Cell cultures

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 sheep pituitary, the localization of prolactin (PRL) receptors in gonadotrophs and the existence of gonadotroph-lactotroph associations have provided morphological evidence for possible direct effects of PRL on gonadotropin secretion . Here, we investigated whether PRL can readily modify the LH response to GnRH throughout the ovine annual reproductive cycle . Cell populations were obtained from sheep pituitaries during the breeding season (BS) and the nonbreeding season (NBS), plated to monolayer cultures for 7 days, and assigned to receive one of the following treatments: 1) nil (control), 2) acute (90- min) bromocriptine (ABr), 3) chronic (7-day) bromocriptine (CBr), 4) ABr and PRL, 5) CBr and PRL, 6) PRL alone, or 7) thyrotropin-releasing hormone . Cells were treated as described above, with the aim of decreasing or increasing the concentrations of PRL in the culture, and simultaneously treated with GnRH for 90 min . The LH concentrations in the medium were then determined by RIA . GnRH stimulated LH in a dose-dependent manner during both stages of the annual reproductive cycle . During the NBS, single treatments did not significantly affect the LH response to GnRH . However, when PRL was combined with bromocriptine, either acutely or chronically, GnRH failed to stimulate LH release at all doses tested (P < 0.01) . In contrast, during the BS, the LH response to GnRH was not affected by any of the experimental treatments . These results reveal no apparent effects of PRL alone, but an interaction between PRL and dopamine in the regulation of LH secretion within the pituitary gland, and a seasonal modulation of this mechanism.

J Neurochem, 2004 Jan, 88(2), 337 - 48
Modulation of beta-amyloid metabolism by non-steroidal anti-inflammatory drugs in neuronal cell cultures; Gasparini L et al.; Alzheimer disease (AD) is characterized by cerebral deposits of beta-amyloid (Abeta) peptides, which are surrounded by neuroinflammatory cells . Epidemiological studies have shown that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD . In addition, biological data indicate that certain NSAIDs specifically lower Abeta42 levels in cultures of peripheral cells independently of cyclooxygenase (COX) activity and reduce cerebral Abeta levels in AD transgenic mice . Whether other NSAIDs, including COX-selective compounds, modulate Abeta levels in neuronal cells remains unexploited . Here, we investigated the effects of compounds from every chemical class of NSAIDs on Abeta40 and Abeta42 secretion using both Neuro-2a cells and rat primary cortical neurons . Among non-selective NSAIDs, flurbiprofen and sulindac sulfide concentration-dependently reduced the secretion not only of Abeta42 but also of Abeta40 . Surprisingly, both COX-2 (celecoxib; sc-125) or COX-1 (sc-560) selective compounds significantly increased Abeta42 secretion, and either did not alter (sc-560; sc-125) or reduced (celecoxib) Abeta40 levels . The levels of betaAPP C-terminal fragments and Notch cleavage were not altered by any of the NSAIDs, indicating that gamma-secretase activity was not overall changed by these drugs . The present findings show that only a few non-selective NSAIDs possess Abeta-lowering properties and therefore have a profile potentially relevant to their clinical use in AD.

Neurochem Int, 2004 May, 44(6), 401 - 11
The neuroprotective effects of estrogen in SK-N-SH neuroblastoma cell cultures; Ba F et al.; Estrogen has been considered to be a neuroprotectant and a neuromodulator in many neuronal cell lines and tissue preparations . The protective effects of estrogen may be mediated through classical estrogen receptors (ERs), or may be due to its anti-oxidant properties which are independent of receptors . The current studies show that 17beta-estradiol (E2) is neuroprotective against beta-amyloid protein 25-35 (Abeta)-, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-, high density culture condition-, and serum deprivation-induced neuronal death in SK-N-SH human neuroblastoma cells . SK-N-SH cells express ERbeta, but not ERalpha, as detected by Western blot analysis . Among all the insults, MPTP, high density culture and serum deprivation induce apoptotic cell death in this cell system as detected by ELISA determination of mono/oligonucleosomes and DNA laddering, while Abeta induces necrotic cell death . The protective effects of E2 are abolished by the addition of tamoxifen and ICI 182,780 in the MPTP treated cells, but not in the other models, suggesting that the effect of E2 in the MPTP model is probably associated with activation of ERbeta . The addition of ICI 182,780 shows a mitogenic effect in SK-N-SH cells in the presence of E2 in control culture or in the Abeta treated groups . Also, ICI 182,780 induced expression of ERalpha . Collectively, the current studies suggest that E2 is neuroprotective in apoptotic and necrotic death induced by multiple insults in SK-N-SH human neuroblastoma cells . Involvement of ER is insult type dependent . ICI 182,780 is able to influence the expression of ERs, probably through upregulation of ERalpha when ERbeta is totally antagonized.

In Vitro Cell Dev Biol Anim, 2003 Sep-Oct, 39(8-9), 343 - 7
Single-nucleotide polymorphism of the urokinase-plasminogen activator gene during aging and transformation of human diploid kidney cell cultures; Beqaj SH et al.; Single-nucleotide polymorphisms (SNPs) are differences in the nucleotide sequence of a specific gene from different individuals . The frequency at which SNPs occur varies among individuals, is gene dependent, and may be influenced by the aging process or by mechanisms that result in cell transformation . Urokinase-plasminogen activator (uPA) is a serine protease that is important in embryonic development, aging, and the onset of pathogenic conditions . The frequency of SNP and the stability of the SNPs in the uPA gene have not been defined with regard to processes that are associated with cellular aging or transformation . In this study, the complete nucleotide sequence has been determined for the gene encoding uPA from 26 human diploid kidney cell lines . The frequency of SNP occurrence within the uPA gene and whether this frequency changed during cellular aging, or after cell transformation, were determined . The results demonstrated three donor-dependent SNPs . One SNP was located at base pair 422, which is in the region of the gene responsible for encoding the high-molecular weight domain of uPA (HMW-uPA) . The other SNPs were located at base pairs 691 and 822, both of which are in the region of the gene responsible for encoding the low-molecular weight domain of uPA (LMW-uPA) . Single-nucleotide polymorphisms were not detected in the portion of the gene responsible for encoding the uPA secretion signal . Leucine or proline would be encoded at amino acid 141 of HMW-uPA as the result of an SNP at base pair 422 . The SNP detected at base pair 691 would encode for lysine or glutamine at amino acid 231 of LMW-uPA . The SNP detected at base pair 822 would not change the encoded asparagine located at position 274 of the protein . The SNPs identified in this study were donor dependent and were not altered during cellular aging, or by changes in karyology due to spontaneous transformation of the cell line . These results demonstrate that the integrity of the uPA gene is stable and not subject to alterations that accompany cell aging or transformation.

Cell Biol Toxicol, 2003 Aug, 19(4), 227 - 42
Rotating wall vessel as a new in vitro shear stress generation system: application to rat coronary endothelial cell cultures; Morin JP et al.; In this paper, we describe a simple new design for the application of controlled, top-hat profiled wall shear stress forces in a way that is independent of hydrostatic pressure and oxygen tension, based on a rotating wall vessel system . This system has been applied to the culture of rat coronary endothelial cells obtained with a Langendorff-derived procedure isolation . Endothelial cells are immunopurified on the basis of RECA expression, and conservation of endothelial phenotype has been assessed on the basis of morphology, RECA and von Willebrand factor expressions and diI-Ac-LDL uptake . Shear stress induced by the rotating wall vessel was measured using a mathematical formula specifically designed for this type of model, and its impact on coronary endothelial cells was evaluated . Shear stress produced cell orientation parallel to the flux direction, elevated NO production and decreased monocyte adhesion . Cells were kept viable and functional for at least 4 days under shear . This simple design allows the handling and management of numerous vials in parallel and appears to be suitable for large-scale studies of both the acute and chronic impact of modulation of the physico-chemical environment on endothelial cell physiology and function.

Rev Belge Med Dent, 2003, 58(3), 189 - 96
{Importance of cell cultures in biocompatible dental materials research}; Nahid M et al.; The need for biocompatible materials implies the necessity of toxicity testing . The toxicity of a dental material can be evaluated by in vitro tests, by animal experimentation including usage tests, and by clinical studies in humans . In vitro studies are mainly performed to evaluate the cytotoxicity (cell damage) or the genotoxicity (specific DNA damage or chromosomal aberration) of a dental material . Cell or tissue cultures play an important role because they allow a maximum standardization . This paper describes their application and interpretation of results in biocompatibility tests.

Protein Expr Purif, 2003 Nov, 32(1), 126 - 34
Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology; Catzel D et al.; Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described . Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding . As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology . Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH . Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90% . This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield . As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins.

Biotechnol Lett, 2003 Nov, 25(21), 1805 - 9
The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture; Takahashi M et al.; Sericin is the silk protein that covers fibroin fibers and functions as a 'glue' in the cocoons of silkworms, and its most abundant component, Ser1, contains repeats of Ser- and Thr-rich 38 amino acid residues . The viability of Sf9 insect cells was 20, 57 and 49% on the fifth day and 41, 91 and 70% on the ninth day after serum deprivation in the presence of no additives, 3000 microg sericin hydrolysate and 350 microg SerD (the peptide containing the two repetitive units) ml(-1), respectively . Thus, the sericin samples were useful in preventing cell death and promoting cellular growth after acute serum deprivation.

Biol Sci Space, 2003 Oct, 17(3), 194 - 5
Evaluation of cell culture flasks designed for experiment under altered gravity-vector conditions; Gyotoku J et al.; Cell culture flasks applicable for altered gravity conditions, such as centrifugation, clino-rotation or microgravity in space, were manufactured for trial . The flask has flat polystyrene surface for monolayer culture and gas-permeable film window on the opposite face . The space in-between consists the culture chamber to be filled with liquid medium . To reduce the water loss and bubble formation in the culture fluid, another gas permeable window was placed on top to form a space where distilled water may be filled . The double-decker culture flask can be used for both space and ground-based experiments in common.

Environ Mol Mutagen, 2003, 42(4), 258 - 73
Three origins of phiX174 am3 revertants in transgenic cell culture; Malling HV et al.; Transgenic systems for measuring mammalian mutagenesis often use recoverable viral vectors . We hypothesize that mutations in these transgenic systems can arise from three different origins of DNA damage and replication errors and that these three origins of mutations (in vivo, ex vivo, and in vitro) can be differentiated in the PhiX174 am3, cs70 single burst assay (SBA) on the basis of burst size (BS) . In vivo mutations are fixed in the animal, ex vivo mutations are fixed in bacterial cells during recovery of the phage, and in vitro revertants arise during the first replications of nonmutant phages under selective conditions . PX-2 cells, derived from a homozygous embryo of a PhiX174 transgenic mouse, were treated with vehicle or N-ethyl-N-nitrosourea (ENU) . An algorithm was developed to estimate the BS that resulted in the highest induced revertant frequency; the estimate was 56 . In vivo revertants were defined as having BS >55, ex vivo revertants as having a BS of 13-56, and in vitro revertants as having a BS of <14 . The frequencies of in vivo revertants at 0, 100, and 200 mg/kg ENU were 0.06, 0.36, and 4.10 x 10(-6) (dose response, P = 0.004); ex vivo revertants were 0.36, 0.46, and 0.41 x 10(-6) (P = 0.37), and in vitro revertants were 0.39, 0.46, and 0.41 x 10(-6) (P = 0.55), respectively . These results show that only in vivo revertants reflect mutagen treatment . They also provide a basis for identifying PhiX174 am3 revertants induced in vivo and may increase the sensitivity of the assay for in vivo mutation.

ALTEX, 2003, 20(4), 275 - 81
Alternatives to the use of fetal bovine serum: serum-free cell culture; Gstraunthaler G; Serum is commonly used as a supplement to cell culture media . It provides a broad spectrum of macromolecules, carrier proteins for lipoid substances and trace elements, attachment and spreading factors, low molecular weight nutrients, and hormones and growth factors . The most widely used animal serum supplement is fetal bovine serum, FBS . Since serum in general is an ill-defined component in cell culture media, a number of chemically defined serum-free media formulations have been developed in the last two decades . Besides modern cell biological advances in cell and tissue culture and efforts towards a standardisation of cell culture protocols in Good Cell Culture Practice, in addition, considerable ethical concerns were raised recently about the harvest and collection of fetal bovine serum . Thus, in order to decrease the annual need for bovine fetuses in terms of the 3Rs through any reduction in the use or partial replacement of serum, as well as in terms of an improvement of cell and tissue culture methodology, serum-free cell culture represents a modern, valuable and scientifically well accepted alternative to the use of FBS in cell and tissue culture.

Brain Res Mol Brain Res, 2003 Dec 12, 120(1), 9 - 21
Striatal neurons but not nigral dopaminergic neurons in neonatal primary cell culture express endogenous functional N-methyl-D-aspartate receptors; Lui PW et al.; Developmental expression of N-methyl-D-aspartate (NMDA) receptor subunits were determined and compared in striatal and nigral neurons in neonatal primary cell cultures . In striatal neurons, NR1, NR2A and NR2B mRNAs and immunoreactivity, and NR2D mRNA were found and the maximal levels of NR1 mRNA and immunoreactivity expression were found at 6 day-in-vitro (DIV) . NMDA receptors found at this stage in striatal neurons are likely to contain NR1 plus NR2A, NR2B and NR2D subunits . In nigral neurons, NR1 and NR2B mRNAs and immunoreactivity, and NR2D mRNA were found and the maximal level of NR1 immunoreactivity expression was found at 10 DIV . Unlike striatal neurons, NMDA receptors found in nigral neurons are likely to contain NR1 plus NR2B and NR2D subunits only . NMDA-induced toxicity assays showed that striatal neurons were most susceptible to cell death at around 10 DIV but nigral neurons were not susceptible to NMDA-induced cell death at all stages . In addition, patch clamp analysis revealed that functional NMDA receptors could only be found in striatal neurons but not in nigral dopaminergic neurons in vitro . The present results indicate that striatal and nigral neurons are programmed to express distinct NMDA receptor subunits during their endogenous development in cell cultures . Despite dopaminergic neurons in culture display NMDA receptor subunits, functional NMDA receptors are not assembled . The present findings have demonstrated that dopaminergic neurons in vitro may behave very differently to their counterparts in vivo in terms of NMDA receptor-mediated responses . Our results also have implications in transplantations using dopaminergic neurons in vitro in treatments of Parkinson's disease.

Fiziol Zh, 2003, 49(5), 105 - 11
Myelination and demyelination processes in the rat cerebellum cell culture: an electron microscopic study; Pivneva TA et al.; We observed manifestations of the myelination process in dissociated culture of the cerebellar tissue of newborn rats and modifications of the structure of myelin sheaths after treating the culture with a demyelinating factor, blood serum of patients suffering from multiple sclerosis (MS) . On day in vitro (DIV) 26, in the control myelin sheaths of the axons demonstrated the closest resemblance to those observed in vivo, and we selected this term for inducing demyelination . Addition of the serum of MS patients to the culturing medium evoked rapid (in 3-6 hours) dramatic changes in the ultrastructure of myelin sheaths; these were a decrease in the number of the lamellae, their splitting and invagination, formation of vesicles, etc . The serum of MS patients in an acute stage of the disease exerted more intensive demyelination effects than that of patients in a remission stage.

Appl Environ Microbiol, 2003 Dec, 69(12), 7377 - 84
Detection of infectious adenovirus in cell culture by mRNA reverse transcription-PCR; Ko G et al.; We have developed and evaluated the reverse transcription (RT)-PCR detection of mRNA in cell culture to assay infectious adenoviruses (Ads) by using Ad type 2 (Ad2) and Ad41 as models . Only infectious Ads are detected because they are the only ones able to produce mRNA during replication in cell culture . Three primer sets for RT-PCR amplification of mRNA were evaluated for their sensitivity and specificity: a conserved region of late mRNA transcript encoding a virion structural hexon protein and detecting a wide range of human Ads and two primer sets targeting a region of an early mRNA transcript that specifically detects either Ad2 and Ad5 or Ad40 and Ad41 . The mRNAs of infected A549 and Graham 293 cells were recovered from cell lysates with oligo(dT) at different time periods after infection and treated with RNase-free DNase to remove residual contaminating DNA, and then Ad mRNA was detected by RT-PCR assay . The mRNA of Ad2 was detected as early as 6 h after infection at 10(6) infectious units (IU) per cell culture and after longer incubation times at levels as low as 1 to 2 IU per cell culture . The mRNA of Ad41 was detected as soon as 24 h after infection at 10(6) IU per cell culture and at levels as low as 5 IU per cell culture after longer incubation times . To confirm the detection of only infectious viruses, it was shown that no mRNA was detected from Ad2 and Ad41 inactivated by free chlorine or high doses of collimated, monochromatic (254-nm) UV radiation . Detection of Ad2 mRNA exactly coincided with the presence of virus infectivity detected by cytopathogenic effects in cell cultures, but mRNA detection occurred sooner . These results suggest that mRNA detection by RT-PCR assay in inoculated cell cultures is a very sensitive, specific, and rapid method by which to detect infectious Ads in water and other environmental samples.

Arch Inst Pasteur Tunis, 2000, 77(1-4), 67 - 72
{Characterization of a Cl . Perfringens type D strain, isolated in the field and optimization of epsilon toxin biosynthesis in a cell culture}; Maaroufi A et al.; A field strain of cl . perfringens, named Dt001, was isolated from kidney of ovine enterotoemia case . The isolate characterized as Cl . perfringens, type D was based on its cultural and biochemical characters and its factors of virulence . The strain was very toxinogenic and well adapted to culture conditions of biofermentation when the parameters related to ptt, incubation time, substrat .. . were optimized . Thus, the use of carbon source as polymer (destrine), the continuous control of pH allowed improvement of the rate of biosynthesis of Epsilon toxine by 10 times . The study of the immunogenicity of the isolate showed that preparations of anacultures were more immunogenic then those of anatoxine type . The fact that the two forms of epsilon antigens (protoxin and active toxin) show similar immune response in rabbits, indicates that the proteolytic action of trypsin is limited only to the toxic sites and does not affect the immunogenic epsitopes of the toxin . It also suggests a molecular organization of epsilon toxin in which the immunogenic epsitopes and the toxin sites are apart . The biotechnological performances and the immunogenicity and toxinogenical of the Dt001 isolate are in favor of its possible use as a component of an inactivated vaccine against enterotoxenia.

J Pharm Sci, 2004 Jan, 93(1), 108 - 23
Compositional effects of cationic lipid/DNA delivery systems on transgene expression in cell culture; Wiethoff CM et al.; Studies of the contribution of various physical properties of cationic lipid/DNA complexes (CLDCs) to their observed transgene expression in vitro were conducted using cationic liposomes composed of the cationic lipids 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and dimethyldioctadecylammonium bromide (DDAB), with or without equimolar amounts of cholesterol (CHOL) or 1,2-dioleoylphosphatidylethanolamine (DOPE) . The relative degree of luciferase expression by CLDCs is dependent on a complex relationship between net charge of the CLDC as well as previously reported properties, such as membrane fluidity and curvature of the cationic bilayer . Assessments were made of the role of these physical properties on CLDC stability in the extracellular medium, the extent of DNA cellular association, and membrane disruption activity . The efficiency of luciferase expression from negatively charged CLDCs is greatly improved by incorporation of DOPE . This result correlates with enhanced resistance to inhibition of gene delivery by heparan sulfate, increased cellular association of DNA, and enhanced membrane disruption activity . Luciferase expression by positively charged CLDCs is greatly reduced by incorporating equimolar amounts of CHOL and DOPE . This result occurs is in spite of increased resistance to heparan sulfate-mediated inhibition of gene delivery, increased DNA cellular association, and enhanced membrane disruption activity . The observed CLDC compositional effects on luciferase expression along with observed effects on the delivery process suggest that a better understanding of the kinetics and specific routes of gene delivery is necessary .

Biophys J, 2003 Dec, 85(6), 3659 - 65
Stochastic model of autocrine and paracrine signals in cell culture assays; Batsilas L et al.; Autocrine signaling systems are commonly studied under cell culture conditions . In a typical cell culture assay, a layer of liquid medium covers a random two-dimensional dispersion of cells, which secrete ligands . In a growing number of experiments, it is important to characterize the spatial range of autocrine and paracrine cell communication . Currently, the spatial distribution of diffusing signals can be analyzed only indirectly, from their effects on the intracellular signaling or physiological responses of autocrine cells . To directly characterize the spatial range of secreted ligands, we propose a stochastic model for autocrine cell cultures and analyze it using a combination of analytical and computational tools . The two main results derived within the framework of this model are 1), an expression for the fraction of autocrine trajectories, i.e., the probability for a ligand to be trapped by the same cell from which it has been secreted; and 2), an expression for the spatial distribution of trapping points of paracrine trajectories . We test these analytical results by stochastic simulations with efficient Brownian dynamics code and apply our model to analyze the spatial operation of autocrine epidermal growth factor receptor systems.

Mutat Res, 2003 Dec 9, 542(1-2), 105 - 16
Heat shock proteins hsp32 and hsp70 as biomarkers of an early response? In vitro induction of heat shock proteins after exposure of cell culture to carcinogenic compounds and their real mixtures; Rossner P Jr et al.; Heat shock proteins (hsp) are a highly conserved group of proteins that are synthesized as a response to different forms of stress (heat, toxic chemicals, diseases, non-physiological pH changes) . Because of their high sensitivity to changes in the environment, these proteins were suggested as possible early biomarkers of exposure in ecotoxicological studies . The purpose of the present study was to check the suitability of hsp 32 and hsp70 as biomarkers of in vitro exposure to environmentally relevant carcinogens: polycyclic aromatic hydrocarbons (PAHs), their nitro-derivates, aromatic amines, acrylonitrile (ACN) and the mixture of organic compounds adsorbed onto ambient airborne particles (extractable organic matter, EOM).The expression of hsp 32 and hsp70 was studied in human diploid lung fibroblasts (HEL cells) and human monocytic leukaemia cells (THP-1 cells) incubated in vitro with different concentrations of dibenzo{a,l}pyrene (DB{a,l}P), 1-nitropyrene, (NP), 4-aminobiphenyl (ABP), ACN and EOM for different periods of time . The incubation of cells with DB{a,l}P, NP, ABP and EOM did not result in increased levels of hsp 32 or hsp70, either in dose- or time-dependent manner . ACN induced the expression of hsp 32 as well as hsp70 in HEL and THP-1 cells, which probably reflects its ability to induce oxidative stress . We conclude that hsp 32 and hsp70 are not suitable biomarkers of an early exposure to PAHs, their nitro-derivates, aromatic amines or EOM under the conditions used.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Dec 25, 798(2), 265 - 74
Metabolism of verapamil: 24 new phase I and phase II metabolites identified in cell cultures of rat hepatocytes by liquid chromatography-tandem mass spectrometry; Walles M et al.; Verapamil is a widely prescribed calcium antagonist, but suffers from extensive first pass metabolism . Despite its frequent use in drug metabolism a complete understanding of its metabolic pathway is still lacking . We thus investigated verapamil's metabolism in cultures of primary rat hepatocytes and isolated metabolites from cell culture media by solid phase extraction (SPE) . In detail, we investigated their structure in multiple liquid chromatography-mass spectrometry (LC-MSn) experiments and found 25 phase I and 14 phase II metabolites . We showed many metabolites to be produced by oxidative dealkylation, and several yet unknown metabolites were identified that stem from hydroxylation and dealkylation reactions . Furthermore, we identified an array of glucuronides and, additionally, a glucoside . Finally, we investigated the enantioselective biotransformation of verapamil and found preferential metabolism of the S-enantiomers . In conclusion, this illustrates again the true complexity of verapamil's disposition.

Metab Eng, 2003 Oct, 5(4), 230 - 45
A comparison of the properties of a Bcl-xL variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures; Figueroa B Jr et al.; The overexpression of bcl-2 and its homologues is a widely used strategy to inhibit apoptosis in mammalian cell culture systems . In this study, we have evaluated the Bcl-2 homologue, Bcl-x(L) and compared its effectiveness to a Bcl-x(L) mutant lacking most of the non-conserved unstructured loop domain, Bcl-x(L)Delta (deletion of amino acids 26 through 83) . The cell line, Chinese hamster ovary (CHO), was genetically modified to express constitutively Bcl-x(L) or the Bcl-x(L) variant and subjected to model apoptotic insults including Sindbis virus (SV) infection, gradual serum withdrawal, and serum deprivation . When cells were engineered to overexpress Bcl-x(L)Delta, cell death due to the SV was inhibited, and Bcl-x(L)Delta provided comparable protection to the wild-type Bcl-x(L) even though expression levels were much lower for the mutant . Furthermore, the cells expressing Bcl-x(L)Delta continued to proliferate following infection while CHO-bcl-x(L) ceased proliferation immediately following infection . As a result, total production of a heterologous protein encoded on the SV was highest in cell lines expressing Bcl-x(L)Delta . Cells expressing the variant Bcl-x(L) also continued to proliferate and showed increased viable cell numbers following gradual serum withdrawal . In contrast, wild-type Bcl-x(L) expressing CHO cells were found to arrest growth but maintain viability following serum withdrawal . Interestingly, CHO cells expressing Bcl-x(L)Delta were also able to recover and return to rapid growth rates much faster than either the wild-type CHO-bcl-x(L) or CHO following the replenishment of fresh complete medium containing 10% FBS . Confocal imaging of yellow fluorescent protein (YFP) fused to the N terminus of Bcl-x(L) and Bcl-x(L)Delta indicated dense aggregates of the Bcl-x(L)Delta while the wild-type protein was distributed throughout the cell in a manner resembling transmembrane localization . As an alternative to complete removal of the loop domain, Bcl-x(L) variants were created in which aspartate residues containing potential caspase recognition sites within the loop domain of Bcl-x(L) were removed . Cell populations expressing various Bcl-x(L)-Asp mutants were exposed to an apoptotic spent medium stimulus, and the cells expressing these Bcl-x(L) variants provided increased viabilities as compared to cells containing wild-type Bcl-x(L) protein . These studies indicate that modification of anti-apoptotic genes can affect multiple cellular properties including response to apoptotic stimuli and cell growth . This knowledge can be valuable in the design of improved apoptosis inhibitors for biotechnology applications.

Neuron, 2003 Oct 30, 40(3), 447 - 9
Lost in space: misregulated positional cues create tripotent neural progenitors in cell culture; Stiles CD; In a widely held view of vertebrate CNS development, neurons, astrocytes, and oligodendrocytes arise from a common tripotent progenitor cell . However, tripotent progenitors have never been detected in developing embryos . In this issue of Neuron, Gabay et al . show that tripotent progenitors can be created in vitro by deregulation of normal dorsoventral positional cues.

J Neurooncol, 2003 Oct, 65(1), 77 - 85
Combination fusion protein therapy of refractory brain tumors: demonstration of efficacy in cell culture; Liu TF et al.; Primary brain tumors including anaplastic astrocytomas and glioblastoma multiforme are difficult to treat because of their locally invasive nature and relative resistance to chemotherapy and radiation therapy . Novel agents that can kill multi-drug resistant tumor cells and reach tumor cells at distant sites in the brain are needed . One such class of agents is fusion proteins which consist of brain-tumor-selective peptide ligands fused to peptide toxins . The ligand directs the protein to the glioma cell surface; the peptide toxin is then internalized into the cell, translocates to the cytosol and catalytically inactivates protein synthesis leading to cell death . Fusion proteins are toxic to multi-drug resistant brain tumor cells . Because of the large molecular weight of these molecules, a unique delivery system has been developed--convection-enhanced delivery (CED) . The method creates a bulk flow which supplements diffusion and achieves drug concentrations in the brain parenchyma orders of magnitude greater than by systemic administration . Patients with recurrent glioma treated with individual fusion protein CED have obtained clinical remissions lasting years . However, toxicities to normal brain have been observed and relapses ultimately occurred . To address the clinical need of these patients and improve upon the therapeutic index observed to date with single fusion protein CED, we generated a novel fusion protein DAB389EGF and tested it in combination with another active fusion protein, IL13PE38QQR . We observed potent glioma cytotoxicity with each fusion protein and synergistic toxicity with the combination . Further, brain tumor cells showed heterogeneous expression of individual receptors suggesting that the combination--DAB389EGF and IL13PE38QQR may show improved efficacy and should undergo further preclinical development for therapy of patients with relapsed high-grade gliomas.

Virology, 2003 Nov 25, 316(2), 191 - 201
Clinical isolates of GB virus type C vary in their ability to persist and replicate in peripheral blood mononuclear cell cultures; George SL et al.; GB virus C/hepatitis G virus (GBV-C) replication in vitro is inefficient and inconsistent . In this study, clinical isolates of GBV-C were evaluated using peripheral blood mononuclear cell (PBMC) based culture methods . Isolates varied consistently in their ability to persistently replicate, and yield increased in cells grown without PHA/IL-2 stimulation . The deduced polyprotein sequence of an isolate that replicated well was determined (GenBank AY196904) and compared to 20 full-length GBV-C sequences . Fourteen of the 16 unique amino acid polymorphisms identified were in the coding regions for nonstructural proteins associated with interferon resistance and RNA replication . These data indicate that clinical GBV-C isolates vary in their ability to persist in culture, do not require PHA/IL-2 stimulation, and that sequence variability in key regulatory regions may affect growth in PBMC cultures . Since GBV-C appears to inhibit HIV replication in a coinfection model, these studies should facilitate determination of the mechanism of this interaction.

