ALTEX, 2004, 21 Suppl 3, 57 - 64 Validation of cell culture models for the intestine and the blood-brain barrier and comparison of drug permeation; Bock U et al.; Cell culture models are useful tools to study the uptake of drugs across the barriers of the human body, like the intestine, the skin or the blood-brain barrier . Cell-based in vitro models not only help to reduce the number of animals used but are also much faster to perform, more cost effective and give more reproducible data than animal studies . Given the increasing number of new drugs and chemicals under development, there is an urgent need for the establishment of such in vitro models . However, the validity of such in vitro models is reflected by its ability to accurately predict the behaviour of a substance at the corresponding in vivo barrier . Here, we compare a well-established cell culture model for the intestine, based on Caco-2 colon carcinoma cells, with a primary cell culture model of the blood-brain barrier . We find that Caco-2 cells and cells of the blood-brain barrier have different barrier properties . Therefore, cells used for cell-based assays should be derived from the corresponding tissue to reflect the in vivo barrier characteristics. Neurobiol Dis, 2004 Apr, 15(3), 563 - 72 Cell death, glial protein alterations and elevated S-100 beta release in cerebellar cell cultures following mechanically induced trauma; Slemmer JE et al.; Recent studies in vivo have shown that cells of the cerebellum, and particularly Purkinje neurons (PNs), are susceptible to damage following traumatic brain injury (TBI) . To investigate more closely the effects of TBI at the cellular level, we subjected cerebellar cell cultures to injury using an in vitro model of stretch-induced mechanical trauma and found increased cell damage and neuronal loss with increasing levels of injury and time post-injury . The release of neuron-specific enolase and S-100 beta were also elevated after injury . Compared to our previous findings in hippocampal cells, S-100 beta levels were much higher in cerebellar cultures after injury, suggesting that cells from different brain regions show variable responses to mechanical trauma . Lastly, the addition of exogenous S-100 beta to uninjured cerebellar cells caused no overt change in cell viability or overall neuronal number; there were, however, fewer calbindin-positive PNs, similar to findings after stretch injury. J Neurochem, 2004 Apr, 89(2), 454 - 63 Anti-PrP antibodies block PrPSc replication in prion-infected cell cultures by accelerating PrPC degradation; Perrier V et al.; The use of anti-PrP antibodies represents one of the most promising strategies for the treatment of prion diseases . In the present study, we screened various anti-PrP antibodies with the aim of identifying those that would block PrP(Sc) replication in prion-infected cell culture . Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein, and SAF61 directed against PrP amino acid residues (144-152), not only inhibited PrP(Sc) formation in prion-infected neuroblastoma cells but also decreased the PrP(C) levels in non-infected N2a cells . In addition, treatment with both SAF34 and SAF61 antibodies decreased PrP(C) and PrP(Sc) levels in the cells synergistically . In the presence of both antibodies, our results showed that the mode of action which leads to the disappearance of PrP(Sc) in cells is directly coupled to PrP(C) degradation by reducing the half-life of the PrP(C) protein. J Neurochem, 2004 Apr, 89(2), 375 - 82 Carnosine uptake in rat choroid plexus primary cell cultures and choroid plexus whole tissue from PEPT2 null mice; Teuscher NS et al.; PEPT2 is functionally active and localized to the apical membrane of rat choroid plexus epithelial cells . However, little is known about the transport mechanisms of endogenous neuropeptides in choroid plexus, and the role of PEPT2 in this process . In the present study, we examined the uptake kinetics of carnosine in rat choroid plexus primary cell cultures and choroid plexus whole tissue from wild-type (PEPT2(+/+)) and null (PEPT2(-/-)) mice . Our results indicate that carnosine is preferentially taken up from the apical as opposed to basolateral membrane of cell monolayers, and that basolateral efflux in limited . Transepithelial flux of carnosine was not distinguishable from that of paracellular diffusion . The apical uptake of carnosine was characterized by a high affinity (K(m) = 34 microM), low capacity (V(max) = 73 pmol/mg protein/min) process, consistent with that of PEPT2 . The non-saturable component was small (K(d) = 0.063 microL/mg protein/min) and, under linear conditions, was only 3% of the total uptake . Studies in transgenic mice clearly demonstrated that PEPT2 was responsible for over 90% of carnosine's uptake in choroid plexus whole tissue . These findings elucidate the unique role of PEPT2 in regulating neuropeptide homeostasis at the blood-cerebrospinal fluid interface. Eur J Hum Genet, 2004 Jul, 12(7), 513 - 20 Features of chromosomal abnormalities in spontaneous abortion cell culture failures detected by interphase FISH analysis; Lebedev IN et al.; Cytogenetic analysis of reproductive wastage is an important stage in understanding the genetic background of early embryogenesis . The results of conventional cytogenetic studies of spontaneous abortions depend on tissue culturing and are associated with a significant cell culture failure rate . We performed interphase dual-colour FISH analysis to detect chromosomal abnormalities in noncultured cells from two different tissues-cytotrophoblast and extraembryonic mesoderm-of 60 first-trimester spontaneous abortions from which cells had failed to grow in culture . An original algorithm was proposed to optimize the interphase karyotype screening with a panel of centromere-specific DNA probes for all human chromosomes . The overall rate of numerical chromosomal abnormalities in these cells was 53% . Both typical and rare forms of karyotype imbalance were found . The observation of six cases (19%) of monosomy 7, 15, 21 and 22 in mosaic form, with a predominant normal cell line, was the most unexpected finding . Cell lines with monosomies 21 and 22 were found both in cytotrophoblast and mesoderm, while cells with monosomy 7 and 15 were confined to the cytotrophoblast . The tissue-specific compartmentalization of cell lines with autosomal monosomies provides evidence that the aneuploidy of different human chromosomes may arise during different stages of intrauterine development . The effect of aneuploidy on selection may differ, however, depending on the specific chromosome . The abortions also revealed a high frequency of intratissue chromosomal mosaicism (94%), in comparison with that detected by conventional cytogenetic analysis (29%; P<0.001) . Confined placental mosaicism was found in 25% of the embryos . The results of molecular cytogenetic analysis of these cell culture failures illustrate that the diversity and phenotypic effects of chromosomal abnormalities during the early stages of human development are underestimated. J Exp Bot, 2004 May, 55(399), 1003 - 12 Epub 2004 Mar 26. Jasmonate and ethylene signalling and their interaction are integral parts of the elicitor signalling pathway leading to beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures; Zhao J et al.; Roles of jasmonate and ethylene signalling and their interaction in yeast elicitor-induced biosynthesis of a phytoalexin, beta-thujaplicin, were investigated in Cupressus lusitanica cell cultures . Yeast elicitor, methyl jasmonate, and ethylene all induce the production of beta-thujaplicin . Elicitor also stimulates the biosynthesis of jasmonate and ethylene before the induction of beta-thujaplicin accumulation . The elicitor-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of jasmonate and ethylene biosynthesis or signal transduction . These results indicate that the jasmonate and ethylene signalling pathways are integral parts of the elicitor signal transduction leading to beta-thujaplicin accumulation . Methyl jasmonate treatment can induce ethylene production, whereas ethylene does not induce jasmonate biosynthesis; methyl jasmonate-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of ethylene biosynthesis and signalling, while blocking jasmonate biosynthesis inhibits almost all ethylene-induced beta-thujaplicin accumulation . These results indicate that the ethylene and jasmonate pathways interact in mediating beta-thujaplicin production, with the jasmonate pathway working as a main control and the ethylene pathway as a fine modulator for beta-thujaplicin accumulation . Both the ethylene and jasmonate signalling pathways can be regulated upstream by Ca(2+) . Ca(2+) influx negatively regulates ethylene production, and differentially regulates elicitor- or methyl jasmonate-stimulated ethylene production. Arch Virol, 2004 Apr, 149(4), 779 - 89 Epub 2004 Jan 05. Rubella virus P90 associates with the cytokinesis regulatory protein Citron-K kinase and the viral infection and constitutive expression of P90 protein both induce cell cycle arrest following S phase in cell culture; Atreya CD et al.; In utero infection of developing fetus by Rubella virus (RV) causes cell division inhibition of critical precursor cells in organogenesis, CNS-associated birth defects and induction of apoptosis in cell culture . The underlying mechanisms of RV-induced congenital abnormalities are not known . Here, we identified a novel interaction between RV replicase P90 protein and a cytokinesis-regulatory protein, the Citron-K kinase (CK), in a yeast two-hybrid cDNA library screen . Aberrations in cytokinesis and subsequent apoptosis do occur in specific cell types when the CK gene is knocked out or, its regulatory function is perturbed . Our analysis found that full-length P90 binds CK and in RV-infected cells P90 colocalizes with CK in the cytoplasm . Furthermore, during RV infection as well as cellular expression of P90 alone, we identified a discrete subpopulation of cells containing 4N DNA content, indicating that these cells are arrested in the cell cycle following S phase, suggesting that cellular expression of viral P90 during RV infection perturbs cytokinesis . Previous reports by others established that RV infection leads to apoptosis in cell culture . These observations together taken to the fetal organogenesis level, favor the idea that RV P90, by binding to cellular CK, invokes cell cycle aberrations resulting in the cell- and organ-specific growth inhibition and programmed cell death during RV infection in utero, which commonly is referred to as RV-induced teratogenesis. Glia, 2004 Apr 15, 46(2), 218 - 23 Microglia enhance dorsal root ganglion outgrowth in Schwann cell cultures; Hynds DL et al.; Transplantation of cellular populations to facilitate regrowth of damaged axons is a common experimental therapy for spinal cord injury . Schwann cells (SC) or microglia grafted into injury sites can promote axonal regrowth of central projections of dorsal root ganglion (DRG) sensory neurons . We sought to determine whether the addition of microglia or microglia-derived secretory products alters DRG axon regrowth upon cultures of SC . Rat DRG explants were grown on monolayers consisting of either SC, microglia, SC exposed to microglia-conditioned medium (MCM), or co-cultures with different relative concentrations of microglia . Image analysis revealed that, compared to SC alone, the extent of neurite outgrowth was significantly greater on SC-microglia co-cultures . Immunocytochemistry for extracellular matrix molecules showed that microglial cells stained positively for growth-promoting thrombospondin, whereas laminin and the inhibitory chondroitin sulfate proteoglycans (CSPGs) were localized primarily to SC . Notably, immunoreactivity for CSPGs appeared reduced in areas associated with DRG outgrowth in co-cultures and SC exposed to MCM . These results show that microglia or their secreted products can augment SC-mediated DRG regrowth in vitro, indicating that co-grafting SC with microglia provides a novel approach to augment sensory fiber regeneration after spinal cord injury . J Biol Chem, 2004 Jun 11, 279(24), 25474 - 82 Epub 2004 Mar 22. Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture; Ma Y et al.; The hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme of HCV RNA replication . We previously identified five novel residues of NS5B in a JK-1 isolate indispensable for RdRP activity in vitro (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S . (2001) Hepatology 33, 728-737) . We addressed the role of these residues in HCV RNA replication using a HCV replicon system derived from an M1LE isolate (Kishine, H., Sugiyama, K., Hijikata, M., Kato, N., Takahashi, H., Noshi, T., Nio, Y., Hosaka, M., Miyanari, Y., and Shimotohno, K . (2002) Biochem . Biophys . Res . Commun . 