechanism behind this hypolipidemic effect is unknown . This study aimed at distinguishing between the main possibilities (increased elimination or decreased production of lipoproteins) using HepG2 cell cultures . Addition of ACTH to the cell culture medium selectively down-regulated apoB mRNA expression and apoB secretion in a dose-dependent manner . At 100 pmol/liter ACTH, the apoB mRNA level was about 40% lower than in the untreated cells, and the secretion of apoB into the medium was decreased to a similar extent . The expression and secretion of other apolipoproteins (apoA-I, apoE, and apoM), however, were not affected by ACTH . Under normal culture conditions the level of secretion of apoB from HepG2 cells is quite low . In the presence of 0.4 mmol/liter oleic acid secretion of apoB increased 3-fold, but this phenomenon was not seen in ACTH-treated cells . Binding and internalization of radiolabeled low density lipoprotein (LDL) by HepG2 cell, as well as LDL-receptor mRNA and scavenger receptor B-I mRNA levels, were not influenced by ACTH . In conclusion, ACTH directly and selectively down-regulated the production and secretion of apoB in HepG2 cell cultures, suggesting that a principal mechanism behind the cholesterol-lowering effect of ACTH in vivo may be a decreased production rate of apoB-containing lipoproteins from the liver. In Vitro Cell Dev Biol Anim, 2001 May, 37(5), 303 - 9 Tumor necrosis factor-alpha binding in porcine primary stromal-vascular cell cultures; Tchoukalova YD et al.; The binding characteristics of tumor necrosis factor-alpha receptors (TNFRs) in primary stromal-vascular cultures from fat tissue of 7-d-old pigs were analyzed . Cells were plated and maintained in 10% fetal bovine serum from day 0 to day 3 and then switched to serum-free medium from day 3 to day 6 to induce lipid filling . On days 3 and 6 of culture, some of the cells were lysed for ligand and immunoblotting and the remainder subjected to competitive and inhibitory-binding assays . Media from day 6 of culture were subjected to ligand and immunoblotting . Competitive binding analysis showed one-site bindings, with IC50s in the nanomolar and Kds in the picomolar ranges, that were not significantly different at both time-points of measurement . However, the Bmax decreased significantly with differentiation . Preincubation with antibody against TNF receptor type 1 (TNFR1) or TNF receptor type 2 reduced the specific binding by 95 and 15%, respectively, suggesting a dominating role of TNFR1 in 125I-labeled TNFalpha (125I-TNFalpha) binding . This was further supported by ligand blotting of cell lysates . Ligand and immunoblotting of cell lysates indicated that TNFalpha utilizes both types of surface receptors and their isoforms which were not modified during differentiation . Ligand blotting of media revealed soluble receptors with high Mr implying the formation of multimers . Immunoblotting suggested the presence of both types of TNFRs, but a greater abundance of soluble TNFR1 . Also, it indicated the additional formation of smaller oligomers from both types of soluble receptors suggesting higher affinity of larger multimers for 125I-TNFalpha. Cell Physiol Biochem, 2001, 11(4), 197 - 202 Cytosolic Ca(2+) levels and DNA synthesis of human umbilical vein endothelial cell cultures are unresponsive to thrombopoietin treatment; Metzen E et al.; The hormone thrombopoietin (TPO) induces proliferation of megakaryocytic progenitors and augments agonist-induced mobilization of Ca(2+) in platelets . Because the action of TPO is not restricted to the megakaryocytic lineage, we studied the occurrence of TPO receptor mRNA and protein, and effects of TPO on cytosolic Ca(2+) levels and DNA synthesis in human umbilical vein endothelial cell cultures (HUVECs) . Polymerase chain reaction following reverse transcription (RT-PCR) of total mRNA revealed that TPO receptor (MPL) mRNA was expressed only at low level in our samples . TPO receptor protein was not detectable in HUVEC lysates investigated by immunoprecipitation and immunoblotting . In contrast to vascular endothelial growth factor (VEGF), TPO did neither alter fura2 fluorescence as a measure of cytosolic Ca(2+) levels nor increase 5-bromo-2'-deoxyuridine incorporation into DNA of HUVECs . In conclusion, our data demonstrate that HUVECs are neither structurally nor functionally responsive to TPO . Nature, 2001 Aug 16, 412(6848), 739 - 43 Antibodies inhibit prion propagation and clear cell cultures of prion infectivity; Peretz D et al.; Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy . In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions . Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction . We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc . Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner . In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection . The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC . Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting. Cancer Res, 2001 Aug 15, 61(16), 5969 - 73 A novel human cell culture model for the study of familial prostate cancer; Yasunaga Y et al.; Research into molecular and genetic mechanisms underlying familial prostate cancer would be greatly advanced by in vitro models of prostate tumor cells representing primary tumors . We have successfully established an immortalized human prostate epithelial cell culture derived from primary tumors of familial prostate cancer patients with telomerase . The actively proliferating early-passaged 957E cells were transduced through infection with a retrovirus expressing the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT) . A high level of telomerase activity was detected in 957E/hTERT cells, but not in 957E cells . 957E/hTERT cells are currently growing well at passage 40, whereas 957E cells senesced at passage 5 . 957E/hTERT cells exhibit epithelial morphology . Expression of an androgen-regulated prostate specific homeobox gene NKX3.1 and an epithelial cell-specific cytokeratin 8, but not prostate specific antigen or androgen receptor, was detected in 957E/hTERT cells . Prostatic stem cell antigen and p16 were also expressed in this line . 957E/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor beta1, potent inhibitors of prostate epithelial cell growth . Chromosome analysis showed that the 957E/hTERT cell line (passage 10) was near diploid human male (XY), with most chromosome counts in the 44-46 range . However, there was random loss of chromosomes 8, 13, X, Y, and alteration in chromosome 4q . The late passage 957E/hTERT cell line (passage 32) was karyologically similar to the early passage 957E/hTERT cell line (passage 10) and also had the same alteration of 4q observed in the early passage 957E/hTERT cell line (passage 10) as well as a trisomy of chromosome 20 . The well-characterized human cancer lines derived from such patients will be useful for the identification and characterization of prostate cancer susceptibility genes . This is the first documented case of an established human prostate cancer cell line from primary tumor of a familial prostate cancer patient. Antimicrob Agents Chemother, 2001 Sep, 45(9), 2510 - 6 Specific inhibition of human immunodeficiency virus type 1 (HIV-1) integration in cell culture: putative inhibitors of HIV-1 integrase; Vandegraaff N et al.; To study the effect of potential human immunodeficiency virus type 1 (HIV-1) integrase inhibitors during virus replication in cell culture, we used a modified nested Alu-PCR assay to quantify integrated HIV DNA in combination with the quantitative analysis of extrachromosomal HIV DNA . The two diketo acid integrase inhibitors (L-708,906 and L-731,988) blocked the accumulation of integrated HIV-1 DNA in T cells following infection but did not alter levels of newly synthesized extrachromosomal HIV DNA . In contrast, we demonstrated that L17 (a member of the bisaroyl hydrazine family of integrase inhibitors) and AR177 (an oligonucleotide inhibitor) blocked the HIV replication cycle at, or prior to, reverse transcription, although both drugs inhibited integrase activity in cell-free assays . Quercetin dihydrate (a flavone) was shown to not have any antiviral activity in our system despite reported anti-integration properties in cell-free assays . This refined Alu-PCR assay for HIV provirus is a useful tool for screening anti-integration compounds identified in biochemical assays for their ability to inhibit the accumulation of integrated HIV DNA in cell culture, and it may be useful for studying the effects of these inhibitors in clinical trials. EMBO J, 2001 Aug 15, 20(16), 4443 - 53 HBV infection of cell culture: evidence for multivalent and cooperative attachment; Paran N et al.; Hepadnaviruses do not infect cultured cells, therefore our knowledge of the mechanism of the early stages of virus-cell interaction is rather poor . In this study, we show that dimethylsulfoxide (DMSO)-treated HepG2 hepatoblastoma cells are infected efficiently by serum-derived hepatitis B virus (HBV) as monitored by viral gene expression and replication markers . To measure virus attachment, a variety of HBV surface proteins (HBsAgs) were conjugated to polystyrene beads and their capacity to attach cells was visualized and quantified by light microscopy at a single-cell resolution . Remarkably, DMSO increases the attachment efficiency by >200-fold . We further identify the QLDPAF sequence within preS1 as the receptor-binding viral domain epitope . Interestingly, a similar sequence is shared by several cellular, bacterial and viral proteins involved in cell adhesion, attachment and fusion . We also found that the small HBsAg contains a secondary attachment site that recognizes a distinct receptor on the cell membrane . Furthermore, we provide evidence in support of multivalent HBV attachment with synergistic interplay . Our data depict a mechanistic view of virus attachment and ingestion. Prenat Diagn, 2001 Jul, 21(7), 529 - 39 Embryonic and fetal globins are expressed in adult erythroid progenitor cells and in erythroid cell cultures; Lau ET et al.; The understanding of human hemoglobin ontogeny during development is of biological and clinical importance . Molecular and immunocytological techniques were used to study the expression of embryonic zeta (zeta), epsilon (epsilon), and fetal gamma (gamma) globin genes in newborn cord blood, peripheral blood from men, pregnant and non-pregnant women, and in vitro mononuclear cell cultures . We have shown that embryonic and fetal globin mRNA and peptides are expressed in cultured erythroid cells and in circulating blood cells from newborns, adult non-pregnant women and from men . The findings suggest that during erythroid cell differentiation in newborns and adults, there is a transient recapitulation of sequential globin chain expression as found during embryonic and fetal development . Furthermore, these findings underscore the need for caution in using embryonic and fetal globin chains as markers to identify erythroid cells of fetal origin in maternal circulation for prenatal diagnosis . In Vivo, 2001 May-Jun, 15(3), 227 - 31 Myxoid malignant fibrous histiocytoma of the uterus: a case with immunohistochemical, ultrastructural and tumor cell culture studies; Kiyozuka Y et al.; The clinical and pathological features, including ultrastructural and immunohistochemical findings, of a primary myxoid malignant fibrous histiocytoma of the uterus in a 60-year-old woman are reported . Microscopically, the principal feature of the tumor was a hypocellular area with diffuse degeneration, containing thin-walled curvilinear vessels, in which hyperchromatic small spindle and stellate cells, sometimes with vacuolated cytoplasm, were found . The transplanted tumor of primary cultured cells in nude mice presented as a prominent myxoid stroma confirming the histological structure of the primary tumor . Immunohistochemically, the presence of epithelial or heterogenous mesenchymal tumor components or cells of smooth muscle derivation were excluded and the tumor cells were positive for vimentin, CD 68, alpha 1-antitrypsin and alpha 1-antichymotrypsin . Ultrastracturally, pseudopodia and filopodia at the cell membrane and intracytoplasmic lysosomal granules were common . The patient had debulking surgery but died 38 days after the primary onset with the tumor occupying the entire abdomen and the pelvis. J Endod, 2001 Apr, 27(4), 273 - 7 Regulation of interleukin-6 expression in human dental pulp cell cultures stimulated with Prevotella intermedia lipopolysaccharide; Tokuda M et al.; Interleukin (IL)-6 expression in human dental pulp cell cultures after stimulation with Prevotella intermedia lipopolysaccharide (LPS) was investigated by Northern blot analysis, enzyme immunoassay, and bioassay . The IL-6 mRNA expression began to increase after 1 hr and continued after up to 8 hr of exposure on stimulation with 10 microg/ml of P . intermedia LPS . The bioactivity was dose-dependent on the concentration of P . intermedia LPS (0 to 100 microg/ml) . The IL-6 mRNA expression was inhibited by actinomysin D and super-induced by cycloheximide . Anti-CD14 monoclonal antibody (MY4) inhibited the IL-6 mRNA expression when administered at a 0.5 microg/ml concentration before stimulation with P . intermedia LPS at 1 microg/ml . The immunoregulatory cytokines (interferon-gamma, IL-10, and IL-4) inhibited LPS-induced IL-6 production with a combined treatment . These results suggest the IL-6 expression by pulp cell cultures is CD14-dependent and regulated at the transcriptional level, and a combined treatment with immunoregulatory cytokines may be effective for control of pulpal inflammation due to P . intermedia LPS. Arch Soc Esp Oftalmol, 2001 Aug, 76(8), 471 - 6 {Trifluoperazine's action on fibroblast proliferation in cell cultures . Comparison of different doses}; Izaguirre Roncal LB et al.; PURPOSE: To evaluate the effect of different doses of trifluoperazine (TFP) on fibroblastic proliferation in cell cultures of sclera and conjunctiva . METHODS: 24 brown rabbits were operated on for non-protected sclerectomy and were divided into four groups . Group 1: Subconjunctival injection of 0.5 ml of balanced saline solution (BSS) 24 h before surgery . Group 2: Intraoperative TFP 10(-2)M for five minutes on the sclera in experimental filtrating surgery . Group 3: Subconjunctival injection of 0.5 ml of TFP 10(-3) M 24 h before surgery . Group 4: Subconjunctival injection of 0.5 ml of TFP 10-4M 24 h before surgery . Conjunctiva and sclera tissue samples were taken 1 hour after surgery in the treated area and at 90 degrees from it . All of the samples were cultured in DMEM with 20% fetal calf serum . The presence of atypical cells and fibroblastic growth was assessed at 3, 6, 9 and 12 days . RESULTS: There were statistically significant differences in all the groups and in the times compared to the control group . In the treated conjunctiva, differences were found between the subconjunctiva 10(-3) M and the intraoperative 10(-2) M on the 3rd and 6th days, between the preoperative 10-2 and subconjunctiva 10(-3) M and 10(-4) after the 6th day . In conjunctiva at 90 degrees, there were significant differences between preoperative 10-2 M and subconjunctiva 10-3 after day 6, between the preoperative 10-2 M and subconjunctiva 10(-4) on days 9 and 12 . There were no significant differences among the different treatments in the sclera samples, but there were differences between all them and the control group . CONCLUSIONS: TFP at a dose of 10(-2) and 10(-4) TFP has an inhibitory effect on cellular proliferation in vitro in the rabbit sclera and conjunctiva . All the doses used produce a significant inhibition of cellular proliferation without producing significant disorders in the cellular morphology. J Neurochem, 2001 Aug, 78(3), 499 - 508 Calcium and pituitary adenylate cyclase-activating polypeptide induced expression of circadian clock gene mPer1 in the mouse cerebellar granule cell culture; Akiyama M et al.; Mammalian circadian clock genes Per1 and Per2 are rhythmically expressed not only in the suprachiasmatic nucleus where the mammalian circadian clock exists, but also in other brain regions and peripheral tissues . The induced circadian oscillation of Per genes after treatment with high concentrations of serum or various drugs in cultured cells suggests the ubiquitous existence of the oscillatory mechanism . These treatments also result in a rapid surge of expression of Per1 . It has been shown that multiple signaling pathways are involved in Per1 gene induction in culture cells . We used a dispersed primary cell culture made up of mouse cerebellar granule cells to examine the stimuli inducing the mPer genes and their signaling pathways in neuronal tissues expressing mPer genes . We demonstrated that mPer1, but not mPer2, mRNA expression was dependent on the depolarization state controlled by extracellular KCl concentration in the granule cell culture . Nifedipine treatment reduced mPer1 induction, suggesting that mPer1 mRNA expression depends on intracellular calcium concentration regulated through a voltage-dependent Ca2+ channel . Transient mPer1 mRNA induction was observed after elevating KCl concentration in the medium from 5 mM to 25 mM . This increased expression was suppressed by a calmodulin antagonist, or CaMKII/IV inhibitor, but not by MEK inhibitors . Addition of pituitary adenylate cyclase-activating polypeptide-38 to the medium also induced transient Per1 gene expression . This induction was mimicked by dibutyryl-cAMP and suppressed by a protein kinase A (PKA) inhibitor, but not by MEK inhibitors . These results suggest that Ca2+/calmodulin-dependent protein kinase II/IV- and PKA-dependent pathways are involved in high-KCl and PACAP-induced mPer1 induction, respectively, and neural tissues use multiple signaling pathways for mPer1 induction similar to culture cells. Melanoma Res, 2001 Jun, 11(3), 213 - 8 Impact of interferons on the expression of melanoma-associated antigens in melanoma short-term cell cultures; Hofbauer GF et al.; Some immunotherapeutic approaches based on melanoma-associated antigens rely on in vitro cultivation of melanoma cells . A beneficial effect of interferons has been shown in melanoma . This study aimed to determine whether stimulation of patient-derived melanoma short-term cell cultures using interferon-alpha and -gamma changes the expression pattern of melanoma-associated antigens . Lymph node, skin and brain metastases were cultivated for up to 3 weeks and treated with interferon-alpha, interferon-gamma or mock stimulation . Expression of the melanoma-associated antigens MAGE-3, MelanA/MART-1 and tyrosinase was determined by flow cytometry and compared with the expression pattern of HLA class I molecules . We found consistently enhanced expression of HLA class I molecules, whereas the melanoma-associated antigens showed mixed responses, with moderate induction, suppression or no visible effect . The reaction to interferon stimulation was similar for all the antigens examined within a single melanoma cell culture . In contrast to the HLA class I molecules, which showed induced expression with interferon, the melanoma-associated antigens showed a varied response to interferon stimulation . Differential reaction to interferon stimulation is of importance to immunotherapeutic modalities and might influence progression of the disease . We therefore suggest that evaluation of variation in melanoma-associated antigen expression in the clinical setting may help to identify patients who would profit from adjuvant interferon therapy. J Acquir Immune Defic Syndr, 2001 Jul 1, 27(3), 213 - 21 Apoptotic effects in primary human umbilical vein endothelial cell cultures caused by exposure to virion-associated and cell membrane-associated HIV-1 gp120; Huang MB et al.; During the course of HIV-1 infection, free virus, infected cells, and free HIV-1 proteins circulate within the host, exposing the host endothelium to these viral factors . We have previously presented evidence showing that soluble HIV-1 gp120 protein interacts with chemokine receptors on primary human endothelium and (through those interactions) induces apoptosis as well as other intracellular effects . The current study examines the effect of exposure of vascular endothelium to gp120 IIIb expressed on the surface of Jurkat cells and in the context of viral particles . Apoptosis was observed in human umbilical vein endothelial cell (HUVEC) cultures exposed to gp160-transfected Jurkat cells as well as to virion particles with gp120 on their surface . Additional experiments show that this apoptotic effect was caused by gp120 protein acting through chemokine receptors on the HUVEC surface, primarily the CXCR4 receptor . At higher concentrations of gp120, this lymphotrophic variant, which has been shown to interact predominantly with CXCR4, seems to interact with and induce apoptosis through the CCR5 receptor . Finally, this apoptotic effect in HUVEC cultures occurs at low levels of the inducing agent, gp120, on cell membranes or on virion particles . These results demonstrate that HIV-1 gp120 is capable of interacting with and killing vascular endothelial cells in multiple in vivo contexts. Ann N Y Acad Sci, 2001 Jun, 939, 450 - 8 The anti-Parkinson drug rasagiline and its cholinesterase inhibitor derivatives exert neuroprotection unrelated to MAO inhibition in cell culture and in vivo; Youdim MB et al.; The antiapoptotic and neuroprotective activity of irreversible monoamine oxidase (MAO) B inhibitor, rasagiline {R(+)-N-propargyl-1-aminioindan}, its S-isomer (TVP1022) and TV 3219, a novel anti-Alzheimer cholinesterase-MAO inhibitor drug derived from rasagiline were examined in PC12 cells cultures and in vivo . We found that these drugs have potent antiapoptotic and neuroprotective activities in response to serum and NGF withdrawal in partially neuronally differentiated PC12 cells and prevent the fall in mitochondrial membrane potential, the first step in cell death . Closed head injury studies in mice have shown that both rasagiline and TVP1022 are neuroprotective . All these compounds possess a propargyl moiety, which normally is responsible for irreversible inactivation of MAO, as is seen with rasagiline . However, neither TVP1022 nor TV3219 are MAO inhibitors, both share the antiapoptotic and neuroprotective actions of rasagiline, indicating that MAO inhibition is not a prerequisite for neuroprotection and that the propargyl moiety exhibits intrinsic neuroprotective pharmacological activity that requires identification. Mar Environ Res, 2000 Jul-Dec, 50(1-5), 541 - 4 Development of a fish in vitro cell culture model to investigate oxidative stress and its modulation by dietary vitamin E; George S et al.; When cultured in Dulbecco's minimal essential medium the established epithelioma papulosum cyprini cell line from carp was found to be vitamin E-deficient due to the very low level of vitamin E in the medium and the foetal calf serum used as supplement . The toxicity of oxidative stressors to this cell line was evaluated by means of the neutral red cytotoxicity assay and it was found that an organic hydroperoxide, t-butylhydroperoxide was extremely cytotoxic and that the redox-cycling agents diquat and menadione were less toxic . When grown under vitamin E supplementation (25 microM), the toxicity of these chemicals was reduced by at least an order of magnitude in concentration demonstrating the protective effect of vitamin E . These data show the importance of vitamin E status for interpretation of in vitro and in vivo data and that this in vitro system is useful for mechanistic studies. Mar Environ Res, 2000 Jul-Dec, 50(1-5), 523 - 6 Skin biopsies for cell cultures from Mediterranean free-ranging cetaceans; Marsili L et al.; The aim of this study was to develop a useful method for obtaining viable tissue samples for establishing cell cultures from skin biopsies of free-ranging cetaceans . The skin biopsies were performed by two methods: dart from an air gun and dart from a crossbow . The dart tip was modified to collect tissue . The tissue was kept in tissue culture medium at ambient temperature, then processed within 24 h . Many modifications in culture technique, with respect to conventional culture methods for human fibroblasts, were made . The cultures thus obtained can be used for many purposes, including genetic and toxicological studies . In toxicology they are an alternative in vitro system for studying threatened animals such as marine mammals . In particular, fibroblasts can be used to test the vulnerability of cetaceans and pinnipeds to different environmental contaminants such as organochlorine compounds, heavy metals and polycyclic aromatic hydrocarbons. Neurosci Lett, 2001 Aug 3, 308(2), 115 - 8 Rapid induction of neurotrophin mRNAs in rat glial cell cultures by Semax, an adrenocorticotropic hormone analog; Shadrina MI et al.; The proliferation, differentiation and survival of neuronal and glial cells are affected by a number of neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and others . In a previous study, we observed the effects of 'Semax' (Met-Glu-His-Phe-Pro-Gly-Pro), the physiologically active analogue of adrenocorticotropic hormone(4--10), on neuronal cell survival in vitro . We hypothesized that these effects may be mediated by the regulation of expression of some neurotrophic factors . To test this hypothesis we analyzed NGF and BDNF gene expression in glial cells obtained from the basal forebrain of newborn rats, following