J Obstet Gynaecol Res . 2003 Oct;29(5):361.
Study on the Mechanism of the Bone Formation Process by 17beta-estradiol during Rat Bone Marrow Stromal Cell Culture; Sado T et al.; The aim of this study is to clarify the mechanism of bone formation by using 17beta-estradiol (E2) during rat bone marrow stromal cell culture . Stromal cells were separated from female rat femora (seven weeks old), and were cultured in an Eagle Minimal Essential Medium (MEM) containing 15% fetal calf serum (FCS) . After confluence, the cells were subcultured in a MEM containing 15% FCS (sex steroids removed by charcoal treatment) with dexamethasone, Na betaglycerophosphate and ascorbic acid phosphate for 18 days . They were then cultured in medium containing E2 or insulin-like growth factor-Iota (IGF-Iota) alone or with anti-IGF-Iota antibody . On the 14th day, the formation of white colored nodules showing positive ALP activity in ALP staining and showing calcium deposition in alizarin red S staining was observed in each culture system, indicating the beginning of calcification . The addition of E2 (10-7M or 10-8M), or IGF-Iota (10 and 100 ng/ml) enhanced ALP activity and calcium content significantly, but 17alpha-estradiol did not . Anti-IGF-Iota antibody significantly inhibited enhancing effects of E2 and IGF-Iota on ALP activity and calcium deposition . Incubating with E2 alone (10-8M), the medium IGF-Iota concentration and IGF-I mRNA expression in the cells were significantly increased on days 10~14, but the expression of IGF-Iota receptor mRNA had not changed . These results suggest that E2 participates in bone formation by promoting IGF-Iota production in the bone marrow stromal cells just before calcification starts.

Antiviral Res, 2003 Oct, 60(2), 91 - 102
Replication of the hepatitis C virus in cell culture; Bartenschlager R et al.; Studies of hepatitis C virus (HCV) replication in cell culture have been greatly facilitated by the development of genetically engineered viral genomes that are capable of self-amplifying to high levels in a human hepatoma cell line . Since the original description of this 'replicon' model in 1999, important improvements have been made . Most notably, cell culture adaptive mutations were identified in various non-structural proteins that enhance RNA replication by several orders of magnitude . More recently, the permissiveness of the host cell was determined as an additional important factor contributing to efficient RNA replication . These discoveries allowed the development of transient replication assays, selectable full length genomes and a variety of novel replicons that will be useful for basic studies and facilitate the development of antiviral drugs . Ultimately, the replicon system may help to decipher the molecular basis of interferon-alpha (IFN-alpha) resistance.

Neurotoxicology, 2003 Dec, 24(6), 787 - 96
Morphological effects of neuropathy-inducing organophosphorus compounds in primary dorsal root ganglia cell cultures; Massicotte C et al.; Chick embryo dorsal root ganglia (DRG) cultures were used to explore early pathological events associated with exposure to neuropathy-inducing organophosphorus (OP) compounds . This approach used an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN) . DRG were obtained from 9-day-old chick embryos, and grown for 14 days in minimal essential medium (MEM) supplemented with bovine and human placental sera and growth factors . Cultures were then exposed to 1 microM of the OP compounds phenyl saligenin phosphate (PSP) or mipafox, which readily elicit OPIDN in hens, paraoxon, which does not cause OPIDN, or the DMSO vehicle . The medium containing these toxicants was removed after 12 h, and cultures maintained for 4-7 days post-exposure . Morphometric analysis of neurites was performed by inverted microscopy, which demonstrated that neurites of cells treated with mipafox or PSP but not with paraoxon had decreased length-to-diameter ratios at day 4 post-exposure . Ultrastructural alterations of neurons treated with PSP and mipafox included dissolution of microtubules and neurofilaments and degrading mitochondria . Paraoxon-treated and DMSO control neuronal cell cultures did not show such evident ultrastructural changes . This study demonstrates that chick DRG show pathological changes following exposure to neuropathy-inducing OP compounds.

Apoptosis, 1999 Oct, 4(5), 335 - 55
Role of Ca2+, Mg2+ and K+ ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture; Ishaque A et al.; We have addressed the possibility that Ca2+, Mg2+ and K+ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death . By removing Ca2+, Mg2+ or K+ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability . The differences were explained in terms of a possible reduction in their respective intracellular levels . From several lines of evidence, the deprivation of K+ ions was the most detrimental to cellular growth and viability and induced significant levels of early apoptotic cells . Another effect of this deprivation was to weaken the plasma membranes without causing membrane breakdown; exposure to high agitation rates confirmed fragility of the cell membranes . Removal of Mg2+ caused a reduction in the levels of early apoptotic cells and predisposed cells to high levels of primary necrotic death . The lower levels of apoptotic cells failed to demonstrate the classic nuclear morphology associated with apoptosis, while retaining other apoptotic features . These results highlighted the importance of utilizing several assays for the determination of apoptosis . The absence of Ca2+ appeared to be the mildest insult, but its deprivation did accelerate a significant decline in culture by increasing apoptotic death . Hybridoma cells overexpressing the apoptotic suppressor gene bcl-2 were protected from the predominantly necrosis inducing effects of Mg2+ ion deprivation and apoptosis inducing effects of Ca2+ ion deprivation . However, apoptosis was not as effectively suppressed in bcl-2 cells responding to incubation in K+ free medium . The inclusion of bcl-2 activity in the mechanisms of Ca2+ Mg2+ or K+ deprivation induced cell death emphasizes a close relationship between ionic dissipation and the apoptotic process.

Am J Reprod Immunol, 2003 Sep, 50(3), 220 - 3
Interleukin-2 production in whole blood cell cultures of women undergoing controlled ovarian hyperstimulation for assisted reproduction technology cycles; Orvieto R et al.; PROBLEM: To determine whether human chorionic gonadotropin (hCG) modulates the in vitro release of interleukin (IL-2) from human peripheral lymphocytes and monocytes derived from patients undergoing controlled ovarian hyperstimulation (COH) . METHOD OF STUDY: A large university-based IVF unit was used for the study . Blood was drawn thrice from 12 women undergoing our routine IVF long gonadotropin-releasing-hormone-analog protocol during the COH cycle: (1) day on which adequate suppression was obtained (Day-S); (2) day of or prior to hCG administration (Day-hCG); and (3) day of ovum pick-up (Day-OPU) . At each point of time, blood was tested for sex-steroid levels and then cultured for 72 hr either without (control-culture) or with hCG (hCG-culture) or with mitogenic stimulation by phytohemagglutinin (PHA-culture) . The culture-medium supernatants were tested for IL-2 levels with a commercial sandwich enzyme-linked immunoassay . RESULTS: Whole blood culture IL-2 levels increased significantly during COH until peak E2, and then decreased significantly after hCG administration . IL-2 levels were decreased in the control- and PHA-culture media on Day-OPU compared with Day-hCG . There were no significant correlations between IL-2 levels in the culture media and serum estradiol, progesterone or human chorionic gonadotropin levels . CONCLUSION: Apparently, hCG attenuates IL-2 production by mononuclear cells with and without mitogenic stimulation, irrespective of the estradiol level . This suggests that hCG may indirectly modulate the inflammatory response, resulting in the ovarian hyperstimulation syndrome.

Proteomics, 2003 Oct, 3(10), 1835 - 62
Toward the identification of liver toxicity markers: a proteome study in human cell culture and rats; Thome-Kromer B et al.; The effects of toxic and nontoxic compound treatments were investigated by high resolution custom developed 2-11 pH gradient NEPHGE (non equilibrium pH gradient electrophoresis) two-dimensional electrophoresis . Two models were compared: (i) in vivo rat and (ii) the human cell line HepG2, to test their suitability in a proteomics based approach to identify a toxicity marker . 163 and 321 proteins were identified from the rat liver and the HepG2 proteome . These represent various isoforms of 113 and 194 different NCBI annotated gene sequences, respectively . Nine compounds were selected to induce proteome variations associated with liver toxicity and metabolism . The rat liver proteome database consists of 78 gels, the HepG2 database of 52 gels . Variant proteins were assessed regarding their usefulness as a toxicity marker by evaluating their treatment specificity against multiple control treatments . Thirteen potential toxicity marker proteins were found in rat liver and eight in HepG2 . Catalase and carbamoylphosphate synthetase-1 isoforms were found to be significantly changed after treatment by 4/4 and 3/4 toxic compounds in rat liver, respectively . Aldo-keto-reductase family 1, member C1 was implicated for 3/4 liver cell toxic compounds in HepG2 . Our approach was able to differentiate the quality of potential toxicity markers and provided useful information for an ongoing characterization of more compounds in a wider number of toxicity classes.

Mikrobiol Z, 2003 Jul-Aug, 65(4), 11 - 6
{Invasiveness and cytopathogenicity of Helicobacter pylori in human cell culture}; Konareva OP et al.; Invasive and cytopathogenic properties of clinical strains of Helicobacter pylori in the cell culture Hep-2 have been studied . It has been established that H . pylori possesses high invasive and adhesive capacity . It has been shown that H . pylori takes considerable effect on the indices of mitotic activity, such as mitotic index (MI), level of pathogenic mitoses; it also stimulates the appearance of pathologies connected with the disturbance of mitotic apparatus and damage of chromosomes . H . pylori in the cell culture Hep-2 was observed to evoke a tendency to neoplastic growth, loss of contact inhibition, resulting in the cells piling up on each other with formation of the transformation loci.

Anal Chem, 2003 Nov 15, 75(22), 6043 - 9
A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture; Ibarrola N et al.; Posttranslational modifications are major mechanisms of regulating protein activity and function in vertebrate cells . It is essential to obtain qualitative information about posttranslational modification patterns of proteins to understand signal transduction mechanisms in greater detail . However, it is equally important to measure the dynamics of posttranslational modifications such as phosphorylation to approach signaling networks from a systems biology perspective . Despite a number of advances, methods to quantitate posttranslational modifications remain difficult to implement due to a number of factors including lack of a generic method, elaborate chemical steps, and requirement for large amounts of sample . We have previously shown that stable isotope-containing amino acids in cell culture (SILAC) can be used to differentially label growing cell populations for quantitation of protein levels . In this report, we extend the use of SILAC as a novel proteomic approach for the relative quantitation of posttranslational modifications such as phosphorylation . We have used SILAC to quantitate the extent of known phosphorylation sites as well as to identify and quantitate novel phosphorylation sites.

Am J Physiol Gastrointest Liver Physiol, 2004 Apr, 286(4), G588 - 95 Epub 2003 Nov 13.
Folate deprivation reduces homocysteine remethylation in a human intestinal epithelial cell culture model: role of serine in one-carbon donation; Townsend JH et al.; Little is known about homocysteine metabolism in intestine . To address this question, we investigated homocysteine metabolism under conditions of folate adequacy and folate deprivation in the Caco-2 cell line, a model of human intestinal mucosal cells . Caco-2 cells were cultured in media enriched with {3-(13)C}serine and {U-(13)C(5)}methionine tracers, and enrichments of intracellular free amino acid pools of these amino acids as well as homocysteine, cystathionine, and cysteine were measured by using gas chromatography/mass spectrometry . Homocysteine transsulfuration plus folate-dependent and total remethylation were quantified from these amino acid enrichments . Homocysteine remethylation accounted for 19% of the intracellular free methionine pool in cells cultured with supplemental folate, and nearly all one-carbon units used for remethylation originated from the three carbon of serine via folate-dependent remethylation . Labeling of cystathionine and cysteine indicated the presence of a complete transsulfuration pathway in Caco-2 cells, and this pathway produced 13% of the intracellular free cysteine pool . Appearance of labeled homocysteine and cystathionine in culture medium suggests export of these metabolites from intestinal cells . Remethylation was reduced by one-third in folate-restricted cell cultures (P < 0.001), and only approximately 50% of the one-carbon units used for remethylation originated from the three carbon of serine under these conditions . In conclusion, the three carbon of serine is the primary source of one-carbon units used for homocysteine remethylation in folate-supplemented Caco-2 cell cultures . Remethylation is reduced as a result of folate restriction in this mucosal cell model, and one-carbon sources other than the three carbon of serine contribute to remethylation under this condition.

J Invertebr Pathol, 2003 Oct, 84(2), 119 - 27
Use of cell culture media for cultivation of the mite pathogenic fungi Neozygites tanajoae and Neozygites floridana; Delalibera I Jr et al.; The pathogenic fungus Neozygites tanajoae, one of the most efficient natural enemies of the cassava green mite (CGM) Mononychellus tanajoa in Brazil, was introduced experimentally in Benin in 1998/1999 for the control of CGM . Isolation methods and culture media for in vitro production of N . tanajoae are reported for the first time in this study . Continuous growth of N . tanajoae was achieved using medium NT-1 (IPL-41+5-10% fetal bovine serum+0.3% lactalbumin hydrolysate+0.3% yeastolate) . This medium supported production of N . tanajoae up to 1.53 (+ or - 0.08) x 10(7) hyphal bodies/mL after 8 days . The growth of N . tanajoae from Cruz das Almas, Brazil, was compared to the growth of two Neozygites floridana isolates with wider host ranges from North Carolina, US, and Palmira, Colombia, in 11 cell culture media . We demonstrated that differences in nutritional requirements exist between N . tanajoae and the similar species, N . floridana . N . tanajoae is a particularly fastidious species highly specific to CGM and grows well in few media while N . floridana which is less host specific, grows in a broader range of media, including serum free media . N . floridana isolates produced more than 2 x 10(6) hyphal bodies/mL in > or =7 of the 11 media tested . However, the N . tanajoae isolate reached the same final concentration in only 3 media . Cell densities of N . tanajoae also increased slower than in N . floridana isolates in most media . N . tanajoae differed morphologically from the two N . floridana isolates in vitro . Hyphal bodies of eight N . tanajoae isolates are shorter than hyphal bodies of the two N . floridana isolates . The distinction of these two species was initially proposed based on host specificity, genetic and physiological patterns and is supported by the results presented in this study.

Vaccine, 2003 Dec 12, 22(2), 177 - 84
Post-exposure therapy in mice against experimental rabies: a single injection of DNA vaccine is as effective as five injections of cell culture-derived vaccine; Bahloul C et al.; Two rabies post-exposure therapies were comparatively evaluated: BALB/c mice were challenged at day 0 with rabies virus and then received either a single dose of rabies DNA vaccine administered at day 0, or five doses of cell culture-derived rabies vaccine administered at days 0, 3, 7, 15 and 28 . Both regimens, rapidly triggered protective levels of neutralizing antibodies against rabies virus in vaccinated mice . In addition, one injection of DNA vaccine protected 53% of the challenged mice, compared to 40% of mice protected after five injections of cell culture-derived vaccine . We conclude that rabies post-exposure vaccination in BALB/c mice, based on a single administration of rabies DNA vaccine might be at least as effective as five injections of cell culture-derived vaccine.

Toxicol Lett, 2003 Dec 15, 146(1), 1 - 8
Gliadin cytotoxicity and in vitro cell cultures; Elli L et al.; Interest in celiac disease, a common enteropathy in Europe and the USA (1/200) caused by the dietary ingestion of gluten in susceptible subjects, has increased over the last few years . Its pathogenesis is still not completely clear, but it certainly involves immune-mediated mechanisms . Although a number of studies have been published concerning the role of T cells in inducing intestinal damage, little is known about the early stages in which gliadin (the toxic component of gluten) starts the whole process . In vitro two- and three-dimensional (multicellular spheroid) cell cultures are a simple and useful means of studying the direct cellular effects of gliadin and other "toxic" cereal peptides . Furthermore, in addition to improving our understanding of pathogenetic mechanisms, cell cultures can also be used to test modified peptides that could replace the toxic components present in the foods that celiac patients must avoid.

Clin Exp Rheumatol, 2003 Sep-Oct, 21(5), 587 - 92
Use of human chondrocyte cell cultures to identify and characterize reactive antibodies in rheumatoid arthritis sera; Sabbatini A et al.; OBJECTIVE: To study the reactivity of rheumatoid arthritis (RA) sera with human chondrocyte populations isolated from normal cartilage and expanded in vitro . METHODS: Human articular chondrocytes were cultured as adherent (non-differentiated) cells on plastic dishes or in suspension (differentiated) on dishes previously coated with a thin layer of 1% agarose . Sera from 28 RA patients and 5 paired synovial fluids were tested on lysates from chondrocytes and fibroblasts as control by immunoblot . Antigen expression on the cell membrane was evaluated by flow cytometry in a few sera . RESULTS: In 9/28 RA sera IgG antibodies specific for chondrocyte antigens (97 kDa, 74 kDa, 67 kDa, 60 kDa, 54 kDa, 48 kDa and 37 kDa) were detected . Twelve sera reacted with proteins expressed both on chondrocytes and fibroblasts and 7 with fibroblasts only; two sera had no reactivity . When lysates from adherent or suspension chondrocytes were compared, RA sera reacted with higher intensity and detected more antigens on chondrocytes cultured in suspension . Flow cytometry assay demonstrated that RA sera are able to recognize antigens expressed on the cell membrane of the human chondrocytes . CONCLUSION: Our data indicate that: a) 32% of the RA sera contain antibodies reactive with antigens expressed exclusively by chondrocytes, but this value rises to 75% if antigens expressed both by chondrocytes and fibroblasts are considered; b) the reactivity of fully differentiated chondrocytes in suspension culture is higher than the reactivity of chondrocytes cultured in monolayer; and c) some of the chondrocyte-specific antigens identified are associated with the chondrocyte membrane . Thus, in vitro cultured chondrocytes may be used to study both the specificity and the biological activity of autoantibodies in RA.

Histochem Cell Biol, 2003 Nov, 120(5), 391 - 400 Epub 2003 Nov 05.
Human osteoclasts express different CXC chemokines depending on cell culture substrate: molecular and immunocytochemical evidence of high levels of CXCL10 and CXCL12; Grassi F et al.; Chemokines are important mediators of chemotaxis, cell adherence, and proliferation and exert specific functions in bone remodeling . Despite the potential intriguing role of chemokines in the regulation of osteoclast (OC) functions, little is known about the expression of chemokines and their receptors in human OCs at different stages of differentiation . Therefore, we analyzed the expression of CXC chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5) and ligands (CXCL8, CXCL10, CXCL12 and CXCL13) both at molecular and protein levels, in human OCs grown on plastic or calcium phosphate-coated slides at different stages of differentiation . Real-time PCR showed that CXCR1, CXCR2, CXCR3, CXCR4, CXCR5 and CXCL8 were expressed in undifferentiated cells and significantly decreased during OC differentiation . By contrast, CXCL10 and CXCL12 were strongly upregulated from day 0 to day 8 in cells grown on calcium phosphate-coated slides . Immunocytochemistry showed that OCs grown on plastic expressed CXCR3, CXCR4, CXCR5, CXCL8 and CXCL12, while they were negative for CXCR1, CXCR2 and CXCL10 . Interestingly, both at molecular and protein levels CXCL10 and CXCL12 significantly increased only when cells were differentiated on calcium phosphate-coated slides . These data suggest that the selection of a substrate that better mimics the tridimensional structure of bone tissue, thus favoring OC maturation and differentiation, may be necessary when studying osteoclastogenesis in vitro.

Phytochemistry, 2003 Dec, 64(7), 1229 - 38
LC-NMR and LC-MS analysis of 2,3,10,11-oxygenated protoberberine metabolites in Corydalis cell cultures; Iwasa K et al.; The metabolism of 2,3,10,11-oxygenated protoberberine alkaloids was studied in cell cultures of Corydalis species . Without prior isolation, the structures of the metabolites were determined by LC-MS and LC-NMR analyses . Tetrahydropseudocoptisine alpha-N-metho salt, pseudoprotopine, and pseudomuramine were identified for the first time, and preliminary evidence for metabolic pathways to the formation of these alkaloids were obtained.

Insect Biochem Mol Biol, 2003 Dec, 33(12), 1239 - 47
Juvenile hormone potentiates ecdysone receptor-dependent transcription in a mammalian cell culture system; Henrich VC et al.; Insect development is guided by the combined actions of ecdysteroids and juvenile hormones (JHs) . The transcriptional effects of ecdysteroids are mediated by a protein complex consisting of the ecdysone receptor (EcR) and its heterodimeric partner, Ultraspiracle (USP), but a corresponding JH receptor has not been defined conclusively . Given that the EcR ligand binding domain (LBD) is similar to that of the JH-responsive rat farnesoid-X-activated receptor (FXR), we sought to define experimental conditions under which EcR-dependent transcription could be promoted by JH . Chinese hamster ovary (CHO) cells were transfected with a plasmid carrying an ecdysteroid-inducible reporter gene, a second plasmid expressing one of the three amino-terminal variants of Drosophila EcR or an EcR chimera, and a third plasmid expressing either the mouse retinoid X receptor (RXR), or its insect orthologue, USP . Each of the EcR variants responded to the synthetic ecdysteroid, muristerone A (murA), but a maximal response to 20-hydroxyecdysone (20E) was achieved only for specific EcR combinations with its heterodimeric partner . Notably, the Drosophila EcR isoforms were responsive to 20E only when paired with USP, and only EcRB2 activity was further potentiated by JHIII in the presence of 20E . EcR chimeras that fuse the activator domains from VP16 or the glucocorticoid receptor to the Drosophila EcR DNA-binding and ligand-binding domains were responsive to ecdysteroids . Again, the effects of JHIII and 20E were associated with specific partners of the chimeric EcRs . In all experiments, the LBD of EcR proved to be the prerequisite component for potentiation by JHIII, and in this conformation may resemble the FXR LBD . Our results indicate that EcR responsiveness is influenced by the heterodimeric partner and that both the N-terminal domain of EcR and the particular ecdysteroid affect JHIII potentiation.

J Exp Zoolog A Comp Exp Biol, 2003 Nov 1, 300(1), 76 - 83
Voltage-Gated ion currents of schwann cells in cell culture models of human neurofibromatosis; Fieber LA; K(+) (K) channels play a role in the proliferation of many cell types in normal cells and certain disease states . Several laboratories have studied K currents in cultured Schwann cells from models of the human diseases, neurofibromatosis type 1 (NF1) and neurofibromatosis type 2 (NF2) . These diseases are characterized by the growth of Schwann cell tumors . In all cell culture NF models the K current properties differ in tumor-derived and normal Schwann cells . Depending on the model however, the type of K channel abnormality differs . K channels appear to play a role in the proliferation of Schwann cell cultures of these disease models, because a link has been established between K current blockade and the inhibition of Schwann cell proliferation in NF1 and NF2 . Differences in the proliferation response of normal Schwann cells to K channel blockers suggest that in vitro regulation of proliferation in neoplastic and normal Schwann cells is complex .

J Biochem Biophys Methods, 2004 Jan 30, 58(1), 49 - 58
Quantitative determination of carbon monoxide in cell culture supernatants by spectrophotometric analysis; Stonek F et al.; A simple, sensitive, nonradioactive method for measuring carbon monoxide (CO) in cell culture supernatant is described . Dissolved CO reacts with hemoglobin (Hb) to carboxyhemoglobin (HbCO) in a modified Conway cell . HbCO is quantified by spectrophotometric analysis, and total concentration of CO given in microg CO/l cell culture supernatant is mathematically calculated . Furthermore, we compared our newly developed method with a recently published method . Confluent human umbilical vein epithelial cells (HUVEC) were incubated with 10(-6) M hydrocortisone known to induce heme oxygenase-2 protein and transcript expression for 4 h and CO production was measured . Levels following hydrocortisone treatment were significantly enhanced compared to controls when using our newly developed technique (p<0.05), whereas only a nonsignificant trend could be observed using the recently published method . We conclude that this nonradioactive technique to quantify CO is more sensitive than previous ones, thereby allowing to measure even physiologic quantities of CO in aqueous solutions.

Plasmid, 2003 Nov, 50(3), 230 - 5
Production of functional recombinant tyrosine hydroxylase by the BPV-1 expression plasmids in the cell cultures; Lampela P et al.; Bovine papilloma virus type-1 (BPV-1)-based expression plasmids TkBPVTH and CGalBPVTH encoding the rat tyrosine hydroxylase (TH) enzyme have been designed for the development of gene therapy for experimental Parkinson's disease . The aim of the present work was to examine the transfection of BPVTH plasmids to express a dopaminergic transgene in the monkey CV1-P fibroblast, rat C6 glioma and human NHA astrocyte cell cultures . The biological function of the transgene was estimated by analyzing the production of recombinant TH mRNA and protein, and the synthesis of L-dopa and dopamine . The highest transfection efficiency was obtained using TkBPVTH plasmids (5 microg) . Furthermore, the expression of TkBPVTH plasmids was associated with significant synthesis of TH enzyme and L-dopa in the C6 and NHA cell cultures.

Planta, 2004 Jan, 218(3), 492 - 6 Epub 2003 Nov 01.
Biphenyl synthase from yeast-extract-treated cell cultures of Sorbus aucuparia; Liu B et al.; Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae . The biphenyl aucuparin accumulated in Sorbus aucuparia L . cell cultures in response to yeast extract treatment . Incubation of cell-free extracts from challenged cell cultures with benzoyl-CoA and malonyl-CoA led to the formation of 3,5-dihydroxybiphenyl . This reaction was catalysed by a novel polyketide synthase, which will be named biphenyl synthase . The most efficient starter substrate for the enzyme was benzoyl-CoA . Relatively high activity was also observed with 2-hydroxybenzoyl-CoA but, instead of the corresponding biphenyl, the derailment product 2-hydroxybenzoyltriacetic acid lactone was formed.

J Hematother Stem Cell Res, 2003 Oct, 12(5), 575 - 85
Dendritic cell culture: a simple closed culture system using ficoll, monocytes, and a table-top centrifuge; Celluzzi CM et al.; Dendritic cells (DCs) are potent antigen-presenting cells involved in the induction of T cell-mediated immune responses and as such have emerged as important candidates for cellular-based therapies . Critical to safe clinical use is the easy manipulation of DCs and their precursors in a closed system . We have developed a serum-free, closed culture system applying a simple wash-Ficoll centrifugation method to reduce platelet and red blood cell (RBC) contamination . This procedure optimized adherence of monocytes (44 +/- 10.9% recovery, >85% expressed CD14(+)/CD163(+)) for the generation of DCs from mononuclear cell (MNC) apheresis units . Most RBCs and up to 98% of platelets were removed . Following density sedimentation, cell viability remained high (98 +/- 2%) with only minimal loss of monocytes (3 +/- 3%) . Importantly, Ficoll-treated monocytes retained their ability to differentiate to mature DCs demonstrated by morphology, phenotype (MHC class II(+), CD1a(+), CD80(+), CD86(+), and CD83(+)), ability to stimulate mixed lymphocyte responses (MLR), present antigen, and produce interleukin-12 (IL-12) . Nonadherent CD3(+) (80 +/- 4%) were also isolated for functional assays . Ficoll can be easily incorporated into a simple adherence-based closed system for collection of lymphocytes and adherent monocytes for DC culture . The procedure is relatively fast (effective working time 5-6 h), does not impair monocyte function or induce substantial cell activation, and can be performed economically using equipment found in a typical blood banking environment.

J Infect Dis, 2003 Nov 1, 188(9), 1345 - 51 Epub 2003 Oct 31.
Polymerase chain reaction for detection of herpes simplex virus (HSV) DNA on mucosal surfaces: comparison with HSV isolation in cell culture; Wald A et al.; This study compared the rate of isolation of herpes simplex virus (HSV) from >36000 samples of mucosal secretions obtained from 296 HSV-infected persons versus the rate of detection of HSV DNA, by means of a real-time quantitative polymerase chain reaction (PCR) assay . Overall, HSV was isolated in 3.0% of samples, and HSV DNA was detected in 12.1% of samples . The mean number of HSV DNA copies was 10(4.9) in samples obtained on days when HSV lesions were present and 10(4.4) in samples from days when HSV lesions were absent . There was a linear relationship between the ability to isolate virus in culture and the log number of copies of HSV DNA in the sample; this relationship persisted in samples from men or women, in samples from human immunodeficiency virus-negative or -positive participants, and in samples obtained on days when lesions were present or absent . In home-collected specimens, the ratio of PCR positivity to viral-culture positivity rose from 3.8:1 in the winter to 8.8:1 in the summer months, reflecting the lability of viral-culture specimens transported during warm weather.

Luminescence, 2003 Sep-Oct, 18(5), 249 - 53
Optimization of peroxynitrite-luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide; Chen SL et al.; We established a peroxynitrite-luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide (CS(2)) . Three factors, including exposure time to ozone (Factor A), volume of peroxynitrite (ONOO(-)) solution (Factor B) and luminol concentrations (Factor C) at three levels were selected and the combinations were in accordance with orthogonal design L(9) (3(4)) . Peroxynitrite was generated from the reaction of ozone and 0.01 mol/L sodium azide (NaN(3)) dissolved in carbonic acid buffer solution (pH 11), and it was reacted with luminol to yield chemiluminescence . The peak value, peak time and kinetic curve of the light emission were observed . The selected combination conditions were 50 s ozone, 800 micro L peroxynitrite and 0.001 mol/L luminol solution . Cell culture solution with CS(2) enhanced the emission intensity of chemiluminescence (F = 8.38, p = 0.018) and shortened the peak time to chemiluminescence (F = 139.00, p = 0.0001) . The data demonstrated that this luminol chemiluminescence system is suitable for detecting peroxynitrite in cell culture solutions for evaluating the effect of CS(2) on endothelial cells .

Curr Genet, 2004 Feb, 45(1), 45 - 53 Epub 2003 Oct 29.
Mosaic (MSC) cucumbers regenerated from independent cell cultures possess different mitochondrial rearrangements; Bartoszewski G et al.; Passage of the highly inbred cucumber ( Cucumis sativus L.) line B through cell culture produces progenies with paternally transmitted, mosaic (MSC) phenotypes . Because the mitochondrial genome of cucumber shows paternal transmission, we evaluated for structural polymorphisms by hybridizing cosmids spanning the entire mitochondrial genome of Arabidopsis thaliana L . to DNA-gel blots of four independently generated MSC and four wild-type cucumbers . Polymorphisms were identified by cosmids carrying rrn18, nad5-exon2, rpl5, and the previously described JLV5 deletion . Polymorphisms revealed by rrn18 and nad5-exon2 were due to one rearrangement bringing together these two coding regions . The polymorphism revealed by rpl5 was unique to MSC16 and was due to rearrangement(s) placing the rpl5 region next to the forward junction of the JLV5 deletion . The rearrangement near rpl5 existed as a sublimon in wild-type inbred B, but was not detected in the cultivar Calypso . Although RNA-gel blots revealed reduced transcription of rpl5 in MSC16 relative to wild-type cucumber, Western analyses revealed no differences for the RPL5 protein and the genetic basis of the MSC16 phenotype remains enigmatic . We evaluated 17 MSC and wild-type lines regenerated from independent cell-culture experiments for these structural polymorphisms and identified eight different patterns, indicating that the passage of cucumber through cell culture may be a unique mechanism to induce or select for novel rearrangements affecting mitochondrial gene expression.