293, 993-999) . The five residues of NS5B in M1LE were found to be critical for HCV replication in vivo and also indispensable for RdRP activity in vitro along with purified bacterial recombinant proteins . We also found a chimeric replicon of JK-1 and M1LE in which only the NS5B sequence derived from JK-1 could not replicate in Huh-7 cells . The residues responsible for the phenomenon were mapped by several chimeric and substituted forms of NS5B M1LE and/or JK-1 isolates in the HCV RNA replicon . Two residues, amino acids 220 and 288, were critical, and two residues, amino acids 213 and 231, were important for efficient HCV replication . Mutant JK-1 NS5B harboring all four residues of M1LE was replication-competent in the chimeric replicon and was as efficient as the original M1LE replicon . By comparing the replication competence in vivo and RdRP activity in vitro with various chimeric and mutated versions of NS5B, the HCV replication ability was found to correlate well with the RdRP activity . However, heat- and dilution-sensitive NS5Bs exhibiting weaker RdRP activity in vitro were found to be replication-incompetent, suggesting that HCV replication requires RdRP activity higher than a certain critical threshold. Br J Ophthalmol, 2004 Apr, 88(4), 560 - 5 Human corneal equivalent as cell culture model for in vitro drug permeation studies; Reichl S et al.; AIMS: For the study of transcorneal in vitro permeation of ophthalmic drugs, excised animal cornea or corneal epithelial cell culture are frequently used as a replacement for the human cornea . The main purposes of this study were to reconstruct a complete human organotypic cornea equivalent, consisting of all three different cell types (epithelial, stromal, and endothelial); to test the barrier function of this bio-engineered human cornea using three different model drugs (pilocarpine hydrochloride (PHCl), befunolol hydrochloride (BHCl), and hydrocortisone (HC)); and to determine its usefulness as an in vitro model for prediction of ocular drug absorption into the human eye . METHODS: A multilayer tissue construct was created step by step in Transwell cell culture insert using SV-40 immortalised human endothelial and epithelial cells and native stromal cells (fibroblasts) . Morphology was characterised by light microscopy using routine H&E staining . Scanning electron microscopy was used to evaluate ultrastructural features . Ocular permeation of drugs across the human cornea construct was tested using modified Franz cells and compared with data obtained from excised porcine cornea and previously described porcine cornea constructs . RESULTS: and conclusion: The cornea construct exhibited typical corneal structures such as a monolayer of hexagonally shaped endothelial cells and a multilayered epithelium consisting of seven to nine cell layers with flat superficial cells . The formation of microplicae and microvilli was also confirmed . The human cornea construct showed similar permeation behaviour for all substances compared with excised porcine cornea . However, permeability (permeation coefficients K(p)) of the human cornea equivalent (PHCl 13.4*10(-6) (SD 3.01*10(-6)); BHCl 9.88*10(-6) (SD 1.79*10(-6)); HC 5.41*10(-6) (SD 0.40*10(-6)) cm/s) was about 1.6-1.8 fold higher than excised porcine cornea . Compared with data from the porcine cornea construct the cultivated human equivalent showed a decreased permeability . The reconstructed human cornea could be appropriate to predict drug absorption into the human eye. Arch Facial Plast Surg, 2004 Mar-Apr, 6(2), 88 - 93 The effect of silicone gel on basic fibroblast growth factor levels in fibroblast cell culture; Hanasono MM et al.; BACKGROUND: Topical silicone gel has shown promise in the treatment of hypertrophic and keloid scars . However, its mechanism of action remains undetermined . OBJECTIVE: To investigate whether the presence of silicone alters the secretion of basic fibroblast growth factor (bFGF), a key cytokine involved in the scar formation process . DESIGN: Serum-free fibroblast cell cultures were established from normal, keloid, and fetal skin, which heals without scarring, and exposed to silicone gel . Serial cell counts were performed, and supernatants were collected for bFGF quantification by enzyme-linked immunosorbent assay at 4, 24, 72, and 120 hours . RESULTS: Growth curves were similar and no statistically significant differences in population doubling times were observed between treated and untreated specimens . Statistically significant differences in bFGF levels between treated and untreated normal fibroblasts were observed at 24, 72, and 120 hours after cell culture initiation . Differences in bFGF levels between treated and untreated fetal fibroblasts that approached statistical significance were observed at 72 and 120 hours . CONCLUSIONS: These results suggest that silicone gel is responsible for increased bFGF levels in normal and fetal dermal fibroblasts . We postulate that silicone gel treats and prevents hypertrophic scar tissue, which contains histologically normal fibroblasts, by modulating expression of growth factors such as bFGF . Our data support the hypothesis that substances that favorably influence wound healing do so by correcting a deficiency or overabundance of the growth factors that orchestrate the tissue repair process. Clin Orthop, 2004 Feb, (419), 238 - 44 Human intervertebral disc cell culture for disc disorders; Stern S et al.; Repair of degenerated intervertebral discs by engineered tissue is a clinical challenge in spinal surgery . Prerequisites are cultivation of intervertebral disc cells and determination of their biologic properties . The influence of disc damage in different spinal disorders on the outcome of disc cell cultures has not been discussed previously . This study showed the feasibility of cultivation of cells from damaged human intervertebral discs and the dependence of cellular culture properties on the underlying disc disorder . Human intervertebral disc cells were isolated from disc tissue obtained during surgical procedures for scoliosis, osteochondrosis, and disc herniation . After proliferation in monolayer culture, cells were embedded in a mixed matrix composed of fibrin and hyaluronic acid . Deoxyribonucleic acid content, hydroxyproline content, and proteoglycan synthesis were determined on Days 7, 14, and 21 . In a three-dimensional environment only cells obtained from scoliotic and osteochondrotic discs showed significant deoxyribonucleic acid and proteoglycan synthesis . However, hydroxyproline content increased only in cells from scoliotic discs . The results of this study show that the formation of extracellular matrix components under three-dimensional culture conditions is dependent on the nature of intervertebral disc damage of the tissue processed. J Cell Sci, 2004 Mar 15, 117(Pt 8), 1457 - 68 MyoD enhances BMP7-induced osteogenic differentiation of myogenic cell cultures; Komaki M et al.; The muscle-specific, basic helix-loop-helix transcription factor MyoD can induce cells from other mesenchymal lineages to express a skeletal muscle phenotype . Interestingly, MyoD is initially upregulated in myogenic cells incubated with bone morphogenetic proteins (BMPs), a treatment that induces osteogenic differentiation, suggesting that MyoD has a role in BMP-induced osteogenesis of myogenic cells . This possibility is supported by our observations that muscle satellite cells derived from adult MyoD(-/-) mice show severely impaired osteogenic induction by BMP-7 (osteogenic protein 1; OP-1) as indicated by the decreased gene expression of the bone markers alkaline phosphatase, osteocalcin, Runx2/Cbfa1, and Osterix . Ectopic expression of MyoD increased alkaline phosphatase activity and Osterix mRNA expression in response to BMP treatment . Similarly, ectopic expression of MyoD in the pluripotent mesenchymal cell line C3H10T1/2 increased alkaline phosphatase activity induced by BMP-7 . Transcription assays showed that transfection with a MyoD-expression vector, but not other myogenic basic helix-loop-helix transcription factors (Myf5, myogenin) increased Runx2/Cbfa1 transactivation of a reporter gene construct containing either six OSE sequences in tandem or a single OSE site . This effect was enhanced by BMP treatment . These studies, therefore, demonstrate that the muscle transcription factor MyoD is required for efficient BMP-induced osteogenesis of myogenic cells and indicate that MyoD might exert its effects through co-operative interactions with Runx2/Cbfa1. Biomaterials, 2004 Aug, 25(17), 3621 - 9 New modified polyetheretherketone membrane for liver cell culture in biohybrid systems: adhesion and specific functions of isolated hepatocytes; De Bartolo L et al.; There has been growing interest in innovative materials with physico-chemical properties that provide improved blood/cell compatibility . We propose new polymeric membranes made of modified polyetheretherketone (PEEK-WC) as materials with potential for use in biohybrid devices . PEEK-WC exhibits high chemical, thermal stability and mechanical resistance . Owing to its lack of crystallinity this polymer can be used for preparing membranes with cheap and flexible methods . We compared the properties of PEEK-WC membranes to polyurethane membranes prepared using the same phase inverse technique and commercial membranes . The physico-chemical properties of the membranes were characterised by contact angle measurements . The different parameters acid (gamma+), base (gamma-) and Lifshitz-van der Waals (gammaLW) of the surface free energy were calculated according to Good-van Oss's model . We evaluated the cytocompatibility of PEEK-WC membranes by culturing hepatocytes isolated from rat liver . Cell adhesion and metabolic behaviour in terms of ammonia elimination, urea synthesis and protein synthesis were evaluated during the first days of culture . Liver cells adhered and formed three-dimensional aggregates on the most tested membranes . PEEK-WC membranes promoted hepatocyte adhesion most effectively . Urea synthesis, ammonia elimination and protein synthesis improved significantly when cells adhered to PEEK-WC membrane . The considerable metabolic activities of cells cultured on this membrane confirmed the good structural and physico-chemical properties of the PEEK-WC membrane that could be a promising biomaterial for cell culture in biohybrid devices. Anticancer Res, 2004 Jan-Feb, 24(1), 139 - 44 Beta-galactosidase and alpha-mannosidase inhibit formation of multicellular nodules in breast cancer cell cultures; Arcaro KF et al.; In response to an estrogen, confluent monolayers of MCF-7 cell cultures develop multi-cellular nodules, termed foci . Post-confluent development of foci occurs with physiologic levels of 17beta-estradiol and are inhibited by various anti-estrogens acting through either the estrogen or aryl hydrocarbon receptors . In the present paper we report that disruption of the terminal sugars on membrane receptors results in inhibition of foci . Treatment with 0.013-0.05 units/ml of beta-galactosidase completely inhibited the development of foci while leaving the monolayer of cells intact . Trials with alpha-mannosidase resulted in a similar but less potent inhibition of foci . Lectin-fluorescent conjugates, RCA (Ricinus communis agglutinin), and ConA (Canavalia ensiformis agglutinin) were used to identify membrane surface carbohydrates on MCF-7 cells . Binding of the RCA-fluorescent conjugate was inhibited by co-treatment with galactose or lactose . Binding of ConA-fluorescent conjugate was significantly inhibited by mannose and n-acetyl-glucosamine . This is the first report of inhibition of foci development in MCF-7 cell cultures by disruption of surface carbohydrates on membrane receptors. Zhonghua Zheng Xing Wai Ke Za Zhi, 2003 Nov, 19(6), 450 - 1 {The fibroblast primary cell culture by the split-thickness skin slide technique}; Zhao YM et al.; OBJECTIVE: To acquire lots of cell to culture during the primary cell culture . METHOD: We take the split-thickness skin slide technique to acquire the dissociated fibroblast cell in two big-ear rats . RESULTS: The cell number is above 10(6) from 1 cm x 2 cm split-thickness skin slide and the technique is simple, economic, effectve . CONCLUSION: We think this way is better than other methods, and should be adopted in the primary cell culture, especially in fibroblast transplantation by injection. J Alzheimers Dis, 2004 Feb, 6(1), 31 - 43 A novel highly pathogenic Alzheimer presenilin-1 mutation in codon 117 (Pro117Ser): Comparison of clinical, neuropathological and cell culture phenotypes of Pro117Leu and Pro117Ser mutations; Dowjat WK et al.; A novel presenilin-1 (PS1) mutation (P117S) in an American pedigree is described . We compare clinical, neuropathological and cell culture phenotypes produced by this mutation with another codon 117 mutation that was earlier discovered by our group in a Polish kindred . Both mutations are associated with an unusually severe Alzheimer disease (AD) phenotype, with the onset starting before the third decade of life, rapid disease progression and acute presentation of clinical symptoms . The severity of clinical phenotype was closely correlated with the abundance of pathology: massive deposition of Abeta42 in plaques, severe neurofibrillary degeneration and neuronal loss . When overexpressed in mouse neuroblastoma N2a cells, both mutations caused loss of an ability to promote neurite outgrowth and produced an increase in the ratio of secreted Abeta42/40 amyloid peptides . In stably transfected N2a cell lines only mutant proteins were endoproteolytically cleaved indicating some dependability of this process on the presence of mutation . Taken together, our results show that clinical and cell culture phenotypes produced by these 2 codon 117 mutations are closely related suggesting that the pathogenic action of PS1 may involve effect on neurite outgrowth and endoproteolytic cleavage of the full-length protein . Given the high potency in vivo and in vitro of both codon 117 mutations, this site of PS1 must be particularly important for its normal/pathogenic function. Mol Biotechnol, 2004 Mar, 26(3), 193 - 206 CFTR transgene expression in primary DeltaF508 epithelial cell cultures from human nasal polyps following gene transfer with cationic phosphonolipids; Montier T et al.; Cystic fibrosis (CF) is the most common autosomal lethal recessive disorder in the Caucasian population . The major cause of mortality is lung