in vitro treatment with 'Semax' . We observed changes in mRNA levels for both the NGF and BDNF genes . The greatest increase in expression was found after 30 min of 'Semax' administration . At this time, BDNF mRNA level was increased eight-fold in comparison with control, and NGF mRNA level was increased five-fold. Brain Res, 2001 Jul 20, 908(1), 1 - 9 Differential roles of cyclooxygenase isoforms after kainic acid-induced prostaglandin E(2) production and neurodegeneration in cortical and hippocampal cell cultures; Kim EJ et al.; Prostaglandins, which are cyclooxygenase (COX) products, are pathologically up-regulated, and have been proven to be closely associated with neuronal death . In this study, we investigated a role of COX isoforms (COX-1 and COX-2) in kainic acid-induced neuronal death in cultured murine cortical or hippocampal neurons . In primary cortical neurons, both indomethacin (COX-1/-2 nonselective inhibitor) and aspirin (COX-1 preferential inhibitor) reduced basal and kainic acid-induced PGE(2) production significantly and prevented neuronal cell death after kainic acid treatment . In contrast, NS398 (COX-2 selective inhibitor) had no effect on kainic acid-induced neuronal cell death . In hippocampal neurons, however, COX-2 inhibitors prevented both kainic acid-induced neuronal death and PGE(2) production . COX-2 expression was remarkably up-regulated by kainic acid in hippocampal neurons; whereas in cortical neurons, COX-2 expression was comparatively less significant . Astrocytes were unresponsive to kainic acid in terms of PGE(2) production and cell death . In conclusion, we suggest that the release of PGE(2) induced by kainic acid occurred through COX-1 activity rather than COX-2 in cortical neurons . The inhibition of PGE(2) release by COX-1 inhibitors prevented kainic acid-induced cortical neuronal death, while in the hippocampal neurons, COX-2 inhibitors prevented kainic acid-induced PGE(2) release and hippocampal neuronal death. Histochem Cell Biol, 2001 Jun, 115(6), 499 - 508 Localization of mRNAs encoding peroxisomal proteins in cell culture by non-radioactive in situ hybridization . Comparison of rat and human hepatoma cells and their responses to two divergent hypolipidemic drugs; Kovacs W et al.; A non-radioactive in situ hybridization (ISH) protocol for localization of mRNAs encoding peroxisomal proteins in hepatoma cell lines from humans (HepG2) and rats (MH1C1) is presented . In comparison to a similar procedure reported for tissue sections, the cell culture preparations require only brief fixation in 4% paraformaldehyde and their permeabilization is achieved by a very low concentration (1 microg/ml) of proteinase K . The exclusive localization of transcripts in the cytoplasm of hepatoma cells with the absence of nuclear staining and the completely negative sense controls confirm the specificity of the method . The marked differences in signal intensity between the results of albumin and beta-actin mRNAs which are of high abundance in contrast to moderate to low abundance of peroxisomal mRNAs show the high sensitivity and the wide range of applicability of our protocol . This is also confirmed by divergent results of treatment of hepatoma cell lines with clofibrate and cetaben on mRNA levels of catalase and acyl-CoA oxidase . The ISH results of drug treatment of cell lines are confirmed also by slot blot analysis of total RNA extracts using 32P-labeled probes . Thus the protocol presented here provides a sensitive tool for ISH localization of mRNAs encoding peroxisomal proteins . In combination with immunocytochemistry it may be useful to monitor intercellular differences in expression levels of specific mRNAs in correlation with the abundance of structurally divergent forms of peroxisomes (tubular versus spherical) and their importance in the biogenesis of peroxisomes. Mol Pharmacol, 2001 Aug, 60(2), 348 - 54 Modulation of protein kinase A activation by fibronectin matrix proteins at developing neuromuscular synapses in Xenopus laevis cell cultures; Liou HH et al.; Extracellular matrix proteins, such as fibronectin, laminin, and collagen, have been implicated in a wide variety of cellular properties, which include cell adhesion, migration, differentiation, and proliferation . In this study, we investigated the modulation of protein kinase A (PKA) activity by matrix proteins at developing motoneurons . The cultures of spinal neurons and myotomal cells were prepared from 1-day-old Xenopus laevis embryos . Spontaneous synaptic currents (SSC) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (V(h) = -60 to approximately -65 mV) . Bath application of agents, which directly or indirectly activate PKA, such as forskolin (20 microM), dibutyryl cAMP (DBcAMP) (1 mM), isoproterenol (10 microM), or albuterol (10 microM), significantly increased SSC frequency in cultures grown on fibronectin (FN)-coated substratum, but not on laminin- or collagen-coated glasses . The evoked synaptic currents increased in response to forskolin in neurons grown on FN substratum . Triflavin, an Arg-Gly-Asp-dependent disintegrin, inhibited potentiating action of isoproterenol in neurons grown on FN substratum, suggesting that integrin is involved in the potentiation of the PKA pathway in the regulation of acetylcholine (ACh) release . There is collaboration of neurotrophic factors and the FN matrix in regulating synaptic transmission in response to DBcAMP . Chronic treatment with neurotrophic factors, such as ciliary neurotrophic factor (150 ng/ml), glial cell line-derived neurotrophic factor (30 ng/ml), or neurotrophin-3 (50 ng/ml), enhanced the SSC-increasing action of DBcAMP in neurons grown on FN-coated glasses . These results suggest that the FN matrix potentiates synaptic transmission in response to PKA activation . Neurotrophic factors may collaborate with FN to regulate spontaneous ACh secretion at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular synapses. J Pharmacol Exp Ther, 2001 Aug, 298(2), 441 - 52 Expression of alpha-adrenoceptor subtypes by smooth muscle cells and adventitial fibroblasts in rat aorta and in cell culture; Faber JE et al.; Previous radioligand binding reports of vascular alpha-adrenoceptor (AR) density have been limited to total alpha1- or alpha2-ARs . Studies using whole blood vessel homogenates have not differentiated among receptor or mRNA expression by medial smooth muscle cells (SMCs) versus adventitial fibroblasts (AFBs) . Therefore, we used quantitative reverse transcription-polymerase chain reaction and radioligand binding to measure alpha-AR subtypes in media, adventitia, and cultured SMCs and AFBs from rat aorta . Both media and adventitia expressed alpha1A-, alpha1B-, alpha1D-, and alpha2D-AR mRNAs, but in markedly different abundances . Total alpha1-AR density was the same for media and adventitia (Bmax = 101 +/- 10 versus 96 +/- 16 fmol/mg of protein) . However, densities for alpha1A-, alpha1B-, and alpha1D-AR subtypes in media were 19 +/- 2, 26 +/- 4, and 55 +/- 2%, and in adventitia were 44 +/- 3, 37 +/- 5, and 19 +/- 2% . No alpha2B- or alpha2C-AR transcripts were detected in either layer or in cultured SMCs or AFBs . Total alpha1-AR densities in cultured SMCs and AFBs (Bmax = 111 +/- 4 and 48 +/- 6 fmol/mg of protein, respectively) were similar to media and adventitia, with alpha1B- and alpha1D-AR transcript levels and receptors largely sustained . However, alpha1A- and alpha2D-AR expression in cultured SMCs and AFBs was strongly reduced, compared with media and adventitia, an effect not prevented by 30 different culture conditions . Like SMCs, exposure of AFBs to norepinephrine induced protein synthesis and proliferation of AFBs . This is the first study to quantitate alpha-AR subtype expression in media and adventitia and in cultured SMCs and AFBs . In addition, we report the intriguing finding that AFBs express alpha1-ARs in similar abundance as medial SMCs and that norepinephrine induced them to proliferate. J Clin Pharmacol, 2001 Jul, 41(7), 708 - 14 P-glycoprotein interactions of nefazodone and trazodone in cell culture; Stormer E et al.; This study investigated the effects of nefazodone (NFZ) and trazodone (TZD) on P-glycoprotein (P-gp) activity and expression in cell culture . NFZ and TZD showed no differential transport between the basolateral to apical and apical to basolateral direction across Caco-2 cell monolayers . Transport in either direction was not affected by verapamil . NFZ was a potent inhibitor (IC50 = 4.7 microM) of rhodamine123 (Rh123) B to A transport across Caco-2 cell monolayers, while TZD had minimal effect . Following 72-hour exposure of LS180V cells to NFZ and TZD (10 microM), a twofold increase in immunoreactive P-gp was observed . Rh123 accumulation into these cells was reduced to 65% and 74% of control by NFZ and TZD (10 microM), respectively . It was concluded that differential rates of transport of NFZ and TZD in Caco-2 cells were not evident . However, NFZ is an inhibitor of P-gp activity at clinically relevant in vivo concentrations and may have the potential to increase bioavailability of coadministered compounds that are substrates for transport . Concentrations of NFZ and TZD achieved in the intestine after chronic oral dosing may induce P-gp expression and reduce absorption of coadministered drugs. Arkh Patol, 2001 May-Jun, 63(3), 48 - 50 {Comparative evaluation of clinical, cytological and cell culture methods of candida vulvovaginitis diagnosis}; Lipova VA et al.; 98 patients with Candida vulvovaginitis (CVV) were examined clinically, cytologically and culturally . Three clinical forms of CVV were distinguished: acute and subacute (26 and 23 patients, respectively)--before treatment, and remission (49 patients)--after treatment . The clinical diagnosis of CVV before the treatment was confirmed cytologically and/or in cultures (87.7%), in remission cytologically (4%), by cultures (20 patients) . Candida in the phase of budding and pseudomycelium were prevalent in acute CVV (84.4%) . In a subacute form Candida occurred in 30.3%, while in the remaining cases blastospores were observed . Significant correlation between the intensity of Candida colonies growth and phase of development on the mucous membrane was not found . Compared to the cultural method, the cytological technique is more cost effective, faster and simpler. Plant Sci, 2001 Jul, 161(2), 315 - 322 Regulation of anthocyanin biosynthesis in UV-A-irradiated cell cultures of carrot and in organs of intact carrot plants; Hirner AA et al.; Two cell lines of Daucus carota are known to differ with respect to anthocyanin accumulation . cDNA clones encoding enzymes involved in anthocyanidin biosynthesis, namely phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219) and leucoanthocyanidin dioxygenase (LDOX; EC 1.14.11.-), were isolated from libraries derived from cell cultures . Northern blot analysis of anthocyanin-accumulating (DCb) and non-accumulating (DCs) cell cultures of carrot showed that the anthocyanin pathway in these anthocyanin-free DCs cells is blocked . The expression of CHS1, DFR1 and LDOX is not detectable . However, F3H and DFR2 behave differently . In the European wild carrot (Daucus carota ssp . carota) the structural genes coding for the enzymes responsible for anthocyanin biosynthesis are strongly expressed in organs which accumulate anthocyanins . Only the dark-purple coloured petals of the central flowers of the inflorescence and to a certain extent the white flowers and the leaves but not the stems and the roots transcribe these genes . To study the effect of anthocyanins as UV-screens the expression of a protein indispensable for cell proliferation like alpha-tubulin (TUB) was monitored. Clin Experiment Ophthalmol, 2001 Jun, 29(3), 138 - 42 Phenotypic analyses of limbal epithelial cell cultures derived from donor corneoscleral rims; Barnard Z et al.; Grafted cultures of limbal epithelial cells aid repair of the corneal epithelium, but their phenotype is unclear . In this study, the phenotype of cultures that were similar in age to those used clinically were analysed . Limbal epithelial cells were isolated from donor corneoscleral rims and grown in various media, including those designed for keratinocytes . Successful cultures in each medium developed predominantly small (10 microm) tightly packed cells . Immunocytochemistry and western blotting revealed expression of keratins 3, 14 and 19 . Expression of these keratins in situ was confirmed by immunohistochemistry . Basal limbal epithelial cells were positive for keratins 14 and 19, and suprabasal cells were positive for keratin 3 . However intense staining for keratin 14 was also observed at the inner cut edge of corneoscleral rims . These findings demonstrate the potential importance of keratins 14 