World J Urol, 2003 Nov, 21(5), 320 - 4 Epub 2003 Oct 29.
Androgen and estrogen receptors in the human corpus cavernosum penis: immunohistochemical and cell culture results; Schultheiss D et al.; Despite the central and peripheral effects of androgens on the nervous system, the local effects of androgens in the corpus cavernosum penis and their importance for erectile function is still unclear . In this study corpus cavernosum biopsies of eight adult potent patients, aged 19-63 years, undergoing penile deviation surgery (group A) and 12 patients undergoing male-to-female transsexual surgery (group B) were immunostained for nuclear androgen and estrogen-alpha receptors . Additionally, primary corpus cavernosum endothelial cell cultures were obtained from six transsexual patients and exposed to testosterone, dihydrotestosterone, estradiol and progesterone likewise for 7 days . Total cell count was performed and cell metabolic activity was measured by a tetrazolium salt-based assay . Androgen and estrogen-alpha receptors were detected in stromal as well as in endothelial cells . Of all cell nuclei, 74.9% (SD 16.4) in group A and 63.5% (SD 17.1) in group B were positively stained for androgen receptors . The respective percentage of estrogen receptors was 11% (SD 9.5) and 21.2% (SD 12.6) . An age-dependent difference in receptor distribution was not observed in either group . In the cell culture system only cultures exposed to testosterone and dihydrotestosterone showed a dose-dependent increase of cell metabolic activity compared to the cultures supplemented with estradiol and progesterone . The significant and age-independent high androgen and low estrogen-alpha receptor distribution found in both groups suggests a possible peripheral effect of androgens at the level of the corpus cavernosum penis in adult humans . This is supported by the observed effect of testosterone and dihydrotestosterone on cell count and endothelial cell metabolism in our cell culture system . The role of estrogens remains unclear.

Antivir Chem Chemother, 2003 Jul, 14(4), 217 - 23
Antiviral activity of CTC-8 against herpes simplex virus (HSV-1) in cell culture: evidence for a selective antiviral effect via a cellular mechanism; Fitzmaurice T et al.; A synthetic programme produced a series of compounds related to natural prostaglandins, which are known to affect the growth of a number of viruses . Several of the compounds showed potent biological activity including antiviral effects . The compound CTC-8 {(S)-4-tert-butyldimethylsilyloxy-2-cyclopenten-1-one} contains the cyclopentenone ring of prostaglandin A1, but the extended side chains common to the prostaglandin family are truncated . The present study demonstrates that CTC-8 inhibits HSV-1 replication in cell culture at sub-toxic concentrations . The antiviral effect was evidenced by reduction in infectious virus yield, although the compound was not effective in the standard plaque-reduction assay . Time-of-addition studies and other experiments provide a possible explanation for these results by suggesting that the antiviral activity is confined to a single cycle . Under the standard conditions of high-multiplicity infection in BHK cells it was notable that CTC-8 is most effective when added for a short period 6-8 h post-infection . Furthermore, multiple passage of HSV-1 in the presence of CTC-8 did not result in the selection of resistant mutants . The results of these and other experiments are consistent with the hypothesis that the mechanism by which CTC-8 inhibits virus replication involves a cellular target . These results encourage further research into the therapeutic potential of this series of compounds.

J Androl, 2003 Nov-Dec, 24(6), 812 - 21
Characterization of membrane rafts isolated from rat sertoli cell cultures: caveolin and flotillin-1 content; Evans WE 4th et al.; Membrane rafts from Sertoli cell cultures were isolated as detergent-insoluble glycosphingolipid-enriched (DIG) fractions on the basis of their enriched content of glycosphingolipids and cholesterol and the resulting insolubility in 1% Triton X-100 and their low buoyant density . Because lipid rafts have been implicated in numerous cell functions, including cell signaling and sites for actin/membrane attachment, studies were initiated to characterize Sertoli cell rafts . This study reports the distribution of the raft structural proteins, caveolin and flotillin-1, implicated in raft microdomain organization . Methods employed included the immunoblotting of cell lysates and detergent-insoluble glycosphingolipid-enriched (DIG) fractions, the immunofluorescent microscopy of peritubular myoid cell (PMC) cultures and cryostat-sectioned testis, and the immunohistochemical staining of paraffin-embedded sections following microwave antigen retrieval techniques . Sertoli cells and Sertoli DIG fractions were found to lack the common raft-associated protein, caveolin, a marker protein for caveolae, but they are enriched in the 48-kd protein, flotillin-1, a protein also implicated in raft formation, cell signaling, and cell motility . Since the primary cell contaminant of Sertoli cell cultures is the PMC, these cells, along with spermatogenic cell fraction (SPGC), were also examined for caveolin and flotillin-1 content . The PMCs contained significant concentrations of both caveolin and flotillin-1 . PMCs in culture exhibited a punctate caveolin staining pattern at the cell surface characteristic of a caveolar location . These data support the idea that the pinocytotic vesicles observed in PMCs are caveolae . PMCs also show a perinuclear location for caveolin characteristic of a Golgi location . Cryostat sections of rat testis showed a marked concentration of caveolin in the PMCs . The PMC location of caveolin was also confirmed by the immunohistochemical staining of sections from paraffin-embedded rat testis following microwave antigen retrieval techniques . Similar experiments showed a more ubiquitous, stage-specific distribution of flotillin-1 among testicular cell types.

Mol Cell Endocrinol, 2003 Oct 31, 208(1-2), 1 - 10
Estrogen inhibits cell cycle progression and retinoblastoma phosphorylation in rhesus ovarian surface epithelial cell culture; Wright JW et al.; Estrogen promotes the growth of some ovarian cancer cells at nanomolar concentrations, but has been shown to inhibit growth of normal ovarian surface epithelial (OSE) cells at micromolar concentrations (1 microg/ml) . OSE cells express the estrogen receptor (ER)-alpha, and are the source of 90% of ovarian cancers . The potential sensitivity of OSE cells to estrogen stresses the importance of understanding the estrogen-dependent mechanisms at play in OSE proliferation and transformation, as well as in anticancer treatment . We investigated the effects of estradiol on cell proliferation in vitro, and demonstrate an intracellular locus of action of estradiol in cultured rhesus ovarian surface epithelial (RhOSE) cells . We show that ovarian and breast cells are growth-inhibited by micromolar concentrations of estradiol, and that this inhibition correlates with estrogen receptor expression . We further show that normal rhesus OSE cells do not activate ERK or Akt in response to estradiol, nor does estradiol block the ability of serum to stimulate ERK or induce cyclin D expression . Contrarily, estradiol inhibits serum-dependent retinoblastoma protein (Rb) phosphorylation and blocks DNA synthesis . This inhibition does not formally arrest cells, and is reversible within hours of estrogen withdrawal . Our data are consistent with growth inhibition by activation of Rb and indicate that sensitivity to hormone therapy in anticancer treatment can be modulated by cell cycle regulators downstream of the estrogen receptor.

Mol Cell Biochem, 2003 Oct, 252(1-2), 379 - 85
Inducible expression of long-chain acyl-CoA hydrolase gene in cell cultures; Takagi M et al.; A long-chain acyl-CoA hydrolase, BACH, is markedly distributed in the brain and localized in neurons . However, the physiological significance of BACH is unclear . To study the gene function, we expressed the mouse BACH gene in C3H 10T1/2 fibroblastic cells using a mifepristone (RU486)-inducible gene expression system . A cell clone, 10T-S6/44, was generated by stable transfection of two plasmids encoding a mifepristone-dependent transactivator and an inducible transgene product, BACH with a C-terminal MYC-tag (BACH-MYC) . The transgene expression in the 10T-S6/44 cells was tightly regulated by mifepristone . Induction of BACH-MYC and an increase in palmitoyl-CoA hydrolase activity were observed in the cells treated with 3 x 10(-11) M mifepristone and reached maximal levels at a concentration of 1 x 10(-9) M for 48 h . The growth rate of cells showing the maximal induction of BACH-MYC was reduced, whereas phospholipid synthesis was unchanged . These results suggested that BACH affects specific cellular systems and functions, but not all acyl-CoA-utilizing processes.

Brain Res, 2003 Nov 21, 991(1-2), 96 - 103
Circadian rhythm and photic control of cAMP level in chick retinal cell cultures: a mechanism for coupling the circadian oscillator to the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, in photoreceptor cells; Ivanova TN et al.; Arylalkylamine N-acetyltransferase (AANAT) is the penultimate and key regulatory enzyme in the melatonin biosynthetic pathway . In chicken retina in vivo, AANAT is expressed in a circadian fashion, primarily in photoreceptor cells . AANAT activity is high at night in darkness, low during the daytime, and suppressed by light exposure at night . In the present study, we investigated the circadian and photic regulation of adenosine 3',5'-monophosphate (cAMP) in cultured retinal cells entrained to a daily light-dark (LD) cycle, as well as the role of Ca(2+) and cAMP in the regulation of AANAT activity . Similar to AANAT activity, cAMP levels fluctuate in a daily fashion, with high levels at night in darkness and low levels during the day in light . This daily fluctuation continued with reduced amplitude in constant (24 h/day) darkness (DD) . These changes in cAMP appear to be causally related to control of AANAT activity . Adenylyl cyclase and protein kinase A inhibitors suppress the nocturnal increase of AANAT in DD, while 8Br-cAMP augments it . The nocturnal increase of AANAT activity also involves Ca(2+) influx, as it is inhibited by nitrendipine, an inhibitor of L-type voltage-gated channels, and augmented by Bay K 8644, a Ca(2+) channel agonist . The effect of Bay K 8644 was antagonized by the adenylyl cyclase inhibitor MDL 12330A, suggesting a link between Ca(2+) influx, cAMP formation, and AANAT activity in retinal cells . Light exposure at night, which rapidly suppresses AANAT activity, also suppressed cAMP levels . The effect of light on AANAT activity was reversed by Bay K 8644, 8Br-cAMP, and the proteasome inhibitor lactacystin . These results indicate a dynamic interplay of circadian oscillators and light in the regulation of cAMP levels and AANAT activity in photoreceptor cells.

Eur Cell Mater, 2001 Jul 12, 2, 1 - 9
The influence of pore geometry in cp Ti-implants--a cell culture investigation; Stangl R et al.; Biocompatibility testing of differently structured titanium implants was performed using an in vitro test system of a newly established human fetal osteoblastic cell line (hFOB 1.19) . Cell adhesion of osteoblastic cells on the different porous geometries and the suitability of a copper vapor laser system for surface structuring was tested with the following parameters: cell-number, cell viability, alkaline phosphatase expression . The analysis of the cell culture results demonstrated that 25 microm and 200 microm porous geometries showed similar or even better results than the negative control of polystyrene; there was no sign of toxic effects . However, the 100 microm porous geometry showed an impressive negative influence on the calculated parameters . The reason for this effect is unclear . The series with 50 microm, 300 microm, 400 microm and 500 microm showed a comparable, intermediate effect on the cell culture with respect to the different parameters . However, the results were worse than with the 25 and 200 microm porous geometry . In conclusion, the 25 microm and 200 microm porous geometry seems to have the most positive effect on the human osteoblastic cell line hFOB 1.19.

Bone, 2003 Oct, 33(4), 673 - 84
Endothelin-1 promotes osteoprogenitor proliferation and differentiation in fetal rat calvarial cell cultures; von Schroeder HP et al.; Endothelin-1 (ET-1), a peptide produced by vascular endothelial cells, has been suggested to be one of the signaling factors between vascular and osteoblastic cells during bone growth and remodeling . The osteoinductive effects of ET-1 were tested on fetal rat calvaria which have the ability to form bone nodules in culture . ET-1 (10(-10) to 10(-6) M) dose-dependently increased cell proliferation . The effect of ET-1 (10(-8) M) on proliferation was greater than that of dexamethasone (Dex; 10(-8) M) . ET-1 also increased the number of bone nodules by 146% over untreated cells, which coincided with a 3.1-fold increase in alkaline phosphatase activity . Limiting dilution assays showed that ET-1 treatment increased the number of osteoprogenitors (CFU-AP and CFU-OB) beyond what would be expected by a proliferative effect alone, indicating that ET-1 also stimulated osteoblast differentiation . Osteocalcin mRNA expression was upregulated as shown by Northern blot analysis . Using cDNA microarray analysis, ET-1 treatment resulted in an expression profile that included an upregulation of 163 genes and expressed sequence tags . Simultaneous addition of ET-1 and Dex to the medium further increased the number of bone nodules and alkaline phosphatase activity over either treatment alone . Our results show that ET-1 promotes both osteoblastic proliferation and differentiation and that the effects of ET-1 and Dex on differentiation are cooperative.

Bone, 2003 Oct, 33(4), 652 - 9
Lovastatin inhibits adipogenic and stimulates osteogenic differentiation by suppressing PPARgamma2 and increasing Cbfa1/Runx2 expression in bone marrow mesenchymal cell cultures; Li X et al.; The mechanism whereby lovastatin can counteract steroid-induced osteonecrosis and osteoporosis is poorly understood . We assessed the effect of lovastatin on a multipotential cell line, D1, which is capable of differentiating into either the osteoblast or the adipocyte lineage . The expression of bone cell and fat cell transcription factors Cbfa1/Runx2 and PPARgamma2, respectively, were determined . 422aP2 gene expression was analyzed . Osteocalcin promoter activity was measured by cotransfecting the cells with the phOC-luc and pSV beta-Gal plasmids . Lovastatin enhanced osteoblast differentiation as assessed by a 1.8x increase in expression of Cbfa1/Runx2 and by a 5x increase in osteocalcin promoter activity . Expression of PPARgamma2 was decreased by 60% . By enhancing osteoblast gene expression and by inhibiting adipogenesis, lovastatin may shunt uncommitted osteoprogenitor cells in marrow from the adipocytic to the osteoblastic differentiation pathway . Future evaluation of lovastatin and other lipid-lowering drugs will help determine their potential as therapeutic agents for osteonecrosis and osteoporosis.

Biotechnol Adv, 1991, 9(1), 31 - 49
Biotechnology and aquaculture: the role of cell cultures; Bols NC; Cell culturing complements recombinant DNA technology in the application of biotechnology to aquaculture . Cell cultures can be prepared from the three main groups of multicellular organisms in aquaculture: fish, shellfish, and seaweeds . These cultures can contribute indirectly to the successful farming of these organisms by providing basic insights into how their growth, reproduction, and health can be understood and manipulated . Finally, they can be a direct source of diverse biochemical products for use in aquaculture, medicine and the food industry.

Biotechnol Adv, 1990, 8(4), 729 - 39
Manufacture of biopharmaceutical proteins by mammalian cell culture systems; Tolbert WR; In the last several years, dramatic advances have been in the development of new biopharmaceuticals including monoclonal antibodies for diagnosis and treatment and such genetically engineered proteins as tPA, Factor VIIIc, erythropoietin and soluble CD4, an anti-AIDS protein . Currently, there are several hundred such candidate drugs in human clinical trials . In most cases, these protein-based drugs will require manufacture by mammalian cell culture due to the inability of lower organisms to properly glycosylate, fold, make correct disulfide bonds and secrete active biomolecular forms . The need for large scale production from cell culture will greatly increase as more of the products in clinical trials are approved for commercial production . This will require significant reduction in manufacturing costs per gram, concomitant with increased capacity to hundreds or perhaps even thousands of kilograms annually . As an example, Invitron's multi-reactor manufacturing facility has operated at greater than one-half million liters per year and has experience with more than 250 mammalian cell lines for producing protein drug products.

Biotechnol Adv, 2002 May, 20(2), 101 - 53
Plant cell cultures: Chemical factories of secondary metabolites; Rao SR et al.; This review deals with the production of high-value secondary metabolites including pharmaceuticals and food additives through plant cell cultures, shoot cultures, root cultures and transgenic roots obtained through biotechnological means . Plant cell and transgenic hairy root cultures are promising potential alternative sources for the production of high-value secondary metabolites of industrial importance . Recent developments in transgenic research have opened up the possibility of the metabolic engineering of biosynthetic pathways to produce high-value secondary metabolites . The production of the pungent food additive capsaicin, the natural colour anthocyanin and the natural flavour vanillin is described in detail.

J Vet Diagn Invest, 2003 Sep, 15(5), 407 - 17
Infectious salmon anemia virus RNA in fish cell cultures and in tissue sections of atlantic salmon experimentally infected with infectious salmon anemia virus; Moneke EE et al.; Current understanding of the etiopathogenesis of infectious salmon anemia (ISA) virus (ISAV) infection in fish comes mostly from virus detection in homogenized tissues taken from ISA-suspected mortalities . This study combined in situ hybridization (ISH) and histology to demonstrate viral RNA transcripts in different fish cell lines infected with ISAV and in tissues collected during the clinical phase of ISAV infection in Atlantic salmon . For this, a riboprobe to mRNA transcripts of ISAV RNA segment 8 was shown to detect viral mRNA in ISAV-infected TO, CHSE-214, and SHK-1 cell cultures . Specific hybridization was initially detected exclusively in the nuclei of infected cells, which is consistent with the nuclear transcription of orthomyxoviruses . For use of the riboprobe on fish tissues fixed in paraformaldehyde or formalin, the conditions used to permeabilize tissues before ISH (Proteinase K or Tween 20) were first optimized . Tissues were collected 15-20 days after challenge from 7 fresh mortalities of Atlantic salmon parr (approximately 20 g) showing severe gross and microscopic lesions, consistent with ISAV infection . Reverse transcription-polymerase chain reaction on tissue pools confirmed the presence of ISAV in each of the 7 fish . Of the tissues examined in each fish, the heart and liver consistently showed the strongest hybridization signal and, therefore, the most in situ virus, which was located in the endothelium of small blood vessels and in macrophage-like cells.

Protein Pept Lett, 2003 Aug, 10(4), 386 - 95
Proliferative activity of neokyotorphin-related hemoglobin fragments in cell cultures; Sazonova OV et al.; alpha-Hemoglobin fragments alpha-(133-141), alpha-(134-141), alpha-(135-141), alpha-(137-141), alpha-(134-140), alpha-(133-138), alpha-(134-140) and alpha-(137-138) stimulate L929 tumor cell proliferation, alpha-(134-141) being the most active . alpha-(134-141) stimulates proliferation of M3 melanoma cells, murine embryonic fibroblasts, primary cultures of red bone marrow and spleen cells . In L929 cells the effect of alpha-(134-141) is cell density independent; in M3 cells alpha-(137-141) and alpha-(134-141) are most active at density 10,000 cells/well (96 well plate) independently on FBS content.

Mol Diagn, 2003 Mar, 7(1), 41 - 3
Detection of Chlamydia pneumoniae in the cerebrospinal fluid of patients with multiple sclerosis by combination of cell culture and PCR : no evidence for possible association; Chatzipanagiotou S et al.; Background: During the course of multiple sclerosis (MS) intrathecal oligoclonal IgGs are present in the cerebrospinal fluid (CSF) . The intracellular human pathogen Chlamydia pneumoniae may play a role either as a causative pathogenetic agent in the disease, or C . pneumoniae-infected MS patients could be immunologically less able to clear the agent from the central nervous system (CNS).Methods: CSF samples were studied in 100 individuals - 70 MS patients and 30 age-matched controls with other neurological diseases . CSF was taken by lumbal puncture; cell cultures were performed by the cell vial technique, followed by a 4-day incubation at 37 degrees C . A nested PCR was performed.Results: C . pneumoniae was detectable in the CSF of only 2.9% of the MS patients and none of control patients (with no significant difference between the MS patients and controls) . IgG antibodies were positive in only 1.43% of the MS patients and 3.33% of the controls . IgA antibodies were positive in 6.66% of the control patients and none of the patients were positive for IgM antibodies . There was no statistically significant difference between the two groups of patients with respect to the three antibody classes.Conclusions: The results confirm the high leave of controversy surrounding a possible link between C . pneumoniae and MS, and the matter requires further thorough investigation.

Neuro Endocrinol Lett, 2003 Jun-Aug, 24(3-4), 224 - 6
Direct effects of cocaine-amphetamine-regulated transcript (CART) on pituitary hormone release in pituitary cell culture; Baranowska B et al.; OBJECTIVE: Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the rat brain, especially in the hypothalamic nuclei and in the anterior pituitary . The aim of this study was to evaluate the effects of CART on pituitary hormone release in pituitary cell culture . MATERIAL AND METHODS: The pituitary hormone release from pituitary cell culture after CART administration was investigated . Concentrations of LH, FSH, PRL, TSH and GH were measured with RIA methods . RESULTS: CART in all doses (1 nMol, 10 nMol, 100 nMol) stimulated prolactin (PRL) release and inhibited TSH release . CART administration caused a dose dependent decrease in LH release . CART did not change GH release from cultured pituitary cells . CONCLUSION: CART may affect directly pituitary hormones release in the cell culture.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12063 - 8 Epub 2003 Sep 30.
Nuclear localization of pyrrole-imidazole polyamide-fluorescein conjugates in cell culture; Best TP et al.; A series of hairpin pyrrole-imidazole polyamide-fluorescein conjugates were synthesized and assayed for cellular localization . Thirteen cell lines, representing 11 human cancers, one human transformed kidney cell line, and one murine leukemia cell line, were treated with 5 microM polyamide-fluorescein conjugates for 10-14 h, then imaged by confocal laser scanning microscopy . A conjugate containing a beta-alanine residue at the C terminus of the polyamide moiety showed no nuclear localization, whereas an analogous compound lacking the beta-alanine residue was strongly localized in the nuclei of all cell lines tested . The localization profiles of several other conjugates suggest that pyrrole-imidazole sequence and content, dye choice and position, linker composition, and molecular weight are determinants of nuclear localization . The attachment of fluorescein to the C terminus of a hairpin polyamide results in an approximately 10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.

Brain Res Dev Brain Res, 2003 Oct 10, 145(1), 71 - 9
Effects of gonadal steroids on pineal morphogenesis and cell differentiation of the embryonic quail studied under cell culture conditions; Haldar C et al.; Receptors for gonadal steroid hormones have been localized in the pineal glands of several vertebrate species . No studies, however, have reported on pineal morphogenesis and cell differentiation following hormonal application in vitro during avian embryonic development . Hormonal regulation of embryonic development is crucial in all vertebrate classes . Although gonadal hormones are known to affect organogenesis in avian embryos and chicks, we wanted to investigate whether gonadal steroids (testosterone and estradiol) have any effect on the morphogenesis and cell differentiation of the avian pineal gland . The steroid hormones had a stimulatory influence on pineal morphogenesis in vitro as evidenced from the radial arrangement of colony-forming cells and the subsequent formation of a follicular-like structure under dispersed-cell culture condition . Administration of testosterone in culture medium significantly promoted the numbers of cells that were positively stained for arginine vasopressin and tyrosine hydroxylase, while estradiol showed only a slight effect . Both of the two steroid hormones significantly decreased the numbers of cells positively stained for serotonin and melatonin . Melatonin released in the culture medium decreased in content within the 24 h following steroid treatment (supported by low immunoreactivity in cultured cells and low level released to the medium) . These results clearly suggest active roles of gonadal steroid hormones on embryonic pineal morphogenesis and cell differentiation and its physiological activity as they do in adult animals.

Phytochem Anal, 2003 Sep-Oct, 14(5), 298 - 305
Analysis of anthraquinones in cell cultures of Cinchona 'Robusta' by HPLC with photodiode array and mass spectrometry detection; Han YS et al.; An HPLC-PAD-ESI/MS method has been developed for the analysis of anthraquinones in cell cultures of Cinchona 'Robusta' . Using a C18 column and gradient elution with a mobile phase system containing acetonitrile, water and trifluoroacetic acid, a satisfactory separation of both anthraquinone glycosides and aglycones in a crude dichloromethane extract could be obtained . Robustaquinone B was identified as a major anthraquinone in the extracts, and another five anthraquinones were tentatively identified from spectroscopic data . A number of minor unknown compounds were detected and were distinguished from the known anthraquinones . An isocratic system for the quantitative determination of robustaquinone B has also been developed.

J Physiol, 2003 Dec 15, 553(Pt 3), 719 - 28 Epub 2003 Sep 26.
Potentiation of quantal secretion by insulin-like growth factor-1 at developing motoneurons in Xenopus cell culture; Liou JC et al.; Although evidence suggests that insulin-like growth factor (IGF) plays an important role in the development and growth of the nervous system, the effect of IGF-1 in the regulation of neurotransmitter release in the peripheral nervous system remains unknown . Here we show that acute application of IGF-1, a factor widely expressed in developing myocytes, dose-dependently enhances the spontaneous acetylcholine (ACh) secretion at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch clamp recording . We studied the role of endogenously released IGF-1 by examining the effect of IGF-1 antibody on the frequency of spontaneous synaptic currents (SSCs) at high-activity synapses, and found SSC frequency was markedly reduced at these high-activity synapses . The IGF-1-induced synaptic potentiation was not abolished when Ca2+ was eliminated from the culture medium or there was bath-application of the pharmacological Ca2+ channel inhibitor Cd2+, indicating that Ca2+ influxes through voltage-activated Ca2+ channels are not required . Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) or ryanodine receptors effectively occluded the increase of SSC frequency elicited by IGF-I . Treating cells with the phosphoinositide-3 kinase (PI3-K) inhibitors wortmannin or LY294002, and with phospholipase Cgamma (PLCgamma) inhibitor U73122, but not the inhibitor of mitogen-activated protein (MAP) kinase PD98059, abolished IGF-1-induced synaptic potentiation . Taken collectively, these results suggest that endogenously released IGF-1 from myocytes elicits Ca2+ release from IP3- and/or ryanodine-sensitive intracellular Ca2+ stores of the presynaptic nerve terminal . This is done via PI3-K and PLCgamma signalling cascades, leading to an enhancement of spontaneous transmitter release.

Curr Opin Mol Ther, 2003 Aug, 5(4), 389 - 96
Induction of RNA interference using short interfering RNA expression vectors in cell culture and animal systems; Wilson JA et al.; RNA interference, (RNAi) is a phenomenon by which the introduction of double stranded RNA (dsRNA) into cells induces targeted degradation of RNA molecules with homologous sequences . In the laboratory, RNAi has become a valuable tool for analysis of gene function through suppression of specific gene products . In addition, RNAi has shown great promise for use in therapeutic strategies designed to suppress the expression of pathogenic genes . In this review, viral expression and animal model systems that indicate the potential benefits of RNAi will be discussed . The major obstacle for the use of short interfering RNA in a laboratory or clinical setting is the need for efficient and sustained delivery of dsRNA to mammalian cells . Here, current methods that are being used to induce RNAi, both in cultured cells and in animal models, will be described with a focus on some of the most promising applications.

J Neurosci Methods, 2003 Oct 30, 129(2), 169 - 81
Ca2+ channel characteristics in neuroendocrine tumor cell cultures analyzed by color contour plots; Mergler S; Neuroendocrine tumor (NET) cells express several voltage-operated Ca2+ channels (VOCCs) . To address the question, if clinically distinct entities of NETs could be associated by specific patterns of VOCC expression, electrophysiological properties of NET primary cultures derived from consecutive surgical resections and permanent NET cell cultures were determined and analyzed by a color-coding contour diagram method . Using whole-cell patch-clamp technique, electrophysiological data were obtained from human pancreatic (foregut) BON and mouse intestinal (midgut) STC-1 cells as well as from human primary cultivated NET cells from fore- and midgut tumors or liver metastasis . To describe definite Ca2+ channel characteristics, we suggested a color-coding method to depict the contours of time- and voltage-dependence . In this study, we could demonstrate specific Ca2+ channel properties in NET cells from midgut tumors which were not found in NET cells from foregut location . This may be important functionally in respect of different cell biological functions of NET cells such as release of bioamines and neuropeptides . In addition, definite differences between the effect of specific and unspecific Ca2+ channel modulators on NET cells were detected . Although most of this information can be obtained by superimposing current-voltage curves at different times after the onset of the voltage step, the color-coding contour diagrams clearly provides a better visual summary, and hence might be generally quite useful.

Haemophilia, 2003 Sep, 9(5), 598 - 604
HCV recovery from peripheral blood mononuclear cell culture supernatants derived from HCV-HIV co-infected haemophilic patients with undetectable HCV viraemia; Bare P et al.; Hepatitis C viraemia, in 38 human immunodeficiency virus positive (HIV+)/hepatitis C virus positive (HCV+) patients, was determined in haemophilic patients during the 4 years since initiation of highly active antiretroviral therapy (HAART) . Six of 38 patients had persistently HCV-negative viraemia for more than 2 years . No correlation between HCV-negative viraemia and CD4+ T-cell counts, HIV viral load, age, type or severity of haemophilia could be established . Reduced levels of HIV viral load and the immune reconstitution that follows the initiation of HAART were not enough to explain the disappearance of HCV from plasma . Individuals who cleared plasma HCV had significantly higher CD8+ T-cell counts (P=0.0013) (mean +/- SE: 1153 +/- 117.8 cells microL(-1)) than those with HCV-positive viraemia (819.1 +/- 40.72 cells microL(-1)) . Because HCV could maintain a low replication level in peripheral blood mononuclear cells (PBMC), we cultured PBMC of five of six patients with undetectable HCV viraemia . We found four of five HCV RNA-positive cultures . The presence of HCV RNA in our cultures proved that these cells may be an important viral reservoir that could contribute to HCV recurrence in plasma even after long periods of negative viraemia . In summary, our results indicate that in spite of prolonged HCV-negative plasma viraemia, HCV patients that are co-infected with HIV may harbour replication-competent HCV in their PBMC . Therefore, true clearance of HCV infection is difficult to achieve in these patients.