disease, owing to the failure of a functional protein from the cystic fibrosis transmembrane conductance regulator (CFTR) gene . Today, even though the knowledge about the CFTR genomic is extensive, no efficient treatment has been developed yet . In this context, gene therapy represents a potential important advance on condition that it could develop efficient and safe transfection agents . Even though viral vectors have been used in most clinical trials owing to their high transfection efficiency, random integration and immunogenicity are still critical side effects . Consequently, all of these drawbacks brought forth the development of nonviral transfection systems . Although they engender few toxicity and immunogenicity problems, their low transfection efficiency is a hurdle that must be overcome . Over the past decade, we have developed an original family of monocationic lipids, cationic phosphonolipids, whose efficiency has been previously demonstrated both in vitro and in vivo . In this report, we observe that a new cationic phosphonolipid (KLN 30) can lead to the restoration of the CFTR protein following the ex vivo transfection of epithelial cells issuing from a F508 homozygous patient . The transgene expression and the cytotoxicity correlate with the charge ratio of the lipoplex . A kinetic study was performed, and a luminescent signal was detected until 35 d after transfection. Biomacromolecules, 2004 Mar-Apr, 5(2), 505 - 10 Temperature-responsive cell culture surfaces enable "on-off" affinity control between cell integrins and RGDS ligands; Ebara M et al.; In this study, specific interactions between immobilized RGDS (Arg-Gly-Asp-Ser) cell adhesion peptides and cell integrin receptors located on cell membranes are controlled in vitro using stimuli-responsive polymer surface chemistry . Temperature-responsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) (P(IPAAm-co-CIPAAm)) copolymer grafted onto tissue culture grade polystyrene (TCPS) dishes permits RGDS immobilization . These surfaces facilitate the spreading of human umbilical vein endothelial cells (HUVECs) without serum depending on RGDS surface content at 37 degrees C (above the lower critical solution temperature, LCST, of the copolymer) . Moreover, cells spread on RGDS-immobilized surfaces at 37 degrees C detach spontaneously by lowering culture temperature below the LCST as hydrated grafted copolymer chains dissociate immobilized RGDS from cell integrins . These cell lifting behaviors upon hydration are similar to results using soluble RGDS in culture as a competitive substitution for immobilized ligands . Binding of cell integrins to immobilized RGDS on cell culture substrates can be reversed spontaneously using mild environmental stimulation, such as temperature, without enzymatic or chemical treatment . These findings are important for control of specific interactions between proteins and cells, and subsequent "on-off" regulation of their function . Furthermore, the method allows serum-free cell culture and trypsin-free cell harvest, essentially removing mammalian-sourced components from the culture process. Oligonucleotides, 2003, 13(4), 193 - 205 Water-soluble polycationic dendrimers with a phosphoramidothioate backbone: preliminary studies of cytotoxicity and oligonucleotide/plasmid delivery in human cell culture; Maszewska M et al.; A series of water-soluble polycationic dendrimers with a phosphoramidothioate backbone (P-dendrimers) was studied in human cell culture . Preliminary studies have shown that P-dendrimers of series 1 and 2, possessing N,N-diethyl-ethylenediamine hydrochloride functions at the surface, show rather moderate cytotoxicity toward HeLa, HEK 293, and HUVEC cells in a standard MTT assay in serum-containing medium, generally lower than lipofectin . The experiments of cellular uptake have shown the necessity for the presence of serum for transfection with P-dendrimers of series 1 and 2 . These compounds efficiently delivered fluorescein-labeled oligodeoxyribonucleotide into HeLa cells in serum-containing medium, but they failed to do so in HUVEC cell culture . The dendrimers were found to be successful mediators of transfection of the HeLa cells with a DNA plasmid containing the functional gene of enhanced green fluorescent protein (EGFP). Neurochem Res, 2004 Jan, 29(1), 127 - 34 Regulation by insulin and insulin-like growth factor of 2-deoxyglucose uptake in primary ependymal cell cultures; Verleysdonk S et al.; Ependymal cells have been reported to express the facilitative glucose carriers GLUT1, GLUT2, and GLUT4, as well as glucokinase . They are therefore speculated to be part of the cerebral glucose sensing system and may also respond to insulin with alterations in their glucose uptake rate . A cell culture model was employed to study the functional status of ependymal insulin-regulated glucose uptake in vitro . Insulin increased the uptake of the model substrate 2-deoxyglucose (2-DG) dependent on the insulin concentration . This was due to a near doubling of the maximal 2-DG uptake rate . Insulin-like growth factor (IGF-1) was at least 10 times more potent than insulin in stimulating the rate of ependymal 2-DG uptake, suggesting that IGF-1, rather than insulin, is the physiological agonist regulating glucose transport in ependymal cells . The predominant glucose transporter in ependymal cell cultures was found to be GLUT1, which is apparently regulated by IGF-1 in ependymal cells. Mol Genet Genomics, 2004 Apr, 271(3), 339 - 46 Epub 2004 Feb 17. Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweet potato and its expression in response to stress; Park SY et al.; A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1, was isolated from cell cultures of sweet potato ( Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated . swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32 . The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform . RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweet potato . The level of swAPX1 RNA progressively increased during cell growth in suspension cultures . In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis . swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 microM), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 microM) or exposure to high temperature (37 degrees C) . In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen ( Pectobacterium chrysanthemi) . These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses. Pflugers Arch, 2004 Jul, 448(4), 462 - 8 Epub 2004 Feb 17. Quantitative phase microscopy: a new tool for measurement of cell culture growth and confluency in situ; Curl CL et al.; Quantitative phase microscopy (QPM) is a recently developed computational approach that provides quantitative phase measurements of specimen images obtained under bright-field conditions without phase- or interference-contrast optics . To perform QPM, an in-focus bright-field image is acquired, together with one positive and one negative de-focus image . An algorithm is then applied to produce a specimen phase map . In this investigation we demonstrate that manipulation of the phase map intensity histogram using novel, non-subjective thresholding and segmentation methods provides enhanced delineation of cells in culture . QPM was utilised to measure the growth behaviour of cultured airway smooth muscle cells over a 92-h period . There was a high degree of correlation between parallel QPM-derived confluency measurements and haemocytometry-derived counts of airway smooth muscle cells over this time period . Using QPM, translucent cells can be visualised with improved cell boundary definition allowing precise and reproducible measurements of cell culture confluency . Quantitative phase imaging provides a rapid, optically simple and non-destructive approach for measurement of cellular morphology . Further development of the QPM-based analysis methodology has the potential to provide even more refined measures of cellular growth. Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2317 - 22 Cells adapted to high NaCl have many DNA breaks and impaired DNA repair both in cell culture and in vivo; Dmitrieva NI et al.; Acute exposure of cells in culture to high NaCl damages DNA and impairs its repair . However, after several hours of cell cycle arrest, cells multiply in the hypertonic medium . Here, we show that, although adapted cells proliferate rapidly and do not become apoptotic, they nevertheless contain numerous DNA breaks, which do not elicit a DNA damage response . Thus, in adapted cells, Mre11 exonuclease is mainly present in the cytoplasm, rather than nucleus, and histone H2AX and chk1 are not phosphorylated, as they normally would be in response to DNA damage . Also, the adapted cells are deficient in repair of luciferase reporter plasmids damaged by UV irradiation . On the other hand, the DNA damage response activates rapidly when the level of NaCl is reduced . Then, Mre11 moves into the nucleus, and H2AX and chk1 become phosphorylated . Renal inner medullary cells in vivo are normally exposed to a variable, but always high, level of NaCl . As with adapted cells in culture, inner medullary cells in normal mice exhibit numerous DNA breaks . These DNA breaks are rapidly repaired when the NaCl level is decreased by injection of the diuretic furosemide . Moreover, repair of DNA breaks induced by ionizing radiation is inhibited in the inner medulla . Histone H2AX does not become phosphorylated, and repair synthesis is not detectable in response to total body irradiation unless NaCl is lowered by furosemide . Thus, both in cell culture and in vivo, although cells adapt to high NaCl, their DNA is damaged and its repair is inhibited. Biotechnol Appl Biochem, 2004 Dec, 40(Pt 3), 261 - 9 Detection and quantification of the human IgG4 half-molecule, HL, from unpurified cell-culture supernatants; Deng L et al.; The presence and absence of inter-heavy-chain disulphide linkages contribute to the existence of the tetrameric (H(2)L(2)) and half (HL) human IgG molecules, respectively {Schuurman, Perdok, Gorter and Aalberse (2001) Mol . Immunol . 38, 1-8} . Reduced effector response in the human IgG4 subclass presents an alternative therapeutic platform in a monoclonal-antibody (mAb) development program . During the initial cell-selection stage, titres of the recombinant human antibody present in crude cell-culture supernatants are determined by ELISA, a technique requiring nanogram quantities of mAb . In the case of an IgG4 antibody, this material is represented mainly by the combination of the tetrameric (H(2)L(2)) and dimeric (HL) forms of the antibody . The determination of concentrations or ratios of tetramer and dimer usually requires at least one chromatographic purification step, and thus frequently this is evaluated later in the mAb development process when the number of potential clones has been reduced . In the present paper we describe a Western-blot-based method that detects and quantifies IgG4 half-molecules, HL, from crude cell-culture supernatants without purification so that H(2)L(2)/HL ratios can be included as a part of early clonal evaluation along with the screening of mAb titres . This method was demonstrated (1) to have a linear HL detection range of 0.5-10 ng, (2) to require microlitre volumes of culture and (3) to react specifically with human IgG4 produced from hybridoma and Chinese-hamster ovary cell cultures . Moreover, this protocol is applicable to evaluate and monitor potential H(2)L(2)/HL variations as a result of changes during the process-development stage of a mAb development program. Acta Otolaryngol, 2004 Jan, 124(1), 30 - 5 All-trans retinoic acid induces mucociliary differentiation in a human cholesteatoma epithelial cell culture; Choi JY et al.; OBJECTIVES: Retinoic acid (RA) can prevent keratin formation and induce mucous differentiation in epithelia . In this study, we attempted to induce keratinizing squamous epithelium from human cholesteatoma epithelial (HCE) cells using an air-liquid interface (ALI) technique . We also examined the effect of RA on the phenotype of keratinizing HCE cells . MATERIAL AND METHODS: HCE cells were cultured in RA-free defined media at an ALI or in a submerged state . We examined the morphological differences between ALI and submerged cultures, and histologically investigated the changes of phenotype after RA treatment . We also determined the effect of RA on the mRNA expressions of the cornifin-alpha and mucin genes as indicators of squamous and mucous differentiation, respectively . RESULTS: Using an ALI technique, we were able to differentiate HCE cells into a keratinizing squamous epithelium . When we treated the keratinizing HCE cells with RA, the morphological phenotype progressively changed into mucociliary epithelium . In addition, the expression of cornifin-alpha mRNA was suppressed, and the expressions of mucin gene 5AC (MUC5AC) and MUC5B mRNA increased progressively with RA treatment . CONCLUSION: We successfully developed a culturing system for keratinizing differentiation of HCE cells using the ALI technique in a defined medium . Our study also clearly showed that RA treatment led to mucociliary differentiation of HCE cells. J Anim Sci, 2004 Feb, 82(2), 429 - 37 Recruitment and differentiation of intramuscular preadipocytes in stromal-vascular cell cultures derived from neonatal pig semitendinosus muscles; Hausman GJ et al.; The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte recruitment and expression of CCAAT/enhancing binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) proteins in stromal-vascular (SV) cell cultures derived from neonatal subcutaneous adipose tissue and semitendinosus muscles . One adipose tissue SV cell culture and one semitendinosus muscle SV cell culture were established from each of six young pigs (5 to 7 d of age) . Conventional SV cell-culture procedures were used to digest adipose and muscle tissue and to harvest and culture adipose and muscle SV cells . Muscles were digested after the removal of all visible connective tissue from the excised muscle . One hour after seeding, muscle SV cell cultures were rinsed and refed new media to remove debris and insoluble muscle protein . The SV cell cultures were double-stained for lipid and the AD-3 antibody, a preadipocyte marker, at 1, 3, and 6 d and were double-stained for lipid and C/EBPalpha or PPARgamma at d 6 . Preadipocytes were randomly distributed and not clustered after 1 d in muscle and adipose SV cultures . Regardless of treatment, relative and absolute fat cell numbers were lower (P < 0.05) in muscle than in adipose-SV cell cultures . The DEX treatments produced similar magnitudes of increase in relative and absolute preadipocytes and adipocytes in muscle- and adipose-SV cultures . Several extracellular matrix substrata had no influence on adipogenesis in muscle-SV cell cultures . These studies indicate that muscle-SV cultures are characterized by a low number of adipocytes under basal conditions and a low number of glucocorticoid-responsive preadipocytes. Proc Natl Acad Sci U S A, 2004 Mar 2, 101(9), 3202 - 7 Epub 2004 Feb 18. Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex; Suzuki M et al.; Dehydroepiandrosterone (DHEA) is a neurosteroid with potential effects on neurogenesis and neuronal survival in humans . However, most studies on DHEA have been performed in rodents, and there is little direct evidence for biological effects on the human nervous system . Furthermore, the mechanism of its action is unknown . Here, we show that DHEA significantly increased the growth rates of human neural stem cells derived from the fetal cortex and grown with both epidermal growth factor (EGF) and leukemia inhibitory factor (LIF) . However, it had no effect on cultures grown in either factor alone, suggesting a specific action on the EGF/LIF-responsive cell . Precursors of DHEA such as pregnenolone or six of its major metabolites, had no significant effect on proliferation rates . DHEA did not alter the small number (<3%) of newly formed neuroblasts or the large number (>95%) of nestin-positive precursors . However, the number of glial fibrillary acidic protein-positive cells, its mRNA, and protein were significantly increased by DHEA . We found both N-methyl-d-aspartate and sigma 1 antagonists, but not GABA antagonists, could completely eliminate the effects of DHEA on stem cell proliferation . Finally we asked whether the EGF/LIF/DHEA-responsive stem cells had an increased potential for neurogenesis and found a 29% increase in neuronal production when compared to cultures grown in EGF/LIF alone . Together these data suggest that DHEA is involved in the maintenance and division of human neural stem cells . Given the wide availability of this neurosteroid, this finding has important implications for future use. Tree Physiol, 1990 Sep, 6(3), 317 - 27 Selection and physiology of cell cultures of Douglas-fir grown under conditions of water stress; Leustek T et al.; Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) cell cultures sampled 3, 6, or 9 days after subculture in nutrient medium were able to survive subsequent subculture in a medium containing 15% polyethylene glycol (PEG) (M(r) 6000-8000) (-1.21 MPa), whereas cell sampled 12 or 16 days after subculture in nutrient medium became senescent when transferred to a medium containing 15% PEG . Cells sampled after subculture for 3, 6, or 9 days in nutrient medium had lower fresh weight/dry weight ratios, lower osmotic potentials, smaller cell diameters, and higher turgor pressures than cells sampled after 12 or 16 days subculture in nutrient medium . Cells surviving subculture to a medium containing 15% PEG did not increase in dry weight for 5 weeks even though the medium was exchanged every 7 days . After 5 weeks, however, dry weight growth resumed and reached 75% of the level attained by control cells grown on PEG-free medium . Long-term growth on a medium containing 15% PEG (PEG-selected cells) could only be sustained if the medium was supplemented with 30 mM glutamine . The PEG-selected cells grew in small clusters, were isodiametric, and had chlorophyll contents 50% higher than unselected cells . The PEG-selected cells also showed lowered cellular osmotic potentials, presumably due to osmoregulation . Turgor pressures of PEG-selected cells were greater than or equal to those of unselected cells. Placenta, 2004 Feb-Mar, 25(2-3), 153 - 65 A primary cell culture system for human cytotrophoblasts of proximal cytotrophoblast cell columns enabling in vitro acquisition of the extra-villous phenotype; Nagamatsu T et al.; Cytotrophoblast (CT) differentiation into the extra-villous phenotype is a crucial process in initiating their invasion into the decidua and thereby developing the placenta . However, how CTs differentiate into extra-villous CTs (EVCTs) is not fully elucidated . To address this, a suitable culture model for CTs has been long-sought . But this has been hampered by annoying problems such as; cell aggregation, in vitro syncytialization, low plating efficiency, etc . The aim of this study is to develop a culture system in which CTs differentiate into EVCTs . CTs were isolated from the first trimester placenta using density gradient separation and immuno-depletion using anti-CD9 antibody to remove contaminating fibroblasts and EVCTs . The resultant isolated CTs were found to have the character similar to poorly differentiated CTs comprising proximal cytotrophoblastic cell columns as confirmed by immunocytochemical and flowcytometric analyses . When cultured on type 4 collagen-coated plates in culture media containing low calcium concentration, CTs neither aggregated nor syncytialized, remaining mononuclear and monolayer state . Interestingly, cultured CTs gradually upregulated integrin alpha1, CD9, and human leukocyte antigen (HLA)-G; the known markers specific for EVCTs invading into the decidua diffusely . Hence, the CT culture system provides a sophisticated experimental model in which highly purified CTs acquire the extra-villous phenotype without syncytialization. J Reprod Dev, 2003 Feb, 49(1), 45 - 53 Matrix-metalloproteinases-2 and -9 production in bovine endometrial cell culture; Hashizume K et al.; In vitro cell culture is a convenient tool for studying cellular mechanisms . In the present study, production of matrix-metalloproteinases (MMPs) in bovine endometrial (containing both epithelial and stromal cells) monolayer cells was examined . Blastocysts attached to the endometrial cells in a monolayer culture were examined for their effects on MMP-2 production . Initial attachment of blastocysts to the monolayer inhibited MMP-2 production by endometrial cells . But once trophoblast cells began to migrate into the endometrial cell layer, MMP-2 production increased, and at the same time MMP-9 production also became evident in the medium . In order to understand how blastocysts affected MMP-2 production, we examined the effect of progesterone, estradiol, insulin-like growth factors (IGFs), tumor necrosis factors (TNFs), and interferon-tau (IFN-tau) supplementation . It was IFN-tau that inhibited the production of MMP-2 . In addition, progesterone at a lower dose appeared to inhibit MMP-2 production . Both TNF-alpha and TNF-beta strongly stimulated the production of MMP-2 and MMP-9, whereas IGFs had no effect . Based on these findings, it appears that conceptus has the capacity to inhibit MMP activity. Biophys Chem, 2004 Feb 15, 107(3), 221 - 7 Ligand trapping in epithelial layers and cell cultures; Berezhkovskii AM et al.; We analyze a stochastic model that describes receptor-mediated ligand trapping in epithelial layers and cell culture assays . In both cases, the problem is reduced to diffusion of a Brownian particle between the partially absorbing and reflective surfaces . We derive an analytical expression for the spatial distribution of the trapping points and identify the domains of applicability of the two limiting regimes . We conclude that a thin layer approximation is applicable for ligand trapping in epithelial layers while a typical cell culture experiment is appropriately described within an infinite layer approximation. Transplantation, 2004 Feb 15, 77(3), 379 - 85 Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface; Nishida K et al.; BACKGROUND: Limbal stem-cell deficiency by ocular trauma or diseases causes corneal opacification and visual loss . Recent attempts have been made to fabricate corneal epithelial graft constructs, but the technology is still evolving . We have developed a novel cell-sheet manipulation technology using temperature-responsive culture surfaces to generate functional, cultivated corneal epithelial cell sheet grafts . METHODS: Human or rabbit limbal stem cells were cocultured with mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes at 37 degrees C . Cell sheets were harvested from the dishes after 2 weeks by reducing temperature to 20 degrees C . Histologic analyses, immunoblotting, and colony-forming assay were performed to characterize the cell sheets . Autologous transplantation was undertaken to reconstruct the corneal surfaces of rabbits with experimentally induced limbal stem cell deficiencies . RESULTS: Multilayered corneal epithelial sheets were harvested intact simply by reducing the temperature, without the use of proteases . Cell-cell junctions and extracellular matrix on the basal side of the sheet, critical to sheet integrity and function, remained intact . A viable population of corneal progenitor cells, close in number to that originally seeded, was found in the sheets . Harvested sheets were easily manipulated, transplantable without any carriers, and readily adhesive to corneal stroma so that suturing was not required . Corneal surface reconstruction in rabbits was highly successful . CONCLUSIONS: Cell sheet engineering technology allows us to create intact, transplantable corneal epithelial cell sheets that retain stem cells from limbal stem cells expanded ex vivo . Our research indicates highly promising clinical capabilities for our bioengineered corneal epithelial sheet. Parasitol Res, 2004 Apr, 92(6), 453 - 8 Epub 2004 Feb 12. Effects of toltrazuril and ponazuril on the fine structure and multiplication of tachyzoites of the NC-1 strain of Neospora caninum (a synonym of Hammondia heydorni) in cell cultures; Darius AK et al.; Rhesus monkey kidney cell cultures were used to propagate tachyzoites of the NC-1 strain of Neospora caninum (syn . Hammondia heydorni) . The infected cell cultures were incubated for 4-12 h in media containing 0, 1, 10 or 100 microg/ml of either toltrazuril or ponazuril . The effects were studied by light and electron microscopy . Drug dosages of at least 30 microg/ml were needed to eliminate the parasites . Ponazuril was found (with respect to the reduction of the number of parasites) to be less effective at dosages of 30 microg/ml compared to toltrazuril . However, the damage to the tachyzoites being incubated in 30 microg toltrazuril or ponazuril seen by electron microscopy was so significant that it was surely lethal . The initial damage occurred within the apicoplast and the tubular mitochondrion in all cases,thus destroying two of the most important cell organelles. Mar Biotechnol (NY), 2001 Jun, 3(Supplement 1), S196 - 202 Cell cultures and retroviral particles from a tumor of a moray eel; Buck C et al.; Until recently, fish cell culture primarily has been useful only in the propagation and study of epidemic viruses significant to the fishing industry . Such fish cell lines derived were developed by appropriating classical techniques of mammalian cell culture, with serum as the major growth supplement . Using an approach in which culture medium is formulated in a cell-type-specific manner with minimal serum and a variety of synergistic supplements, several fish cell lines have been derived that may serve multiple uses . We established cell lines from a potentially tumorous skin lesion of a green moray eel (Gymnothorax funebris) and control tissues, and identified putative retroviral particles in the medium from the tumor cells that are not present in medium from cultures of normal cells from the same eel . The relationship between the virus and the cause of the tumor is not clear, but the genomic structure of this virus should provide useful information in understanding the evolution of retroviruses in general. Biochem Biophys Res Commun, 2004 Mar 5, 315(2), 517 - 24 Immunocytochemical localization and expression of heme oxygenase-1 in primary astroglial cell cultures during differentiation: effect of glutamate; Li Volti G et al.; Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide (CO), and biliverdin . We investigated subcellular localization of HO-1 using confocal laser scanning microscopy (CLSM) and the expression by Western blot in primary astroglial cells during differentiation and after exposure to glutamate (100microM) . CLSM analysis of immunostained HO-1 in cultured astroglial cells during differentiation showed an increase of fluorescence between 7 and 14 days and a decrease between 14 and 21, although HO-1 peaked at 14 days it remained at high levels . The distribution of HO-1 protein undergoes modification in the various cellular compartments . Furthermore, localization of the protein in untreated astrocytes at 7 days appeared prevalently