and 19 as markers of epithelial cell differentiation in the human cornea. Am J Physiol Endocrinol Metab, 2001 Aug, 281(2), E269 - 74 Involvement of thioredoxin in the regulation of growth hormone secretion in rat pituitary cell cultures; Hata I et al.; We report here an examination of the effect of thioredoxin (TRX) on the secretion of growth hormone (GH) from rat anterior pituitary cells in vitro . Treatment of rat pituitary cells with growth hormone-releasing factor (GRF), but not GH, led to a significant increase in intracellular TRX protein levels . GRF, recombinant human TRX (rhTRX), and a combination thereof were all shown to induce immediate GH secretion from pituitary cells, as evidenced by perifusion experiments . RhTRX, but not other reducing agents such as beta-mercaptoethanol and N-acetyl-L-cysteine, augmented GRF-stimulated and -unstimulated GH secretion from rat pituitary cells in a dose-dependent manner . RhTRX did not significantly affect the GH mRNA expression of pituitary cells stimulated in the presence or absence of GRF . In addition, rhTRX-augmented GH secretion was not significantly affected by the presence of cycloheximide . Collectively, these findings suggest that TRX is induced by stimulation with GRF and plays a regulatory role in GH secretion from rat anterior pituitary cells by enhancing the secretion of stored GH, rather than by the synthesis of GH. J Nephrol, 2001 May-Jun, 14(3), 198 - 203 Mesangial cell cultures; Mene P; Cell culture of glomerular mesangial cells (MC) has been available to most renal research laboratories since the early 80s . Key to a large number of studies on the biochemistry and molecular biology of the glomerulus, MC in culture have extensive analogies with this in vivo rather undifferentiated intercapillary cell population . They proliferate in response to mitogens and growth factors, can be growth-arrested by withdrawal of serum or 3D culture in collagen gels, synthesize an extracellular matrix that includes interstitial collagens, and display most markers of mesangial origin, including a functional contractile apparatus . As proliferation and matrix synthesis/degradation in vitro are regulated by cytokines and growth factors, cultured cells are an ideal tool for studying pathophysiological events such as mesangial expansion, scarring, and glomerulosclerosis . Current techniques for MC isolation and culture are reviewed, with several methodological issues relevant to the characterization, propagation and long-term maintenance of functional clones. Neuroendocrinology, 2001 Jul, 74(1), 43 - 54 Rapid stimulatory effects of brain-derived neurotrophic factor and neurotrophin-3 on somatostatin release and intracellular calcium rise in primary hypothalamic cell cultures; Marmigere F et al.; Although the long-lasting effects of neurotrophins have been extensively studied, less data are available on their rapid effects, especially on peptide release . In the present report, we investigated rapid effects of neurotrophins on somatostatin release and on intracellular calcium concentration ({Ca(2+)}(i)) in primary cultures of hypothalamic neurons . RT-PCR experiments revealed mRNA expression of the three high-affinity neurotrophin receptors tyrosine kinase (Trk) TrkA, TrkB and TrkC, indicating potential responses to their preferential ligands: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3), respectively . We demonstrated that BDNF, and to a lesser extent NT-3, induced significant time- and concentration-dependent somatostatin release, while NGF was devoid of any effect . BDNF or NT-3 induction of somatostatin release was inhibited by the Trk inhibitors K-252a and genistein, whereas K-252b, a less effective inhibitor, had no effect . BDNF- and NT-3-induced somatostatin release depended upon extra- and intracellular Ca(2+) since it was completely abolished in the presence of the Ca(2+) chelators BAPTA (bis-(alpha-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid) or BAPTA-AM (bis-(alpha-aminophenoxy)-ethane-N,N,N',N'-tetraacetoxymethylester), respectively . In addition, BDNF and NT-3 induced a sustained and rapid increase in {Ca(2+)}(i) which depended on the extracellular Ca(2+) concentration . MK-801 (dizocilpine) and tetrodotoxin (TTX) entirely blocked neurotrophin-evoked somatostatin release and {Ca(2+)}(i) rise in response to BDNF and NT-3 application in most neurons . Neurotrophin-induced {Ca(2+)}(i) rise was completely blocked by K-252a . The present results are consistent with: (1) an indirect effect of neurotrophins on somatostatin release via endogenous glutamate release and subsequent NMDA receptor activation, (2) a major indirect effect of neurotrophins on Ca(2+) rise in hypothalamic neurons which very likely occurs through NMDA receptor activation . Taken altogether, these results indicate that BDNF and NT-3 can rapidly affect the activity of hypothalamic neurons . Mund Kiefer Gesichtschir, 2001 May, 5(3), 166 - 72 {Mechanical stimulation of osteoblasts in cell culture}; Meyer U et al.; BACKGROUND: Mechanical loading of bone is known to play a crucial role in bone remodeling and regeneration . Whereas the clinical effects of mechanically modulated bone healing have been extensively studied, less is known about the underlying mechanisms on a cellular level . This study was aimed at investigating the effects of uniaxial strains on osteoblast-like cells in culture . Mechanical loading was applied in physiological and hyperphysiological magnitudes . Nonstimulated cultures served as controls . RESULTS: Cultured primary bovine periosteal cells exhibited phenotypic features of osteoblast-like cells . Application of physiological strains (2,000 mu strain) led to a bone-specific expression of extracellular matrix proteins (osteonectin, osteocalcin, collagen type I) . Hyperphysiological loads (10,000 mu strain) were associated with an increased synthesis of proteoglycans . Proliferation of cells was higher than the controls at 10,000 mu strain and showed no difference from physiologically loaded osteoblasts . DISCUSSION: Our study demonstrates that physiological loading of osteoblast-like cells enhances the regenerative capacity of bone, whereas hyperphysiological loads may impair bone regeneration. Int J Parasitol, 2001 Aug, 31(10), 1048 - 55 Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture; Hijjawi NS et al.; This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle genotypes) in the HCT-8 cell line . Furthermore, for the first time the complete life cycle was perpetuated in vitro for up to 25 days by subculturing . The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-reinfection cycle of C . parvum . This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro . In addition, thin-walled oocysts of the cattle genotype were infective for ARC/Swiss mice but similar oocysts of the human genotype were not . This culture system will provide a model for propagation of the complete life cycle of C . parvum in vitro. J Appl Toxicol, 2000 Dec, 20 Suppl 1, S77 - 80 Exposure of human epidermal keratinocyte cell cultures to sulfur mustard promotes binding of complement C1q: implications for toxicity and medical countermeasures; Cowan FM et al.; Sulfur mustard (HD)-increased proteolytic activity, HD-enhanced expression of Fc receptor (FcR) on human epidermal keratinocytes (HEK) and associated inflammatory responses may contribute to HD pathology . Like the FcR, the first component of the classical complement (C') cascade, C1q, binds to the Fc region of antibody to mediate inflammatory responses . Complement C1q binds specifically to the C1q receptor (C1qR) on the blebs of apoptotic human keratinocytes and is proposed as a cell surface marker for apoptosis . Assays by fluorescent antibodies demonstrated significantly enhanced binding of C1q to HEK cell cultures exposed to HD . The cell populations of HEK that showed enhanced C1q binding also demonstrated an intermediate uptake of propidium iodide that was greater than in viable unexposed cells but less than in dead cells . The HD-enhanced C1q binding was concentration-dependent, negative by flow cytometry or weakly positive by digital scanning microscopy at 100 microM and positive by both methods at 300 microM . Binding of C1q was also time-dependent, weakly positive at 8 h, and positive at 16 and 24 h after HD exposure . The HD-increased C1qR that binds C1q to the surface of HEK might be a contributing mechanism or a marker for the inflammation and vesication associated with HD exposure. Phytochemistry, 2001 Jul, 57(6), 1013 - 22 A heartwood pigment in Dalbergia cell cultures; Czako M et al.; In an extensive survey of the genera Baphia, Caesalpinia, Dalbergia, Haematoxylon, and Pterocarpus, we have identified a number of species whose cell cultures accumulated pigments similar to those in heartwood . Thirteen rosewood (Dalbergia) species produced a purple quinonemethide pigment in the callus that was apparently identical between the species . The pigment was first purified from D . retusa cell culture and its structure was elucidated by mass, infrared, and detailed 1H and 13C NMR and NOE spectroscopic studies including 2D experiments (COSY, NOESY, HMQC, and HMBC) . Retusapurpurin A (1a) is a C(30) isoflavan of novel skeleton whose formation can be rationalized to occur via regioselective oxidative coupling of an isoflavan to 4,4'-dihydroxy-2'-methoxychalcone . Retusapurpurin A was also isolated from D . parviflora heartwood and cell culture indicating that stress metabolism pathways that are shared with heartwood-type secondary metabolism subpathways are initiated in Dalbergia cell cultures . Therefore, Dalbergia cell cultures afford a good model system for studying heartwood-type metabolic differentiation. Plast Reconstr Surg, 2001 Jul, 108(1), 104 - 13 Stimulated healing of recalcitrant wounds by topical application of enriched cell culture medium: a clinical report; Lindenbaum ES et al.; This study was designed to test the efficacy of enriched cell culture medium as a wound dressing . The rationale was to create within the wound space an optimal microenvironment, conducive to cellular proliferation, vascular granulation tissue formation, and epithelialization . This study was performed on various wounds that failed to respond to previous conventional treatments.A total of 288 wounds were within the inclusion criteria, with only contaminated and neoplastic wounds excluded . Most of the patients (80 percent) were ambulatory, and the wounds were examined by the attending physician once every 7 to 14 days at an outpatient clinic . The remaining 20 percent of patients were admitted to the study while hospitalized.Cell culture medium MCDB, supplemented with insulin, thyroxin, and growth hormone, was gelled . The gel was self-applied once a day to freshly washed wounds, covered with a gauze pad, and anchored with netting.Healing started 7 to 14 days after the initiation of treatment with enriched cell culture medium . However, the criterion for success of the treatment was determined on complete wound closure, which was achieved in 189 of 288 wounds (65.6 percent) . Wound closure was correlated with the initial wound volume, stage, and origin . The average time required for closure of wounds caused by systemic pathologies (n = 181) and those based on regional status (n = 107) were 12.0 and 4.4 weeks, respectively, compared with 290 and 10.3 weeks of the previous conventional treatment . In 19 extensive wounds, when vascularized granulation tissue was established, a successful surgical closure was attained.Most wounds of patients who did not continue the enriched cell culture medium treatment (34.4 percent) manifested reduced wound volume, ranging from 11 to 98 percent of initial volume . Discontinuation of treatment was associated with difficulties in reaching the clinic for the weekly examination, rather than for reasons directly related to the treatment itself, and occurred significantly earlier during the treatment period.Thus, enriched cell culture medium was effective in stimulating wound healing in recalcitrant wounds . The healing was rapid with minimum scarring and pain . No side effects or allergic reactions were reported or observed. Immunol Invest, 2001 Feb, 30(1), 1 - 15 Cell culture conditions affect LPS inducibility of the inflammatory mediators in J774A.1 murine macrophages; Cohly H et al.; Peripheral blood monocytes and tissue macrophages contribute significantly to the inflammatory response . Bacterial lipopolysaccharide (LPS) has profound effects on these cells including, but not limited, to differentiation into macrophages, production of reactive nitrogen and oxygen species and secretion of pro-inflammatory cytokines . Herein, we describe a variant of the J774A.1 murine macrophage line that is reversibly resistant