SADJ, 2003 Jun, 58(5), 183 - 6, 188
Presenting an in vitro cell culture model to determine the cytotoxic effect of benzoyl peroxide vapours; Bester MJ et al.; Benzoyl peroxide (BP) is an initiator of polymerisation used in the synthesis of methyl methacrylate dental materials, and is a known allergen that causes allergic contact dermatitis during occupational exposure, especially in dentists and dental personnel . The eyes of dental technicians, dentists and even patients with an allergic history are the first level of exposure, and therefore complaints of allergic reactions of the eyes are usually noted . In this article the authors used a modification direct cell culture testing method to assess the cytotoxic potential of BP . L929 mouse fibroblasts as well as cells from human limbal eye rings were exposed to BP vapours in the experiments . The MTT and crystal violet assays were also employed to determine cell numbers and viability . Results indicated that there is an exponential decrease in cell viability . After exposure of five minutes, cell viability had already decreased by 20 and 40% for vapours derived from either a 10 or 20 microliters aliquot of BP, respectively . After a further five minutes, cell viability decreased by a further 10% with a statistical difference form the control of p < 0.01 . Results indicated that BP vapours are cytotoxic to mouse L929 and human eye fibroblasts . Furthermore, the authors concluded that the permanent L929 mouse fibroblast cell line can provide valuable information regarding the cytotoxic effects of dental products and associated compounds that form toxic vapours.

Arch Toxicol, 2003 Nov, 77(11), 621 - 9 Epub 2003 Sep 24.
Activities of drug metabolizing enzymes in bovine colon epithelial cell cultures; Birkner S et al.; The metabolic competence of cultured bovine colon epithelial cells was evaluated by determining activities of phase I and II enzymes in colonocytes cultured for different intervals (maximum of 10 days) compared with activities measured in freshly isolated cells . Cytochrome p50 1A1-associated 7-ethoxyresorufin O-deethylase (EROD) activity was detectable in freshly isolated colonocytes and in colon cells maintained in culture for up to 5 days . In contrast to liver samples, cytochrome p50 3A4-associated 7-benzyloxyresorufin O-debenzylase (BROD) activity was not detectable in bovine colon cells . Prostaglandin H synthase-mediated production of prostaglandin E(2) was found in freshly isolated and also in cultured colonocytes . Both isoenzymes (COX 1 and COX 2) were detected in cultured cells . To examine phase II metabolic potency, activities of N-acetyltransferases 1 and 2, of phenol and amino sulfotransferases, of glutathione S-transferases alpha, mu, pi and theta and of UDP-glucuronyltransferase were measured . N-Acetyltransferase (NAT) activity (substrate p-aminobenzoic acid, PABA, a diagnostic substrate for the human NAT-1 enzyme) was stable under culture conditions and during the observed culture period comparable to that of freshly isolated cells . In contrast, sulfamethazine, a specific substrate for NAT-2, was not acetylated, neither in bovine colon cells nor in bovine liver samples . Whereas activity of amino sulfotransferase (substrate 2-naphthylamine) decreased continuously during the entire culture period, the activity of phenol sulfotransferase (substrate 1-naphthol) decreased only slowly . Activity of total glutathione S-transferases (alpha, mu, and pi) (substrate 1-chloro-2,4-dinitrobenzene) decreased after 2 days in culture, but was stable during the following culture period . Activity of glutathione S-transferase theta (substrate epoxy-3-nitrophenoxypropane) changed during the culture period . At the beginning and the end (after 10 days) of the culture period maximum activity was measured . Activity of UDP-glucuronyltransferase increased during the culture period reaching a maximum after 7 days . The results show that cultured bovine epithelial colon cells express several enzyme activities required for the biotransformation of xenobiotics.

Invest Ophthalmol Vis Sci, 2003 Oct, 44(10), 4550 - 8
Basic fibroblast and epidermal growth factors stimulate survival in adult porcine photoreceptor cell cultures; Traverso V et al.; PURPOSE . To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas . METHODS . Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium . Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF . Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways . RESULTS . Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase . Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1 . Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter . In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments . Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF . Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone . There was no detectable selective loss of rods or cones in the experimental model . Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands . CONCLUSIONS . The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.

Am J Reprod Immunol, 2003 Jul, 50(1), 90 - 7
Stimulatory effect of the herbal medicine Keishi-bukuryo-gan on a cytokine-induced neutrophil chemoattractant, in rat ovarian cell culture; Yasui T et al.; PROBLEM AND METHOD OF STUDY: We investigated the effects of Keishi-bukuryo-gan, a Japanese herbal medicine, and its crude ingredients in relation to the production of cytokine-induced neutrophil chemoattractant (CINC/gro), interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), which are known to stimulate the secretion of CINC/gro in the ovulatory process, and the effects of Keishi-bukuryo-gan with those of Toki-shakuyaku-san, which has been shown to have an effect on the ovary . We cultured whole ovarian dispersates from immature (3-week-old) female rats with Keishi-bukuryo-gan, Toki-shakuyaku-san and crude ingredients of Keishi-bukuryo-gan . The contents of CINC/gro, IL-1beta and TNFalpha in the cultured media were determined by enzyme-linked immunosorbent assay (ELISA) . RESULTS: Keishi-bukuryo-gan stimulated the secretion of CINC/gro in a dose-dependent manner, and the secretion of CINC/gro into the culture medium increased significantly at concentrations of Keishi-bukuryo-gan of 10 and 100 microg/mL (P < 0.001) . The stimulatory effect of Keishi-bukuryo-gan on the production of CINC/gro is significantly (P < 0.001) stronger than that of Toki-shakuyaku-san at the same concentrations of 100 microg/mL . In addition, Keishi-bukuryo-gan stimulated the secretion of IL-1beta in a dose-dependent manner, while it did not stimulate the secretion of TNFalpha even at a concentration of 100 microg/mL . Moutan Cortex, Paeoniae Radix and Persicae Semen, which are crude ingredients of Keishi-bukuryo-gan, enhanced the secretion of CINC/gro significantly (P < 0.01) in cultured whole ovarian dispersates . CONCLUSIONS: These results show that Keishi-bukuryo-gan can stimulate the secretion of CINC/gro as well as the production of IL-1beta and that this stimulatory effect of Keishi-bukuryo-gan was significantly stronger than that of Toki-shakuyaku-san in immature rat ovarian cell culture.

Bioprocess Biosyst Eng, 2003 Nov, 26(1), 1 - 10 Epub 2003 Sep 20.
Improvement of a mammalian cell culture process by adaptive, model-based dialysis fed-batch cultivation and suppression of apoptosis; Frahm B et al.; Both conventional and genetic engineering techniques can significantly improve the performance of animal cell cultures for the large-scale production of pharmaceutical products . In this paper, the effect of such techniques on cell yield and antibody production of two NS0 cell lines is presented . On the one hand, the effect of fed-batch cultivation using dialysis is compared to cultivation without dialysis . Maximum cell density could be increased by a factor of approximately 5-7 by dialysis fed-batch cultivation . On the other hand, suppression of apoptosis in the NS0 cell line 6A1 bcl-2 resulted in a prolonged growth phase and a higher viability and maximum cell density in fed-batch cultivation in contrast to the control cell line 6A1 (100)3 . These factors resulted in more product formation (by a factor of approximately 2) . Finally, the adaptive model-based OLFO controller, developed as a general tool for cell culture fed-batch processes, was able to control the fed-batch and dialysis fed-batch cultivations of both cell lines.

Arch Virol, 2003 Sep, 148(9), 1815 - 25
Mutation in the 1D gene (VP1) of Foot-and-mouth disease virus serotype Asia1 during serial cytolytic infections in cell culture; Manoj Kumar R et al.; Changes in the nucleotide sequence of the 1D gene of two vaccine strains (IND 63/72 and IND 491/97) of Foot-and-mouth disease virus (FMDV) serotype Asia1 during serial cytolytic infections in cell culture have been analyzed . Sequence comparisons revealed a majority of transition mutations in IND 491/97 . The mutation frequency of the 1D gene of IND 491/97 was about 4.5 to 6.0 fold higher than that of IND 63/72 . At the amino acids 40-60 and 140-160 regions the mutation frequency was higher compared to the whole VP1 . Both viruses showed a constant change at certain residues of the G-H loop region with an accumulation of amino acid replacements during serial cytolytic passages in cell culture . The critical residues (145 and 153) identified previously using mAbs recognizing trypsin-sensitive epitopes were not substituted in the absence of immune selection but changes were observed at positions 142 and 148 . Non-reactivity of IND 63/72 after 50(th) passage level onwards with a panel of mAbs indicated an alteration in the antigenic specificity of the virus . Comparison of amino acid sequences in the entire capsid coding region of the naturally occurring field isolates with that of the 50(th) and 100(th) passage level viruses of IND 63/72 revealed that the residues 56 and 74 of VP2 could be involved in mAb binding . The results suggest that fixation of amino acid replacements occurs in VP1 of Asia1 virus, which could play an important role in antigenic variation by modulating different antigenic epitopes located on the surface of the virus.

J Cell Physiol, 2003 Nov, 197(2), 225 - 31
Role of insulin-like growth factor binding protein (IGFBP)-3 in TGF-beta- and GDF-8 (myostatin)-induced suppression of proliferation in porcine embryonic myogenic cell cultures; Kamanga-Sollo E et al.; Both transforming growth factor (TGF-beta) and growth and development factor (GDF)-8 (myostatin) affect muscle differentiation by suppressing proliferation and differentiation of myogenic cells . In contrast, insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cells . In vivo, IGFs are found in association with a family of high-affinity insulin-like growth factor binding proteins (IGFBP 1-6) that affect their biological activity . Treatment of porcine embryonic myogenic cell (PEMC) cultures with either TGF-beta(1) or GDF-8 suppressed proliferation and increased production of IGFBP-3 protein and mRNA (P < 0.005) . An anti-IGFBP-3 antibody that neutralizes the biological activity of IGFBP-3 reduced the ability of either TGF-beta(1) or GDF-8 to suppress PEMC proliferation (P < 0.005) . However, this antibody did not affect proliferation rate in the presence of both TGF-beta(1) and GDF-8 . These data show that IGFBP-3 plays a role in mediating the activity of either TGF-beta(1) or GDF-8 alone but not when both TGF-beta(1) and GDF-8 are present . In contrast to findings in T47D breast cancer cells, treatment of PEMC cultures with IGFBP-3 did not result in increased levels of phosphosmad-2 . Since TGF-beta and GDF-8 are believed to play a significant role in regulating proliferation and differentiation of myogenic cells, our current data showing that IGFBP-3 plays a role in mediating the activity of these growth factors in muscle cell cultures strongly suggest that IGFBP-3 also may be involved in regulating these processes in myogenic cells.

J Pharm Sci, 2003 Oct, 92(10), 1957 - 67
The effect of cell culture conditions on saquinavir transport through, and interactions with, MDCKII cells overexpressing hMDR1; Williams GC et al.; MDCK cells are cultured using wide-ranging conditions and can produce variable results . To develop a standard protocol for studying saquinavir transport using MDCKII cells, stably transfected MDCKII cells overexpressing human Pgp (MDCKII-PGP) and MDCKII wild-type cells (MDCKII/wt) were used to evaluate the combined effects of seeding density (6.9 x 10(5) or 5 x 10(4) cells/cm2), substratum (polycarbonate +/- collagen coating) and saquinavir presence on monolayer integrity, Pgp expression, and saquinavir transport . The saquinavir efflux ratio (ratio of BL --> AP/AP --> BL permeability) for MDCKII-PGP cells (6.9 x 10(5) cells/cm2) was 57 with variable mannitol permeabilities . Consistent mannitol permeabilities and higher saquinavir efflux ratios were obtained with 5 x 10(4) cells/cm2 on polycarbonate (78) or collagen-coated polycarbonate (126) . The MDCKII/wt saquinavir efflux ratio was 9 . Saquinavir presence increased paracellular permeability for all treatments relative to cells seeded onto collagen-coated membranes . Collagen coating caused increased Pgp expression and saquinavir efflux ratios correlated (r2 = 0.96) with Pgp expression levels {MDCKII-PGP (on collagen-coated polycarbonate) > MDCKII-PGP (on polycarbonate) > MDCKII/wt (on collagen-coated polycarbonate)} . These results directly and quantitatively link interrelated differences in cell culture conditions to changes in monolayer integrity, transporter expression, and active transport; and emphasize the critical application of controls in cell culture models .

Brain Res Bull, 2003 Sep 30, 61(5), 547 - 53
Primary cell culture of suprachiasmatic nucleus; Ren D et al.; In mammals, the suprachiasmatic nucleus (SCN) contains a biological clock that drives circadian rhythms in vivo and in vitro . Primary dissociated neuronal culture is a useful research tool, which allows cell-by-cell morphological and physiological study of the SCN . A long-term primary dissociated SCN neuron culture is the prerequisite to understanding how neural activity and morphology interact in the SCN . The essential details of recent effective SCN culture methods are reviewed, including preparation of cells, medium and substrate, maintenance of cultures, and characterization of cultured SCN neurons . This technique is growing in importance, especially with the advent of multi-electrode array (MEA) recording.

Eur J Pharm Sci, 2003 Sep, 20(1), 107 - 13
Epidermal cell culture model derived from rat keratinocytes with permeability characteristics comparable to human cadaver skin; Marjukka Suhonen T et al.; The permeability characteristics of an organotypic epidermal culture model derived from rat epidermal keratinocytes, ROC, and isolated human cadaver epidermis, HEM, were compared . Rat epidermal keratinocyte (REK) cell line was grown for 3 weeks on collagen gel in the absence of feeder cells in culture inserts at an air-liquid interface . Transdermal permeabilities of 18 compounds ranging from 92 to 504 in molecular weight and from -4.3 to 3.9 in log of octanol-water partition coefficient, charged or uncharged, were measured in the culture model and isolated human epidermis . The REK organotypic culture model (ROC) provided a close estimate of human epidermal permeabilities over the whole range of the solutes used with on the average of 2-fold higher permeability coefficients (range 0.3-5.2) than those obtained from isolated human cadaver epidermis . The easily maintained and reproducible ROC model may be useful in screening transepidermal drug permeabilities together with possessing potential for research on dermal formulations, irritation, toxicity and gene therapy.

Eur J Pharm Sci, 2003 Sep, 20(1), 99 - 106
Paracellular and passive transcellular permeability in immortalized human corneal epithelial cell culture model; Toropainen E et al.; A cell culture model of human corneal epithelium (HCE-model) was recently introduced {Invest . Ophthalmol . Vis . Sci . 42 (2001) 2942} as a tool for ocular drug permeation studies . In this study, passive permeability and esterase activity of the HCE-model were characterised . Immortalised human corneal epithelial cells were grown on collagen coated filters under air-lift . The sensitivity of transcellular permeability to lipophilicity was tested in studies using nine beta-blockers . The size selectivity of the paracellular route was investigated using 16 polyethylene glycol oligomers (PEG) . An effusion-like approach was used to estimate porosity and pore sizes of the paracellular space in HCE membrane . Permeability and degradation of fluorescein diacetate to fluorescein in HCE-cells was used to probe the esterase activity of the HCE-model . Drug concentrations were analyzed using HPLC (beta-blockers), LC-MS (PEGs), and fluorometry (fluorescein) . Permeabilities were compared to those in the excised rabbit cornea . Penetration of beta-blockers increased with lipophilicity according to a sigmoidal relationship . This was almost similar to the profile in excised cornea . No apical to basolateral directionality was seen in the permeation of beta-blockers . Paracellular permeability of the HCE-model was generally slightly higher than that of the excised rabbit cornea . The HCE-model has larger paracellular pores, but lower pore density than the excised cornea, but the overall paracellular space was fairly similar in both models . The HCE-model shows significant esterase activity (i.e . fluorescein diacetate was converted to free fluorescein) . These data on permeability of 27 compounds demonstrate that the barrier of the HCE-model closely resembles that of the excised rabbit cornea . Therefore, the HCE-model is a promising alternative corneal substitute for ocular drug delivery studies.

Dis Aquat Organ, 2003 Aug 4, 55(3), 253 - 8
Propagation of infectious yellow head virus particles prior to cytopathic effect in primary lymphoid cell cultures of Penaeus monodon; Assavalapsakul W et al.; Yellow head virus is a highly pathogenic agent that can cause a fatal disease in several species of penaeid shrimps . Using a primary cell culture and an in vitro quantal assay (TCID50), this study sought to determine the propagation profile of yellow head virus after inoculation at a low multiplicity of infection in the lymphoid tissue (oka organ) of Penaeus monodon . Detectable levels of virus were present as early as 24 h post-inoculation . Maximal viral yields were reached by 4 d post-infection, approximately 24 h after the onset of a detectable cytopathic effect, which was normally observable at 3 d post-inoculation . The methodology provides a useful tool for studying yellow head virus-host-cell interactions.

Neurochem Int, 2004 Jan, 44(2), 107 - 18
Apolipoprotein E protects against oxidative stress in mixed neuronal-glial cell cultures by reducing glutamate toxicity; Lee Y et al.; Apolipoprotein E (ApoE) deficiency has been shown to adversely affect outcome after transient cerebral ischemia and head trauma . Since oxidative stress contributes to these injuries, the ability of ApoE to reduce irreversible oxidative damage was studied in primary mixed neuronal-glial cell cultures . Cells (13-16 days in vitro) were exposed to 50 microM hydrogen peroxide (H2O2) for 30 min, and toxicity was determined by the release of lactate dehydrogenase (LDH) 24 h after exposure . The presence of recombinant human ApoE2 (100, 300, or 1000 nM) in the culture media partially protected against oxidative injury . This protection was not reversed by pre-treatment with receptor associated protein . The NMDA receptor antagonist, MK-801, also provided partial protection against H2O2 toxicity . The degree of protection was similar to that conferred by ApoE treatment . The protective effects of ApoE and MK-801 were not additive; no ApoE protection was observed in cultures treated with MK-801 prior to H2O2 exposure . ApoE treatment had no effect on H2O2 stimulated glutamate release, but did increase the rate of glutamate uptake via the high affinity glutamate transporter in H2O2 treated cultures . Pre-treatment with ApoE also conferred partial protection against glutamate-induced LDH release . Taken together, these findings suggest that ApoE protects mixed neuronal-glial cell cultures against irreversible oxidative injury from H2O2 by reducing secondary glutamate excitotoxicity.

Plant Physiol, 2003 Sep, 133(1), 361 - 7
Dehydroascorbate uptake activity correlates with cell growth and cell division of tobacco bright yellow-2 cell cultures; Horemans N et al.; Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been proven to correlate with differences in growth or cell cycle progression . Here, a possible correlation between growth and the activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated . Protoplasts were isolated from a tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA transport was tested with (14)C-labeled ASC . Ferricyanide (1 mM) or dithiothreitol (1 mM) was included in the test to keep the external (14)C-ASC in its oxidized respectively reduced form . Differential uptake activity was observed, correlating with growth phases of the cell culture . Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in the presence of dithiothreitol did not . The enhanced DHA uptake was not due to higher endogenous ASC levels that are normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake . Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mM DHA . In addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle . An increase in uptake activity was observed during M phase and the M/G1 transition . These experiments are the first to show that DHA transport activity into plant cells differs with cell growth . The relevance of the data to the action of DHA and ASC in cell growth will be discussed.

Eur J Pharm Biopharm, 2003 Sep, 56(2), 183 - 7
Increased efficacy of acyclovir-loaded microparticles against herpes simplex virus type 1 in cell culture; de Jalon EG et al.; Acyclovir is one of the most effective and selective agents against viruses of the herpes group . In order to increase its antiviral activity, acyclovir loaded microparticles, prepared by an O/W solvent evaporation method were developed . Their antiviral activity against herpes simplex virus type 1 (HSV-1) and toxicity were evaluated on Vero cells and then compared with those presented by a drug solution . The 50% inhibitory concentration (IC(50)) values for acyclovir loaded microspheres determined by plaque reduction assays at 48 and 96 h, were found to be 1.06 +/- 0.01 microM and 0.15 +/- 0.03 microM, respectively, while the equivalent values obtained for an acyclovir solution were 1.28 +/- 0.04 microM at 48 h and 0.27 +/- 0.02 microM at 96 h . These results indicate that acyclovir shows a higher antiviral activity, against herpes simplex virus type 1, when this drug was loaded in microparticles rather than as a drug solution, especially after 96 h of incubation . The toxicity of these microparticles was determined by the MTT test at 48 and 96 h . At 48 h only a small toxicity was found (cell viability ranged from 72 to 82%, with the higher concentration tested) and it could not be attributed to the microparticles, since the acyclovir control solution showed similar toxicity values . However, after 96 h a higher toxicity was observed with acyclovir microparticles as well as with the unloaded ones (cell viability located between 60 and 70%) . In summary, acyclovir-loaded microparticles have shown to be promising carriers for the effective delivery of acyclovir in the treatment of HSV-1 infections in cells so they can have a potential use in vivo.

Bioorg Med Chem, 2003 Sep 15, 11(19), 4171 - 8
Nitrobenzocyclophosphamides as potential prodrugs for bioreductive activation: synthesis, stability, enzymatic reduction, and antiproliferative activity in cell culture; Li Z et al.; In efforts to obtain potential anticancer prodrugs for gene-directed enzyme prodrug therapy using Eschericia coli nitroreductase, a series of four benzocyclophosphamide analogues were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation . All four analogues were found to be stable in phosphate buffer at pH 7.4 and 37 degrees C and were good substrates of E . coli nitroreductase with half lives between 7 and 24 min at pH 7.0 and 37 degrees C . However, only two analogues 6a and 6c, both with a benzylic oxygen in the phosphorinane ring para to the nitro group, showed a modest 33-36-fold enhanced cytotoxicity in E . coli nitroreductase-expressing cells . These results suggest that good substrate activity and the para benzylic oxygen are required for activation by E . coli nitroreductase . Compounds 6a and 6c represent a new structure type for reductive activation and a lead for further modification in the development of better analogues with improved selective toxicity to be used in gene-directed enzyme prodrug therapy.

Biomaterials, 2003 Nov, 24(25), 4573 - 83
Retinal pigment epithelium cell culture on surface modified poly(hydroxybutyrate-co-hydroxyvalerate) thin films; Tezcaner A et al.; There is currently no effective treatment for the retinal disorders caused by retinal pigment epithelium (RPE) degeneration . Transplantation of allografts is the main strategy towards correction of this malady . Tissue engineering could offer hope and involve the use of biodegradable polymeric templates to replace diseased or lost RPE . In this study PHBV8 film was chosen as a temporary substrate for growing retinal pigment epithelium cells as an organized monolayer before their subretinal transplantation . The surface of the PHBV8 film was rendered hydrophilic by oxygen plasma treatment to increase the reattachment of D407 cells on the film surface . Power and duration was changed, from 50 W, 10 min to 100 W, 20 min during plasma treatment . The effect of these two parameters on surface hydrophilicity, morphology, topography, surface composition of PHBV8 thin films was studied using AFM, SEM, and phase contrast microscopy . The effect of changes in surface characteristics on cell reattachment, spreading and cell growth rate was investigated . It was found that as the treatment level was increased the surface hydrophilicity increased and roughness was decreased probably due to ablation . The PHBV8 film treated with 100 W 10 min was found to be the most suitable for 24 h reattachment of D407 cells . The cells were also grown to confluency as an organized monolayer suggesting PHBV8 film as a potential temporary substrate for subretinal transplantation to replace diseased or damaged retinal pigment epithelium.

Pharm Res, 2003 Aug, 20(8), 1210 - 24
Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium; Troutman MD et al.; PURPOSE: The purpose of this work was to elucidate the asymmetric effect of P-gp on modulation of absorptive and secretory transport of compounds across polarized epithelium, to develop experimental parameters to quantify P-gp-mediated modulation of absorptive and secretory transport, and to elucidate how P-gp-mediated modulation of transport is affected by passive diffusion properties, interaction of the substrate with P-gp, and P-gp expression . METHODS: The permeability of a set of P-gp substrates was determined in absorptive and secretory directions in Madine-Darby Canine kidney (MDCK), Caco-2, and MDR-MDCK monolayers . The transport was also determined in the presence of GW918, a non-competitive P-gp inhibitor, to quantify the permeability without the influence of P-gp . From these two experimental permeability values in each direction, two new parameters, absorptive quotient (AQ) and the secretory quotient (SQ), were defined to express the functional activity of P-gp during absorptive and secretory transport, respectively . Western blot analysis was used to quantify P-gp expression in these monolayers and in normal human intestinal . RESULTS: P-gp expression in Caco-2 and MDR-MDCK monolayers was comparable to that in normal intestine, and much less in MDCK cells . For all models, the substrates encompassed a wide range of apparent permeability due to passive diffusion (PPD) . The parameters AQ and SQ, calculated for all compounds, assessed the attenuation in absorptive and enhancement of secretory transport, respectively, normalized to the permeability due to passive diffusion . Analysis of these parameters showed that 1) P-gp affected absorptive and secretory transport differentially and 2) compounds could be stratified into distinct groups with respect to the modulation of their absorptive and secretory transport by P-gp . Compounds could be identified whose absorptive transport was either strongly affected or poorly affected by changes in P-gp expression . For certain compounds, AQ values showed parabolic relationship with respect to passive diffusivity, and for others AQ was unaffected by changes in passive diffusivity . CONCLUSIONS: The relationship between attenuation of absorptive transport and enhancement of secretory transport of compounds by P-gp is asymmetric, and different for different sets of compounds . The relationship between attenuation of absorption by P-gp and passive diffusivity of compounds, their interaction potential with P-gp, and levels of P-gp expression is complex; however, compounds can be classified into sets based on these relationships . A classification system that describes the functional activity of P-gp with respect to modulation of absorptive and secretory transport was developed from these results.

Biophys J, 2003 Sep, 85(3), 1938 - 47
FTIR spectroscopy demonstrates biochemical differences in mammalian cell cultures at different growth stages; Mourant JR et al.; We have observed differences in the infrared spectra of viable fibroblast cells depending on whether the cells were in the exponential (proliferating) or plateau (nonproliferating) phase of growth . The spectral changes were observed even after correcting for cell number and volume, ruling out these trivial explanations . Several of the changes occurred for both transformed and normal cell lines and were greater for the normal cell line . The biochemical basis of the spectral changes was estimated by fitting the cell spectra to a linear superposition of spectra for the major biochemical components of mammalian cells (DNA, RNA, protein, lipids, and glycogen) . The ratios of RNA/lipid and protein/lipid increased when the cells were in the exponential phase compared to the plateau phase of growth . The fits of cell spectra to individual biochemical components also demonstrated that DNA is a relatively minor spectroscopic component as would be expected biochemically . Contrary to other reports in the literature, our data demonstrate that determining DNA content or structure using Fourier transform infrared spectroscopy data is difficult due to the relatively small amount of DNA and the overlap of DNA bands with the absorption bands of other biochemical components.

Diabetes, 2003 Sep, 52(9), 2315 - 20
An insulin-degrading enzyme inhibitor decreases amylin degradation, increases amylin-induced cytotoxicity, and increases amyloid formation in insulinoma cell cultures; Bennett RG et al.; Amylin (islet amyloid polypeptide) is the chief component of the islet amyloid found in type 2 diabetes, and amylin fibril precursors may be cytotoxic to pancreatic beta-cells . Little is known about the prevention of amylin aggregation . We investigated the role of insulin-degrading enzyme (IDE) in amylin degradation, amyloid deposition, and cytotoxicity in RIN-m5F insulinoma cells . Human (125)I-labeled amylin degradation was inhibited by 46 and 65% with the addition of 100 nmol/l human amylin or insulin, respectively . (125)I-labeled insulin degradation was inhibited with 100 nmol/l human amylin, rat amylin, and insulin (by 50, 50, and 73%, respectively) . The IDE inhibitor bacitracin inhibited amylin degradation by 78% and insulin degradation by 100% . Amyloid staining by Congo red fluorescence was detectable at 100 nmol/l amylin and was pronounced at 1,000 nmol/l amylin treatment for 48 h . Bacitracin treatment markedly increased staining at all amylin concentrations . Bacitracin with amylin caused a dramatic decrease in cell viability compared with amylin alone (68 and 25%, respectively, at 10 nmol/l amylin) . In summary, RIN-m5F cells degraded both amylin and insulin through a common proteolytic pathway . IDE inhibition by bacitracin impaired amylin degradation, increased amyloid formation, and increased amylin-induced cytotoxicity, suggesting a role for IDE in amylin clearance and the prevention of amylin aggregation.

Neurochem Res, 2003 Sep, 28(9), 1307 - 13
Effects of ginsenosides on carbachol-stimulated formation of inositol phosphates in rat cortical cell cultures; Lee JH et al.; We examined the effect of ginseng total saponins (GTS) on phosphoinositide metabolism stimulated by activation of muscarinic receptor using rat cortical cultures . Carbachol stimulated formation of {3H}inositol phosphates ({3H}InsPs) by 3.3-fold over basal level in {3H}inositol-prelabeled cells . Pretreatment of GTS inhibited formation of {3H}InsPs evoked by carbachol by 70%-90% . Addition of GTS alone had no effect on the basal formation of {3H}InsPs . The inhibitory effect of the GTS on carbachol-stimulated formation of {3H}InsPs was dose- and time-dependent . IC50 was 6.0 +/- 2.8 microg/ml . We also examined the effect of GTS on {3H}InsP1, {3H}InsP2, or {3H}InsP3 formation evoked by carbachol . Although GTS had no effect on the basal {3H}InsP1, {3H}InsP2, or {3H}InsP3 formation, pretreatment of GTS inhibited {3H}InsP1, {3H}InsP2, or {3H}InsP3 formation evoked by carbachol, respectively . Addition of individual ginsenosides such as ginsenoside Rb1, Rc, Rd, Re, or Rg2 had no effect on the basal formation of {3H}InsPs, whereas pretreatment of ginsenoside Rb2, Rc, Rd, Re, Rf, Rg1 or Rg2 inhibited formation of {3H}InsPs evoked by carbachol by 79%-89% . The results suggest that the inhibitory effect of GTS and its individual ginsenosides on carbachol-stimulated formation of {3H}InsPs in cortical neurons could be one pharmacological action of Panax ginseng.