localized in the cytosol and in the perinuclear region . In contrast, at 14 and 21 days, fluorescence detection suggests that HO-1 was present also in the nucleus, and in the nucleoli . Fluorescence intensity significantly increased in glutamate-treated astrocytes during all development stages and the protein appeared in the cytosol, in the nucleus and in the nucleoli . The involvement of AMPA/Ka receptors was studied in glutamate-treated astroglial cells at 14 days by the preincubation of the cells with GYKI 52466, a specific receptor inhibitor, of AMPA/Ka receptor demonstrating the involvement of these receptors . Western blot analysis of HO-1 confirmed the CLSM results . Our results demonstrate that changes in HO-1 protein expression and localization in primary cultured astroglial cells may be part of the underlying mechanisms involved in brain development as well as in neurodegenerative diseases. Biotechnol Prog, 2004 Jan-Feb, 20(1), 338 - 45 The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors; Sin A et al.; Whole animal testing is an essential part in evaluating the toxicological and pharmacological profiles of chemicals and pharmaceuticals, but these experiments are expensive and cumbersome . A cell culture analog (CCA) system, when used in conjunction with a physiologically based pharmacokinetic (PBPK) model, provides an in vitro supplement to animal studies and the possibility of a human surrogate for predicting human response in clinical trials . A PBPK model mathematically simulates animal metabolism by modeling the absorption, distribution, metabolism, and elimination kinetics of a chemical in interconnected tissue compartments . A CCA uses mammalian cells cultured in interconnected chambers to physically represent the corresponding PBPK . These compartments are connected by recirculating tissue culture medium that acts as a blood surrogate . The purpose of this article is to describe the design and basic operation of the microscale manifestation of such a system . Microscale CCAs offer the potential for inexpensive, relatively high throughput evaluation of chemicals while minimizing demand for reagents and cells . Using microfabrication technology, a three-chamber ("lung"-"liver"-"other") microscale cell culture analog (microCCA) device was fabricated on a 1 in . (2.54 cm) square silicon chip . With a design flow rate of 1.76 microL/min, this microCCA device achieves approximate physiological liquid-to-cell ratio and hydrodynamic shear stress while replicating the liquid residence time parameters in the PBPK model . A dissolved oxygen sensor based on collision quenching of a fluorescent ruthenium complex by oxygen molecules was integrated into the system, demonstrating the potential to integrate real-time sensors into such devices. Biotechnol Prog, 2004 Jan-Feb, 20(1), 316 - 23 Development of a microscale cell culture analog to probe naphthalene toxicity; Viravaidya K et al.; Prediction of human response to drugs or chemicals is difficult as a result of the complexity of living organisms . We describe an in vitro model that can realistically and inexpensively study the adsorption, distribution, metabolism, elimination, and potential toxicity (ADMET) of chemicals . A microscale cell culture analog (microCCA) is a physical replica of the physiologically based pharmacokinetics (PBPK) model . Such a microfabricated device consists of a fluidic network of channels to mimic the circulatory system and chambers containing cultured mammalian cells representing key functions of animal "organ" systems . This paper describes the application of a two-cell system, four-chamber microCCA ("lung"-"liver"-"other tissue"-"fat") device for proof-of-concept study using naphthalene as a model toxicant . Naphthalene is converted into reactive metabolites (i.e., 1,2-naphthalenediol and 1,2-naphthoquinone) in the "liver" compartment, which then circulate to the "lung" depleting glutathione (GSH) in lung cells . Such microfabricated in vitro devices are potential human surrogates for testing chemicals and pharmaceutics for toxicity and efficacy. ASAIO J, 2004 Jan-Feb, 50(1), 9 - 14 Effects of basic fibroblast growth factor and transforming growth factor-beta on maturation of human pediatric aortic cell culture for tissue engineering of cardiovascular structures; Fu P et al.; Optimal in vitro conditions are necessary for the development of a strong, well structured, and functional tissue engineered cardiovascular structure eventually designed for implantation . To further optimize in vitro conditions for cell proliferation and extracellular matrix formation in tissue engineering of cardiovascular structures, in this study, ascorbic acid and growth factors as additives to standard cell culture medium were evaluated for their effect on tissue development in vitro . Biodegradable polymer patches {polyglycolic acid (PGA) coated with poly-4-hydroxybutyrate (P4HB)} were seeded with human pediatric aortic cells and cultured for 7 and 28 days . Group A was cultured with standard medium (DMEM with 10% fetal calf serum and 1% antibiotics) supplemented with ascorbic acid; group B was cultured with standard medium plus ascorbic acid and basic fibroblast growth factor (bFGF); group C was cultured with standard medium adding ascorbic acid and transforming growth factor (TGF) . Analysis of the cell seeded polymer constructs included DNA assay, collagen assay, and histologic and immunohistochemical examination for cell proliferation and collagen formation . After 7 and 28 days of culture, group B and group C showed a significantly higher DNA content compared with group A . The addition of bFGF (group B) led to a markedly higher collagen synthesis after 28 days of culture compared with the additives in groups C and A . The histologic and immunohistochemical examination also revealed a more dense, organized tissue development with pronounced matrix protein formation in the tissue engineered structures in group B after 28 days of culture . When seeded on to the polymeric scaffold, human vascular cells proliferate and form organized cell tissue after 28 days of culture . The addition of bFGF and ascorbic acid to the standard medium enhances cell proliferation and collagen synthesis on the biodegradable polymer, which leads to the formation of more mature, well organized tissue engineered structures. J Cardiovasc Electrophysiol, 2003 Oct, 14(10 Suppl), S229 - 36 Spontaneous initiation and termination of complex rhythms in cardiac cell culture; Bub G et al.; INTRODUCTION: Complex cardiac arrhythmias often start and stop spontaneously . These poorly understood behaviors frequently are associated with pathologic modification of the structural heterogeneity and functional connectivity of the myocardium . To evaluate underlying mechanisms, we modify heterogeneity by varying the confluence of embryonic chick monolayer cultures that display complex bursting behaviors . A simple mathematical model was developed that reproduces the experimental behaviors and reveals possible generic mechanisms for bursting dynamics in heterogeneous excitable systems . METHODS AND RESULTS: Wave propagation was mapped in embryonic chick myocytes monolayers using calcium-sensitive dyes . Monolayer confluence was varied by plating cultures with different cell densities and by varying times in culture . At high plating densities, waves propagate without breaks, whereas monolayers plated at low densities display spirals with frequent breaks and irregular activation fronts . Monolayers at intermediate densities display bursting rhythms in which there is paroxysmal starting and stopping of spiral waves of activity . Similar spatiotemporal patterns of activity were also observed as a function of the time in culture; irregular activity dominates the first 30 hours, followed by repetitive bursting dynamics until 54 hours, after which periodic target patterns or stable spirals prevail . In some quiescent cultures derived from older embryos, it was possible to trigger pacemaker activity following a single activation . We are able to reproduce all of these behaviors by introducing spatial heterogeneity and varying neighborhood size, equivalent to cell connectivity, in a spontaneous cellular automaton model containing a rate-dependent fatigue term . CONCLUSION: We observe transitions from irregular propagating waves, to spiral waves that spontaneously start and stop, to target waves originating from localized pacemakers in cell culture and a simple theoretical model of heterogeneous excitable media . The results show how physiologic properties of spontaneous activity, heterogeneity, and fatigue can give rise to a wide range of different complex dynamic behaviors similar to clinically observed cardiac arrhythmias. Appl Microbiol Biotechnol, 2004 Jul, 65(1), 18 - 24 Epub 2004 Jan 31. Monitoring the progress of infection and recombinant protein production in insect cell cultures using intracellular ATP measurement; Olejnik AM et al.; Several monitoring methods used to predict viable cell density have been the subject of extensive studies, including oxygen uptake rate, carbon dioxide evolution rate, optical density, NADH-dependent fluorescence and relative permittivity measurement . We propose intracellular ATP determination by bioluminescence assay to monitor the progress of baculovirus infection and recombinant protein production in insect cell cultures . We found that the ATP content in viable cells increased after virus addition . The increase in the ATP level was observed until the maximum recombinant protein accumulation was reached . At maximum product yield, the specific ATP content significantly decreased . Results obtained in both batch and fed-batch cultures demonstrated that the specific ATP level could be considered as a good indicator of recombinant protein productivity . Monitoring the cellular ATP content after viral infection makes it possible to define the optimum time for product harvest . The main advantage of applying the ATP assay as an index of the progress of infection and recombinant protein synthesis is its short time and sensitivity. Toxicol In Vitro, 2004 Apr, 18(2), 153 - 63 Toxicology investigations with cell culture systems: 20 years after; Zucco F et al.; From almost 20 years the "in vitro" model has gained a wide ground in toxicological investigation, providing advanced tools, reliable protocols, mechanistic information . These advancements have been done thanks to different approaches, addressed at improving chemical testing and validating procedures, at exploring the cellular and molecular basis of toxicity, at studying the modifications that xenobiotics undergo in the cellular environment . In this review the most advanced cellular models, the mechanisms of cell death, the techniques to monitor gene activation, following chemical exposure, is highlighted . Moreover the more recent in vitro models to approach the biotransformation issue will be presented. Med Princ Pract, 2004 Mar-Apr, 13(2), 91 - 4 Assessment of Chlamydia trachomatis prevalence by cell culture and transcription-mediated amplification in symptomatic women; Cicek C et al.; OBJECTIVE: The frequency of Chlamydia trachomatis in women with mucopurulent discharge was determined by a cell culture technique and a transcription-mediated amplification (TMA) assay in endocervical swab specimens . SUBJECTS AND METHODS: Endocervical swab specimens were obtained from 116 symptomatic patients with genitourinary complaints or abdominal pain . All of the women were married, with an age range of between 19 and 44 (median 29) years . The cell culture assay was used in all specimens . For 75 specimens the TMA assay was also performed . RESULTS: Positive cell culture test results were obtained in 6 (5.2%) patients . Among 75 specimens, 2 were positive by both TMA and culture assays, while 1 specimen was positive only by the culture assay . Of those positive for C . trachomatis, 5 were in the 19- to 25-year age group, and 1 was in the >25-year age group . All of the patients with positive results were of low socioeconomic status . CONCLUSIONS: This study revealed a relatively low rate of C . trachomatis infections in symptomatic married women in Turkey . A commercial TMA assay failed to identify all positive patients, in contrast to a 'gold standard' culture assay used in patients having such infections . In Vitro Cell Dev Biol Anim, 2003 Jul-Aug, 39(7), 291 - 6 Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-gamma-producing CHO cell line; Burteau CC et al.; A strong tendency is currently emerging to remove not only serum but also any product of animal origin from animal cell culture media during production of recombinant proteins . This should facilitate downstream processing and improve biosafety . One way consists in the fortification of protein-free nutritive media with plant protein hydrolysates . To investigate the effects of plant peptones on mammalian cell cultivation and productivity, CHO 320 cells, a clone of CHO K1 cells genetically modified to secrete human interferon-gamma (IFN-gamma), were first adapted to cultivation in suspension in a protein-free medium . Both cell growth and IFN-gamma secretion were found to be equivalent to those reached in serum-containing medium . Eight plant peptones, selected on the basis of their content in free amino acids and oligopeptides, as well as molecular weight distribution of oligopeptides, were tested for their ability to improve culture parameters . These were improved in the presence of three peptones, all having an important fraction of oligopeptides ranging from 1 to 10 kDa and a small proportion of peptides higher than 10 kDa . These peptones do not seem