to multiple effects of LPS when cultured in different types of media . J774A.1 cells are adherent and spread out when cultured in DMEM/F12; however, when cultured in RPMI 1640, the cells are rounded and relatively non-adherent . Different types of tissue culture plates, sera, and media supplements were not responsible for these changes . We examined LPS-induced reactive nitrogen species using the Greiss reagent . J774A.1 cells cultured in RPMI exhibit a 5-fold increase in nitrites in culture supernatants after LPS stimulation whereas those in DMEM/F12 do not . Zinc staining of total cellular protein of cells in COHLY ET AL . RPMI and DMEM/F12 electrophoresed on a SDS-PAGE showed noticeable banding differences . LPS-induced cytokine gene expression was studied by RT-PCR . LPS induced TNF-alpha, IL-1alpha, IL-1beta, and sIL-1Ra in cells cultured in RPMI but not those cultured in DMEM/F12 with the exception of TNFalpha . This report shows that environmental factors contained in the culture medium alone can reversibly alter the biochemical nature of monocytes and macrophages. Photodermatol Photoimmunol Photomed, 2001 Jun, 17(3), 126 - 9 Ultraviolet-filtering properties of commonly used tissue cell culture plasticware; Brown DB et al.; BACKGROUND: Fluorescent sunlamps are a common source of ultraviolet radiation (UVR) for photobiology research . However, these lamps emit a significant amount of biologically "irrelevant" wavelengths that, if not removed, can drastically skew results and perhaps lead to mistaken conclusions regarding human photobiology . The use of a cellulose triacetate sheet (Kodacel) to filter the shorter ultraviolet wavelengths has become the accepted standard in photobiology . Over time, the transmission characteristics of this sheet may be altered due to photochemical changes . In addition, in vitro experiments utilizing filtered fluorescent sunlamps require the removal of plastic tissue cell culture lids, increasing the possibility of contamination . METHODS: We evaluated the transmission characteristics of various commercially available plastic lids used in tissue cell cultures . In addition, we used a biological system containing the human elastin promoter/chloramphenicol acetyltransferase reporter gene construct to compare the effects of filtering from these plastic lids . RESULTS: Here, we demonstrate that the transmission of UVR and the biological response through plastic culture dish lids is similar to that of Kodacel . CONCLUSION: Although this is an improvement for in vitro experiments, further improvements can be made using more realistic UVR sources, e.g . UVA-340 lamps, which mimic the short wavelengths of sunlight. Biomed Tech (Berl), 2001 May, 46(5), 133 - 6 {Development of an incubation system for an inverted microscopy for long-term observation of cell cultures using chamber slides}; Feicht W et al.; Trifunctional bispecific antibodies open up new immunological possibilities in tumour treatment . Prior to clinical application, comprehensive investigations using animal models and in vitro examinations need to be done . To investigate long-term interactions between various immunologically active blood cells and individual tumour cells in the presence of antibodies, we developed an incubation system for experimental cell cultures on an inverted microscope . The system consists of a perspex box with a central moisture chamber with integrated water reservoir, external air circulation heating, and a CO2 supply . The sterile cell cultures are located in the wells of a slide positioned within a depression in the water reservoir . The newly developed incubation system enables continuous observation over the long term of experiments under optimal cell cultures conditions in combination with modern video techniques. In Vitro Cell Dev Biol Anim, 2001 Apr, 37(4), 251 - 8 Renal cell cultures for the study of growth factor interactions underlying kidney organogenesis; Mattii L et al.; The present study was performed in four renal cell lines to evaluate their capability to: (1) produce and express transforming growth factor alpha (TGFalpha), its respective receptor, the epidermal growth factor receptor (EGFr) and the small G protein, RhoA, and (2) exhibit morphogenetic properties when grown on Matri-cell substrates . The cell lines were derived from normal (Madin-Darby canine kidney cells), embryonic (SK-NEP-1 and 293 cells), and cancerous (human renal adenocarcinoma cells) kidneys . TGFalpha messenger ribonucleic acid, evaluated by a nonradioactive in situ hybridization technique, was found to be expressed in all the cell lines . Large amounts of TGFalpha peptide were observed in all four cell lines, while EGFr was highly expressed only in cancerous ACHN and embryonic-tumor SK-NEP-1 cells . RhoA peptide was found in appreciable amounts in SK-NEP-1 and 293 cells (compared to the other two cell lines) . The morphogenetic properties of the four cell lines were assessed, by culturing them on Matri-cell dishes: SK-NEP-1 cells alone were found to grow in three-dimensional structures forming clusters and worm-like cellular aggregates . This feature was displayed by SK-NEP-1 cells but not by the other three cell lines, and may be connected with the contemporary presence of RhoA, EGFr, and TGFalpha found in significant amounts only in the SK-NEP-1 cell line. Dis Aquat Organ, 2001 May 4, 45(1), 19 - 24 Characterisation of ISAV proteins from cell culture; Griffiths S et al.; The characterisation of 2 infectious salmon anemia virus (ISAV) proteins is described . Proteins were harvested from ISAV-infected Chinook salmon embryo (CHSE)-214 cell culture by continuous elution denaturing gel electrophoresis, enabling the harvest of specific molecular weight fractions . Through the use of a polyclonal antiserum to ISAV, it was possible to identify a potentially autolytic major antigen of 72 kDa and a glycosylated protein of approximately 38 kDa which varied in size depending on cell line compatibility . N-terminal amino acid sequencing of the glycosylated proteins suggests that it is encoded by segment 6 of the ISAV genome . Further, sequence analysis of the glycosylated protein account for the variable molecular weight and may explain differences in host cell compatibility. Aust Endod J, 1999 Aug, 25(2), 70 - 5 Investigations on a cell culture medium for storage and transportation of avulsed teeth; Pohl Y et al.; Non-physiologic storage of avulsed teeth leads to a high incidence of root resorption, resulting in poor prognosis . This study investigated the suitability of specially composed cell culture media for storage of extracted teeth for up to 48 hours . Autoradiographic investigations revealed that the proliferative activity of periodontal ligament (PDL) cells of teeth stored in cell culture medium for up to 48 hours increased with storage time . Studies on proliferation of PDL cells after storage of teeth in different media for up to 24 hours demonstrated that the proliferative activity is dependent on the composition of the medium . Immunohistochemical investigations with markers for cell proliferation revealed that pulp cells of extracted immature teeth show numerous proliferations after storage for up to 24 hours in a special cell culture medium but few proliferations after storage in Hanks Balanced Salt Solution (HBSS) . The investigations indicate that a special cell culture medium can preserve cell viability of PDL cells adhering to extracted teeth for at least 48 hours . The in vitro results are confirmed by a case presented: After storage of two upper central incisors for 36 hours in the cell culture medium the teeth could be successfully reimplanted after extraoral insertion of titanium posts into the root canal (auto-alloplastic reimplantation) . Clinical and radiological follow-up examinations for 12 months revealed normal periodontal healing. Indian J Exp Biol, 2000 Dec, 38(12), 1192 - 200 Cell culture of biopsied endometriomas after danazol/hormonal therapy: a study of growth features and fertility effects; Stevenson AF et al.; The growth features of cells from endometriomas biopsied from patients who had been treated with Danazol or with hormones have not been studied in vitro . Danazol is a versatile drug and despite its recognised efficacy in controlling endometriosis, little is known about is cytotoxicity and mechanism of action . Culture of the biopsied endometriomas permitted qualitative cytotoxic assessments by way of comparison with cultured normal uterine endometrial cells treated in vitro with Danazol . The growth characteristics were studied in monolayer and collagen gel cultures . Cytopathology was characterised by light and electron microscopy . Since endometriosis is associated with infertility in women, data from in vitro fertilisation (IVF) were analysed with respect to treatment modalities as compared with cases suffering fallopian occlusion . Danazol reduced pregnancy chances . Two factors may be responsible: (a) altered follicle development resulting in poor oocyte quality (b) reduced nidation because of long-lasting endometrial cytotoxicity . The experimental findings reported here support the latter explanation . Consequently, Danazol therapy should be conditional; patients wishing to achieve pregnancy should preferably receive hormonal therapy. Hum Mol Genet, 2001 Jun 1, 10(12), 1307 - 15 Geldanamycin activates a heat shock response and inhibits huntingtin aggregation in a cell culture model of Huntington's disease; Sittler A et al.; Huntington's disease (HD) is a progressive neurodegenerative disorder with no effective treatment . Geldanamycin is a benzoquinone ansamycin that binds to the heat shock protein Hsp90 and activates a heat shock response in mammalian cells . In this study, we show by using a filter retardation assay and immunofluorescence microscopy that treatment of mammalian cells with geldanamycin at nanomolar concentrations induces the expression of Hsp40, Hsp70 and Hsp90 and inhibits HD exon 1 protein aggregation in a dose-dependent manner . Similar results were obtained by overexpression of Hsp70 and Hsp40 in a separate cell culture model of HD . This is the first demonstration that huntingtin protein aggregation in cells can be suppressed by chemical compounds activating a specific heat shock response . These findings may provide the basis for the development of a novel pharmacotherapy for HD and related glutamine repeat disorders. J Immunol Methods, 2001 Aug 1, 254(1-2), 183 - 90 Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells; Marin V et al.; Endothelial cells play a key role in prominent immunological and pathological processes such as leukocyte trafficking, inflammation, atheroma or cancer cell metastasis . Umbilical veins are probably the most widely used source for human endothelial cells, since they are more easily available than many other vessels, they are free from any pathological process and they are physiologically more relevant than many established cell lines . Here, we describe a standard protocol for preparation, maintenance and quality control of these cells. Aquat Toxicol, 2001 Aug, 53(3-4), 279 - 89 The further development of rainbow trout primary epithelial cell cultures as a diagnostic tool in ecotoxicology risk assessment; Dowling K et al.; The use of short-term cytotoxicity assays for the initial screening of chemicals not only aids in establishing priorities for the selection of chemicals that should be tested in vivo, but also decreases the time in which potential toxicants can be valued . Rainbow trout primary skin epithelial cell cultures are one such assay . Rainbow trout primary skin cell cultures contain two cell types, keratinocytes and goblet mucus cells . Two aquatic pollutants, copper and prochloraz were screened using this cell system . The influence of media composition on the effects of the aquatic pollutants was also studied by testing the chemicals in both serum-containing and serum-free medium and the morphological changes that occurred within the cell cultures recorded . The concentration of copper that causes a reduction of 90% in the residual of day 3 growth of the primary cell culture system was found to be approximately 10 fold more than that of prochloraz . Prochloraz was found to cause a greater reduction in growth area when added to the primary cell culture system in serum-free medium than in serum-containing medium . Copper, in contrast, was found to exert reduced toxicity when added to the test cultures in serum-free medium compared with addition in serum-containing medium . Prochloraz was found to kill the epithelial cells by a process of necrosis . Copper, was found to kill the epithelial cells by both necrosis and apoptosis in a ratio of 2:1 . It was also observed that as the dose of both chemicals increased, the