Vet Microbiol, 2003 Sep 1, 95(3), 159 - 74
Growth of recombinant equine herpesvirus 1 (EHV-1) replaced with passage-induced mutant gene 1 and gene 71 derived from an attenuated EHV-1 in cell cultures and in the lungs of mice; Kirisawa R et al.; The relationship of passage-induced mutant genes 1 and 71 of an attenuated equine herpesvirus 1 (EHV-1) with virulence was analysed by constructing nine recombinant EHV-1 viruses by homologous recombination . Gene 1 or/and gene 71 of a virulent EHV-1 strain, HH1, was replaced by a mutant gene 1 or/and 71 of an attenuated HH1 strain, BK343, respectively . The beta-galactosidase gene of Escherichia coli was inserted within the gene 1 or 71 coding sequence of HH1 to inactivate the genes . Virus replications of these recombinant viruses in cell cultures were similar, but release of the gene 71-inactivated virus from infected cells was delayed compared to that of the other viruses . Plaque sizes of the recombinant viruses were similar to those of HH1, but those of BK343 were significantly smaller, indicating an effect of some unknown factor(s) on viral cell-to-cell spread . The growth abilities of the recombinant viruses with a mutant gene 1 or/and 71 in lungs of mice were similar to those of HH1, but those of gene 71-inactivated viruses were reduced to the level of BK343 and the titers were about 100-times lower than those of the other recombinant viruses . These results indicate that the mutant genes 1 and 71 of BK343 might not confer an attenuated nature to EHV-1.

Chem Commun (Camb), 2003 Aug 21, (16), 2030 - 1
Electrochemical, in vitro and cell culture analysis of integrated redox catalysts: implications for cancer therapy; Giles GI et al.; Integrated catalysts as redox sensitisers towards cancer.

Endocrinology, 2003 Sep, 144(9), 4134 - 43
Stanniocalcin 1 stimulates osteoblast differentiation in rat calvaria cell cultures; Yoshiko Y et al.; Stanniocalcin 1 (STC1) is a mammalian homolog of STC, the fish calcium/phosphate-regulating polypeptide whose functions are only beginning to be elucidated . Recently, we demonstrated that STC1 stimulates, in an autocrine/paracrine fashion, bone mineralization by increasing phosphate uptake in osteoblasts apparently via the functional activity of the sodium-dependent phosphate transporter, Pit1 . We have now assessed the role of STC1 on osteoblast development in fetal rat calvaria (RC) cell cultures . STC1 mRNA and protein were differentially expressed over the time course of cultures, and dexamethasone, a potent stimulator of differentiation in this model, shifted peak STC1 expression levels to earlier times . Overexpression {recombinant human (rh) STC1} and underexpression (antisense oligonucleotides) of STC1 accelerated and retarded, respectively, osteogenic development as well as osteopontin and osteocalcin mRNA expression in mature osteoblast cultures, but not osteoprogenitor cell cultures . Dexamethasone shifted the effective doses required for these effects to higher and lower concentrations of antisense oligonucleotides and rhSTC1, respectively . Concomitantly, rhSTC1 increased both sodium-dependent phosphate uptake and Pit1 gene expression in nodule formation stages, but not in primitive progenitor stages of RC cell cultures . Thus, STC1 accelerates osteoblast development in an autocrine/paracrine manner in the RC cell culture model.

Intervirology, 2003, 46(4), 227 - 31
Improved detection of dengue-1 virus from IgM-positive serum samples using C6/36 cell cultures in association with RT-PCR; Oliveira De Paula S et al.; Dengue is the most important arboviral disease in the world, and its diagnosis is primarily made by serology . Virus isolation has been successful mainly in clinical samples obtained during the acute phase of illness, and is carried out through inoculation of clinical samples into C6/36 cell monolayers followed by the detection of infection by indirect immunofluorescence assay (IFA) . We compared the efficiency of RT-PCR and IFA in the detection of dengue-1 virus after inoculation of C6/36 cells with samples obtained in the convalescent period of dengue infection . Out of 75 IgM-positive samples inoculated into C6/36 cells, 2 were positive by IFA while 17 were positive by RT-PCR . The 2 IFA-positive samples were collected during the acute phase of the illness; 17 positive samples were found by RT-PCR, including the 2 detected by IFA . For both methods, we also investigated the time necessary for viral detection using a fixed dose of 1 x 10(4) viruses/ml . RT-PCR and IFA detected the dengue virus 1 and 4 days after virus inoculation, respectively . The results obtained here indicate that RT-PCR is the most sensitive method in the detection of dengue viruses using C6/36 cells for viral isolation .

Toxicol Lett, 2003 Oct 15, 144(3), 337 - 49
Apoptosis-mediated neurotoxic potential of a planar (PCB 77) and a nonplanar (PCB 153) polychlorinated biphenyl congeners in neuronal cell cultures; Sanchez-Alonso JA et al.; Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic depending on the chemical structure of the congeners . This study investigated the neurotoxic potential of 3,3',4,4'-tetrachlorobiphenyl (TCB) (PCB 77, a non-ortho-substituted planar congener) and 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) (PCB 153, a di-ortho-substituted nonplanar congener) by assessing cell viability and apoptotic cell death in neuronal cell cultures . We have combined morphological and biochemical techniques to establish the relevance of apoptosis in neuronal cell death induced by the two selected PCB congeners . Treatment with both planar and nonplanar congeners caused the loss of cell viability and accelerated apoptosis in a time- and concentration-dependent manner . However, the extent of apoptosis generated was greater for the non-ortho-substituted planar congener (PCB 77) than for the di-ortho-substituted nonplanar congener (PCB 153) . This was correlated with the loss of cell viability since the planar congener was more cytotoxic . Based on our findings, the apoptosis induced by PCBs involves the increase of caspase-3 activity in neuronal cell cultures . It is reasonable to assume that PCB-induced apoptosis may be linked to the neurotoxic effect of these toxicants and that different molecular mechanisms may operate in the induction of apoptosis depending on the planarity or nonplanarity of the PCB congeners.

Antiviral Res, 2003 Aug, 59(3), 143 - 54
Cell culture replication of herpes simplex virus and, or human cytomegalovirus is inhibited by 3,7-dialkoxylated, 1-hydroxyacridone derivatives; Lowden CT et al.; The synthetic acridone compound, 5-chloro-1,3-dihydroxyacridone inhibits herpes simplex virus (HSV) replication by inducing the formation of defective viral (B-type) capsids {Antiviral Res . 53 (2002) 113} . In this report, synthetic elaboration of the 1-hydroxyacridone scaffold coupled with antiviral testing led to the identification of 3,7-dimethoxy-1-hydroxy-acridone (2) as an inhibitor of low multiplicity human cytomegalovirus (HCMV) infection (ED(50) value of 1.4 microM (0.5 microg/ml); greater than 35-fold selectivity) . Compound 2 was inactive against HSV replication and the efficacy as an anti-HCMV agent at higher viral loads was only apparent if host cells were replicated in the presence of the compound prior to infection . Interestingly, the 3,5-dimethoxy regioisomer inhibited cell replication (mean CC(50) 33 microM) and was inactive as a selective anti-herpes agent . A limited parallel synthesis and testing of ten 3,7-dialkoxylated compounds closely related to compound 2 led to the discovery of the 3-ethoxy-, 3-propoxy-, 3-isopropoxy- and 3-allyloxy-derivatives as dual inhibitors of both HSV and HCMV (selectivity of the 3-allyloxy analog was greater than 10- and 36-fold, respectively) . The 3-benzyloxy-derivative was active (ED(50) value of 6.9 microM) against HCMV only . Moreover, the corresponding C-7 variable alkoxylated parallel series were either weakly active or inactive antiviral agents suggesting an apparent requirement for a C-7 methoxy substituent in the active structure . Exploratory mode of action studies showed that dual inhibitors were most active against a low multiplicity HSV infection and potent inhibition of viral release likely contributed to this . Furthermore, suppression of late viral protein synthesis by dual inhibitors did not correlate with anti-HSV activity . On the basis of the present findings, the 1-hydroxyacridone scaffold is further expanded as a useful template for the discovery of investigational anti-herpes agents . As a group, the active 3,7-dialkoxylated compounds likely have diverse mechanisms of action, consequently they are of potential medicinal interest.

Pol J Pharmacol, 2003 Mar-Apr, 55(2), 235 - 8
Differential effect of betulin and betulinic acid on cytokine production in human whole blood cell cultures; Zdzisinska B et al.; Betulin and betulinic acid, plant-derived triterpenoid compounds, have been described to possess anti-inflammatory activity . In this paper, we examine the ability of both compounds to induce and modulate cytokine production in human whole blood cell cultures . The results indicate that betulin is a modest TNF-alpha inducer and also an enhancer of mitogen-induced TNF-alpha production . In contrast to betulin, betulinic acid is a modulator of cytokine production by Th1/Th2 cell subpopulations which slightly enhances IL-10 formation and inhibits IFN-gamma production, reducing IFN-gamma/IL-10 ratio from 3.6 to 2.6.

J Biomed Mater Res A, 2003 Sep 15, 66(4), 880 - 4
The effect of hyaluronan on osteoblast proliferation and differentiation in rat calvarial-derived cell cultures; Huang L et al.; Hyaluronan (or hyaluronic acid, HA) is an essential component of extracellular matrices . It interacts with other macromolecules and plays a predominant role in tissue morphogenesis, cell migration, differentiation, and adhesion . The cell signaling functions of HA are mediated through the CD-44 receptor and are dependent upon the molecular weight of the polymer . We hypothesized that an HA of appropriate molecular weight alone in optimal concentration may induce osteoblast differentiation and bone formation . Enzyme-digested calvarial-derived mesenchymal cells from 2-day-old newborn rats were cultured with the addition of HA of three different molecular weights (2300, 900, and 60 kDa) . We added, 0.5, 1.0, and 2.0 mg/mL HA for each molecular weight to the medium at the first plating of cells . After 7 to 20 days in culture, cell proliferation and differentiation were evaluated by measuring thymidine incorporation, alkaline phosphatase activity, and osteocalcin gene expression . The effects of HA on bone formation were examined by using Alizarin red staining for mineralization . The results showed that low molecular weight HA (60 kDa) significantly stimulated cell growth, increased osteocalcin mRNA expression in a dose-dependent manner, but showed no apparent effects on alkaline phosphatase activity and bone mineralization . On the other hand, high-weight HA (900 and 2,300 kDa) significantly increased all the parameters examined, particularly alkaline phosphatase activity, in a dose-dependent manner and stimulated cell mineralization to 126% and 119% of the controls, respectively, in the 1.0 mg/mL dose . Our findings suggest that HA has a molecular weight-specific and dose-specific mode of action that may enhance the osteogenic and osteoinductive properties of bone graft materials and substitutes due to its stimulatory effects on osteoblasts .

Mar Biotechnol (NY), 2003 Jan-Feb, 5(1), 64 - 9
Initiation of cartilage cell culture from skate (Raja porasa Günther); Fan TJ et al.; Cartilage tissues from the proboscis of skate (Raja porasa Gunther) were used to initiate primary cultures of cartilage cells . Aseptically dissected cartilage tissues were immersed in MEM medium free of fetal bovine serum (FBS), pH 7.6, and minced into small pieces (1 mm3 on average) . After hydrolysis with collagenase II, hyaluronidase, and trypsin for 2 hours at room temperature, the acquired cartilage cells were rinsed twice with 20% FBS-supplemented MEM medium and then inoculated into 25-cm3 cell culture flasks, and incubated at 24 degrees C . The primary cultures were initiated successfully, and the cartilage cells grew gradually into a confluent monolayer at day 10 . Effects of growth factors were also tested in this study, and it was found that 20 ng/ml of basic fibroblast growth factor and 100 ng/ml of insulin-like growth factor II together had the most prominent stimulating effect on the growth and division of cartilage cells in the series of concentration combinations employed . The induced cartilage cells cultured formed a confluent monolayer at day 7.

Nat Med, 2003 Sep, 9(9), 1125 - 30 Epub 2003 Aug 17.
Clonal vaccinia virus grown in cell culture as a new smallpox vaccine; Weltzin R et al.; Although the smallpox virus was eradicated over 20 years ago, its potential release through bioterrorism has generated renewed interest in vaccination . To develop a modern smallpox vaccine, we have adapted vaccinia virus that was derived from the existing Dryvax vaccine for growth in a human diploid cell line . We characterized six cloned and one uncloned vaccine candidates . One clone, designated ACAM1000, was chosen for development based on its comparability to Dryvax when tested in mice, rabbits and monkeys for virulence and immunogenicity . By most measures, ACAM1000 was less virulent than Dryvax . We compared ACAM1000 and Dryvax in a randomized, double-blind human clinical study . The vaccines were equivalent in their ability to produce major cutaneous reactions ('takes') and to induce neutralizing antibody and cell-mediated immunity against vaccinia virus.

Mol Biol Cell, 2003 Aug, 14(8), 3144 - 55 Epub 2003 May 03.
Subcellular localization of LGN during mitosis: evidence for its cortical localization in mitotic cell culture systems and its requirement for normal cell cycle progression; Kaushik R et al.; Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling . In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division . Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used . We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis . Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture . In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins . The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.

J Cancer Res Clin Oncol, 2003 Oct, 129(10), 565 - 8 Epub 2003 Aug 16.
Comparison of human lung cancer/SCID mouse tumor xenografts and cell culture growth with patient clinical outcomes; Anderson TM et al.; Leukemic cell growth in SCID mice has been reported as a predictor of disease relapse . However, there is a paucity of literature regarding xenograft growth and clinical outcomes in non-small cell lung cancer (NSCLC) . Seventy-nine specimens from patients with NSCLC were either subcutaneously implanted into SCID mice and/or placed in tissue culture . Retrospective chart review was correlated with stage, histology, necrosis, disease-free interval, and survival . Tumor xenografts were successfully established with 17 of 37 (46%) tumor biopsy tissues . Thirteen of 59 (22%) specimens grew in cell culture . Patients whose tumors grew in SCID mice had no difference in survival compared to those with no growth ( n=20, p=0.42) . Median survival was 36 months in 13 patients whose tumors grew in cell culture compared to 39 months in 46 patients without growth . Eight of 12 (67%) patients with metastasis showed SCID/human xenograft growth, whereas nine of 25 (36%) without metastases did so ( p=0.08) . Growth of tumor cells in vitro occurred in 11 of 31 (35%) adenocarcinomas, one of 25 (4%) squamous cell carcinomas, and one of three (33%) large cell carcinomas ( p=0.02) . Well or moderately differentiated tumors grew in cell culture in only two of 22 (9%), whereas poorly or undifferentiated tumors grew in 11 of 32 (34%) cases ( p=0.03) . We conclude that neither the ability of a tumor to engraft and grow in SCID mice nor its ability to grow in vitro in cell culture is a reliable predictor of disease outcome or survival in patients with NSCLC . The ability to propagate tumors in vitro appears to be more dependent upon the histological type of tumor and its degree of differentiation.

Hum Reprod, 2003 Sep, 18(9), 1767 - 71
Effect of GnRH analogues on apoptosis and release of interleukin-1beta and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis; Meresman GF et al.; BACKGROUND: The aim of the present study was to evaluate the effect of GnRH analogues on the in-vitro eutopic endometrial cell apoptosis and release of interleukin-1beta (IL-1beta) and vascular endothelial growth factor (VEGF) . METHODS: Biopsy specimens of eutopic endometrium obtained from 16 women with untreated endometriosis and 14 controls were studied . Apoptosis, IL-1beta and VEGF release were evaluated in epithelial endometrial cell cultures after incubation with leuprolide acetate (LA) as GnRH agonist, antide as GnRH antagonist, and a combination of both . The percentage of apoptotic cells was evaluated by the acridine orange-ethidium bromide technique, and IL-1beta and VEGF concentrations were assessed by using commercial enzyme-linked immunosorbent assay (ELISA) kits . RESULTS: We found that LA (100 ng/ml) enhanced apoptosis in endometrial cell cultures from endometriosis patients and controls and this effect was reversed by antide at 10(-7) mol/l . IL-1beta and VEGF release was downregulated by LA in cultures from controls and endometriosis patients . The addition of antide 10(-7) mol/l reversed this inhibition . Endometrial cultures treated with antide at 10(-7) mol/l did not show any significant effects compared with basal conditions . CONCLUSIONS: GnRH agonists appear to have a direct effect in endometrial cells cultures, by enhancing the percentage of apoptotic cells and decreasing the release of pro-mitogenic cytokines such as IL-1beta and VEGF.

Vaccine, 2003 Sep 8, 21(25-26), 4043 - 51
A BHK-21 cell culture-adapted tick-borne encephalitis virus mutant is attenuated for neuroinvasiveness; Goto A et al.; We derived the baby hamster kidney (BHK)-21 cell culture-adapted, tick-borne encephalitis (TBE) virus mutant . To reveal the pathogenicity of the TBE virus, we compared the pathogenicity of the mutant (Oshima Cl-1) and parental (Oshima 5-10) virus in mouse model . The neurovirulence of mutant in mice was identical to that of parent . However, the level of neuroinvasiveness was higher for parent than for mutant . The degrees of viremia and virus titers in the spleen were lower in mice that were inoculated subcutaneously (s.c.) with mutant than in mice that received parent . Unlike parent, mutant was rarely detected in the brains of s.c . inoculated mice . Genetic analysis revealed that mutant had single amino acid substitutions in each of the E and NS5 proteins compared with parent . Furthermore, while mutant infection of BHK-21 cells was inhibited by glycosaminoglycans (GAGs), this was not the case for parent . In summary, the BHK-21-cell-adapted mutant virus showed reduced neuroinvasiveness in mice due to low-level induction of viremia . The attenuation process involved a single amino acid change in the E protein, which may have resulted in the rapid clearance of the virus due to its high affinity for negatively charged molecules in vivo.

Transfusion, 2003 Sep, 43(9), 1309 - 16
Comparison of two apheresis systems for the collection of CD14+ cells intended to be used in dendritic cell culture; Strasser EF et al.; BACKGROUND: Monocytes collected by leukapheresis are increasingly used for dendritic cell (DC) culture in cell factories suitable for DC vaccination in cancer . STUDY DESIGN AND METHODS: Using modified MNC programs on two apheresis systems (Cobe Spectra and Fresenius AS.TEC204), leukapheresis components collected from 84 patients with metastatic malignant melanoma and from 31 healthy male donors were investigated . MNCs, monocytes, RBCs, and platelets (PLTs) in donors and components were analyzed by cell counters, WBC differential counts, and flow cytometry . RESULTS: In 5-L collections, Astec showed better results regarding monocyte collection rates (11.0 vs . 7.4 x 10(6)/min, p = 0.04) and efficiencies (collection efficiency, 51.9 vs . 31.9%; p < 0.001) . Both devices resulted in monocyte yields at an average of 1 x 10(9) (donors) and 2.5 x 10(9) (patients), whereas Astec components contained high residual RBCs . Compared to components with low residual PLTs, high PLT concentration resulted in higher monocyte loss (48 vs . 20%, p < 0.0001) before DC culture . CONCLUSION: The Astec is more efficient in 5-L MNC collections compared to the Spectra . Components with high residual PLTs result in high MNC loss by purification procedures . Thus, optimizing MNC programs is essential to obtain components with high MNC yields and low residual cells as prerequisite for high DC yields.

Brain Res, 2003 Sep 5, 983(1-2), 48 - 57
The effect of oxidative stress on accumulation of apolipoprotein E3 and E4 in a cell culture model of beta-amyloid angiopathy (CAA); Mazur-Kolecka B et al.; Apolipoprotein E (apoE) is a multifunctional molecule that is active during brain development, maintenance, and injury . Allele varepsilon 4 of apoE is recognized as a risk factor for beta-amyloidosis, but the responsible mechanisms are not clear . Recently, we showed that vascular smooth muscle cells (SMCs) from varepsilon 4/ varepsilon 4 carriers are the most susceptible to oxidative protein damage that was associated with the appearance of apoE-Abeta-immunoreactive granules in cells . Here, we demonstrate that apoE4 is more readily accumulated in SMCs treated with ferrous ions than is apoE3 . ApoE accumulated in lysosomes in the form of monomers, dimers, apoE-containing complexes, and apoE fragments . ApoE4 and apoE4-containing complexes persisted in SMCs longer than apoE3 and its complexes . Both isoforms of apoE stimulated formation of apoE-Abeta deposits and increased immobilization of iron in cultures treated with ferrous ions . The accumulation of apoE-Abeta deposits in lysosomes was associated with the appearance of lipid peroxidation products such as malondialdehyde and 4-hydroxynonenal-2-nonenal . The higher cellular accumulation of apoE4 than apoE3 in SMCs exposed to oxidative stress may facilitate development of beta-amyloid angiopathy that is more frequent in varepsilon 4/ varepsilon 4 carriers.

Gene Ther, 2003 Sep, 10(18), 1584 - 91
A comparison of gene repair strategies in cell culture using a lacZ reporter system; Nickerson HD et al.; Synthetic oligonucleotides and DNA fragments of less than 1 kilobase (kb) have been shown to cause site-specific genetic alterations in mammalian cells in culture and in vivo . We have used a lacZ reporter gene system to compare the efficiency of episomal and chromosomal gene repair in human embryonic kidney epithelial cells (HEK293), Chinese Hamster Ovary fibroblasts (CHOK1), human bronchial epithelial cells (16HBE), and mouse embryonic stem (ES) cells . The lacZ gene contains a G to A nucleotide change, (Glu to Lys mutation) that abrogates beta-galactosidase activity . We compared the efficiency of different gene repair methods to correct this mutation and restore beta-galactosidase activity . We evaluated PCR-generated double-stranded DNA fragments of 0.52-1.9 kb, single-stranded DNA oligonucleotides of 20, 35, or 80 bases containing internal phosphorothioate links, and a 68 base RNA:DNA oligonucleotide . All of the oligonucleotides and DNA fragments showed some gene repair ability with an episomal plasmid . Short DNA fragments of 0.52 kb or greater gave the highest frequencies of episomal gene repair while single-stranded DNA oligonucleotides gave the highest frequency of chromosomal repair . In the context of a chromosomal target, antisense DNA oligonucleotides gave 5-fold higher frequencies of gene repair than their sense counterparts . The RNA:DNA chimeric oligonucleotide gave little or no gene repair on either a chromosomal or episomal target.

Eur J Pharm Sci, 2003 Aug, 19(5), 433 - 42
Do cell culture conditions influence the carrier-mediated transport of peptides in Caco-2 cell monolayers?
Behrens I, Kissel T.
Despite the fact that different laboratories have reported large differences in permeability for actively transported substrates, Caco-2 cell monolayers are widely used as in vitro model to study small intestinal drug transport . Therefore, we investigated the effect of cell culture conditions, such as time in culture, membrane support, seeding density and supplements to the medium, on the morphology, the formation of tight junctions, as well as the expression of two peptide transporters (PepT1, HPT1) and the efflux pump, P-glycoprotein (Pgp), in Caco-2 cell monolayers . Tight junction formation was assessed by transepithelial electrical resistance measurements; multi-cell layer formation by confocal laser scanning microscopy, the expression of transporters by RT-PCR and the permeability of the PepT1 substrate, cephradine . Both morphology and the expression of carrier-mediated transporters, varied strongly as a function of culture conditions . An increase of differentiation, as documented by tight, homogeneous cell monolayer formation displaying a strong expression of all carrier-mediated transporters, was found up to 3 weeks post seeding . One week later, multi-layer structures were observed and the expression of Pgp decreased . Polyester and polyethylene terephthalate membrane supports decreased the paracellular transport rates substantially, while collagen-coating of PC inserts showed no influence on the morphology and even increased carrier-mediated transporter expression . An average seeding density of 6x10(4) cells/cm(2) seemed to be most favorable, since lower seeding densities led to thin monolayers with altered tight junctions and higher seeding densities to the formation of multilayers . In summary, the expression of carrier-mediated transporters was strongly affected by the culture conditions . The full differentiation was reached after 21 days on collagen-coated polycarbonate inserts at an initial seeding density of 6x10(4) cells/cm(2).

J Clin Microbiol, 2003 Aug, 41(8), 3687 - 9
New generation of cell culture assay for smallpox vaccine potency; Leparc-Goffart I et al.; The potency of smallpox vaccines produced in the 1970s was tested by titration onto chorioallantoic membranes of fertilized hen eggs (CAM assay) . The potency specification commonly approved for these vaccines was a titer above 10(8) pock-forming units per milliliter . We developed and validated a cell culture titration assay to have a more reliable potency test . The cell titration assay and the CAM assay were tested in parallel on 34 first-generation smallpox vaccine lots . These allowed us to demonstrate that a correlation does exist between the two titration techniques and to determine a new in-house specification for the cell titration method . This in vitro potency assay will allow us to test first-generation smallpox vaccines produced on the skin of living animals but will also give a hint of the potency specification that should be assigned for new generations of cell-derived smallpox vaccines.

J Clin Microbiol, 2003 Aug, 41(8), 3597 - 601
Comparison of SmartCycler real-time reverse transcription-PCR assay in a public health laboratory with direct immunofluorescence and cell culture assays in a medical center for detection of influenza A virus; Habib-Bein NF et al.; A single-tube real-time (fluorogenic) reverse transcription (RT)-PCR with the SmartCycler instrument (SmartCycler RT-PCR) for influenza A virus detection was evaluated with 238 respiratory specimens . Direct immunofluorescence antibody staining (DFA) and primary rhesus monkey kidney cell culture were performed on-site at Yale-New Haven Hospital . Specimens were transported to the Connecticut Department of Public Health Laboratory for real-time RT-PCR . Cell culture detected influenza A virus in all 150 influenza A virus-positive specimens, DFA detected the virus in 148 influenza A virus-positive specimens, and SmartCycler RT-PCR detected the virus 143 influenza A virus-positive specimens . The sensitivity and specificity of RT-PCR were 95.3 and 100%, respectively . The high sensitivity and specificity and the rapid turnaround time made the SmartCycler RT-PCR valuable for the rapid diagnosis of influenza A, especially in a public health laboratory . The closed real-time RT-PCR system avoided cross-contamination possible with RT-PCR and the excessive manipulations required for conventional RT-PCR analysis and saved time and labor as well . In a medical center, rapid diagnosis by DFA was labor intensive but was 98.7% sensitive and 100% specific compared to the results of culture and provided results within 2 h throughout operating hours, helping with bed allocation on admission and patient management.

Gen Comp Endocrinol, 2003 Aug, 133(1), 61 - 70
Dexamethasone induced preadipocyte recruitment and expression of CCAAT/enhancing binding protein alpha and peroxisome proliferator activated receptor-gamma proteins in porcine stromal-vascular (S-V) cell cultures obtained before and after the onset of fetal adipogenesis; Hausman GJ; The present study examined the influence of dexamethasone (DEX) treatment on preadipocyte recruitment and expression of transcription factor proteins in adipose tissue stromal-vascular (S-V) cell cultures from 50 and 75 day old pig fetuses and young pigs . C/EBPalpha, C/EBPdelta, and PPARgamma immunoreactive cells had evenly reactive nuclei and unreactive nucleoli . DEX recruited many more preadipocytes in 75 day than in 50 day fetal S-V cultures . However, DEX did not increase the number of differentiated preadipocytes (lipid+, C/EBPalpha+) in 50 day S-V cultures and only slightly increased this number in 75 day fetal S-V cultures . In fetal cultures, extensive, precocious increases in C/EBPalpha expression (number of reactive cells) by day three were followed by extensive decreases in expression . However, PPARgamma expression was not expressed precociously since preadipocyte lipid accretion and PPARgamma immunoreactivity were strongly linked in fetal and pig S-V cultures . Nevertheless, all cells with lipid in fetal S-V cultures were C/EBPalpha and PPARgamma reactive . DEX increases preadipocyte differentiation in pig S-V cultures and in this study DEX increased PPARgamma expression to a much greater degree in pig than in fetal S-V cultures . These studies suggest that restricted adipogenesis in the pig fetus is attributable to limited DEX induced PPARgamma expression.

Ann Univ Mariae Curie Sklodowska {Med}, 2002, 57(2), 496 - 504
Changes in serum concentrations and in cytokine production by blood cell cultures of a patient with major depression--case report; Dubas-Slemp H et al.; The aim of the paper was to compare the periodical changes in serum cytokine levels and in cytokine production in short-term blood lymphocyte cultures of two persons: the patient with major depression and healthy control . In sera of both persons such cytokines as IL-1 beta, IFN-gamma and IL-6 were detected, but IL-6 level in serum of depressed patient was higher than that observed in healthy control . In both persons examined serum cytokine level changed periodically during 9 days of observation showing rhythmic waves . When cytokines were induced in lymphocyte cultures periodical changes in their production were also observed, but IL-1 alpha, IL-1 beta and IL-12 production was significantly lower in comparison to lymphocytes of healthy control . After 36 days of medical treatment cytokine levels in serum of patient normalized, except for IL-12 . Our results suggest that inconsistence in the data from papers of different authors concerning cytokine production in major depression could result from periodical changes in their level and from the fact that patients were examined at the time when certain cytokine was near its peak or declined significantly . The rhythmic changes in cytokine level should be taken into consideration when the role of cytokines in major depression is examined.