to add significantly to the nutritive potential to basal protein-free nutritive medium . Nevertheless, supplementation of an oligopeptide-enriched wheat peptone improved cell growth by up to 30% and IFN-gamma production by up to 60% in shake-flask experiments . These results suggest that the use of plant peptones with potential growth factor-like or antiapoptotic bioactivities could improve mammalian cell cultivation in protein-free media while increasing the product biosafety. In Vitro Cell Dev Biol Anim, 2003 Nov-Dec, 39(10), 424 - 7 Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing; Liu MY et al.; Polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated . The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans . A total of 36 cell lines representing 13 different species were selected for this study . The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis . The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species . However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis . These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination. Brain Res, 2004 Feb 20, 998(2), 218 - 29 Neutrophils both reduce and increase permeability in a cell culture model of the blood-brain barrier; Inglis VI et al.; This study was carried out to determine the effects that human neutrophils have on permeability across a model of the blood-brain barrier (BBB) formed by primary cultures of bovine brain microvessel endothelial cells (BBMEC) . Transendothelial electrical resistance (TEER) was used to measure changes in permeability across BBMEC monolayers in a dual compartment system, during neutrophil interactions . When neutrophils (5 x 10(6)/ml) were applied to monolayers, TEER increased (permeability decreased) . Adenosine was implicated, since the TEER increase was blocked by adenosine deaminase (1 U/ml) and the adenosine A2 receptor antagonist ZM 241385 (at 10(-6) M but not 10(-8) M, implicating A2B receptors) . Oxygen free radicals were implicated as the TEER increase was blocked by combined catalase (100 U/ml) and superoxide dismutase (60 U/ml) . When a gradient of the bacterial chemoattractant peptide formyl methionyl leucine phenylalanine (fMLP, 10(-7) M) was applied to neutrophils, the TEER decreased (permeability increased), concurrent with migration . When fMLP (10(-7) M) was added to the neutrophils, without migration, no change occurred . The TEER decrease was blocked by loading endothelium with the calcium buffer BAPTA (10 microM) and partially blocked by the serine protease inhibitor aprotinin (20 microg/ml) . Measures to block the potential extracellular triggers heparin binding protein, glutamate, oxygen free radicals and binding to intercellular cell adhesion molecule-1 (ICAM-1) were ineffective . These data indicate that neutrophils both reduce and increase permeability in a cell culture model of the BBB, correlated to their proximity and migration through the endothelium . They explore the role of neutrophils in BBB breakdown, and the formation or amelioration of vasogenic cerebral edema. Biosci Rep, 2003 Aug, 23(4), 169 - 74 Successful reconstruction of damaged ocular outer surface in humans using limbal and conjuctival stem cell culture methods; Sangwan VS et al.; When the ocular outer surface is badly damaged, subsequent corneal transplantation fails due to the absence of basal cells that are needed to support the graft . With the realization that the limbus and the conjunctiva have adult stem cells that can be cultured, it has been possible for us to explant culture these on de-epithelized human amniotic membrane, and to graft the resulting viable and transparent epithelium to 125 needy human patients with success . Ultrastructural, histological, biochemical and immunological assays establish the identity of the cells and the tissue formed. Planta, 2004 May, 219(1), 121 - 31 Epub 2004 Jan 28. Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated Cupressus lusitanica cell cultures; Zhao J et al.; Inositol 1,4,5-trisphosphate {Ins(1,4,5)P(3)} rapidly accumulates in elicited Cupressus lusitanica Mill . cultured cells by 4- to 5-fold over the control, and then it is metabolized . Correspondingly, phospholipase C (PLC) activity toward phosphatidylinositol 4,5-bisphosphate {PtdIns(4,5)P(2)} is stimulated to high levels by the elicitor and then decreases whereas Ins(1,4,5)P(3) phosphatase activity declines at the beginning of elicitation and increases later . These observations indicate that elicitor-induced biosynthesis and dephosphorylation of Ins(1,4,5)P(3) occur simultaneously and that the Ins(1,4,5)P(3) level may be regulated by both PtdIns(4,5)P(2)-PLC and Ins(1,4,5)P(3) phosphatases . Studies on the properties of PLC and Ins(1,4,5)P(3) phosphatases indicate that PLC activity toward PtdIns(4,5)P(2) was optimal at a lower Ca(2+) concentration than activity toward phosphatidylinositol whereas Ins(1,4,5)P(3) phosphatase activity is inhibited by high Ca(2+) concentration . This suggests that Ins(1,4,5)P(3) biosynthesis and degradation may be regulated by free cytosolic Ca(2+) . In addition, a relationship between Ins(1,4,5)P(3) signaling and accumulation of a phytoalexin (beta-thujaplicin) is suggested because inhibition or promotion of Ins(1,4,5)P(3) accumulation by neomycin or LiCl affects elicitor-induced production of beta-thujaplicin . Moreover, ruthenium red inhibits elicitor-induced accumulation of beta-thujaplicin while thapsigargin alone induces beta-thujaplicin accumulation . These results suggest that Ca(2+) released from intracellular calcium stores may mediate elicitor-induced accumulation of beta-thujaplicin via an Ins(1,4,5)P(3) signaling pathway, since it is widely accepted that Ins(1,4,5)P(3) can mobilize Ca(2+) from intracellular stores . This work demonstrates an elicitor-triggered Ins(1,4,5)P(3) turnover, defines its enzymatic basis and regulation, and suggests a role for Ins(1,4,5)P(3) in elicitor-induced phytoalexin accumulation via a Ca(2+) signaling pathway. Neurosci Lett, 2004 Feb 6, 356(1), 5 - 8 Potentiation of mitogenesis in adult rat chromaffin cell cultures by immunosuppressive agent FK506; Powers JF et al.; The immunosuppressive drugs FK506 and cyclosporin inhibit T- and B-lymphocyte proliferation and exert neuritogenic and/or cytoprotective effects on several types of neurons . While the immunosuppressive actions of both drugs are mediated in large part by inhibition of the Ca(2+)-dependent phosphatase, calcineurin, FK506 is known to exert additional effects . In the present study, FK506 is shown to potentiate mitogenic effects of the neurotrophic factor, neurturin, on normal adult rat adrenal chromaffin cells in culture . The effect is not seen with cyclosporin or with a non-immunosuppressive analog of FK506, GPI-1046 . The finding of increased mitogenesis in response to FK506 may have applications to the study of normal and neoplastic neuroendocrine cells and to understanding the development of some types of tumors in transplant patients. Cell Motil Cytoskeleton, 2004 Mar, 57(3), 175 - 85 Positioning and capture of cell surface-associated microtubules in epithelial tendon cells that differentiate in primary embryonic Drosophila cell cultures; Tucker JB et al.; Using primary embryonic Drosophila cell cultures, we have investigated the assembly of transcellular microtubule bundles in epidermal tendon cells . Muscles attach to the tendon cells of previously undescribed epidermal balls that form shortly after culture initiation . Basal capture of microtubule ends in cultured tendon cells is confined to discrete sites that occupy a relatively small proportion of the basal cell surface . These capturing sites are associated with hemiadherens junctions that link the ends of muscle cells to tendon cell bases . In vivo, muscle attachment and microtubule capture occur across the entire cell base . The cultured tendon cells reveal that the basal ends of their microtubules can be precisely targeted to small, pre-existing, structurally well-defined cortical capturing sites . However, a search and capture targeting procedure, such as that undertaken by kinetochore microtubules, cannot fully account for the precision of microtubule capture and positioning in tendon cells . We propose that cross-linkage of microtubules is also required to zip them into apicobasally oriented alignment, progressing from captured basal plus ends to apical minus ends . This involves repositioning of apical minus ends before they become anchored to an apical set of hemiadherens junctions . The proposal is consistent with our finding that hemiadherens junctions assemble at tendon cell bases before they do so at cell apices in both cultures and embryos . It is argued that control of microtubule positioning in the challenging spatial situations found in vitro involves the same procedures as those that operate in vivo . Eur J Pediatr Surg, 2003 Dec, 13(6), 361 - 6 Urothelial mesh--a new technique of cell culture on biomaterials; Hobbiesiefken J et al.; INTRODUCTION: Urogenital malformations, trauma or tumours may demand surgical reconstruction in children . Cell culture is an important technology in biomaterial research and tissue engineering . Tissue-engineering of urothelial organs is of interest in children, because the number of complications and re-operations may be reduced . Actually, monolayer cultures of urothelium are used for tissue engineering and biocompatibility testing . A culture system that more closely mimics the physiologic environment of the urothelium would be of interest . The aim of this study was to determine the biological and mechanical characteristics of urothelial mesh cultured in vitro . METHODS: Meshes containing urothelium, lamina propria, and submucosal tissue were generated using a skin mesh graft cutter . Meshes were cultured in 6-well plates, on collagen I/III, polydioxanone/polylactic acid and silicone matrices . Cell morphology was examined by inversion microscopy, histology, and scanning electron microscopy . It was compared to urothelium cultured by methods reported in the literature . To define the basic mechanical properties, meshes were extended longitudinally by a servohydraulic testing machine and strain diagrams generated . RESULTS: Urothelium was reproducibly cultured from meshes . Cell growth could be induced onto fibrillary collagen, polydioxanone-polylactic acid matrices and shaped polyurethane surfaces . Cells formed confluent layers of flat cells, resembling native urothelium . The meshes have unique mechanical properties, allowing for stable fixation, surgical handling and mechanical stimulation . CONCLUSIONS: Meshes can be used for cell culture on biomaterials . They maintain epithelial-stromal integrity and mechanic stability . The small size of tissue bridges allows in vitro culture for long periods with many potential advantages for tissue engineering and biologic research . Applications are possible both in vitro and in vivo. J Neuroimmunol, 2004 Feb, 147(1-2), 123 - 6 Cannabinoids and morphine differentially affect HIV-1 expression in CD4(+) lymphocyte and microglial cell cultures; Peterson PK et al.; The influence of substances of abuse on the progression of HIV-1 infection is controversial, and pharmacologic factors have been postulated as a potential explanation for conflicting data arising from epidemiological studies and animal models . In the present study, cell culture models of HIV-1 infection were used to test this hypothesis . The synthetic cannabinoid WIN 55,212-2 was found to potently inhibit HIV-1 expression in a concentration- and time-dependent manner in CD4(+) lymphocyte and microglial cell cultures . In sharp contrast, morphine either inhibited or stimulated viral expression, depending upon the time of drug exposure, and marked differences were observed between CD4(+) and microglial cells . Also, WIN 55,212-2 inhibited the stimulatory effect of morphine in HIV-1 infected CD4(+) cells . These in vitro findings support the notion that pharmacologic factors need to be considered in epidemiological studies and animal models that pertain to HIV-1 infection. Brain Res Mol Brain Res, 2004 Jan 5, 120(2), 145 - 50 Survival of motor neuron gene downregulation by RNAi: towards a cell culture model of spinal muscular atrophy; Trulzsch B et al.; Gene silencing with double-stranded RNA (RNAi) has proved useful for gene function studies, and should be especially well suited to studying diseases resulting in embryonal lethality where transgenic animal models are difficult to generate . We are applying this approach to the autosomal recessive disease spinal muscular atrophy (SMA) . SMA is caused by mutations in the survival of motor neuron gene (SMN) . The SMN protein is ubiquitously expressed and plays a role in RNA processing and its reduction in SMA ultimately leads to motor neuron degeneration in the spinal cord . The reasons for this motor neuron selectivity, however, are still unclear . SMN is essential for the viability of most eukaryotic organisms and this has made the generation of animal models of SMA extremely difficult . Here we describe a different approach to study SMN function using RNAi to silence SMN expression in cells . We designed double-stranded small interfering RNA (siRNA) targeted against murine Smn and transfected the murine embryonal terato-carcinoma cell line P19 . The siRNAs reduced both Smn RNA and protein levels in the P19 cells compared to controls . These results illustrate that double-stranded RNA can be an effective gene silencing approach even in a protein that is essential for survival and highly expressed, and it could therefore be a valuable tool to study SMN function. FEBS Lett, 2004 Jan 16, 557(1-3), 33 - 8 Chalcone dimethylallyltransferase from Morus nigra cell cultures . Substrate specificity studies; Vitali A et al.; A new prenyltransferase (PT) enzyme derived from the microsomal fractions of cell cultures of Morus nigra was shown to be able to prenylate exclusively chalcones with a 2',4'-dihydroxy substitution and the isoflavone genistein . Computational studies were performed to shed some light on the relationship between the structure of the substrate and the enzymatic activity . PT requires divalent cations, particularly Mg(2+), to be effective . The apparent K(m) values for gamma,gamma-dimethylallyldiphosphate and 2',4'-dihydroxychalcone were 63 and 142 microM, respectively . The maximum activity of the enzyme was expressed during the first 10 days of cell growth. Acta Biochim Pol, 2003, 50(4), 973 - 84 Degradation of extracellular nucleotides and their analogs in HeLa and HUVEC cell cultures; Gendaszewska-Darmach E et al.; The use of nucleotides and their analogs in the pharmacological studies of nucleotide receptors (P2 class) should be preceded by detailed studies on their degradation connected with ecto-enzymes of a given cell type . In the present studies we have analyzed stability of some phosphorothioate and phosphonate analogs of ATP and ADP in the HeLa epitheloid carcinoma and endothelial HUVEC cells cultures . Our studies have revealed that ecto-nucleotide pyrophosphatase (E-NPP) is one of the main enzymes involved in the extracellular degradation of ATP and other nucleotides in the HeLa cells . On the other hand, the ecto-ATPDase is responsible for the hydrolysis of extracellular nucleotides in human endothelial cell cultures, while the E-NPP-like enzymes of the HUVEC cells are not essential to this degradation . The concerted action of the aforementioned ecto-enzymes and nucleotide pyrophosphatase, 5'-nucleotidase and adenosine deaminase present in fetal bovine serum (FBS) supplied to the culture medium, results in partial or complete degradation of the phosphorothioate (ATPgammaS) and phosphonate analogs of adenosine nucleotides (alpha,beta-methylene-ATP and beta,gamma-methylene-ATP) in the cell cultures . Only ADPbetaS appears to be resistant to these enzymes . The influence of some nucleotides and their analogs on the proliferation of the HeLa cells in presence or absence of FBS is also discussed. J Virol Methods, 2004 Mar 15, 116(2), 133 - 8 Development of an in situ hybridisation procedure for the detection of sole aquabirnavirus in infected fish cell cultures; Alonso MC et al.; An in situ hybridisation (ISH) technique has been developed to detect sole aquabirnavirus in infected fish cell lines bluegill fibroblast (BF-2), EPC, and chinook salmon embryo cells (CHSE-214) . A 613 bp cDNA probe for viral RNA coding for a fragment of VP2 protein was generated by reverse transcription polymerase chain reaction (RT-PCR) using infectious pancreatic necrosis virus (IPNV) specific DNA primers . Infected cells were strongly labelled, and no non-specific reaction was observed in non-infected cells used as negative controls . The specificity of the probe was examined by testing it against a range of IPNV serotypes such as Ab, Sp and VR-299 . The ISH technique was compared with the immunofluorescence procedure to determine the sensitivity of detection of sole aquabirnavirus in BF-2 cells . The probe used in the ISH technique detected weak positivity at 8h post-inoculation (p.i.) in the cytoplasm of infected BF-2 cells inoculated with 10(3) TCID50/ml, whilst the labelling appears at 24h p.i . when the immunofluorescence technique was applied . At all other time intervals the results were equivalent. J Matern Fetal Neonatal Med, 2003 Oct, 14(4), 261 - 6 Cocaine affects prostaglandin production in human umbilical cord cell cultures; Mastrogiannis DS et al.; OBJECTIVE: To investigate the possible effects of cocaine on prostacyclin and prostaglandin (PG) E2 production from endothelial cells derived from human umbilical cord . STUDY DESIGN: First-passaged endothelial cells derived from the umbilical vein were incubated with various doses of cocaine, procaine and lidocaine and 24 h later the supematants were assayed for prostacyclin metabolites 6-keto-PGF1alpha and PGE2 . Cocaine concentrations tested were 0, 10, 100, 500 and 1000 microg/ml . RESULTS: Cocaine produced a dose-dependent reduction in prostacyclin and PGE2 production from endothelial cells (p) < 0.05) . Acetylcholinesterase (a possible detoxifier of cocaine) abolished the effect of cocaine on prostacyclin production . Procaine, an esterol-type anesthetic, produced a similar effect on prostacyclin production, an effect not observed with lidocaine . CONCLUSION: It is speculated that, when present in high concentrations, cocaine may affect vascular tone by inhibition of endothelial cell prostacyclin and PGE2 release. J Eukaryot Microbiol, 2003, 50 Suppl, 689 - 90 Mode of action of ponazuril against Toxoplasma gondii tachyzoites in cell culture; Mitchell SM et al.; Toxoplasma gondii is an important apicomplexan parasite of humans and other warm-blooded animals . Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States . We investigated the mode of action of ponazuril against developing RH strain T . gondii tachyzoites in African green monkey kidney cells . Host cells were infected with 2.0 x 10(5) tachyzoites and treated with 5 microg/ml ponazuril . Cultures were fixed and examined by transmission electron microscopy 3 days after treatment . Ponazuril interfered with normal parasite division . This led to the presence of multinucleate schizonts stages . Up to six tachyzoites were observed partially budded from the surface of these schizonts . Large vacuoles developed in these schizonts and they eventually degenerated. Br J Cancer, 2004 Jan 26, 90(2), 476 - 82 Variation in RNA expression and genomic DNA content acquired during cell culture; Hiorns LR et al.; Specific chromosomal abnormalities are increasingly recognised to be associated with particular tumour subtypes . These cytogenetic abnormalities define the sites of specific genes, the alteration of which is implicated in the neoplastic process . We used comparative genomic hybridisation (CGH) to examine DNA from different breast and ovarian cancer cell lines for variations in DNA sequence copy number compared with the same normal control . We also compared different sources of the MCF7 breast line by both CGH and cDNA expression arrays . Some of the differences between the subcultures were extensive and involved large regions of the chromosome . Differences between the four subcultures were observed for gains of 2q, 5p, 5q, 6q, 7p, 7q, 9q, 10p, 11q, 13q, 14q, 16q, 18p and 20p, and losses of 4q, 5p, 5q, 6q, 7q, 8p, 11p, 11q, 12q, 13q, 15q, 19p, 19q, 20p, 21q, 22q and Xp . However, few variations were found between two subcultures examined, 5 months apart, from the same initial source . The RNA arrays also demonstrated considerable variation between the three different subcultures, with only 43% of genes expressed at the same levels in all three . Moreover, the patterns of the expressed genes did not always reflect our observed CGH aberrations . These results demonstrate extensive genomic instability and variation in RNA expression during subculture and provide supportive data for evidence that cell lines do evolve in culture, thereby weakening the direct relevance of such cultures as models of human cancer . This work also reinforces the concern that comparisons of published analyses of cultures of the same name may be dangerous. Glia, 2004 Feb, 45(3), 258 - 68 Glial activation modulates glutamate neurotoxicity in cerebellar granule cell cultures; Perez-Capote K et al.; We studied the influence of glial cells on the neuronal response to glutamate toxicity in cerebellar granule cell cultures . We compared the effect of glutamate on neuronal viability in neuronal vs . neuronal-glial cultures and determined this effect after pretreating the cultures with the lipopolysaccharide (LPS) of Escherichia coli, agent widely used to induce glial activation . Morphological changes in glial cells and nitric oxide (NO) production were evaluated as indicators of glial activation . We observed that glutamate neurotoxicity in neuronal-glial cultures was attenuated in a certain range of glutamate concentration when compared to neuronal cultures, but it was enhanced at higher glutamate concentrations . This enhanced neurotoxicity was associated with morphological changes in astrocytes and microglial cells in the absence of NO production . LPS treatment induced morphological changes in glial cells in neuronal-glial cultures as well as NO production . These effects occurred in the absence of significant neuronal death . However, when LPS-pretreated cultures were treated with glutamate, the sensitivity of neuronal-glial cultures to glutamate neurotoxicity was increased . This was accompanied by additional morphological changes in glial cells in the absence of a further increase in NO production . These results suggest that quiescent glial cells protect neuronal cells from glutamate neurotoxicity, but reactive glial cells increase glutamate neurotoxicity . Therefore, glial cells play a key role in the neuronal response to a negative stimulus, suggesting that this response can be modified through an action on glial cells . Endocrinology, 2004 May, 145(5), 2283 - 90 Epub 2004 Jan 15. Estrogen Induces Neuropeptide Y (NPY) Y1 receptor gene expression and responsiveness to NPY in gonadotrope-enriched pituitary cell cultures; Hill JW et al.; We showed previously that neuropeptide Y1 receptor (Y1R) expression is increased in the hypothalamus on proestrus afternoon and that this up-regulation of Y1R mRNA may permit neuropeptide Y (NPY) to facilitate release of the preovulatory GnRH surge . Because NPY also modulates LH release directly, we examined steroid regulation of Y1R expression in the female rat anterior pituitary . Treatment of female rats with estrogen in vivo decreased the levels of Y1R mRNA in the whole pituitary gland . In lactotrope/somatotrope-enriched pituitary cells separated by unit gravity sedimentation, 17beta-estradiol (E(2)) treatment likewise suppressed Y1R expression . In contrast, E(2) elevated Y1R mRNA in gonadotrope-enriched cell populations, indicating that estrogen regulates Y1R mRNA expression differently in gonadotropes vs . other pituitary cell types . After exposure to E(2), NPY augmented GnRH-induced LH release from gonadotrope-enriched cells in a manner requiring Y1R activation . Without steroid exposure, this augmentation disappeared, and with progesterone alone, NPY reduced GnRH-induced LH release . In addition, NPY inhibited prolactin secretion from primary pituitary cells in a steroid-free environment, but not in the presence of estrogen . These findings demonstrate that E(2) can directly up-regulate gonadotrope responsiveness to NPY and suggest that this action is mediated at least in part by E(2)'s ability to stimulate Y1R gene expression in gonadotropes . Our observations are consistent with the idea that this regulatory mechanism represents a component of E(2)'s positive feedback actions in pituitary gonadotropes . The biological importance of E(2)'s opposite effects on Y1R expression in other pituitary cell types remains to be determined. Br J Biomed Sci, 2003, 60(4), 217 - 20 Toxoplasma gondii from liquid nitrogen for continuous cell culture: methods to maximise efficient retrieval; Mavin S et al.; This study aims to increase the efficiency of continuous growth of Toxoplasma gondii in HeLa cells from tachyzoite stocks frozen in liquid nitrogen . Freezing and retrieval of tachyzoites for continuous cell culture requires more stringent protocols than those published for animal culture . The freezing and retrieval conditions are optimised so that a quality harvest (> or = 1 x 10(6) tachyzoites/mL, > or = 90% viability) can be produced using T . gondii recovered from liquid nitrogen as fast and reliably as possible . Retrieval success rate increased from 36% to 100% . An improved freezing procedure using chilled reagents and freshly harvested parasites, and adoption of an effective recovery protocol with retrieval of 3 x 10(7) tachyzoites into 75 cm2 flasks, change of maintenance media after six hours and subsequent blind passage all contributed to this success . The result is faster and more dependable production of T . gondii for diagnostic and experimental use. J Neurocytol, 2003 May, 32(4), 373 - 80 5-HT2a receptors in rat sciatic nerves and Schwann cell cultures; Gaietta GM et al.; Pharmacological approaches and optical recordings have shown that Schwann cells of a myelinating phenotype are activated by 5-HT upon its interaction with the 5-HT(2A) receptor (5-HT(2A)R) . In order to further characterize the expression and distribution of this receptor in Schwann cells, we examined rat sciatic nerve and cultured rat Schwann cells using probes specific to 5-HT(2A)R protein mRNA . We also examined the endogenous sources of 5-HT in rat sciatic nerve by employing both histochemical stains and an antibody that specifically recognizes 5-HT . Rat Schwann cells of a myelinating phenotype contained both 5-HT(2A)R protein and mRNA . In the healthy adult rat sciatic nerve, 5-HT(2A)Rs were evenly distributed along the outermost portion of the Schwann cell plasma membrane and within the cytoplasm . The most prominent source of 5-HT was within granules of the endoneurial mast cells, closely juxtaposed to Schwann cells within myelinating sciatic nerves . These results support the hypothesis that the 5-HT receptors expressed by rat Schwann cells in vivo are activated by the release of 5-HT from neighboring