number of goblet cells contained in the cell cultures decreased . A PAS stain was carried out to determine if the goblet cells were exocytosing their contents onto the cell culture surface . It was found that as chemical exposure increased the number of cells expressing positivity for mucus also increased . The results of this study add further evidence to support that primary cell cultures are a very appropriate model for toxicity risk assessment. Exp Dermatol, 2001 Jun, 10(3), 184 - 92 Inhibition of keratinocyte growth in cell culture and whole skin culture by mast cell mediators; Huttunen M et al.; Mast cells are suggested to participate in regenerative processes, but their influence on epithelialization and wound healing has not been well studied . Since mast cells can be found in contact with epidermis in chronic inflammatory skin diseases and venous ulcers, the effect of mast cells on keratinocyte growth was studied . Keratinocytes were cultured in serum-free conditions with (complete medium) or without (basal medium) epidermal growth factor (EGF) and bovine pituitary extract (BPE) to reach subconfluence in a 24-well plate, and the cells were treated with different mast cell mediators histamine, heparin and tryptase, or lysate from HMC-1 cells, a human leukemic mast cell line . Whole skin cultures were used as a model for in vitro wounds to study the effect of mast cells on epithelial outgrowth from skin specimens . Histamine inhibited 3H-thymidine incorporation of keratinocytes dose-dependently by 29% at 1 mM, and 89% at 5 mM histamine . In whole skin culture, histamine inhibited epithelial outgrowth dose-dependently by 64% already at 0.1 mM histamine and maximally (91%) at 1 mM histamine . Heparin inhibited 3H-thymidine incorporation dose-dependently by up to 33% at 2 microg/ml in the absence, but not in the presence, of EGF/BPE . In contrast, in whole skin culture, heparin first inhibited the epithelial outgrowth by up to 27% at 2 microg/ml, but then reversed the inhibition to 30% stimulation at 200 microg/ml . Skin tryptase (0.0285 to 2.85 microg/ml) with or without heparin (0.5 to 20 microg/ml) did not affect thymidine incorporation in keratinocytes . Lysate from HMC-1 cells, but not that from control, neuroblastoma cells, inhibited 3H-thymidine incorporation in keratinocytes dose-dependently, and maximal (47%) inhibition was reached with 16,700 lysed HMC-1 cells/ml . In whole skin culture, HMC-1 lysate inhibited the epithelial outgrowth by up to 36% at 67,000 lysed cells/ml . The results show that mast cells and their mediators are inhibitory to keratinocyte 3H-thymidine incorporation and epithelial outgrowth in vitro, although, the inhibitory effect of histamine was seen at high concentrations suggesting a requirement for close morphologic vicinity of mast cells to keratinocytes . Thus, mast cells are assumed to control epidermal regeneration and to impair epithelialization of chronic ulcers. Acta Ophthalmol Scand, 2001 Jun, 79(3), 309 - 12 A murine cell culture model for post-trabeculectomy anfibrotic treatment: Induction of apoptosis by Cyclosporin; Cristofanilli M et al.; PURPOSE: Experimental trials aimed at the research of selective antifibrotic agents are under development for the alternative treatment of glaucoma patients who are usually considered high-risk post-surgical individuals after trabeculectomy . Authors present here an in vitro model system for the treatment of post-trabeculectomy patients . The study is aimed at the evaluation of different drugs in a mouse fibroblast model . METHODS: The antifibrotic activity of Cyclosporin A, Interferon 2alpha, 5-Fluorouracyl was investigated on 3T6 cells in culture . Cell viability and proliferation was assessed after drug treatment . Molecular analysis of DNA degradation was evaluated by means of radioactive labeling and gel electrophoresis . RESULTS: The three drugs were shown to affect cell proliferation and viability in a differential fashion . However, only Cyclosporin A was able to control cell proliferation, inducing apoptosis . This phenomenon was reduced by supplementation of trolox, a compound known to inhibit programmed cell death . These results strongly suggest that this model system might be useful as a test of pharmacological functionality . CONCLUSION: A rapid and efficient model system is described for the assessment of cell viability and proliferation after treatment with agents of potential pharmacological use . Cyclosporin A induces a significant apoptosis . This is important for the negative control of fibrotic degeneration in post-trabeculectomy that is required for successful surgery in glaucoma patients . Therefore, Cyclosporin A might become a clinically interesting drug for the antifibrotic treatment of post-trabeculectomy. Pharmacol Res, 2001 Mar, 43(3), 241 - 4 The effects of dantrolene alone or in combination with nimodipine in glutamate-induced neurotoxicity in cerebellar granular cell cultures of rat pups; GepdIremen A et al.; Despite the existence of some positive and negative reports on dantrolene in ischemic states, combined application of an endoplasmic reticulum Ca2+ release inhibitor and a calcium channel blocker has not yet been elucidated . In the present study, we have investigated the role of dantrolene in subsequent doses alone or in coexistence with the dihydropyridine calcium antagonist nimodipine (10(-4) M concentration) in glutamate-induced (10(-7) M) neurotoxicity in cerebellar granular cell cultures of rat pups . Glutamate induced neuronal cell death at a concentration of 10(-7) M . Despite the fact that none of the groups tested were able to reverse cell death to control values, dantrolene was found to be effective in preventing glutamate toxicity in cerebellar cultures of rat pups . The protective effect of dantrolene potentialized in combination with nimodipine at all doses tested . The most effective dose of dantrolene was found to be 10(-4)M in combination with nimodipine . As a result, both extracellular and internal calcium stores play important roles in the genesis of neuronal cell death induced by glutamate . Intensive Care Med, 2001 Apr, 27(4), 686 - 93 Critical illness polyneuropathy: clinical findings and cell culture assay of neurotoxicity assessed by a prospective study; Druschky A et al.; OBJECTIVE: First, to evaluate the role of typical intensive care-related conditions like sepsis, prolonged ventilation, drug effects and metabolic disorders in the pathogenesis of critical illness polyneuropathy (CIP); second, to investigate the possible significance of patient serum neurotoxicity assessed by an in vitro cytotoxicity assay with respect to CIP development . DESIGN: Prospective study . SETTING: Neurological intensive care unit . PATIENTS AND PARTICIPANTS: Twenty-eight patients who were on mechanical respiratory support for at least 4 days during a 21-month study period . RESULTS: Diagnosis of CIP was established by clinical and electrophysiological examination in 16 (57%) of 28 patients . Patients were investigated on days 4, 8 and 14 of mechanical ventilation . Two of 16 CIP patients had clinical signs of polyneuropathy at initial examination . Factors that correlated significantly with the development of CIP were: the multiple organ failure score on day 8 of ventilation, the total duration of respiratory support, the presence of weaning problems and the manifestation of complicating sepsis and/or lung failure . The in vitro toxicity assay showed serum neurotoxicity in 12 of 16 CIP patients . Electrophysiological investigations yielded false positive results of the toxicity assay in six patients (not developing CIP) and false negative results in four patients (developing clinical and electrophysiological signs of CIP) . Statistical analysis did not reveal a significant correlation between the diagnosis of CIP and the finding serum neurotoxicity . CONCLUSION: The results support the hypothesis of a multi-factorial aetiopathogenesis of CIP . We observed serum neurotoxicity in the majority of CIP patients, indicating the possible involvement of a so far unknown, low-molecular-weight neurotoxic agent in CIP pathogenesis. Biomaterials, 2001 Jul, 22(13), 1713 - 9 Characterization of collagen gel solutions and collagen matrices for cell culture; Sheu MT et al.; The influence of glutaraldehyde as a crosslinking agent to increase the strength of collagen matrices for cell culture was examined in this study . Collagen solutions of 1% were treated with different concentrations (0-0.2%) of glutaraldehyde for 24 h . The viscoelasticity of the resulting collagen gel solution was measured using dynamic mechanical analysis (DMA), which demonstrated that all collagen gel solutions examined followed the same model pattern . The creep compliance model of Voigt-Kelvin satisfactorily described the change of viscoelasticity expressed by these collagen gel solutions . These crosslinked collagen gel solutions were freeze-dried to form a matrix with a thickness of about 0.2-0.3 mm . The break modulus of these collagen matrices measured by DMA revealed that the higher the degree of crosslinking . the higher the break modulus . The compatibility of fibroblasts isolated from nude mouse skin with these collagen matrices was found to be acceptable at a cell density of 3 x 10(5) cells/cm2 with no contraction, even when using a concentration of glutaraldehyde of up to 0.2%. Hybridoma, 2001 Apr, 20(2), 123 - 9 Immunological characterization of a mucin-associated protein from hamster tracheal epithelial cell culture; Park Y et al.; Airway mucins are high molecular mass (>10(6) dalton) glycoproteins with various types of associated molecules including glycoproteins, lipoproteins, and lipids . The study of mucin-associated proteins is limited largely due to the lack of specific probes . In this study, we produced a monoclonal antibody, MAbHT10, against a 190-kDa mucin associated-protein by immunizing mice with hamster airway mucin purified in nondissociative condition . Using HT10, the 190-kDa mucin-associated protein was characterized immunologically . The 190-kDa mucin-associated protein is glycoprotein and HT10 recognized carbohydrate containing portion of the protein . The association of 190-kDa protein with mucin is strong enough that heat and detergent treatment is required to dissociate it from mucin as evidenced by gel filtration chromatography, Western blot, enzyme-linked immunoadsorbent assay (ELISA), and co-immunoprecipitation . The expression of the 190-kDa protein is increased with the development of hamster tracheal epithelial cells in culture, but showed differences with the pattern of the regulation of mucin expression . Adenosine triphosphate (ATP), a known strong mucin secretagogue, dose-dependently increased mucin release but caused only marginal increase in the release of the 190-kDa protein . The MAb should be useful in the structural and functional analysis of the 190-kDa mucin-associated proteins in physiological and pathological situations such as chronic airway diseases. Microbios, 2001, 105(411), 111 - 8 Detection of herpes simplex and varicella zoster viruses in clinical specimens using direct immunofluorescence and cell culture assays; Meqdam MM et al.; Patients (33 in toto) with a clinical diagnosis of herpes infections (simplex, zoster or chickenpox) were investigated for the presence of herpes simplex virus (HSV) and varicella zoster virus (VZV) in skin samples, using direct immunofluorescence and cell culture assays . Five patients with nonherpetic vesiculobullous disorders were included as negative controls . Of the 33 patients, nineteen (57.6%) were positive for HSV or VZV and fourteen (42.4%) were negative . Five controls were all negative for HSV or VZV . Of the nineteen positive patients, HSV was isolated from eight (42.1%) patients, by both direct immunofluorescence and cell culture assays . VZV was isolated from eleven (57.9%) patients, eleven (100%) by direct immunofluorescence assay, and six (54.5%) by cell culture assays . HSV was isolated from one patient clinically diagnosed as chickenpox (VZV), but otherwise the positive laboratory results were concordant with the clinical diagnosis . For epidemiological studies, atypical cases and immunocompromised patients the clinical diagnosis should be confirmed in the laboratory. Tissue Cell, 2001 Apr, 33(2), 154 - 60 Role of water-soluble matrix fraction, extracted from the nacre of Pinctada maxima, in the regulation of cell activity in abalone mantle cell culture (Haliotis tuberculata); Sud D et al.; In mollusks, the mantle is responsible for the secretion of an organic matrix that mineralizes to form the shell . A model of mantle cell culture has been established from the nacreous gastropod Haliotis tuberculata . First, viability of cells, quantified by