Arch Dermatol Res, 2003 Sep, 295(5), 190 - 8 Epub 2003 Aug 01.
The mesenchymal substrate influences the epithelial phenotype in a three-dimensional cell culture; Merne M et al.; Cell-matrix interactions are thought to influence epithelial structure, growth, and differentiation . Three-dimensional cell cultures were used to study the effects of the composition of the dermal equivalent on the morphology of epithelium grown from HaCaT skin keratinocytes . Three commercial preparations, a basement membrane preparation from a tumor (matrix 1), two preparations consisting of collagen type I and III (matrix 3 and 4) and a noncommercial preparation containing collagen type I (matrix 2) were investigated . The effects of fibroblasts of different origin (vaginal mucosa, oral buccal mucosa, and skin) were investigated in connection with matrix 4 . The histomorphology and expression of the proteins PCNA, Ki-67, p53, p21, pankeratin, involucrin, cytokeratin 10 (Ck10), Ck17, Ck19 and collagen type IV were evaluated . Three-dimensional cultures of HaCaT cells gave rise to an epithelium with an immature and hyperproliferative character, showing active proliferation with intense PCNA staining . Both matrix 1 and matrix 2 resulted in an epithelium with budding into the matrix and some degree of layering . These epithelia showed only scattered Ck17 and Ck19 expression but a low terminal differentiation potential as indicated by scattered Ck10 and involucrin staining . The epithelia of cocultures with matrices 3 and 4 were positive for Ck17 and Ck19 . However, the epithelium on matrix 3 showed strong expression of the terminal differentiation marker Ck10 and involucrin . This methodological study provides evidence of the importance of standardization of the composition of the matrix to avoid confounding effects on epithelial morphology and protein expression in studies using a three-dimensional epithelial culture model.

Int Immunopharmacol, 2003 Sep, 3(9), 1301 - 12
Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus; Han SB et al.; We investigated the mechanism of the immunomodulatory action of polysaccharide (ASP) isolated from a cell culture of Acanthopanax senticosus . ASP was found to directly increase the proliferation and differentiation of B cells, and the cytokine production of macrophage, but not the proliferation and cytokine production of T cells . Since ASP cannot penetrate the cell membrane due to its large molecular mass, such cellular activation may be caused by the surface binding of ASP to receptors expressed on B cells and macrophages . The possibility that TLRs, which are known to be involved in immune-related responses, may be the receptor(s) of ASP was investigated . The immunomodulating activities of ASP on the B cells and macrophages of C3H/HeJ mice, expressing a defective toll-like receptor (TLR)-4, were decreased versus the corresponding cells from C3H/HeN mice . In addition, the activities of ASP on B cells and macrophages were significantly reduced by treating the cells with antibodies to TLR4 and TLR2 prior to ASP, suggesting that both of them are the possible receptors of ASP . The ligation of TLRs induced by ASP was able to activate mitogen-activated protein kinases (MAPKs), such as Erk1/2, p38 and JNK, and the transcription factor NF-kappaB . Although ASP was shown to activate the TLR signaling cascades in the same manner as lipopolysaccharide (LPS), these two could be differentiated by the finding that polymyxin B (PMB), a specific inhibitor of LPS, did not significantly affect the activities of ASP on B cells and macrophages . Taken together, our results demonstrate that ASP, isolated from a cell culture of A . senticosus, activates B cells and macrophages by interacting with TLRs and leading to the subsequent activation of mitogen-activated protein kinases and NF-kappaB.

Prostate, 2003 Sep 15, 57(1), 57 - 65
Increased growth factor production in a human prostatic stromal cell culture model caused by hypoxia; Berger AP et al.; BACKGROUND: Local hypoxia may be one of the triggers of embryonic reawakening of the stroma and subsequent hyperplastic growth in the prostate . Using a cell culture model of human prostatic stromal cells, we investigated the effects of hypoxia on activation of hypoxia-inducible factor 1 (HIF 1) and on the production of growth factors . METHODS: Primary prostatic stromal cells were grown in normal and hypoxic (1% O(2)) atmosphere . Activation of HIF 1 was evaluated after different time intervals by Western blot . Induced secretion of growth factors VEGF, FGF-7, TGF-beta, IL 8, and FGF-2 were analyzed by ELISA . To confirm the in vitro findings we also performed immunohistochemistry of HIF 1alpha as well as pro-collagen I, collagens I, III, and IV in the benign tissue of radical prostatectomy specimens . RESULTS: HIF 1 is activated in a time-dependent manner, already starting 1 hr after exposure of stromal cells to hypoxic conditions . Secretion of VEGF, FGF-7, TGF-beta, FGF-2, and IL 8 is increased under hypoxic in vitro conditions in comparison to normoxia . Levels of TGF-beta, VEGF, and IL 8 were rapidly and statistically significantly increased in the supernatant of hypoxic cells . Consistent with the in vitro findings, immunohistochemistry of HIF 1alpha in (benign prostatic hyperplasia) BPH tissue revealed strong HIF 1alpha nuclear staining in hyperplastic areas . No difference was observed in the collagen pattern between hyperplastic and normal prostate tissue . CONCLUSIONS: Prostatic stromal cells respond to hypoxia by upregulation of secretion of several growth factors suggesting that hypoxia can trigger prostatic growth . Therefore, hypoxia might be a key factor contributing to the pathogenesis of BPH .

J Virol, 2003 Aug, 77(16), 8793 - 800
Adaptation of Puumala hantavirus to cell culture is associated with point mutations in the coding region of the L segment and in the noncoding regions of the S segment; Nemirov K et al.; We previously developed a model for studies on hantavirus host adaptation and initiated genetic analysis of Puumala virus variants passaged in colonized bank voles and in cultured Vero E6 cells . With the data presented in this paper, the sequence comparison of the wild-type and Vero E6-adapted variants of Puumala virus, strain Kazan, has been completed . The only amino acid substitution that distinguished the two virus variants was found in the L protein, Ser versus Phe at position 2053 . Another mutation found in the L segment, the silent transition C1053U, could result from the selection of a variant with altered L RNA folding . Nucleotide substitutions observed in individual L cDNA clones, most of them A-->G and U-->C transitions, suggested that the population of L RNA molecules is represented by quasispecies . The mutation frequency in the L segment quasispecies appeared to be similar to the corresponding values for the S and M quasispecies . Analysis of the cDNA clones with the complete S segment sequences from passage 20 confirmed our earlier conclusion that the cell-adapted genotype of the virus is represented mostly by variants with mutated S segment noncoding regions . However, the spectrum of the S segment quasispecies appeared to be changing, suggesting that, after the initial adaptation (passages 1 to 11), the viral population is still being driven by selection for variants with higher fitness.

Methods Cell Biol, 2003, 71, 129 - 56
Working with Xenopus spinal neurons in live cell culture; Gomez TM et al.; Neurons from the Xenopus spinal cord are highly versatile and easily manipulated, making them an ideal model system to answer questions regarding the cellular and molecular basis of early neural development and function . Xenopus has been a productive model system in studies ranging from axon growth and guidance to synaptic plasticity . Exogenous molecules, such as proteins, fluorescent tracers, and nucleic acids, can be injected into early blastomeres to load tracers in all neurons or into late blastomeres to target specific classes of neurons based on established lineage maps . Xenopus spinal neurons also provide an excellent culture system, as neurons extend processes on a variety of substrata and develop at room temperature in minimal salt solutions . Live fluorescent neurons can be imaged for hours with fluorescence microscopy at room temperature in static cultures without neurotrophic support or serum . This highly reduced culture system minimizes variables that can confound interpretation of results . Cultures can be prepared at various stages of development as dissociated neurons or as spinal cord explants . Both excitatory and inhibitory neurons develop in culture, and synaptic contacts among neurons and between neurons and nonneuronal targets form naturally . The simple anatomy and rapid rostral-to-caudal development of the Xenopus spinal cord also make this an excellent in vivo model system to analyze axon guidance by identifiable classes of neurons . This chapter focuses on techniques that exploit both in vitro and in vivo qualities of this system.

Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 151 - 5 Epub 2003 Mar 13.
Enhancement of taxol production and release in Taxus chinensis cell cultures by ultrasound, methyl jasmonate and in situ solvent extraction; Wu J et al.; This study evaluates the use of a novel mechanical stimulus, ultrasound (US), and a putative chemical elicitor, methyl jasmonate (MJ), combined with in situ solvent extraction (two-phase culture), to enhance taxol production by Taxus chinensis cells in suspension culture . The volumetric taxol yield was increased 1.5- to 1.8-fold with 2 min US treatment once or twice during a 4-week culture period, about 5-fold with 60-120 microM MJ, and 7- to 9-fold by in situ solvent extraction of taxol with dibutyl phthalate (DBP) (11% v/v) . The percent of extracellular taxol or taxol release was also significantly increased . The combined use of US (day 5 or 9) and MJ treatment (day 7) resulted in taxol yields 20-50% higher than each of the treatments used alone . The most favorable strategy for taxol production was the application of US or MJ treatment, followed by in situ solvent extraction, giving rise to a taxol yield of 33-35 mg/l, about 17-fold higher than the control, at 1.9 mg/l . It was found that the organic solvent DBP, as well as US and MJ, stimulated the enzyme activity of secondary metabolic pathways, which was partially responsible for the enhanced taxol production.

Anesthesiology, 2003 Aug, 99(2), 368 - 75
Neuroprotective effects of propofol in a model of ischemic cortical cell cultures: role of glutamate and its transporters; Velly LJ et al.; BACKGROUND: During cerebral ischemia, excess of glutamate release and dysfunction of its high affinity transport induce an accumulation of extracellular glutamate, which plays an important role in neuronal death . The authors studied the relationship among propofol neuroprotection, glutamate extracellular concentrations, and glutamate transporter activity in a model of ischemic cortical cell cultures . METHODS: Thirteen-day-old primary cortical neuronal-glial cultures were exposed to a 90-min combined oxygen-glucose deprivation (OGD) in an anaerobic chamber, followed by reoxygenation . Propofol was added only during the OGD period, and its effect was compared to that of the N-methyl-d-aspartate receptor antagonist dizocilpine (MK-801) . Twenty-four hours after the injury, cell death was quantified by lactate dehydrogenase release and cell viability by reduction of 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT) . Extracellular concentrations of glutamate in culture supernatants and glutamate uptake were performed at the end of OGD period by high-performance liquid chromatography and incorporation of l-{3H}glutamate into cells, respectively . RESULTS: At clinically relevant concentrations (0.05-10 microm), propofol offered protection equivalent to that of MK-801 . It significantly reduced lactate dehydrogenase release and increased the reduction of MTT . At the end of the ischemic injury, propofol was able to reverse the OGD-induced increase in glutamate extracellular concentrations and decrease of glutamate uptake . The inhibition of the glial GLT1 transporter by 3-methyl-glutamate did not further modify the effect of propofol on glutamate uptake, suggesting that GLT1 was not the major target of propofol . CONCLUSION: Propofol showed a neuroprotective effect in this in vitro model of OGD, which was apparently mediated by a GLT1-independent restoration of the glutamate uptake impaired during the injury.

Diabetes, 2003 Aug, 52(8), 2007 - 15
Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture; Gao R et al.; We have reproduced a previously described method for the in vitro generation of endocrine cells in adult human pancreatic tissue culture . The aim of this study was to characterize the nature of pancreatic progenitor cells and to identify the factors necessary for their differentiation in this model . During monolayer expansion, two types of cells proliferated sequentially; first cytokeratin 19 (CK19)-positive ductal epithelial cells and then nestin-positive fibroblastoid cells . After the bromodeoxyuridine-labeled cells were traced in differentiated islet buds, some of the proliferating ductal cells had differentiated into endocrine cells, whereas nestin-positive cells could not give rise to endocrine tissue . Serum-free culture was found to be an absolute requirement for the endocrine differentiation to occur . Also, overlay of the cells with Matrigel was essential, whereas nicotinamide had a potentiating effect . The in vitro-generated islet buds released insulin in response to glucose nearly as efficiently as native islets . When transplanted under the kidney capsule of nude mice, only one of five grafts demonstrated further growth with foci of both endocrine and exocrine differentiation . Our results support the previous notion that pancreatic progenitor cells represent a subpopulation of ductal epithelial cells . No evidence was found for the development of endocrine cells from nestin-positive stem cells.

Biotechnol Lett, 2003 Mar, 25(5), 381 - 5
Conjugation of heparin into carboxylated pullulan derivatives as an extracellular matrix for endothelial cell culture; Na K et al.; A carboxylated pullulan, for use as a structural material for a number of tissue engineering applications, was synthesized and conjugated with heparin . By immobilization of heparin to pullulan, endothelial cells (ECs) attached on the heparin-conjugated pullulan were more aggregated than when attached to other pullulan derivatives . Attachments were 50, 45, 49, and 90% for a polystyrene dish, pullulan acetate, carboxylated pullulan, and heparin-conjugated pullulan, respectively . Heparin-conjugated pullulan inhibited the proliferation of smooth muscle cells (SMCs) in vitro . Heparin-conjugated pullulan material can thus be used for the proliferation of vascular ECs and to inhibit the proliferation of SMCs.

Clin Nutr, 2003 Aug, 22(4), 391 - 9
Exogenous nucleosides alter the intracellular nucleotide pool in hepatic cell cultures . Implications in cell proliferation and function; Arnaud A et al.; BACKGROUND & AIMS: Dietary nucleotides are reported to influence the growth and functioning of the liver . The objective of the study was to evaluate the uptake and incorporation of exogenous nucleosides by hepatic cells, and the potential implications for cell proliferation and function . METHODS: Liver stellate cell line CFSC-2G and primary hepatocytes in single and mixed cultures were exposed to mixtures of nucleosides and the concentrations of nucleoside derivatives were determined in the cultures, by high-performance liquid chromatography . Cell proliferation (DNA synthesis, cell cycle) and function (adenylate charge, albumin content, mitochondrial succinate dehydrogenase activity) were also evaluated . RESULTS: The exogenous nucleosides increased the intracellular concentrations of UTP, UDP-glucose, CDP-choline and NAD(+), in the single cultures of CFSC-2G and hepatocytes . Modification of the intracellular nucleotide pool paralleled changes in cell functional status, as indicated by increased adenylate charge and albumin content in hepatocyte cultures and in their co-cultures with CFSC-2G, and by increased succinate dehydrogenase activity in hepatocytes . CONCLUSION: Exogenous nucleosides were taken up by CFSC-2G and hepatocytes, which modified the intracellular concentrations of nucleotides, improved the functional status of hepatocytes, and partially restored the impaired adenylate charge of the co-cultures.

Brain Res Mol Brain Res, 2003 Jul 23, 115(2), 187 - 95
Ca2+-independent phospholipases A2 and production of arachidonic acid in nuclei of LA-N-1 cell cultures: a specific receptor activation mediated with retinoic acid; Antony P et al.; The LA-N-1 cell nucleus contains Ca2+-independent phospholipase A2 (PLA2) activity hydrolyzing plasmenylethanolamine (PlsEtn) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PtdEtn) . These enzymes hydrolyze glycerophospholipids to produce arachidonic acid and lysoglycerophospholipids . The treatment of LA-N-1 cell cultures with all-trans retinoic acid (atRA) results in time- and dose-dependent stimulation of PlsEtn-PLA2 and PtdEtn-PLA2 activities in the nuclear fraction . PLA2 activities in the non-nuclear fraction (microsomes) are not affected by atRA, whilst the pan retinoic acid receptor (RAR) antagonist, BMS493, blocks the PLA2 activities in the nuclear fraction . This indicates that the stimulation of PLA2 activities is a receptor-mediated process . Treatment of LA-N-1 cell cultures with cycloheximide has no effect on basal PLA2 activities . However, atRA-mediated stimulation of PLA2 activities in LA-N-1 cell nuclei is partially inhibited by cycloheximide indicating that this decrease in PLA2 activity is due to a general decreased protein synthesis . Our results also support earlier studies in which atRA induces morphologic differentiation through the stimulation of PLA2-generated second messengers such as arachidonic acid and eicosanoids.

J Biomater Appl, 2003 Jul, 18(1), 63 - 78
Surface reactivity of calcium phosphate based ceramics in a cell culture system; John A et al.; Surface reactivity of Calcium Phosphate materials--Hydroxyapatite (HA), Tricalcium Phosphate (beta-TCP), Hydroxyapatite-Tricalcium Phosphate (HA-TCP) were elucidated in a cell culture system . MG-63 osteoblast-like cells were seeded onto the ceramic discs to evaluate changes in the cell morphology and functionality with respect to the different substrates . The dissolution and re-precipitation of calcium phosphate phases on the surface of the discs in the culture medium was found to be prominent on beta-TCP when compared with HA . Low calcium (Ca), magnesium (Mg) and alkaline phosphatase (ALP) levels and high phosphorous (P) levels in the medium of beta-TCP were observed . This indicated that P must have leached out into the medium from beta-TCP and Ca in turn deposited from the medium onto beta-TCP resulting in the apatite phase transformation . The low ALP activity in beta-TCP medium is however an indication of low osteoblastic activity . Under the phase contrast microscope, the osteoblast cells around HA material were found to be confluent and viable, while in the vicinity of beta-TCP only cellular debris was observed . In the case of HA-TCP, only a few viable cells surrounded the material amidst the debris . Scanning electron microscopy revealed numerous cells on the surface of HA showing different cell behaviour like anchorage, attachment, adhesion and spreading in the early time period as the surface was only slightly disturbed with re-crystallisation . But with time the entire surface of HA had changed due to precipitation and re-crystallization which did not support cell behaviour while the cells surrounding the material showed normal growth . On the contrary, cells were scarcely observed on the entirely changed surface of beta-TCP and HA-TCP even from the earlier days of the culture and the morphology of cells surrounding the material too started changing . These results establish that HA promoted the activity of osteoblast cells . HA surface remained unaltered for some time, while the surface of beta-TCP underwent dissolution of surface ions and resulted in the re-crystallization of apatite over the surface . The resulting changes in the surrounding milieu of beta-TCP with high phosphate and low Ca levels probably was responsible for the death of the cells.

J Plant Physiol, 2003 Jun, 160(6), 607 - 14
Regulation of anthraquinone biosynthesis in cell cultures of Morinda citrifolia; Stalman M et al.; Cell cultures of Morinda citrifolia L . are capable of accumulating substantial amounts of anthraquinones . Chorismate formed by the shikimate pathway is an important precursor of these secondary metabolites . Isochorismate synthase (EC 5.4.99.6), the enzyme that channels chorismate into the direction of the anthraquinones, is involved in the regulation of anthraquinone biosynthesis . Other enzymes of the shikimate pathway such as deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) do not play a regulatory role in the process . The accumulation of anthraquinones is correlated with isochorismate synthase activity under a variety of conditions, which indicates that under most circumstances the concentration of the branchpoint metabolite chorismate is not a rate-limiting factor . Anthraquinone biosynthesis in Morinda is strongly inhibited by 2,4-D, but much less by NAA . Both auxins inhibit the activity of isochorismate synthase proportionally to the concomitant reduction in the amount of anthraquinone accumulated . However, the correlation between enzyme activity and rate of biosynthesis is less clear when the activity of the enzyme is very high . In this case, a limiting concentration of precursor may determine the extent of anthraquinone accumulation . Partial inhibition of chorismate biosynthesis by glyphosate leads to less anthraquinone accumulation, but also to a reduction in ICS activity . The complexity of the interference of glyphosate with anthraquinone biosynthesis is illustrated by the effect of the inhibitor in cell cultures of the related species Rubia tinctorum L . in these cells, glyphosate leads to an increase in anthraquinone content and a concomitant rise in ICS activity . All data indicate that the main point of regulation in anthraquinone biosynthesis is located at the entrance of the specific secondary route.

Genes Dev, 2003 Aug 1, 17(15), 1855 - 69 Epub 2003 Jul 17.
Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation; Ayyanathan K et al.; Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation . Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9) . To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain . We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing . In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter . Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation . Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable.

Inflamm Res, 2003 Jun, 52(7), 283 - 6
PAF-receptor in inflammatory versus non inflammatory human epidermis, cell cultures and embryonal cells; Bayerl C et al.; OBJECTIVE: PAF-R (platelet activating factor-receptor) has been found on human keratinocytes to bind PAF, a proinflammatory phospholipid . We aimed to study PAF-R in a range of dermal cell lines and in samples of normal and psoriatic human skin to learn about its further role in humans . METHODS: PAF-R was labeled immunocytochemically, histochemically and additionally studied with western blotting in human keratinocytes, human fibroblasts, embryonal keratinocytes, tumor cell lines and samples of normal and psoriatic human skin . RESULTS: Keratinocytes from adult and embryonal epidermis of the 20th week of pregnancy showed a low level of labeling for PAF-R, but 3 +/- 0.05% of plantar keratinocytes from adults were positive as were 4.2 +/- 0.05% of embryonal plantar keratinocytes from the 21st week of pregnancy . In fibroblasts from adult and embryonal epidermis the protein was expressed at low levels . Western blotting revealed PAF-R positive bands at 67 k.Da in normal human skin and psoriasis, in psoriasis additionally at 45 k.Da . A 68 k.Da band was found in the colon cancer line HT 29 (control), and HaCaT cells, in embryonal keratinocytes additionally at 116 k.Da . CONCLUSIONS: PAF-R seems not to be important for embryonal or adult fibroblasts . In embryonal keratinocytes it is turned on after the 21st pregnancy week in a few cells seen as a band of 67 k.Da and at 116 k.Da, the latter is not found in adult keratinocytes . An additional 45 k.Da band of PAF-R was found in psoriasis that might stand for a truncated receptor . In the epithelial tumor cell line HaCaT and the HT29 colon cancer cell line PAF-R characterizes the anti-apoptotic effect of this receptor propagating tumor proliferation.

Am J Obstet Gynecol, 2003 Jul, 189(1), 22 - 7
Estrogen and progesterone receptor subtype expression in normal and malignant ovarian epithelial cell cultures; Li AJ et al.; OBJECTIVE: Epidemiologic data suggest that the malignant transformation of ovarian epithelium may be linked to altered steroid hormone homeostasis . STUDY DESIGN: Estrogen receptor-alpha, estrogen receptor-beta, progesterone receptor A, and progesterone receptor B messenger RNA and protein expression were evaluated by reverse transcriptase-polymerase chain reaction and Western blot analysis in primary cell cultures of human ovarian surface epithelium (n = 23 cultures) and Cedars-Sinai ovarian cancer (n = 23 cultures) . RESULTS: The ratio of estrogen receptor-alpha/estrogen receptor-beta messenger RNA expression was 10 times higher in primary ovarian cancer cultures (9.94 +/- 3.90) than in normal ovarian surface epithelium cultures (1.00 +/- 0.16, P =.04) . Estrogen receptor-alpha/estrogen receptor-beta protein ratio in primary ovarian cancer cultures (2.13 +/- 0.43) was twice that of normal human ovarian surface epithelium cultures (1.00 +/- 0.13, P =.05) . Individual estrogen receptor-alpha and estrogen receptor-beta messenger RNA and protein expression were not significantly different . Progesterone receptor B protein levels in primary ovarian cancer cultures (2.08 +/- 0.42) were twice that of normal surface ovarian epithelium cultures (1.00 +/- 0.10, P =.04), although differences in progesterone receptor B messenger RNA and progesterone receptor A protein expression were not observed . CONCLUSION: Malignant ovarian epithelial cells demonstrated multiple alterations in the expression of sex steroid hormone receptors.

Photochem Photobiol Sci, 2003 Jun, 2(6), 660 - 7
Zinc octa-n-alkyl phthalocyanines in photodynamic therapy: photophysical properties, accumulation and apoptosis in cell cultures, studies in erythrocytes and topical application to Balb/c mice skin; Kaestner L et al.; Two octa-substituted phthalocyanines, namely 1,4,8,11,15,18,22,25-octakis(decyl)phthalocyaninato zinc(II) (ZnODPc) and 1,4,8,11,15,18,22,25-octakis(pentyl)phthalocyaninato zinc(II) (ZnOPPc), were investigated for their use in photodynamic therapy (PDT) after topical application . Both substances exhibited favourable properties as photosensitisers in vitro: absorption maxima around 700 nm with absorption coefficients of about 190000 (M(-1) cm(-1)), a singlet oxygen quantum yield of 0.47 +/- 0.02 (ZnODPc), and good accumulation in keratinocytes and fibroblasts . Cell death after phthalocyanine-photosensitisation appeared to occur mainly via apoptosis . The in vivo experiments demonstrated a good accumulation of the phthalocyanines after topical application in a tetrahydrofuran-azone formulation onto the dorsal skin of Balb/c mice: {(4.6-4.7) +/- 1.0}% of deposited dye could be recovered after 3 h from deposition . ZnODPc showed significantly better skin-photosensitising properties than ZnOPPc and is therefore a potential candidate for the treatment of psoriasis.

J Med Virol, 2003 Sep, 71(1), 7 - 17
Importance of amino acid 216 in nonstructural protein 2B for replication of hepatitis A virus in cell culture and in vivo; Graff J et al.; Clinical isolates of hepatitis A virus (HAV) replicate inefficiently in cell culture unless mutations are acquired throughout the genome . An Ala-to-Val substitution in the nonstructural protein 2B (2B-216) was known to have a major impact on replication in cell culture . Analysis of chimeric viruses confirmed that the 2B-A{216}V change was critical for efficient replication and that Leu or Ile could substitute for Val . Viruses containing Val, Ile, or Leu at 2B-216 all replicated with similar kinetics in cell culture, whereas the virus containing Ala at this position grew 10- to 20-fold less efficiently . In contrast, in vivo, virus with either Ala or Val at 2B-216 replicated equally efficiently when tested in a chimpanzee and in tamarins, and each amino acid was stably maintained . Attempts to complement wild-type 2B in trans with adapted 2B provided by co-infection with a second viable HAV mutant failed to enhance replication of the virus containing the wild-type 2B sequence .

Urol Oncol, 2003 Mar-Apr, 21(2), 117 - 22
Expression of inducible nitric oxide synthase in paired neoplastic and non-neoplastic primary prostate cell cultures and prostatectomy specimen; Wang J et al.; Nitric oxide (NO) is an important signaling molecule for ischemia, inflammation, angiogenesis, immune response, and cell growth and differentiation . It has recently been shown that increased production of NO within various human cancers may contribute to tumor angiogenesis, tumor growth and metastasis, and tumor-related immune suppression . NO can be produced by several NO synthases (NOS), including inducible synthase (iNOS), which is expressed during cell activation and produces NO in larger quantity and for a longer period of time than non-inducible NOSs . In this study, we examined the expression levels of iNOS mRNA and protein in prostate adenocarcinoma using a paired nonneoplastic and neoplastic primary prostate cell culture system and related prostatectomy specimens . Six pairs of neoplastic and nonneoplastic primary prostate cell cultures were established from radical prostatectomy specimens based on homogeneity of the originating tumor and the nonneoplastic tissue . Radioactive reverse transcriptase polymerase chain reaction and subsequent quantitative analysis of iNOS mRNA were performed on the cultures using beta-actin as an internal control . Immunohistochemical studies with an anti-iNOS monoclonal antibody were performed on the corresponding formalin-fixed paraffin-embedded prostatectomy tissue sections . We observed marked patient-to-patient variation in "normal" levels of iNOS mRNA . However, all six neoplastic cultures showed moderately to markedly higher mRNA levels than did their paired nonneoplastic cultures . In addition, iNOS protein levels were significantly higher in paraffin-embedded prostate cancer tissue sections than in adjacent nonneoplastic tissue . Overexpression of iNOS mRNA and protein levels is present in moderately differentiated prostate adenocarcinoma and may contribute to prostate cancer angiogenesis, tumor growth, and tumor-related immunosuppression.

J Neurosci, 2003 Jul 9, 23(14), 6123 - 31
Inhibitory but not excitatory cortical neurons require presynaptic brain-derived neurotrophic factor for dendritic development, as revealed by chimera cell culture; Kohara K et al.; To address questions of whether endogenous BDNF acts differentially on inhibitory and excitatory neurons, and through what routes, we used chimera culture of cerebral cortical neurons derived from BDNF-/- mice and another type of transgenic mice that express green fluorescence protein and BDNF . Presynaptic BDNF transferred to both types of neurons, GABA-synthesizing enzyme-positive and -negative neurons . The latter neurons were confirmed to be glutamatergic with immunocytochemistry . Dendritic development of the former inhibitory neurons was promoted by endogenous BDNF transferred from presynaptic, excitatory neurons . In contrast, dendritic development of excitatory neurons was not related to the presence or absence of presynaptic BDNF, suggesting that BDNF acts on inhibitory neurons through an anterograde, transsynaptic route so as to promote dendritic development, whereas this is not the case in excitatory neurons.

Respir Physiol Neurobiol, 2003 Jul 16, 136(2-3), 131 - 9
In vitro intermittent hypoxia: challenges for creating hypoxia in cell culture; Baumgardner JE et al.; Intermittent hypoxia has been implicated in morbidities associated with sleep apnea, and may be a novel cellular signal for inflammation {J . Appl . Physiol . 90 (2001) 1986} . Standard cell culture has two major limitations for studying the effects of steady-state P(O(2)) and intermittent hypoxia . First, convective mixing in the culture media can be variable, making precise control of cellular P(O(2)) difficult . Second, diffusion of oxygen through the culture media slows changes in cellular P(O(2)) after rapid changes in the gas phase P(O(2)) . Our estimates of diffusional transients for standard cell culture suggest significant restrictions in the ability to cycle P(O(2)) at frequencies relevant to intermittent hypoxia . We present a novel system for forced convection cell culture with adherent cells inside capillary tubing . Steady state cellular P(O(2)) is regulated to an accuracy of approximately 1 Torr . The response time for cycling of P(O(2)) is less than 1.6 sec . This system is ideally suited for studies of intermittent hypoxia in adherent cells.

Laryngorhinootologie, 2003 Jun, 82(6), 408 - 15
{Influence of rhinologic usual and unusual drugs on fibroblasts from nasal polyps in cell culture}; Ostwald J et al.; BACKGROUND: Nasal polyposis is treated by surgery and/or by medication but in parts without permanent remission . Fibroblasts and their proliferation are involved in the complex mechanism of polyp genesis . Therefore we have analysed the influence of 12 medications on fibroblasts from nasal polyps growing in vitro . METHOD: Nasal polyps, obtained during usual surgical procedure, are enzymatically digested and cultured in serum containing media . The growing cells are identified as fibroblasts using flow cytometry with a AS02-FITC antibody (Dianova) . The analysis is achieved with 5 - 6 different fibroblast cultures in each medicament tested, mostly using concentrations of the active substance from 0.006 to 1.333 mg/ml . The fibroblasts are cultured 4 days in the presence of active substances or as controls . Finally the cells are trypsinated and counted . RESULTS: Mometason, Beclomethason, Fluticason, Verapamil and Timolol are the group with the strongest reduction of fibroblasts . Mometason shows a reduction to 6 % of controls at a concentration of 30 micro g/ml whereas the reduction at this concentration amounts to 30 - 60 % in the other members of this group . Mesazalin, Methylprednisolone and Pentoxifylline demonstrate the smallest influence; Prednisolon-21-hydrogen-succinate, Pilocarpin, Piroxicam and Diclofenac show an effect on a middle level . CONCLUSIONS: A strong reduction of fibroblasts from nasal polyps in vitro is possible with usual rhinological medicaments but also with unusual substances in this field.