mast cells. J Neurochem, 2004 Feb, 88(3), 708 - 16 NR2A induction and NMDA receptor-dependent neuronal death by neurotrophin-4/5 in cortical cell culture; Choi SY et al.; We have previously shown that prolonged exposure to neurotrophins induces oxidative neuronal death . In the present study, we further examined the cascades involved in neurotrophin-4/5 (NT-4/5)-induced neuronal death . Exposure of mature cortical cultures for 48 h to NT-4/5 induced neuronal death through TrkB activation . The NT-4/5-induced neuronal death was largely attenuated by addition of MK-801, indicating a critical role for NMDA receptors . Western blots revealed the induction of NR2A by NT-4/5 . In addition, levels of phospho-NR2A and 2B increased, suggesting the upregulation of the NMDA receptor function . Whereas glutamate levels in the media changed little, levels of D-serine and L-glycine, co-agonists at NMDA receptors, increased significantly following NT-4/5 treatment . Exposure to NT-4/5 resulted in the activation of Src and extracellular signal-regulated kinase-1/2 (Erk-1/2) . Their inhibitors blocked NR2A induction and phosphorylation as well as neuronal death induced by NT-4/5 . In addition, Egr-1 was induced in an Src- and Erk-1/2-dependent manner . Anti-sense oligodeoxynucleotides to egr-1 attenuated NR2A induction as well as neuronal death . Although induction of NADPH oxidase and neuronal nitric oxide synthase (nNOS) contributes to NT-4/5-induced neuronal death, inhibition of their activity did not reduce NR2A induction . Conversely, blockade of NMDA receptors did not attenuate induction of NADPH oxidase or nNOS . These results indicate that two events are largely independent of each other . Our results demonstrate that the signaling cascade of TrkB leads to increase in NMDA receptor activity . Whereas this cascade may play an important role in the modulation of NMDA receptors in physiologic conditions, in the context of TrkB overactivation, it may contribute to neuronal death. J Vet Med B Infect Dis Vet Public Health, 2003 Dec, 50(10), 505 - 9 Use of a quantitative TaqMan-PCR for the fast quantification of mycobacteria in broth culture, eukaryotic cell culture and tissue; Lewin A et al.; The quantification of slow-growing mycobacteria such as Mycobacterium tuberculosis or M . bovis from in vitro and in vivo samples is complicated by their long generation time, their ability to form aggregates, and their capacity to persist in a state of dormancy . We compared different methods for the establishment of growth curves for broth cultures of M . bovis bacille Calmette-Guerin (BCG) . A quantitative TaqMan-PCR yielded results comparable with those obtained by protein quantification and measurement of the ATP content of the cultures . The quantitative TaqMan-PCR furthermore turned out to be particularly suitable for the measurement of multiplication of BCG within eukaryotic cells . Furthermore, it is a fast method allowing an estimation of the mycobacterial load in tissue long before colony counts can be obtained. Neurotox Res, 2003, 5(6), 425 - 32 Cell culture protection and in vivo neuroprotective capacity of flavonoids; Dajas F et al.; Flavonoids are an important group of recognized antioxidants ubiquitous in fruits, vegetables and herbs . There are epidemiological evidences for the stroke-protecting capacity of flavonoids and while the neuroprotective power of complex extracts rich in flavonoids like those of Ginkgo biloba, green tea or lyophilized red wine have been demonstrated in several studies, neuroprotection by individual flavonoids has been poorly studied in vivo . The neuroprotective capacity of individual flavonoids was studied in PC12 cells in culture and in a model of permanent focal ischemia (permanent Middle Cerebral Artery Occlusion - pMCAO) . In the in vivo experiments, flavonoids were administered in lecithin preparations to facilitate the crossing of the blood brain barrier . The simultaneous incubation of PC12 cells with 200 micro M hydrogen peroxide (H2O2) and different flavonoids for 30 min resulted in a conspicuous profile: quercetin, fisetin, luteolin and myricetin significantly increased cell survival while catechin, kaempherol and taxifolin did not . Quercetin was detected in brain tissue 30 min and 1 h after intraperitoneal administration . When one of the protective flavonoids (quercetin) and one of those that failed to increase PC12 cell survival (catechin) were assessed for their protective capacity in the pMCAO model, administered i.p . 30 min after vessel occlusion, quercetin significantly decreased the brain ischemic lesion while catechin did not . It is concluded that when administered in liposomal preparations, flavonoids structurally related to quercetin could become leads for the development of a new generation of molecules to be clinically effective in human brain ischemia. Biosens Bioelectron, 2004 Feb 15, 19(7), 741 - 7 Application of on-chip cell cultures for the detection of allergic response; Matsubara Y et al.; In this report, the development of a microfluidic cell chip for monitoring allergic response is described . A rat basophilic leukemia cell line (RBL-2H3), a tumor analog of rat mucosal mast cells, has been used as a model to observe its allergic response upon antigen stimulus . The cells were cultivated on a poly(dimethylsiloxane) (PDMS) chip, the surface of which was modified by several methods . The PDMS chip, which comprised a cell cultivation chamber and microfluidic channels, was fabricated by conventional molding methods . In order to detect the allergic response, a fluorescent dye, quinacrine, was introduced inside the cell compartment that included histamine . The cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with anti-DNP IgE . When exocytosis events occurred, the microfluidic system detected the fluorescent signal of quinacrine, which was released from RBL-2H3 cells by using a photomultiplier tube (PMT) fitted onto a microscope. Vopr Virusol, 2003 Nov-Dec, 48(6), 30 - 3 {Possibilities of culturing mutant variants of Ade12 adenoviruses in 293 cell culture}; Sergeev AN et al.; The results of polymerase chain reaction and of DNA sequencing of the Adel2 mutant variant of adenovirus serotype 5, passaged 10 times and capable of selectively infecting and lysing the p53-deficient human tumor cells, are indicative of a high stability of its genotype and of the phenotypic properties acquired by it in successive passage on 293 cells . The absence of admixtures of wild-type adenovirus was clearly shown in the cultivation and passage processes . It was revealed in an experimental analysis of virus-productive properties of the studied continuous cell culture 293 by using the method of multilayer cultivation, that the maximal Adel2 yield is obtained at the 50% cytopathic effect . Virus doses, that are effective for cell-culture contamination, are within a range of 100-10 TCPE50 per cell . In order to spare the viral material, the infecting dose of 10 TCPE50 per cell was chosen to infect a cell monolayer. Int Arch Allergy Immunol, 2003 Dec, 132(4), 373 - 9 Cytokine production, lymphocyte proliferation and T-cell receptor Vbeta expression in primary peripheral blood mononuclear cell cultures from nickel-allergic individuals; Cederbrant K et al.; BACKGROUND: Clinical history and patch test constitute the two cornerstones in the diagnosis of nickel (Ni) allergy . Due to technical and interpretative limits of the patch test, the in vitro lymphocyte transformation test (LTT) has been developed for confirming contact allergy; however, most studies show an overlap in lymphocyte proliferation between Ni-allergic and nonallergic subjects using the LTT . The aim of this study was to see if the secretion of cytokines, especially interleukin (IL)-10 and IL-17, or the use of T-cell receptor (TCR) Vbeta families in Ni-stimulated primary peripheral blood mononuclear cell (PBMC) cultures might be more useful for discriminating between allergic and nonallergic subjects . METHODS: Ni(2+)-stimulated primary PBMC cultures derived from female subjects diagnosed as Ni-allergic (n = 5) or nonallergic (n = 5) on the basis of a positive or negative patch test were assessed for cell proliferation by tritiated thymidine incorporation and for production of interferon-gamma, IL-4, IL-10 and IL-17 in the culture supernatant by ELISA . The immunophenotype and TCR-Vbeta family affiliation of the Ni(2+)-induced lymphoblasts were determined by flow cytometry . RESULTS: Lymphocytes from Ni-allergic individuals challenged with a high and a low concentration of Ni showed significantly higher cell proliferation than lymphocytes from nonallergic individuals, but all subjects showed a positive LTT result (stimulation index >2) . We found a significantly higher release of IL-10 in Ni(2+)-treated cultures from Ni-allergic compared with nonallergic subjects that provided better separation between individuals in the two groups than did lymphocyte proliferation . The proliferating lymphoblasts were predominantly CD4+, and in 2 of the 5 Ni-allergic subjects, but in none of the 5 nonallergic subjects, the CD4+ lymphoblasts showed a dominance of TCR-Vbeta17 . CONCLUSIONS: Determination of IL-10 production in primary PBMC cultures is a potentially promising in vitro method for discrimination of Ni allergy in females, as compared with cell proliferation . Biochem Biophys Res Commun, 2004 Jan 23, 313(4), 915 - 21 Wnt proteins promote neuronal differentiation in neural stem cell culture; Muroyama Y et al.; Wnt signaling is implicated in the control of cell growth and differentiation during CNS development from studies of mouse and chick models, but its action at the cellular level has been poorly understand . In this study, we examine the in vitro function of Wnt signaling in embryonic neural stem cells, dissociated from neurospheres derived from E11.5 mouse telencephalon . Conditioned media containing active Wnt-3a proteins are added to the neural stem cells and its effect on regeneration of neurospheres and differentiation into neuronal and glial cells was examined . Wnt-3a proteins inhibit regeneration of neurospheres, but promote differentiation into MAP2-positive neuronal cells . Wnt-3a proteins also increase the number of GFAP-positive astrocytes but suppress the number of oligodendroglial lineage cells expressing PDGFR or O4 . These results indicate that Wnt-3a signaling can inhibit the maintenance of neural stem cells, but rather promote the differentiation of neural stem cells into several cell lineages. J Biomed Mater Res A, 2004 Feb 1, 68(2), 360 - 4 Pitfalls in the detection of lipid vectors in neural cell culture and in brain tissue; Rizk T et al.; Lipid particles (liposomes and lipid-coated microbubbles) are currently studied as vectors for drug delivery to the central nervous system . The visualization of these particles is usually based on their labeling with a lipophilic fluorescent dye (3,3'-dioctadecycloxacarbocyanine perchlorate) or staining with Oil Red O . The purpose of this article was to highlight the difficulties and pitfalls encountered with the use of these techniques in the detection of lipid particles in neural cell cultures and in brain tissue . In vitro and in vivo studies were conducted on different neural cell cultures (rat and human tumors, microglial cells) and animal models of brain lesion (lipopolysaccharide and quinolinic acid-induced lesion, induced brain tumor) . The cells or brain slices were observed with optical microscopy after staining with Oil Red O, fluorescent microscopy, or scanning electron microscopy . Intra and extracytoplasmic lipid particles (stained with Oil Red O or autofluorescent or visualized by scanning electron microscopy) were naturally found in the cells and tissues studied . Intracytoplasmic lipid microparticles were present in tumoral and microglial cells . These lipid microparticles were also observed with some extracytoplasmic lipid droplets in the induced brain lesions . These images could be misinterpreted as lipid vectors if the cells or animals would have been treated with such a vector . J Neuroimmunol, 2004 Jan, 146(1-2), 114 - 25 IL-1 beta-mediated neuropeptide and immediate early gene mRNA induction is defective in Lewis hypothalamic cell cultures; Wei R et al.; We previously found that Lewis (LEW/N) hypothalamic cells respond to interleukin-1beta (IL-1beta) with reduced corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) peptide synthesis and secretion compared to Fischer (F344/N) cells . To investigate whether this peptide hyporesponsiveness in LEW/N cells is secondary to their deficient mRNA expression, temporal mRNA expression patterns of CRH, AVP, and several hypothalamic neuropeptides induced by IL-1beta in LEW/N and F344/N hypothalamic dissociated cell cultures were delineated by quantitative real-time polymerase chain reaction (RT-PCR) . To investigate the molecular mechanisms underlying neuropeptide mRNA induction in cells of both strains, temporal mRNA expression patterns of immediate early genes (IEGs) and several signal transduction-associated molecules were also examined . We found that LEW/N hypothalamic cells were hyporesponsive to IL-1beta induction of neuropeptide and IEG mRNA, while LEW/N cells transcribed more IL-1 receptor and inducible nitric oxide synthase (iNOS) compared to F344N/N cells, suggesting that LEW/N and F344/N hypothalamic cells are differentially activated by IL-1beta. Biol Reprod, 2004 May, 70(5), 1299 - 305 Epub 2003 Dec 26. Effects of prolactin on the luteinizing hormone response to gonadotropin- releasing hormone in primary pituitary cell cultures during the ovine annual reproductive cycle; Gregory SJ et al.; In the shee