the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) reduction assay, was monitored in order to determine a cell density and a time-culturing period in order to investigate biomineralization processes in vitro . During the first 11 days of culture, an increase of MTT response demonstrated an activation of cultured cells mitochondrial activity as confirmed by the total protein content assay . The effect of a water-soluble extract from the organic matrix of Pinctada maxima (WSM) was tested on this cell culture system for 11 days-period exposure . WSM reduced the global viability of mantle cells in a dose-dependent way which corresponded to a cell death . Alkaline phosphatase activity normalized to total protein content increased in the presence of WSM . This increase may be due to an activation of cells and a selection of one (or a few) cell type(s) . Further investigations will help us to determine this selectivity issue. Free Radic Biol Med, 2001 Jun 15, 30(12), 1418 - 25 Light-dependent generation of reactive oxygen species in cell culture media; Grzelak A et al.; Cell culture media (RPMI 1640, Dulbecco's Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5'-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS) . The production of ROS was light dependent . The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin . These observations point to exposure of cells to ROS under in vitro culture conditions. Biochim Biophys Acta, 2001 May 28, 1539(1-2), 85 - 92 Effect of the beta(3)-adrenergic agonist Cl316,243 on functional differentiation of white and brown adipocytes in primary cell culture; Klaus S et al.; We investigated the effect of the specific beta(3)-adrenergic receptor agonist CL 316,243 (CL) on proliferation and functional differentiation of the Siberian hamster (Phodopus sungorus) white and brown preadipocytes in primary cell culture . Proliferation of both white and brown preadipocytes was stimulated by a general beta-adrenergic agonist (isoproterenol) but not by CL . Lipolysis of differentiated white and brown adipocytes was stimulated similarly by CL with maximum effect at 10 nM . Thermogenic properties of cells were assessed by immunodetection of UCP-1, the brown adipocyte specific uncoupling protein, and measurement of cytochrome c oxidase (COx) activity as an index of mitochondrial capacity . UCP-1 content was largely increased by CL in BAT but not in WAT cultures . Basal UCP-2 mRNA levels were similar in WAT and BAT cultures and increased by both CL and isoproterenol . COx activity of BAT cultures was twice as high as that of WAT cultures but in neither cell culture system could it be increased by beta-adrenergic stimulation . We suggest (i) that white and brown preadipocyte proliferation is increased in vitro via beta1 or beta(2), but not beta(3)-adrenergic pathways, (ii) that white and brown preadipocytes represent different cell types, and (iii) that in vitro beta-adrenergic stimulation it is not sufficient to induce complete thermogenic adaptation of brown adipocytes. J Neurosci Methods, 2001 May 30, 107(1-2), 47 - 61 The dispersed cell culture as model for functional studies of the subcommissural organ: preparation and characterization of the culture system; Schoniger S et al.; The subcommissural organ (SCO) is an enigmatic secretory gland of the brain, which is believed to be derived from ependymal (glial) precursor cells . We here developed a dispersed cell culture system of the bovine SCO as an approach to functional analyses of this brain gland . Tissue of the bovine SCO obtained from the slaughterhouse was papain dissociated either directly after dissection or after preparation of SCO explants . The latter had been maintained for 4-6 weeks in organ culture . The dispersed cells were cultured for up to 14 days and continuously tested for their secretory state by immunostaining of their secretory product . With respect to the morphology of the SCO cells (shape, processes, nucleus), no difference was found between the culture of freshly dissociated SCOs and that of dissociated SCO explants . In all cases, the dissociation caused a dedifferentiation; typical elongated cells were formed increasingly after 1 day of culture . Thereafter, only the cellular size increased, whereas the shape and the viability of the cells remained unchanged . Proliferating SCO cells were never observed . The culture obtained from fresh SCO tissue contained more glia cells and fibrocytes than the culture prepared from SCO explants . The proliferation of glia cells and fibrocytes was suppressed by blocking the mitotic activity with cytosine-beta-D-arabino furanoside (CAF) . The cytophysiological features of the cultured dispersed cells of both origins did not differ as demonstrated by classical histology, by immunocytochemistry for the secretory products of the SCO, by the characteristics of calcium influx into the cytoplasm ({Ca2+}i) and cyclic adenosine monophosphate (cAMP) after stimulation with adenosine-5-triphosphate, substance P or serotonin, and by the activation of the transcription factor cAMP-responsive element-binding protein . Because of the maintenance of their viability, their capacity to release the secretory product into the culture medium, their receptive capacity, and their signal transduction pathways, we conclude that the dispersed cell culture system, especially that obtained from SCO explants, represents an appropriate and useful model for functional studies of the mammalian SCO. Biosci Biotechnol Biochem, 2001 Apr, 65(4), 853 - 60 Increases of secondary metabolite production in various plant cell cultures by co-cultivation with cork; Yamamoto H et al.; Cork tissues increased secondary metabolite production of various plant cell cultures in a different manner from those of conventional elicitors . In Sophora flavescens and Glycyrrhiza glabra cultured cells, cork tissues increased the amounts of both lipophilic and hydrophilic flavonoids without affecting the cell growth, although elicitors such as copper ion and yeast extracts showed a clear inhibition of cell growth with the increasing amount of these lipophilic ones . The validity of this effect of cork tissues covered a wide range of aromatic compounds produced by suspension cell cultures derived from diverse plant species . Woody tissues of Japanese cypress had a very similar effect to that of cork . Partial purification of cork tissues suggested that the production-stimulating factor was present in the hemicellulose B fraction that was not included in the dedifferentiated cultured tissues. Biotechnol Prog, 2001 May-Jun, 17(3), 403 - 6 Utilization of an alternative carbon source for efficient production of human alpha(1)-antitrypsin by genetically engineered rice cell culture; Terashima M et al.; Human alpha(1)-antitrypsin was produced by genetically engineered rice cells using promoter and signal peptide of a rice alpha-amylase isozyme . Batch and continuous cultures were employed to investigate the effects of alternative carbon sources on the alpha(1)-antitrypsin production . While this expression system is inducible by sugar depletion, we have found that the productivity of alpha(1)-antitrypsin increased 2.4- to 3.4-fold, compared with the control medium without carbon source, in medium containing an alternative carbon source, such as pyruvic acid and glyoxylic acid . The accumulated alpha(1)-antitrypsin in the medium containing pyruvic acid reached 18.2-24.2 mg/g-dry cell in 50-70 h by batch culture. Chem Pharm Bull (Tokyo), 2001 May, 49(5), 639 - 41 Synthesis of styrenes through the biocatalytic decarboxylation of trans-cinnamic acids by plant cell cultures; Takemoto M et al.; A novel method for producing styrenes from trans-cinnamic acids was developed . When trans-cinnamic acid was incubated with plant cell cultures at room temperature, styrene was obtained . 4-Hydroxy-3-methoxystyrene (2a), 3-nitrostyrene (2f) and furan (2g) were synthesized quantitatively. Appl Environ Microbiol, 2001 Jun, 67(6), 2484 - 8 Best viral elution method available for quantification of enteroviruses in sludge by both cell culture and reverse transcription-PCR; Monpoeho S et al.; The aim of this study was to select one or several virus extraction techniques that enable simultaneous detection of enterovirus genomes and infectious particles in different types of urban sludge . Eight techniques were compared by using 16 different liquid and solid sludge samples . The numbers of infectious enteroviruses in cell cultures were determined by using the most-probable-number method . The enterovirus genome was quantified by a single-tube reverse transcription-PCR using TaqMan technology . The results were statistically analyzed by Friedman's test, a nonparametric test for analysis of randomized block data using only ranks in terms of extraction technique efficiency . Two techniques seemed to yield higher viral titers as determined by simultaneous detection by cell culture and PCR . The first involved a 10% beef extract solution at pH 9 and sonication; the second involved a 0.3 M NaCl-7% beef extract solution at pH 7.5 followed by Freon treatment . In solid sludge, no significant differences were observed among the eight techniques tested . Both of the best techniques can be used for simultaneous detection of infectious enterovirus particles and genomes in any type of urban sludge. Vet Dermatol, 2001 Apr, 12(2), 75 - 80 In vitro release of interferon-gamma by trichophytin-stimulated whole blood cell cultures from ringworm-vaccinated and control calves experimentally inoculated with Trichophyton verrucosum; Lund A et al.; Of a group of seven calves, four were vaccinated against bovine ringworm with the vaccine Ringvac bovis LTF-130 (Alpharma, Oslo, Norway), while three calves were left unvaccinated . All calves were inoculated epicutaneously with a virulent strain of Trichophyton verrucosum . Clinical signs were recorded . In response to stimulation with trichophytin, in vitro interferon-gamma (IFN-gamma) production in whole blood cell cultures was assessed in samples obtained pre- and post-vaccination and pre- and post-inoculation . A commercial enzyme immunoas say kit was used to measure IFN-gamma levels (Bovigam, CSL, Victoria, Australia) . Control calves developed typical ringworm lesions, whereas vaccinated calves did not . Following vaccination, release of IFN-gamma in whole blood cell cultures indicated the presence of circulating trichophytin-specific lymphocytes . After inoculation with T . verrucosum, IFN-gamma production was demonstrated in samples from both vaccinated and control calves . This study showed that vaccination with Ringvac bovis LTF-130 elicits a protective immune response suggesting involvement of the cellular branch of the immune system . Experimental infection of naive nonvaccinated calves with T . verrucosum, also indicated stimulation of a cell-mediated immune response essential for resolution of lesions. J Virol, 2001 Jun, 75(12), 5614 - 26 Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures; Ciufo DM et al.; Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8 . KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin . Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators . Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein . Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC . Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions . Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures . In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens . We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein . However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection . The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations. Vet Immunol Immunopathol, 2001 May 10, 79(1-2), 41 - 52 Differential cytokine mRNA expression in swine whole blood and peripheral blood mononuclear cell cultures; Yancy H et al.; The kinetics of interleukin-2 (IL-2), IL-6, IL-8 and IL-10 gene expression in concanavalin A (Con A)-activated whole blood (WB) and peripheral blood mononuclear cell (PBMC) cultures were examined using reverse transcriptase-polymerase chain reaction (RT-PCR) . Unstimulated PBMC or WB cultures failed to show increases in basal cytokine PCR amplicon levels for any cytokine examined . PBMC cultures demonstrated peak expression of IL-2, IL-6, IL-8 and IL-10 mRNA levels at 12, 24, 24 and 6h, respectively . WB cultures exhibited peak IL-2, IL-6, IL-8 and IL-10 mRNA levels at 24, 12, 6 and 24h, respectively . PBMC cultures consistently exhibited higher levels of IL-2 mRNA at all times examined than did WB cultures . WB cultures consistently had higher levels of IL-6 mRNA than PBMC cultures . IL-8 and IL-10 protein levels in PBMC cultures were first detected 12h after stimulation and continued to increase in concentration through 48h . In WB cultures, IL-8 and IL-10 protein levels were first noted at 12 and 6h, respectively . WB culture IL-8 and IL-10 levels quickly reached equilibrium after being detected and remained at levels lower than those noted in PBMC cultures . These results show WB cultures represent an approach with reduced cost and time when compared to traditional cell culture and isolation methods . It may also produce an in vitro test system that more closely resembles in vivo conditions. J Chromatogr A, 2001 Apr 13, 913(1-2), 341 - 7 Determination of pyruvate and lactate in primary liver cell culture medium during hypoxia by on-line microdialysis-liquid chromatography; Wu YS et al.; A microdialysis sampling device was constructed for the measurement of pyruvate and lactate in primary liver cell culture medium during hypoxia . It was composed of a Petri dish, a dialysis membrane and two transmission tubes within a hypoxia chamber . The dialysis membrane was located in the Petri dish such that it was immersed in the culture medium . Dialysates were collected and introduced by an on-line injector to a liquid chromatographic system for analysis of pyruvate and lactate . The detection limit of this assay was 0.2-2.0 microM with acceptable intra- and inter-assay reproducibilities . In order to validate the assay, primary liver cells were incubated in the Petri dish within a hypoxia chamber in an incubator . The baseline concentrations of pyruvate and lactate in primary liver cell culture medium were 10.6+/-5.6 and 607+/-143 microM, respectively . These levels drastically changed during hypoxia and reperfusion . In conclusion, the present assay provides a sensitive, direct measurement of pyruvate and lactate in culture medium while minimizing pretreatment procedures for sample preparation. Biotechniques, 2001 May, 30(5), 1010 - 4 96-well plate-based method for total collagen analysis of cell cultures; Fenwick SA et al.; Total collagen assays are often laborious and use large quantities of consumables . We have developed a new method of assaying total 3H-proline-labeled collagen from cultured cells . Cells and media are harvested from 96-well plates directly onto fiberglass filtermats and counted in the Wallac 1205 flat-bed scintillation counter (BetaPlate) . The assay was validated by comparison with a traditional total collagen assay . The resulting assay provides a rapid one-step method for quantifying collagen synthesis, which, unlike many collagen assays, does not require extensive dialysis or precipitation of proteins. Menopause, 2001 May-Jun, 8(3), 216 - 21 Effect of an estrogen/statin combination on biochemical markers of endothelial function in human coronary artery cell cultures; Mueck AO et al.; OBJECTIVE: The combination of an estrogen with a statin for therapy of postmenopausal women is of interest because both substance classes exert beneficial effects on the lipid profile . However, both substance classes also elicit positive direct effects on the vasculature . Therefore in the present in vitro study an estrogen/statin combination was investigated for its effect on biochemical markers of endothelial function . MATERIAL AND METHODS: In endothelial cell cultures from human coronary arteries, the effect of estradiol/fluvastatin, alone and in equimolar combinations, were tested at the concentrations 0.01, 0.1, and 1 microM . The vasodilator prostacyclin, the vasoconstrictor endothelin, endothelial nitric oxide synthase (responsible for synthesis of the vasodilator nitric oxide), the procoagulatory factor plasminogen activator inhibitor-1, and the monocyte chemoattractant protein were chosen as markers . RESULTS: The estradiol/fluvastatin combination was able to increase prostacyclin production (25-100%) in an additive manner . The reduction of endothelin synthesis in the range of 21-46% was higher with the combination than with the monosubstances; the reduction, however, was not statistically significant . The expression of endothelial nitric oxide synthase was not significantly increased by the combination compared with the monosubstances, however, a tendency to an additive increase was observed . For the synthesis of plasminogen activator inhibitor-1, no significant changes were seen for either the monosubstances or the combination . The synthesis of monocyte chemoattractant protein-1 was decreased by the combination between 21% and 40%; the decrease, however, was not statistically significant compared with the effect of the monosubstances . The effective estradiol concentrations are higher than can be achieved by replacement therapy; in contrast, fluvastatin was effective at concentrations that can be reached during clinical treatment . CONCLUSIONS: These results indicate that an estrogen/statin combination exerts beneficial effects on the vasculature that seem to be superior to the effects of the monosubstances . The changes found for the biochemical markers can improve endothelial function . The presented results should encourage the performance of clinical studies in cardiovascular risk patients with estrogen/statin combinations. J Chromatogr A, 2001 Apr 13, 913(1-2), 341 - 7 Determination of pyruvate and lactate in primary liver cell culture medium during hypoxia by on-line microdialysis-liquid chromatography; Wu YS et al.; A microdialysis sampling device was constructed for the measurement of pyruvate and lactate in primary liver cell culture medium during hypoxia . It was composed of a Petri dish, a dialysis membrane and two transmission tubes within a hypoxia chamber . The dialysis membrane was located in the Petri dish such that it was immersed in the culture medium . Dialysates were collected and introduced by an on-line injector to a liquid chromatographic system for analysis of pyruvate and lactate . The detection limit of this assay was 0.2-2.0 microM with acceptable intra- and inter-assay reproducibilities . In order to validate the assay, primary liver cells were incubated in the Petri dish within a hypoxia chamber in an incubator . The baseline concentrations of pyruvate and lactate in primary liver cell culture medium were 10.6+/-5.6 and 607+/-143 microM, respectively . These levels drastically changed during hypoxia and reperfusion . In conclusion, the present assay provides a sensitive, direct measurement of pyruvate and lactate in culture medium while minimizing pretreatment procedures for sample preparation. Synapse, 2001 Jul, 41(1), 65 - 70 Postsynaptic mechanism may contribute to inhibitory acetylcholine effect on GABAergic synaptic transmission in hippocampal cell cultures; Storozhuk MV et al.; The effect of acetylcholine (ACh) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections . Spontaneous IPSCs and IPSCs evoked by extracellular stimulation of a single presynaptic neuron were recorded . ACh inhibited the evoked IPSCs in most of the connections, although facilitation was also observed . Regardless of inhibitory or facilitatory effects on the evoked IPSCs, an enhanced spontaneous synaptic input to the postsynaptic neurons was usually observed . ACh-induced changes in the evoked IPSCs were usually accompanied by changes in paired pulse depression (PPD), which are thought to reflect presynaptic mechanisms of modulation . However, the time course of PPD changes did not always match that of the IPSC changes, suggesting a contribution of other, possibly postsynaptic, mechanism(s) . To analyze this possibility, effects of ACh on responses to direct application of exogenous GABA were studied . In a proportion of the neurons (40%) ACh reversibly decreased GABA responses, indicating that postsynaptic mechanisms may also contribute to the inhibitory ACh effect on GABAergic transmission . We conclude that several different modulatory mechanisms of ACh action participate in the regulation of GABAergic transmission at the level of synaptic connection of a single GABAergic neuron . J Physiol, 2001 May 15, 533(Pt 1), 215 - 26 Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture; Meissner JD et al.; The addition of cyclosporin A (500 ng ml(-1)) - an inhibitor of the Ca2+-calmodulin-regulated serine/threonine phosphatase calcineurin - to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA . In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was found . The level of enzyme activity of CS was also not affected . When the Ca2+ ionophore A23187 (4 x 10(-7) M) was added to the medium, a partial fast-to-slow transformation occurred . The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased . Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression . The expression of MHCIIa was also not influenced by cyclosporin A . Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS . The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A . When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast-to-slow transformation was induced . Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression . The nuclear translocation of the calcineurin-regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture . The effects of cyclosporin A demonstrate the involvement of calcineurin-dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore- and electrostimulation-induced fast-to-slow transformations . The data indicate a differential regulation of MHCI, of MHCII and of metabolism . Calcineurin alone is not sufficient to mediate the complete transformation. Vaccine, 2001 May 14, 19(25-26), 3444 - 50 Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains; Tree JA et al.; Different types of microcarriers were assessed for the large-scale culture of influenza virus in the Madin-Darby canine kidney (MDCK) cells . Both porous and solid carriers were examined . A higher titre of influenza A/PR8/34 virus was recovered from cultures using solid (1.3x10(9) PFU per ml) rather than porous carriers (4.0x10(8) PFU per ml) . High titres of virus (1.0x10(9) PFU per ml) were also obtained from roller bottle cultures of MDCK cells and the traditional culture technique using embryonated hens' eggs (3.9x10(9) PFU per ml) . We found that solid carriers composed of dextran with a positive charge are the most suitable carriers for the large-scale growth of influenza A virus in MDCK cells using serum-free media. Biotechnol Bioeng, 2001 Jun 20, 73(6), 522 - 9 Isolation of a recombinant antibody from cell culture supernatant: continuous annular versus batch and expanded-bed chromatography; Giovannini R et al.; Annular chromatography represents a crossflow approach to chromatographic separations, that allows the continuous separation of multicomponent mixtures . The potential of the method for continuous bioseparation has been discussed for some time, however, we demonstrate for the first time the processing of a complex feed (cell culture supernatant) taken from an actual (bio)process . Moreover, while previously published applications of annular chromatography concentrated on noninteractive (gel filtration) or nonspecific (ion exchange) chromatography, we show the possibility of continuous annular affinity chromatography . In particular, a commercially available preparative continuous annular chromatography (P-CAC) system was used to purify a recombinant antibody (human IgG(1)-kappa) from CHO cell culture supernatants by (pseudo)affinity chromatography on hydroxyapatite (HA) and rProtein A . Methods developed using small (2 mL) batch columns could be directly transferred to the P-CAC, where they yielded similar results in terms of final product quality . Yields were between 87% and 92% in the case of HA and between 77% and 82% in the case of rProtein A chromatography . DNA removal was nearly quantitative in all cases . Concomitantly, the antibody fraction of the total protein content was raised by one order of magnitude in HA and by a factor of 50 by rProtein A chromatography . In addition, a novel HA material (particle diameter -120 microm) was investigated, which was compatible with expanded-bed applications . However, the final purity of the antibody thus obtained and also the yields (<70%) were less than satisfactory . Bone, 2001 May, 28(5), 474 - 83 Tumor necrosis factor-alpha cooperates with receptor activator of nuclear factor kappaB ligand in generation of osteoclasts in stromal cell-depleted rat bone marrow cell culture; Komine M et al.; A member of the tumor necrosis factor (TNF) family, receptor activator of nuclear factor kappaB ligand (RANKL; also known as ODF, OPGL, and TRANCE), plays critical roles in osteoclast differentiation and activation in the presence of macrophage colony-stimulating factor (M-CSF) . Recently, TNF-alpha has also been shown to induce the formation of multinucleated osteoclast-like cells (MNCs) in the presence of M-CSF from mouse macrophages . We demonstrated that mononuclear preosteoclast-like cells (POCs) w