Mol Ther, 2003 Jul, 8(1), 130 - 42
Evidence of protein transduction but not intercellular transport by proteins fused to HIV tat in retinal cell culture and in vivo; Cashman SM et al.; The human immunodeficiency virus type-1 Tat protein is known to exit virally infected cells and enter the nucleus of adjacent uninfected cells . This property has been mapped to an 11-amino-acid protein transduction domain (PTD) . When the PTD of Tat is fused to heterologous proteins and added exogenously to cells, the fusion peptide is able to demonstrate protein transduction across plasma membranes . Recent reports indicate that endogenously expressed Tat fusion peptides can demonstrate intercellular transport and improve biodistribution of therapeutic protein in the context of adenovirus vectors . Intercellular transport and protein transduction have not been observed in some studies and in the former have been attributed to an artifact of fixation . We have attempted to resolve these studies using an approach that unambiguously distinguishes cells that express Tat fusion protein from those that receive it from their environment . We find no evidence of intercellular transport in the context of an adenovirus vector in cell culture or in vivo . Instead, we find that Tat fusion peptides are down regulated in terms of expression not only in the context of adenovirus vectors, but also when expressed from transfected plasmid DNA . However, when Tat fusion peptides are released from cells by degradation of the plasma membrane, the fusion peptides demonstrate protein transduction without the need for cell fixation, indicating a unidirectional transport of Tat fusion proteins across the plasma membrane . Our data are consistent with previously reported studies and help to explain the apparently different results obtained from several different laboratories.

J Tongji Med Univ, 1999, 19(3), 233 - 6
Effects of the novel GH secretogogue, hexarelin on GH secretion and phosphatidylinositol hydrolysis by human pituitary somatotrophinomas in cell culture; Liu Q et al.; The effects of the novel GH-releasing hexapeptide, Hexarelin, on cultured human pituitary somatotrophinomas were investigated . Hexarelin (0.01-100 nmol/L) dose-dependently stimulated GH secretion up to 4.6-fold . Maximal effects occurred with 10 nmol/L . These effects were very similar to those observed with GHRP-6 . The effects of Hexarelin were reduced by phloretin, an inhibitor of protein kinase C (PKC) . The rate of phosphatidylinositol (PI) hydrolysis was markedly increased by Hexarelin in a dose-dependent manner . These results demonstrated that Hexarelin could directly stimulate GH secretion by human pituitary somatotrophs in a PKC-dependent manner, probably via activation of the PI transduction system.

Mikrobiyol Bul, 2002 Jul-Oct, 36(3-4), 301 - 8
{Sensitivities of various cell cultures for the isolation of enteroviruses}; Ozkaya E et al.; In the present study, the sensitivities of HEp-2 (human epithelioma), RD (rhabdomyosarcoma) and L20B (mouse cells, that have receptors for human polioviruses) cell cultures have been evaluated and compared, for the isolation and identification of enteroviruses from the stool and cerebrospinal fluid samples of patients with acute flask paralysis and aseptic meningitis, which were examined between the years 1999-2000, in Refik Saydam Institute of Hygiene Center, Virology, Tissue Culture and Enterovirus Laboratory . Of a total of 1663 samples, 131 viral strains were isolated, and 120 of them were identified as enteroviruses, and 11 as adenoviruses . The isolation rates of 48 Sabin-like polioviruses from HEp-2, RD and L20B cell lines were found similar, as 83.3%, 87.5% and 91.6%, respectively . All of 47 Echovirus strains were isolated from RD cells, all of 13 Coxsackie type B strains were isolated from HEp-2 cells, and all of 12 non-polio enteroviruses were isolated from RD cells . All of 11 adenovirus strains that have been grown in Hep-2 cells, were thought to be occasionally isolated due to the passage of viruses to gastrointestinal tract, and excreted via stool, thus having no clinical significance for these patients . As a result, it was concluded that, all of these three cell lines and especially L20B were sensitive for polioviruses, RD cell line being more sensitive for Echovirus, and HEp-2 cell line being more sensitive for Coxsackie type B virus strains.

Exp Cell Res, 2003 Jul 15, 287(2), 199 - 208
Time course of actin cytoskeleton stiffness and matrix adhesion molecules in human bronchial epithelial cell cultures; Doornaert B et al.; Human bronchial epithelial (HBE) cells adhere to underlying extracellular matrix (ECM) via integrin-type transmembrane receptors . Integrins link the ECM to the cytoskeleton (CSK), establishing a mechanical continuum by which forces are transmitted between the outside and the inside of the cells . The present study investigates the time course of global and actin CSK stiffness of HBE cells (16HBE14o-) growing on various matrix substrates as a function of culture time until confluence, and the concomitant time course of F-actin and adhesion molecule distribution . Our results showed a progressive increase in actin CSK stiffness from cell seeding to confluence, related to acquisition of highly polymerized cortical and cytosolic F-actin organization and up-regulation of certain matrix ligands, such as beta 1-, alpha 5-, and alpha v-integrin subunit expression . Moreover, compared to fibrillar type I collagen, reticular type IV collagen used as matrix substrate, appeared to amplify actin CSK stiffness of HBE confluent cells probably in relation to up-regulation of alpha 3-integrin subunit . Taken together, these results support the concept of a close interaction among actin CSK stiffness, structural actin organization, specific integrin molecule involvement, cell spreading, and extracellular matrix.

J Neurosci Res, 2003 Jul 15, 73(2), 141 - 55
Cell-specific expression pattern of monocarboxylate transporters in astrocytes and neurons observed in different mouse brain cortical cell cultures; Debernardi R et al.; Evidence suggests that lactate could be a preferential energy substrate transferred from astrocytes to neurons . Such a process implies the presence of specific monocarboxylate transporters on both cell types . Expression of MCT1 and MCT2, two isoforms of the monocarboxylate transporter (MCT) family, was studied in enriched cultures of mouse cortical astrocytes or neurons . It was observed that, at both the mRNA and the protein levels, astrocytes strongly expressed MCT1 but had very little if any MCT2 . By contrast, neurons had high amounts of MCT2 mRNA, although MCT1 mRNA was also detected . Double immunofluorescent labelings with appropriate markers confirmed the cell-specific preference in the expression of MCT1 and MCT2, but they revealed that a subset of neurons expresses low to moderate levels of MCT1 . Parallel immunocytochemical stainings of cultured neurons with the presynaptic marker synaptophysin showed that MCT2 expression is correlated with synaptic development . Although MCT2 and synaptophysin were not colocalized, their distribution was similar, and they were often closely apposed, suggesting that MCT2 could be associated with postsynaptic terminals . Interaction between astrocytes and neurons, as occurring in layered cultures, did not modify the levels of MCT1 and MCT2 expression or their distribution and cell-specific preference under the conditions used . However, a close apposition between neurites and MCT1-expressing astrocytic processes was apparent and developed as cultures evolved . In addition to providing an extensive description of MCT distribution in cultured cells, our data underscore the potential of such preparations for future studies on the regulation of MCT expression .

Brain Res, 2003 Jul 18, 978(1-2), 213 - 22
Glutamate-dependent glutamine, aspartate and serine release from rat cortical glial cell cultures; Rao TS et al.; Glia play a pivotal role in glutaminergic excitatory neurotransmission in the central nervous system by regulating synaptic levels of glutamate and by providing glutamine as the sole precursor for the neurotransmitter pool glutamate to neurons through the glutamate-glutamine cycle . In the present investigation, we examined the influence of glutamate application on glutamine, serine and aspartate release from rat cortical glial cultures . The glial glutamate transporters rapidly cleared exogenously applied glutamate and this was accompanied by rapid increases in aspartate and glutamine, and a delayed increase in serine levels in the glial-conditioned medium . While glutamate-induced increases in glutamine and serine were sustained for up to 24 h, increases in aspartate lasted only for up to 6 h . The glutamate-induced increases in aspartate and glutamine were dependent both on the concentration and the duration of glutamate stimulus, but were largely insensitive to the inhibition of non-N-methyl-D-aspartate receptors or the metabotropic glutamate receptor 5 . Inhibition of the glutamate transporter function by L-trans-pyrrolidine 2,4-dicarboxylate decreased the rate of glutamate uptake but not completely abrogated the uptake process, and this resulted in the attenuation of rate of glutamate induced glutamine synthesis . Dexamethasone treatment increased serine and glutamine levels in conditioned medium and increased glutamate induced glutamine release suggesting an upregulation of glutamine synthase activity . These results further substantiate coupling between glutamate and glutamine, and shed light on glutamate-dependent release of serine and aspartate, which may further contribute to excitatory neurotransmission.

J Chromatogr A, 2003 May 9, 996(1-2), 173 - 80
Isolation of chlorinated fatty acid methyl esters derived from cell-culture medium and from fish lipids by using an aminopropyl solid-phase extraction column; Akesson-Nilsson G; An aminopropyl-based solid-phase extraction technique was used for isolation of chlorinated fatty acids in lipids . A range of different chlorinated fatty acids was eluted in a small volume of solvent (4 ml) and the recoveries of the different species and isomers were quantitative . Only 1% of the vastly dominating unchlorinated fatty acid methyl esters were recovered in the fractions containing the chlorinated fatty acid methyl esters . This method makes it possible to isolate and detect > or = 1 microg of a chlorinated fatty acid methyl ester in 1 g of lipid.

Neurotox Res, 2002 Mar, 4(2), 161 - 3
Neurotoxicity of some MAO inhibitors in adult rat hypothalamic cell culture; Hurtado-Guzman C et al.; Monoamine oxidase-A (MAO-A) {amiflamine (AMF) and 4-methylthioamphetamine (MTA)} and MAO-B (L-deprenyl) inhibitors were found to be cytotoxic in a concentration-dependent manner for RCHT cells derived from adult rat hypothalamus . The cytotoxic effects were increased when the inhibitors were co-incubated with dicoumarol and especially with 25 micro M AMF+100 micro M dicoumarol (2.5-fold; P <0.001) . The treatment of RCHT cells solely with AMF induced a marked decrease in the expression of DT-diaphorase mRNA.

Placenta, 2003 Jul, 24(6), 638 - 47
Caffeine enhances the expression of the angiotensin II Type 2 receptor mRNA in BeWo cell culture and in the rat placenta; Tanuma A et al.; Although chronic caffeine exposure during pregnancy has been shown to affect fetal growth, adverse effects of caffeine on embryogenesis are not only well understood, but also controversial . We have used gene chip technology in an attempt to identify to what extent, if any, caffeine could possibly alter gene expressions in the cytotrophoblast-like cell line BeWo . Few down-regulated genes were found; most of the genes were up-regulated, suggesting that chronic caffeine exposure during the gestational period could exert certain influences on embryogenesis . The highest up-regulated gene expression of BeWo cells by caffeine was angiotensin II type 2 (AT(2)) receptor gene . We focused the genes of the renin-angiotensin system (RAS), angiotensin II type 1 (AT(1)) and AT(2)receptors and angiotensin I converting enzyme, for study on caffeine's responsive gene expression in BeWo cells and in the placentae of pregnant rats that were fed a diet supplemented with caffeine (2 mg/100 g body weight) during gestation, and analysed the gene expressions using RT-PCR and LightCycler system . A significantly increased AT(2)receptor gene expression and a slight decreased AT(1)receptor gene expression demonstrated the caffeine's effect to the placental RAS.

J Ocul Pharmacol Ther, 2003 Jun, 19(3), 265 - 70
Release of nitric oxide by N-nitropyrazoles in rabbit lacrimal gland cell culture; Xuan B et al.; PURPOSE: In order to prove the effects of N-nitropyrazoles to increase ocular blood flow and to facilitate retinal function after ischemia are due to nitric oxide (NO) release, eleven N-nitropyrazoles which are known NO donors were used to test their ability to produce NO in rabbit lacrimal gland (RLG) cell cultures . METHODS: Nitropyrazoles (0.3-3 microg/ml) were added into RLG cell culture and the NO produced was determined with nitrate-nitrite colorimetric assay . RESULTS: All nitropyrazoles (11 compounds) tested produced NO in various degrees with the highest degree by 13 fold over control (DN-5) and the lowest level by only 2.8 fold over control (DN-7) . CONCLUSION: The results indicated that NO is involved in ocular blood flow increase and retinal function recovery after ischemia by N-nitropyrazoles . However, there is no potency relationship between in vitro data and in vivo data, which is understandable because the experimental environments are different.

Med Microbiol Immunol (Berl), 2004 Nov, 193(4), 205 - 8 Epub 2004 Nov.
Supernatants from human cytomegalovirus (HCMV)-infected retinal glial cells increase transepithelial electrical resistance in a cell culture model: evidence of HCMV immune escape in the eye?
Scholz M, Margraf S, Menon S, Schuller A, Doerr HW, Cinatl J.
The underlying mechanisms leading to persistence of human cytomegalovirus (HCMV) in the immune privileged retina are not fully understood . This in vitro study was done to evaluate the influence of HCMV-infected retinal glial cells on epithelial barrier functions . Glial cells derived from human eyes were cultured and infected with the clinical HCMV isolate Hi91 . Supernatants of mock (GS(mock)) and Hi91 (GS(Hi91)) -infected glial cells were collected at 72 h post inoculation and used for incubation of CaCo-2 cells grown in transwell chambers . Transepithelial electrical resistance (TER) was analyzed as a measure of epithelial integrity . Virus-free GS(Hi91 )but not GS(mock) increased TER from 250 Omega/cm(2) to more than 1,000 Omega/cm(2 )within 2 h . Increased TER values were measured up to 48 h ( n=3) . No changes in TER were observed when conditioned supernatants from HCMV-infected human foreskin fibroblasts were used . No evidence of GS(Hi91)-induced modification of beta-catenin (zonula adherens) or occludin and ZO-1 (zonula occludens) was found . Our results suggest that HCMV-infected glial cells may support epithelial barrier functions by a yet unknown mechanism . Our findings may help to explain the ocular persistence of HCMV and the maintenance of ocular immune privilege early in infection.

Diabetologia, 2003 Jul, 46(7), 956 - 60 Epub 2003 Jun 21.
Induction of adiponectin gene expression in human myotubes by an adiponectin-containing HEK293 cell culture supernatant; Staiger H et al.; AIMS/HYPOTHESIS: Adiponectin, an adipocytokine known to be down-regulated in obesity-linked disorders, is considered to be a potential key mediator of insulin sensitivity . In this study, we asked whether adiponectin is able to regulate ten selected genes possibly associated with insulin sensitivity in human skeletal muscle cells . METHODS: To this end, we treated in vitro differentiated human myotubes with the culture supernatant of HEK293 cells stably transfected with human recombinant adiponectin and assessed gene expression by RT-PCR . Intracellular adiponectin protein was quantified by radioimmunoassay and visualized by Western blotting . RESULTS: In contrast to the control supernatant, the adiponectin-containing supernatant consistently induced expression of adiponectin mRNA in human myotubes from eight different donors (mean increase: 90-fold over control; n=8, p<0.001) . This increase in mRNA was paralleled by a rise in intracellular adiponectin protein (mean increase: 8.3-fold over control; n=4, p<0.05) . Expression of the other nine candidate genes was not altered . In human skin fibroblasts and HepG2 cells, the adiponectin-enriched supernatant did not induce relevant amounts of adiponectin mRNA . CONCLUSIONS/INTERPRETATION: In conclusion, we show here that adiponectin gene expression is specifically inducible in skeletal muscle cells.

Exp Eye Res, 2003 Jul, 77(1), 35 - 49
Differential protein expression in lens epithelial whole-mounts and lens epithelial cell cultures; Ong MD et al.; PURPOSE: Lens fibergenesis is a problem in several types of cataract and in the posterior capsular opacification following cataract surgery . To correct improper fiber differentiation or to prevent unwanted growth on the posterior capsule following cataract surgery requires a thorough understanding of normal and abnormal fiber formation . To this end, studies were initiated to characterize fiber differentiation in the bovine lens and in lens epithelial cell cultures . METHODS: Indirect immunofluorescence and immunoblot analysis were employed to study the expression of vimentin, beta-crystallin, gamma-crystallin, filensin, aquaporin 0 and the Na, K-ATPase catalytic subunit isoforms (alpha1, alpha2, alpha3) in bovine lens epithelium whole-mounts as well as lens epithelial cell cultures propagated in medium containing 10% bovine serum or in medium supplemented with bovine serum concentrations < or =4% . RESULTS: Three distinct cell types were observed in the bovine lens epithelium . The cells of the central zone were identified by a polarized distribution of two distinct Na, K-ATPase catalytic subunit isoforms, alpha1 to the apical (fiber side) and alpha3 to the basal (aqueous humor side) membranes . Lateral to the polarized central zone, was the germinative zone of cells, best characterized by perinuclear vimentin basket-like structures and the loss of polarized Na, K-ATPase catalytic subunit isoforms . Lateral to the germinative zone were the cells of the transition zone (meridinal rows) where expression of the lens specific proteins beta-crystallin, gamma-crystallin, filensin and aquaporin 0 as well as the lens fiber-, adipocyte- and brain glia-specific Na, K-ATPase catalytic subunit, alpha2 are expressed . The cultured cells propagated in medium supplemented with 10% serum bore no resemblance to any of the cells of the bovine lens epithelium whole-mounts . The cells propagated in the medium supplemented with the lower bovine serum levels resembled the differentiating fibers of the transition zone of the bovine lens epithelium whole-mounts as well as superficial cortical fibers . CONCLUSIONS: Since the low-serum lens epithelial cell cultures bear a remarkable resemblance to early differentiating fibers, they are reasonable models for the study of early fiber differentiation or prevention of differentiation . The culture conditions employed do not yield the polarized cells of the central zone . Nor has the function of these polarized cells in lens fluid, nutrient and ion homeostasis been determined.

Neurochem Int, 2003 Nov, 43(6), 563 - 71
Early biochemical and histological alterations in rat corticoencephalic cell cultures following metabolic damage and treatment with modulators of mitochondrial ATP-sensitive potassium channels; Reinhardt R et al.; The present study was aimed at characterizing alterations of the nucleotide content and morphological state of rat corticoencephalic cell cultures subjected to metabolic damage and treatment with modulators of mitochondrial ATP-dependent potassium channels (mitoK(ATP)) . In a first series of experiments, in vitro ischemic changes of the contents of purine and pyrimidine nucleoside diphosphates and triphosphates were measured by high performance liquid chromatography (HPLC) and the corresponding histological alterations were determined by celestine blue/acid fuchsin staining . As an ischemic stimulus, incubation with a glucose-free medium saturated with argon was used . Ischemia decreased the levels of adenosine, guanine and uridine triphosphate (ATP, GTP, UTP) and increased the levels of the respective dinucleotides ADP and UDP, whereas the GDP content was not changed . Both 5-hydroxydecanoate (5-HD) and diazoxide failed to alter the contents of nucleoside diphosphates and triphosphates, when applied under normoxic conditions . 5-HD (30 microM) prevented the ischemia-induced changes of nucleotide and nucleoside levels . Diazoxide (300 microM), either alone or in combination with 5-hydroxydecanoate (30 microM) was ineffective . Pyruvate (5 mM) partially reversed the effects of ischemia or ischemia plus 2-deoxyglucose (20mM) in the incubation medium . Diazoxide (300 microM) and 5-HD (30 microM) had no effect in the presence of pyruvate (5mM) and 2-deoxyglucose (20mM) . Staining the cells with celestine blue/acid fuchsin in order to classify them as intact, reversibly or profoundly injured, revealed a protective effect of 5-HD . When compared with 5-HD, diazoxide, pyruvate and 2-deoxyglucose had similar but less pronounced effects . In conclusion, these results suggest a protective role of 5-hydroxydecanoate on early corticoencephalic nucleotide and cell viability alterations during ischemia.

Anticancer Res, 2003 Mar-Apr, 23(2B), 1549 - 54
Tamoxifen enhances apoptotic effect of cisplatin on primary endometrial cell cultures; Drucker L et al.; BACKGROUND: Cisplatin (CDDP) dose-limited by its side-effects is, in some instances, synergistically amplified when combined with tamoxifen (TAM) . TAM has been shown to modulate apoptotic pathways of normal endometrial cells, whereas CDDP induces apoptosis in malignant endometrial cells . Their combined effect on normal or malignant endometrium is as yet unknown . This study aimed to evaluate the combined CDDP and TAM's apoptotic effect on normal endometrial tissue in the context of hormonal milieu . MATERIALS AND METHODS: Primary endometrial cell cultures were established and maintained both in the presence and absence of steroidal hormones . The cultures were treated for 24 hours with 20 microM TAM and 50 microM CDDP as single drugs and in combination . Apoptosis was determined by evaluation of pre G1 cell populations in the cell cycle analysis with flow cytometer . RESULTS AND CONCLUSION: CDDP induced apoptosis in all cultures regardless of hormonal environment, while TAM significantly enhanced CDDP-induced apoptosis in steroidal deficient media in an additive manner . These are novel findings depicting CDDP's effect on normal endometrium, singularly and combined with TAM.

Invest Clin, 2003 Jun, 44(2), 129 - 36
{Increase in nitric oxide concentrations in serum and mononuclear cell cultures from patients with Type II reaction state of Hansen's disease}; Rada E et al.; The word "reaction" is used in leprosy to describe signs and symptoms of acute inflammation . Type II reactions, including erythema nodosum leprosum (ENL) occur in the multibacillary forms of Hansen's disease . Nitric oxide (NO) could play a role in the response of the host, where a high NO production would be involved in acute inflammatory processes . In this paper we evaluate NO production in serum and in the supernatants of mononuclear cell cultures (MNCC), measured indirectly by Griess' method . The results obtained in serum showed that 52% of patients with ENL (15/29) had a production over 30 microM, distributed as follows: 8/15 had a mean concentration of 36.38 +/- ?5.71 microM; 1/15, 70.5 microM and 6/15 had a mean concentration greater than 100 microM (205.97 +/- 5 microM) . Forty eight percent presented nitrite and nitrate levels lower than 30 microM (18.93 +/- 6.15) . Only supernatants of mononuclear cell cultures from ENL patients collected at 120 hours of incubation presented NO production levels higher than 10 microM +/- 6.53, as compared with the supernatants from the stable polar forms of the disease (lepromatous leprosy and tuberculoid leprosy), where values were 2.52 microM +/- 1.18 and 2.69 microM +/- 1.07, respectively . These preliminary results show a different metabolic activity in the group of patients with Type II reaction state (ENL).

Neuroendocrinology, 2003 May, 77(5), 298 - 304
Regulation of follicle-stimulating hormone secretion by the interactions of activin-A, dexamethasone and testosterone in anterior pituitary cell cultures of male rats; Leal AM et al.; This study was designed to evaluate the effects of glucocorticoids and gonadal steroids on the expression of inhibin/activin subunits and follistatin of the anterior pituitary and test the hypothesis that resulting changes in the local activin/inhibin/follistatin tone contribute to steroid effects on follicle stimulating hormone (FSH) production from gonadotropes . In primary cell cultures of male rat anterior pituitaries, dexamethasone (DEX) or testosterone (T) stimulated FSH secretion and FSHbeta mRNA and their effects were additive with activin-A . Follistatin (FS288) and inhibin-A antagonized the rise in FSH secretion both in the absence and presence of exogenous activin-A . Despite the similarity in their action on FSH production, DEX and T had opposite effects on follistatin mRNA levels . Follistatin mRNA levels of cultured rat anterior pituitary cells were elevated upon the addition of DEX but attenuated by T . On the other hand, both DEX and T suppressed inhibin/activin betaB mRNA levels while only DEX affected betaA mRNA . In these cells, activin-A stimulated follistatin and inhibin/activin betaB mRNA levels but had no effect on betaA . Together, DEX and activin-A caused a further increase in follistatin mRNA levels while T attenuated the effect of activin-A alone . Both steroids attenuated the effect of activin-A on betaB mRNA accumulation . These results support the possibility that DEX and T, possibly acting on different subsets of anterior pituitary cells, use distinct mechanisms to modify the local activin/inhibin/follistatin circuitry and thereby upregulate FSH production from the anterior pituitary gonadotropes .

Planta Med, 2003 May, 69(5), 457 - 9
Kinobeon A as a potent tyrosinase inhibitor from cell culture of safflower: in vitro comparisons of kinobeon A with other putative inhibitors; Kanehira T et al.; Kinobeon A is produced from cell cultures of the medicinal plant, safflower . Mushroom tyrosinase activity was inhibited in a concentration-dependent manner when treated with kinobeon A using L-tyrosine or L-3,4-dihydroxyphenylalannine (L-DOPA) as substrates . IC50 values were 22 microM (substrate: L-tyrosine) and 27 microM (L-DOPA) . Inhibition of human tyrosinase activity also increased with increasing concentrations of kinobeon A using L-DOPA as the substrate, with an IC50 value of 2.5 microM . Kinobeon A was a more potent competitive inhibitor than kojic acid, arbutin or L-ascorbic acid for both mushroom and human tyrosinase as determined from Lineweaver-Burk plots . These results suggested that kinobeon A could be a potent natural tyrosinase inhibitor.

Mol Genet Genomics, 2003 Jul, 269(4), 542 - 52 Epub 2003 Jun 12.
Differential expression of six novel peroxidase cDNAs from cell cultures of sweetpotato in response to stress; Park SY et al.; Six peroxidase (POD) cDNAs were isolated from suspension cultures of sweetpotato (Ipomoea batatas) by cDNA library screening, and their expression was investigated with a view to understanding the physiological functions of each POD in relation to environmental stress . The gene products encoded by these cDNAs could be divided into two groups, anionic PODs (SWPA4, SWPA5, SWPA6) and basic PODs (SWPB1, SWPB2, SWPB3), on the basis of the predicted pI values of the mature proteins . RT-PCR analysis revealed that the six POD genes showed diverse expression patterns in various tissues of intact plants, a various stages of growth in suspension cultures, and in leaf tissues exposed to different stresses . The six genes from which they were derived are predominantly expressed in cultured cells of sweetpotato . Thus, transcripts of swpa4 were not detected in any tissues of the intact plant . The genes swpa6 and swpb1 were highly expressed in root tissues, whereas swpa6 and swpb3 were highly expressed in stem tissues . During suspension culture, the expression patterns of the six genes differed from each other . The level of swpa4, swpa5, swpb2 and swpb3 transcripts progressively increased during culture, whereas swpa6 and swpb1 showed high expression levels regardless of the age of the culture . In leaf tissues the six POD genes responded differently to various abiotic stresses . In particular, swpa4 was highly induced by several abiotic stresses, including exposure to hydrogen peroxide (440 mM) or NaCl (100 mM), and wounding of leaf tissues, suggesting that this POD gene is inducible by many stresses . Based on the different expression patterns of these POD genes, we propose that each POD may have different enzymatic properties and physiological functions during cell growth and development.

Life Sci, 2003 Jul 4, 73(7), 849 - 56
Disaccharide analysis of glycosaminoglycans synthesized by cardiac myxoma cells in tumor tissues and in cell culture; Negishi M et al.; We analyzed the main disaccharide units of glycosaminoglycans synthesized by cardiac myxoma cells in vivo and in cell culture using high-performance liquid chromatography after 1-phenyl-3-methyl-5-pyrasolone labeling . Cardiac myxoma tissues contained large amounts of chondroitin-6-sulfate (46%) and hyaluronic acid (32%), along with some chondroitin-4-sulfate (13%), chondroitin (6%), and much less dermatan sulfate (3%) . Cultured cardiac myxoma cells synthesized mainly chondroitin-6-sulfate . The abundant glycosaminoglycans in myxoma tissues may make up the characteristic friable gelatinous matrix which is favorable for embolism and tumor cell growth.

Acta Otolaryngol, 2003 May, 123(4), 466 - 70
Retinoic acid depletion induces keratinizing squamous differentiation in human middle ear epithelial cell cultures; Choi JY et al.; OBJECTIVE: The pathogenesis of cholesteatoma behind an intact tympanic membrane remains controversial . Squamous metaplasia of the middle ear mucosa is thought to be a possible mechanism in such cases . However, to date, no definitive experimental results have proved this association . This study was undertaken to investigate whether normal human middle ear epithelial (NHMEE) cells undergo keratinizing squamous differentiation in a retinoic acid (RA)-deficient culture . MATERIAL AND METHODS: We examined the morphological differences between RA-deficient and -sufficient cultures, and determined the expressions of the mucin gene and cornifin-alpha mRNAs as indicators of mucous and squamous differentiation, respectively . RESULTS: Histomorphologically, the NHMEE cells differentiated into a keratinizing squamous epithelium in RA-deficient cultures . In addition, the expressions of mucin gene 5AC (MUCSAC) and MUC8 mRNAs were suppressed, and the expression of cornifin-alpha mRNA increased progressively as a function of differentiation in RA-deficient cultures . CONCLUSIONS: This study shows that RA depletion induces keratinizing squamous differentiation in NHMEE cell cultures . This finding supports the hypothesis that middle ear cholesteatoma originates from metaplastic middle ear mucosa.

Plant J, 2003 Jun, 34(6), 847 - 55
Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning, functional expression, and site-directed mutagenesis of two polyketide synthases; Liu B et al.; Benzophenone derivatives, such as polyprenylated benzoylphloroglucinols and xanthones, are biologically active secondary metabolites . The formation of their C13 skeleton is catalyzed by benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of Hypericum androsaemum . BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53-63% amino acid sequence identity . Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa . Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone . BPS did not accept activated cinnamic acids as starter molecules . In contrast, recombinant chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic acids . The enzyme shared 60.1% amino acid sequence identity with BPS . In a phylogenetic tree, the two PKSs occurred in different clusters . One cluster was formed by CHSs including the one from H . androsaemum . BPS grouped together with the PKSs that functionally differ from CHS . Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over 4-coumaroyl-CoA.

Am J Physiol Lung Cell Mol Physiol, 2003 Sep, 285(3), L730 - 9 Epub 2003 Jun 06.
IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation; Atherton HC et al.; In addition to a direct proinflammatory role, IL-13 has been demonstrated to induce a goblet cell metaplastic phenotype in the airway epithelium in vivo . We have studied the direct effects of IL-13 (and IL-4) on well-differentiated, air-liquid interface cultures of human bronchial epithelial cells (HBEs) and provide a quantitative assessment of the development of a mucus hypersecretory phenotype induced by these cytokines . Using Alcian blue staining of goblet cells and immunohistochemical detection of MUC5AC, we found that IL-13 (and IL-4) induced increases in the goblet cell density (GCD) of the HBE cultures . The effects of these cytokines were critically dependent on concentration: 1 ng/ml routinely induced a 5- to 10-fold increase in GCD that was associated with a hypersecretory ion transport phenotype . Paradoxically, 10 ng/ml of either cytokine induced a profound reduction in GCD . Removal of EGF from the culture media or treatment of the cells with AG-1478 {a potent inhibitor of EGF receptor tyrosine kinase (EGFR-TK)} demonstrated that the EGFR-TK pathway was key to the regulation of the basal GCD but that it was not involved in the IL-13-driven increase . The IL-13-driven increase in GCD was, however, sensitive to inhibition of MEK (PD-98059, U-0126), p38 MAPK (SB-202190), and phosphatidylinositol (PtdIns) 3-kinase (LY-294002) . These data support the concept that IL-13 is in part able to induce a mucus hypersecretory phenotype through a direct interaction with the airway epithelium and that MAP kinase and PtdIns 3-kinase signaling pathways are involved.

J Surg Res, 2003 Apr, 110(2), 332 - 7
Melatonin and vitamin D3 increase TGF-beta1 release and induce growth inhibition in breast cancer cell cultures; Bizzarri M et al.; BACKGROUND: Evidence has accumulated that 1,25-dihydroxyvitamin D(3) {1,25-(OH)(2)D(3)} is involved in the regulation of the proliferation of breast tumor cells . For complete tumor suppression high hypercalcemic doses of 1,25-(OH)(2)D(3) are needed . The aim of this study was to assess the effect of combined treatment of 1,25-(OH)(2)D(3) at low doses and melatonin (MEL) on the proliferation of estrogen-responsive rat breast cancer cell line RM4 . MATERIALS AND METHODS: RM4 cell proliferation was assessed by {3H}thymidine uptake . The presence of TGF-beta(1) in serum-free conditioned medium was determined by inhibition antibody binding assay . RESULTS: In 17-betaE cultured RM4 cells both MEL and 1,25-(OH)(2)D(3) alone and in combination significantly reduced {3H}thymidine incorporation in a dose-related fashion . MEL by itself was ineffective in inhibiting the FCS-cultured RM4 cells, while 1,25-(OH)(2)D(3) strongly inhibited {3H}thymidine incorporation . Meanwhile, MEL increased the sensitivity of the FCS-cultured RM4 cells to 1,25-(OH)(2)D(3) in the combined regimen, from 20- to 100-fold . MEL significantly enhanced the TGF-beta(1) secretion from RM4 cells and vitamin D(3) increased the TGF-beta(1) secretion in a dose-dependent manner, from 2- to 7-fold . Moreover, a further enhancement of the TGF-beta(1) release was obtained with the combined treatment, but only for low 1,25-(OH)(2)D(3) concentrations . The addition of monoclonal anti-TGF-beta(1) antibody to the medium of RM4 cells exposed to vitamin D(3) alone or in combination with MEL increased the {3H}thymidine uptake compared to the correspondent cells cultured without antibody . CONCLUSIONS: Our data point to a potential benefit of combination therapy with 1,25-(OH)(2)D(3) and MEL in the treatment of breast cancer and suggest that the growth inhibition could be related, at least in part, to the enhanced TGF-beta(1) secretion.

Vet J, 2003 Jul, 166(1), 86 - 92
Comparison of polymerase chain reaction and cell culture for the detection of Chlamydophila species in the semen of bulls, buffalo-bulls, and rams; Amin AS; Two hundred and thirty six semen samples were collected from 120 bulls, 60 buffalo-bulls, and 56 rams located on farms of known history of infection with Chlamydophila species . All semen samples were examined by polymerase chain reaction (PCR) and cell culture techniques for detection of Chlamydophila species . The primers were selected to allow the amplification of all target species in a single reaction by identifying conserved sequences in the omp2 gene . PCR assay detected more positive samples (36) from the semen samples collected from different animal species than were detected by the culture method (21) . The results indicated that all culture-positive semen samples (21) from different species were PCR positive . The detection limit of the PCR assay was determined with DNA extracted from fourfold serial dilution of C . abortus (B577) and C . pecorum (11/88) cultures and found to be 0.25 inclusion-forming units (IFU) per PCR, while the culture method could not detect less than 4 IFU . This is the first report using PCR for the detection of Chlamydophila species in buffalo-bulls' semen and the assay provides a simple, sensitive, rapid, and reliable means for the detection and identification of the organism.

J Gen Virol, 2003 Jun, 84(Pt 6), 1415 - 25
Five unique open reading frames of infectious laryngotracheitis virus are expressed during infection but are dispensable for virus replication in cell culture; Veits J et al.; The chicken alphaherpesvirus infectious laryngotracheitis virus (ILTV) exhibits several unique genetic features including an internal inversion of a conserved part of the unique long genome region . At one end, this inversion is preceded by a cluster of five open reading frames (ORFs) of 335-411 codons, designated ORF A to ORF E, that are not present in any other known herpesvirus genome . In this report we analysed expression of these genes and identified the corresponding viral RNA and protein products . Northern blot analyses showed 3'-coterminal transcripts of ORFs A and B, and monocistronic mRNAs of ORFs C and D . ORF E is part of a 3'-coterminal transcription unit that includes the conserved glycoprotein H and thymidine kinase genes . Monospecific antisera obtained after immunization of rabbits with bacterial fusion proteins allowed detection of the protein products of ORF A (40 kDa), ORF B (34 kDa), ORF C (38 and 30 kDa), ORF D (41 kDa) and ORF E (44 kDa) in ILTV-infected cells . For functional analyses, five virus recombinants possessing deletions within the individual ORFs and concomitant insertions of a reporter gene cassette encoding green fluorescent protein were generated . All virus mutants were replication competent in cell culture, but exhibited reduced virus titres or plaque sizes when compared to wild-type ILTV . These findings indicate that the ILTV-specific ORF A to ORF E genes might be important for virus replication in the natural host organism.

J Med Virol, 2003 Jul, 70(3), 451 - 8
Human herpesvirus-6 modulates RANTES production in primary human endothelial cell cultures; Caruso A et al.; Human herpesvirus 6 (HHV6) is a beta-herpesvirus capable of infecting several cell types from different origins . HHV6 is the etiological agent of exantem subitum and has been associated with several diseases, all characterized by an inflammatory response triggered by chemokines . We show that strain U1102 of HHV6 is able to infect persistently human endothelial cells obtained from umbilical veins, adult aorta and adult heart microvessels, without apparent cytopathic effect . Analysis by in situ PCR showed that HHV6 sequences were present in 20% of HUVEC, 10% of aortic, and 1% of heart microvascular endothelial cells . Regardless of endothelial cell origin, HHV6 infection induced de novo synthesis of the RANTES CC-chemokine . It was found, however, that microvascular endothelial cells, despite their lower susceptibility to HHV6 infection, showed the highest RANTES expression . Chemokine production occurred also in the absence of viral DNA synthesis . Furthermore, RANTES synthesis required an active viral genome, as UV-inactivated HHV6 infection of endothelial cells did not lead to chemokine production . We investigated the expression of HHV6 U51 gene, which encodes a chemokine receptor that is already known to sequester and down modulate RANTES in epithelial cells . HHV6-infected endothelial cells co-expressed RANTES and U51 mRNAs starting from 12 hr up to 48 hr post-infection . Then, RANTES transcripts disappeared whereas U51 messages continued to be expressed . In conclusion, this study highlights the major role of HHV6 in endothelial cell biology and the development of inflammatory processes .

Ann N Y Acad Sci, 2003 Apr, 986, 410 - 5
Adaptation of murine inner medullary collecting duct (IMCD3) cell cultures to hypertonicity; Capasso JM et al.; Recently, we have adapted IMCD3 cell cultures to survive under increasing hypertonic conditions (i.e., 600 and 900 mOsmol/kg H(2)O) . In adapted cells, ATPase activity is increased by one order of magnitude, while the expression of the alpha and beta subunit is increased by a factor of 4 to 5 over controls (300 mOsmol/kg H(2)O) . Corresponding increases in mRNAs were also detected . The gamma subunit has been described as being uniquely expressed in some areas of the kidney, but never in cell cultures (even those derived from kidney tissues) . However, the gamma subunit was detected at the protein and mRNA levels in the adapted IMCD3 cells . In contrast to the alpha and beta subunits, the levels of gamma protein and mRNA expression continue to increase as a function of the media ion concentration . We have also demonstrated that signaling pathways that upregulate the alpha, beta, and gamma subunits are very different . Increasing concentrations of the PI3 kinase inhibitor, LY294002, resulted in a dose-dependent reduction in the expression of the gamma subunit, with total abolition at 10 micro M . However, LY294002 had no significant effect on the expression of the alpha subunit . Inhibition of the JNK2 (but not of the JNK1) pathways by dominant negative transfections abolished the upregulation of the gamma, but not the a subunit . Failure to upregulate the expression of the gamma subunit was associated with a marked decrease in cell viability upon stress.

Biochem Biophys Res Commun, 2003 Jun 6, 305(3), 484 - 7
Lysine hydroxylation of collagen in a fibroblast cell culture system; Uzawa K et al.; The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared . Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB) . The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed . In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains . Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs . A similar profile was observed in the tissue sample of the EDS-VIB patient . These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.

Zhonghua Er Bi Yan Hou Ke Za Zhi, 2001 Dec, 36(6), 409 - 11
{A cell culture model in vitro for studying the permeability of cochlear microvascular endothelial cells in guinea pigs}; Zhang X et al.; OBJECTIVE: To establish an "in vitro" system for studying the permeability of cochlear microvascular endothelial cells . METHODS: Cochlear stria vascularis from guinea pigs were isolated by microsurgery, then cultured in vitro to get the monolayer cells of cochlear microvascular endothelium . The purity and property of cultured cells were identified by immunochemical method . The model in vitro was established by millcell technique of inoculation of the cultured monolayer cells of cochlear microvascular endothelium, then the permeability of this model was studied by 125I-bovine serum albumin . The positive group (brain microvascular endothelial cells), negative group (lung microvascular endothelial cells) and blank group (no cells) were conducted as control . RESULTS: 1 . When cochlear stria vascularis fragment was isolated and cultured for two days, a few culture cells appeared around the fragments, then the amounts of culture cells increased . About ten days later, clusters of culture cells could be seen . Generally, spindle-shaped single culture cells were observed after cloning . When subcultured for eight days, there formed a monolayer tightly packed against the neighbouring cells; these cells showed the cobblestone shape under the microscope . For immunochemistry, over 95% of the cultured cells showed a positive reaction to factor VIII related antigen . 2 . The permeability of 125I-bovine serum albumin in testing group was higher than that in the positive group (P < 0.05) and was lower than that in the negative group and the blank group (P < 0.01) . CONCLUSION: An ideal model in vitro has been established for studying the permeability of cochlear microvascular endothelial cells at molecular level.

Cardiovasc Res, 2003 May 1, 58(2), 478 - 86
Endothelial progenitor cell culture and differentiation in vitro: a methodological comparison using human umbilical cord blood; Eggermann J et al.; OBJECTIVE: Endothelial progenitor cells (EPC) can contribute to vascular repair and targeted tumour therapy . Little is known about generating EPC from human umbilical cord blood . We therefore compared methods for purification of EPC from human umbilical cord blood . METHODS: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation and used either unselected or after CD34 preselection . Unselected mononuclear cells were cultured for 9 days . Culture-dish-adherent (CDAC) and non-adherent (CDNAC) CD34+ cells were cultured separately for 4 weeks . Surface markers were assessed by immunofluorescence staining and FACS analysis . RESULTS: In unselected mononuclear cells, VEGF-R2 and VE-cadherin expression increased up to day 6 . They stained positive with UEA-1 and took up acetylated LDL . Expression of CD45 and CD14 decreased over time, but remained strong . CD133 and CD34 were not expressed . CD34+-CDNAC acquired an endothelial phenotype over time with an increase of VEGFR-2 and von Willebrand factor (vWF) . CD45 and CD14 decreased, while CD34 and the progenitor-cell marker CD133 remained strongly expressed . CD34(+)-CDAC showed a strong increase in VEGFR-2, CD133, CD34 and vWF, while CD14 decreased, and CD45 did not change . CONCLUSION: Putative EPC can be obtained from human umbilical cord blood . When selected for CD34, cells can be differentiated in culture to express markers of mature endothelial cells, while keeping progenitor markers . In contrast, short-term culture of unselected mononuclear cells leads to an endothelioid-monocytoid phenotype devoid of progenitor markers . Thus, the outgrowth from CD34-selected cells appears to be superior to short-term culture of unselected mononuclear cells with regard to endothelial cell-lineage specific differentiation of cells with a progenitor marker profile.

Drug Metab Dispos, 2003 Jun, 31(6), 691 - 3
Basolateral active uptake of nitrofurantoin in the CIT3 cell culture model of lactation; Gerk PM et al.; Nitrofurantoin and other agents are actively transported into human and rodent milk . The purpose of this study was to determine whether nitrofurantoin active transport across mammary epithelia occurs basolaterally or apically, using the CIT3 cell culture model of lactation . The CIT3 model actively transports nitrofurantoin in the basolateral to apical direction . Basolateral to apical permeability {92.9 +/- 6.6 (microl/h)/cm(2)} was differentially decreased by unlabeled nitrofurantoin (250 microM) on the basolateral, apical, or both sides {49.5 +/- 1.8, 57.9 +/- 1.4, or 48.5 +/- 1.6 (microl/h)/cm(2), respectively} . Apical to basolateral permeability {27.6 +/- 1.8 (microl/h)/cm(2)} was increased in the presence of unlabeled nitrofurantoin (250 microM) on the basolateral, apical, or both sides {36.4 +/- 1.5, 39.9 +/- 0.7, 42.4 +/- 1.1 (microl/h)/cm(2), respectively} . These data indicate a basolateral active uptake mechanism for nitrofurantoin, which remains to be identified . This mechanism may influence the exposure of suckling infants to xenobiotics, as well as having potentially toxic effects on the lactating mammary epithelium and possibly altering the nutritional quality of the milk.

Eur J Pharm Biopharm, 2003 May, 55(3), 283 - 9
Mechanistic appraisal of the effects of some protease inhibitors on ciliary beat frequency in a sequential cell culture system of human nasal epithelium; Remigius UA et al.; The aim of this study was to investigate the suitability of a sequential monolayer-suspension culture system as a model to screen subacute effects of drug excipients on ciliary beat frequency (CBF) . The CBF of the cultured cells was measured by computerized microscope photometry . Protease inhibitors (puromycin, bestatin, bacitracin, actinonin and thiomersal) were used as model compounds and the mechanisms of ciliary inhibition were investigated by probing the involvement of arachidonic acid metabolism, guanylate cyclase (cGMP), protein kinase C (PKC) and adenosinetriphosphate (ATP) inhibition . Bestatin concentration-dependently reduced CBF by inhibiting arachidonic acid metabolism, cGMP, PKC and endogenous ATP consumption . Thiomersal and DMSO used for dissolving actinonin reduced CBF (P<0.05) via a non-specific mechanism . Bacitracin (8 mM) and puromycin (135 mM) had no effect on CBF after acute exposure (15-30 min) (P>0.05), but significantly reduced the CBF by approximately 15.0% following daily 15-min exposure for 1 week . This study shows that (i) sequential monolayer-suspension culture system is a valid model to screen both acute and subacute effects of drug excipients on CBF; and (ii) bacitracin, puromycin and actinonin are more cilio-compatible than bestatin and thiomersal and as such are more potentially useful nasal absorption enhancer from ciliotoxicity perspective.

Anim Reprod Sci, 2003 Sep 15, 78(1-2), 25 - 31
Effect of neuropeptide Y on GnRH-induced LH release from bovine anterior pituitary cell cultures derived from heifers in a follicular, luteal or ovariectomized state; Denniston DJ et al.; Objectives were to determine if neuropeptide Y (NPY) had direct effects GnRH induced secretion of LH from the anterior pituitary gland, and if endogenous steroids modulated the effect of NPY . To accomplish these objectives, 15 Hereford heifers were assigned to one of three ovarian status groups: follicular, luteal, or ovariectomized . One animal from each of the three ovarian status groups was slaughtered on each of 5 days and anterior pituitary gland harvested . Anterior pituitary gland cells within ovarian status were equally distributed and randomly assigned to one of three cell culture treatments: no NPY or GnRH (control), 10 nM GnRH, or 100 nM NPY+10 nM GnRH . Anterior pituitary cell cultures were incubated with or without NPY for 4 h and further incubated for an additional 2 h with or without GnRH and supernatant collected for quantification of LH . Treatment of anterior pituitary cell cultures with GnRH or GnRH+NPY did not affect LH release in cultures obtained from follicular (S.E.=5%; P=0.58) or ovariectomized (S.E.=7%; P=0.22) heifers . Both GnRH and GnRH+NPY increased LH release from anterior pituitary cell cultures from heifers in the luteal phase (S.E.=14%; P < or = 0.05) compared to control cultures . Cultures from luteal phase heifers treated with GnRH did not differ from those treated with GnRH+NPY (P=0.34) . These data provide evidence to suggest that effects of NPY on LH release may occur primarily at the level of the hypothalamus.

Free Radic Res, 2003 Apr, 37(4), 391 - 7
Irradiation of cells with ultraviolet-A (320-400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide; Mahns A et al.; Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen . Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H2O2) . Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation . Indeed, the exposure of cell culture media to UVA (30 J/cm2) results in the generation of significant amounts of H2O2, with concentrations of about 100 microM . H2O2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation . From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H2O2 . Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H2O2 generation . It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.

Biomed Environ Sci, 2003 Mar, 16(1), 68 - 82
Genotoxic effects of PAH containing sludge extracts in Chinese hamster ovary cell cultures; Krishnamurthi K et al.; OBJECTIVE: Many studies have been conducted in order to evaluate the genotoxicity of chemicals and waste materials, which utilized in vivo test protocols . The use of animals for routine toxicity testing is now questioned by a growing segment of society . METHODS: Keeping the above fact in mind, we have conducted in the present study the genotoxicity evaluation of oily sludge samples generated from a petroleum refinery and petrochemical industry and ETP sludge from petroleum refinery using DNA damage, chromosomal aberration, p53 protein induction and apoptosis in short term in vitro mammalian Chinese Hamster Ovary cell cultures . RESULTS: It is evident from the results that the oily sludge compounds derived from petroleum refinery and petrochemical industry could cause DNA damage, chromosomal aberration, p53 protein accumulation and apoptotic cell death on exposure to oily sludge extracts in the presence of metabolic activation system (S-9 mix), however, ETP sludge extract could not cause significant genotoxicity in comparison to oily sludge extract and negative control . CONCLUSION: The effect may be attributed to polycyclic aromatic hydrocarbons present in the samples as evidenced from GC-MS.

Endocrinology, 2003 Jun, 144(6), 2388 - 95
Glucosamine induces resistance to insulin-like growth factor I (IGF-I) and insulin in Hep G2 cell cultures: biological significance of IGF-I/insulin hybrid receptors; Sakai K et al.; IGF-I stimulates insulin-like actions directly through its receptor, and it also enhances sensitivity to insulin-mediated effects in vivo . These studies were undertaken to analyze the role of IGF-I, insulin, and insulin/IGF-I hybrid receptors (HRs) in mediating IGF-I and insulin signaling in cells that had been made insulin-resistant by treatment with glucosamine . Human HepG2 cells, which express IGF-I receptors, insulin receptors (IRs), and IGF-I/insulin HRs, were exposed to 20 mM glucosamine; and the effects of IGF-I and insulin in stimulating glycogen synthesis were determined . An overnight exposure to glucosamine markedly attenuated the effects of insulin and IGF-I in stimulating glycogen synthesis . To determine which receptors were mediating this effect, the ability of insulin and IGF-I to stimulate phosphorylation of their respective receptors was analyzed . An 18-h exposure to glucosamine (20 mM) caused a 75% reduction in the ability of IGF-I to phosphorylate its receptor but no change in receptor abundance . Glucosamine also caused a major reduction in insulin-stimulated receptor phosphorylation, although, unlike IGF-I, there was also a 50% reduction in IR abundance . Exposure to glucosamine also resulted in a reduction in the ability of IGF-I or insulin to stimulate phosphorylation of insulin IGF-I/HRs . The combination of insulin plus IGF-I was a more potent stimulus of HR phosphorylation than either agent alone, and this combination was also more potent in partially reversing the inhibitory effect of glucosamine . Taken together, these findings indicate that glucosamine induces a loss of sensitivity to stimulation of insulin, IGF-I, or HR tyrosine kinase activity by insulin or IGF-I . Although insulin is able to partially reverse the effect of glucosamine on IR phosphorylation, it has a very minimal effect on glucosamine-induced inhibition of HR phosphorylation . However, the combination of IGF-I and insulin induces a major increase in HR phosphorylation, even in the presence of glucosamine, suggesting that it is improving the sensitivity of the HR to insulin activation.

J Neuroimmunol, 2003 May, 138(1-2), 25 - 30
Polyclonal IgM influence oligodendrocyte precursor cells in mixed glial cell cultures: implications for remyelination; Stangel M et al.; Polyclonal immunoglobulins for intravenous use (IVIg) are a potent immunomodulator and have been shown to be effective in several immune-mediated diseases . This includes inflammatory demyelinating diseases of the central nervous system (CNS) like multiple sclerosis (MS) . Besides their immunomodulatory function, IVIg have been proposed to enhance remyelination based on studies in the animal model of Theiler's murine encephalomyelitis virus (TMEV) . Disappointingly, recent treatment trials in patients with MS have failed to demonstrate repair of longstanding deficits . Since the clinical trials have used IVIg that contained nearly exclusively IgG, whereas the most pronounced effect in TMEV was seen with IgM, this could be a possible explanation for the negative outcome in the MS trials . Here we have examined the effects of a new polyclonal IgM preparation (IVIgM) on cultured oligodendrocyte precursor cells (OPCs) . To achieve successful remyelination, OPCs proliferate, migrate, and differentiate into mature myelinating oligodendrocytes . IVIgM and commercial IVIg preparations had no influence on proliferation and differentiation of either isolated OPCs or OPCs in coculture with microglia . In contrast, IVIgM inhibited the proliferation of OPCs in mixed glial cultures containing astrocytes and microglia . This was not seen in cultures treated with IVIg, albumin, or interferon-gamma (IFN-gamma), suggesting that this is a specific effect of IVIgM . Differentiation was slightly delayed by IVIgM in mixed glial cultures, but this was not statistically significant and interferon-gamma had a similar effect . These results underline the importance of IgM in influencing OPCs and corroborate the in vivo findings that polyclonal IgM are more potent than IgG in their capacity to influence remyelination . The exact mechanism of how this modulation of OPCs is achieved remains unknown, but a complex interaction among all cells present in the CNS has to be postulated.

Antiviral Res, 2003 Apr, 58(2), 139 - 47
Susceptibility to highly sulphated glycosaminoglycans of human immunodeficiency virus type 1 replication in peripheral blood lymphocytes and monocyte-derived macrophages cell cultures; Bartolini B et al.; In the search for new drugs against human immunodeficiency virus type 1 (HIV-1), the replication of III(B) and BaL strains, and of seven primary isolates from AIDS patients, cultured both in peripheral blood lymphocytes (PBLs) and in monocyte-derived macrophages (MACs), was investigated in the presence of two dermatan sulphate and heparin at 10 microg/ml . The three polysaccharides effectively inhibited the replication of III(B) in PBLs and of BaL in MACs, while producing either a slight inhibition or an unexpected large increase in the replication of the seven primary isolates, especially in MAC cultures . In one case, stimulation was found in PBLs and, at lower doses, also with BaL in MACs . Co-receptor use, adaptation to C8166 T cell line, partial sequence of the gp120 V3 loop, variation in positive charge distribution and number of potential glycosylation sites along the V3 loop were assessed for each strain . No explanation could be found for the different susceptibility of the viruses to the polysaccharides . Their presence probably brings about both inhibitory and stimulatory effects, the final outcome depending on the virus, cells and polysaccharide.

Diagn Microbiol Infect Dis, 2003 May, 46(1), 39 - 47
Monitoring intracellular replication of Chlamydophila (Chlamydia) pneumoniae in cell cultures and comparing clinical samples by real-time PCR; Bonanomi A et al.; Strains of Chlamydophila pneumoniae may be associated with respiratory disease or atherosclerosis . Two real-time quantitative PCR assays targeting the species-specific genes Cpn0278 and ArgR were developed to compare the in vitro growth of respiratory strains AR39 and K6 with that of atherosclerotic strain A03 and to quantify C . pneumoniae in clinical samples . A third real-time PCR assay was designed to assess contamination with Mycoplasma spp . The assays targeting C . pneumoniae detected DNA concentrations corresponding to 10(4) to 10(-4) inclusion-forming units (IFU)/reaction and were highly specific . AR39 exhibited the longest lag phase and period of exponential growth; K6 augmented growth rates at higher inocula; and A03 grew at highest rates . Contamination with Mycoplasma spp . of AR39 and A03 unlikely accounted for growth differences between them . Numbers of IFU in C . pneumoniae-positive respiratory secretions varied within 4 to 5 orders of magnitude . The assays described may prove valuable for pathogenicity studies.

Tissue Eng, 2003 Apr, 9(2), 291 - 9
Dynamic rotational seeding and cell culture system for vascular tube formation; Nasseri BA et al.; Optimization of cell seeding and culturing is an important step for the successful tissue engineering of vascular conduits . We evaluated the effectiveness of using a hybridization oven for rotational seeding and culturing of ovine vascular myofibroblasts onto biodegradable polymer scaffolds suitable for replacement of small- and large-diameter blood vessels . Large tubes (12 mm internal diameter and 60 mm length, n = 4) and small tubes (5 mm internal diameter and 20 mm length, n = 4) were made from a combination of polyglycolic acid/poly-4-hydroxybutyrate and coated with collagen solution . Tubes were then placed in culture vessels containing a vascular myofibroblast suspension (10(6) cells/cm(2)) and rotated at 5 rpm in a hybridization oven at 37 degrees C . Light and scanning electron microscopy analyses were performed after 5, 7, and 10 days . Myofibroblasts had formed confluent layers over the outer and inner surfaces of both large and small tubular scaffolds by day 5 . Cells had aligned in the direction of flow by day 7 . Multiple spindle-shaped cells were observed infiltrating the polymer mesh . Cell density increased between day 5 and day 10 . All conduits maintained their tubular shape throughout the experiment . We conclude that dynamic rotational seeding and culturing in a hybridization oven is an easy, effective, and reliable method to deliver and culture vascular myofibroblasts onto tubular polymer scaffolds.

Pharm Res, 2003 Apr, 20(4), 569 - 75
Mechanistic studies on nonviral gene delivery to the intestine using in vitro differentiated cell culture models and an in vivo rat intestinal loop; Cryan SA et al.; PURPOSE: To identify factors influencing nonviral vector transfection in differentiated CaCo-2 and mucus-secreting coculture, CaCo-2: Ht29GlucH, cell culture models and to compare these in vitro results with in vivo transfection efficiency in rat intestine . METHODS: A range of nonviral vectors including DOTAP, Lipofectin, Superfect, PEI, and polylysine were investigated . CaCo-2 and a mucus-secreting coculture were used at 21 days . Transfection efficiency was assessed using pCMVluc (firefly luciferase) plasmid, and radio-labeled plasmid was used to determine the binding and internalization of plasmid DNA . The in vivo model used was a ligated rat intestinal loop . RESULTS: Transfection levels decreased by over 1000-fold in differentiated models relative to nondifferentiated COS-7 cells and were related to reductions in luciferase production by individual cells . Active internalization of DNA by the differentiated cells decreased . Removal of mucus by the mucolytic agent N-acetylcysteine, from the coculture system significantly reduced (p < 0.05) transfection efficiency . In vivo the transfection efficiency of PEI proved superior to DOTAP . CONCLUSIONS: Nonviral gene delivery to the hostile environment of the intestine is possible . Mechanistic studies using differentiated intestinal cell models aid identification of the rate-limiting steps to transfection and represent a more physiologically relevant approach to predict gene delivery to the intestine.

Eur J Clin Microbiol Infect Dis, 2003 May, 22(5), 300 - 2 Epub 2003 May 09.
Failure to detect Chlamydia pneumoniae by cell culture and polymerase chain reaction in major arteries of 93 patients with atherosclerosis; Bishara J et al.; To detect Chlamydia pneumoniae in punch specimens of the aortic wall of 61 patients undergoing coronary-aortic bypass graft, and carotid atheromas of 32 patients undergoing carotid endarterectomy, cell culture (HEp-2 cells) and two polymerase chain reaction assays in two different laboratories were used . All cultures and polymerase chain reaction tests for Chlamydia pneumoniae were negative . Further studies are required to explore the complex relationship between Chlamydia pneumoniae and atherosclerosis.

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