Mol Reprod Dev, 2002 Jan, 61(1), 67 - 77 Apoptosis in the preimplantation mouse embryo: effect of strain difference and in vitro culture; Kamjoo M et al.; Cell death by apoptosis occurs predominantly in the inner cell mass (ICM) of the blastocyst, the cell population which carries the germ line and gives rise to the foetus . The frequency of apoptosis in blastocysts varies widely within outbred species such as human and cow . We have addressed the basis of this variation by examining the relative influence of strain difference and in vitro culture conditions on apoptosis, using embryos from two different strains of mice (MF1 and C57BL6/CBA) in two different culture media (M16 and kSOM) . In both strains and all crosses apoptosis was first detected by nuclear fragmentation or TUNEL {Terminal deoxynucleotidyl transferase mediated d-UTP nick end-labelling} labelling at the early blastocyst stage . This was true for embryos which had developed in vivo, and in vitro in both M16 and kSOM . The apoptotic index in blastocysts was found to be significantly different between both media and strain (P < 0.0001) . Blastocysts from MF1 x MF1 at equivalent stages had an apoptotic index of 32.4% in M16 and 20.3% in kSOM . Blastocysts from C57BL6/CBA x C57BL6/CBA had an apoptotic index of 19.3% in M16 and 14.4% in kSOM . When embryos of similar cell number were compared, a significantly greater apoptotic index was found for cultured MF1 x MF1 embryos with a cell number between 40 and 59 compared to similar directly flushed C57BL6/CBA embryos (P = 0.001), and MF1 embryos (P < 0.0005) . MF1 x MF1 embryos and C57BL6/CBA x MF1 embryos of 60-79 cells had a greater apoptotic index in M16 than kSOM (P < 0.0005) but the difference between media was not significant for C57BL6/CBA x C57BL6/CBA . When strain was compared MF1 x MF1 embryos of 60-79 cells had a significantly greater apoptotic index than C57BL6/CBA x MF1 in both media (P < 0.0005 M16; P = 0.002 kSOM) and than C57BL6/CBA x C57BL6/CBA in M16 (P = 0.019) . Our data suggest that genetic make-up and the chemical composition of simple medium are equally important in determining the level of apoptosis . Microbiol Res, 2001, 156(4), 387 - 91 Production and characteristics of Debaryomyces hansenii killer toxin; Marquina D et al.; The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied . The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action . Production of the killer toxin was stimulated in the presence of proteins of complex culture media . Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly . Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase . Optimal stability was observed at pH 4.5 and temperatures up to 20 degrees C . Above pH 4.5 a steep decrease of the stability was noted . The activity was hardly detectable at pH 5.1. Int Microbiol, 2001 Mar, 4(1), 27 - 33 Changes in the photosynthetic apparatus of diatoms in response to low and high light intensities; Janssen M et al.; The centric diatom Cyclotella cryptica and two strains of the pennate diatom Phaeodactylum tricornutum were grown under low and high light intensities (300 lux and 3,000 lux) over 4-6 weeks . Growth was monitored by repetitive cell count . The culture media were replaced weekly to avoid morphological and biochemical alterations caused by nutrient depletion . The ultrastructure of the cells was examined by transmission electron microscopy . Alterations in the light-harvesting antenna systems were investigated by Western immunoblotting . Both diatoms reduced the plastid area, i.e . decreased the amount of thylakoid lamellae, under high light intensity . The thylakoids still ran in groups of three with parallel orientation within the chloroplasts . The girdle band lamellae were not affected . The amounts of storage compounds and vacuoles increased . SDS-PAGE of total cell protein followed by Western immunoblotting with antisera directed against subunits of the light-harvesting antenna systems of C . cryptica (cc-antiserum) and the cryptophyte Cryptomonas maculata (cmac-antiserum) revealed that both diatoms reduced the amount of antenna polypeptides under increased light intensity . The cc-antiserum immunodecorated two bands with relative molecular masses (Mr) of 18,000 and 22,000 in C . cryptica . Both decreased under high light conditions to 67.2 +/- 6.1% . Five to seven bands in the Mr range of 14,000-27,000 were recognized in P . tricornutum . They decreased to 83 +/- 5.3% . Furthermore, the immunolabeling pattern for both strains differed under the two light regimes . The cmac-antiserum immunodecorated two polypeptides with Mr of 24,000 and 23,000 in C . cryptica, while both strains of P . tricornutum had five polypeptides in the Mr range of 14,000-24,000 that showed some differences in staining intensities between the two strains and in response to the light intensity applied. Mycoses, 2001 Nov, 44(9-10), 426 - 31 Case report . Trichophyton mentagrophytes var . nodulare causing tinea pedis; Brasch J; The identification of Trichophyton mentagrophytes var . nodulare is described, based on a strain recently isolated from tinea pedis . Different culture media and physiological tests were used in order to compare this strain with related strains . The main characteristics of T . mentagrophytes var . nodulare were its deep yellow-orange pigmentation, which was released from the mycelium, the flat growth of its colonies and the formation of nodular bodies . Supplementation of Sabouraud glucose agar with 3% NaCl reduced the aerial mycelium and stimulated the formation of conidia . Until now an identification of T . mentagrophytes var . nodulare based on DNA-patterns has not been published . Trichophyton mentagrophytes var . nodulare is supposed to be an anthropophilic dermatophyte causing ordinary tinea and onychomycosis . The low number of reports indicates that it is a very rare variety. Dev Biol (Basel), 2001, 106, 455 - 9; discussion 460-1, 465-75 Transmissible spongiform encephalopathies: vaccine issues; Cashman NR; The recent emergence of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob Disease (vCJD) suggests that transmissible spongiform encephalopathies (TSEs) pose an ongoing threat to human and animal health . To avoid iatrogenic transmission of TSEs in vaccines, strategies must be developed to obviate TSE agent infectivity in cellular substrates, cell culture media components and enzymes, and excipients, and to validate the safety of these components and field vaccines efficiently Mycopathologia, 2001, 152(2), 97 - 101 Toxic activity from liquid culture of Colletotrichum acutatum; Jayasinghe CK et al.; Colletotrichum acutatum has become an increasingly important plant pathogen worldwide . With this background, a study was carried out to characterize the toxicity of liquid culture media from different isolates and to identify some properties of the toxic principles . Liquid culture media from all isolates were toxic to rubber leaves and induced the anthracnose symptoms . Toxicity of the culture filtrate was not host specific and toxic substances were thermostable . Acetone soluble fraction of the culture filtrates retained the toxic activity and it was effective even at a concentration of 700 microg dry mycelium mass/ml. Graefes Arch Clin Exp Ophthalmol, 2001 Oct, 239(10), 783 - 7 Testing of corneoscleral discs and their culture media of seropositive donors for hepatitis B and C virus genomes; Sengler U et al.; The prevalence of donors seropositive for hepatitis B virus (HBV) surface antigen (HBsAg) or hepatitis C virus (HCV) antibody (anti-HCV) in western countries is estimated to be 0.5%-1% . There have been only two cases, however, published so far, where hepatitis B was suspected to have been transmitted by penetrating keratoplasty {4} . Concerning HCV, no suspected transmission by keratoplasty has been reported so far . This is also true for the time before serological screening for infectious diseases became mandatory for corneal donors . In the Lions Cornea Bank North Rhine Westfalia, 4.7% (HBV) and 3.2% (HCV) respectively of the corneas of the years 1995 to 1999 were discarded due to a "non-negative serology" . In about 50% of these cases the screening test (ELISA) generated no valid signal and, therefore, a "questionable positivity" was assumed . Since in Germany corneal graft shortage still is a limiting factor for penetrating keratoplasty, this study was to evaluate the detectability of HBV-DNA and HCV-RNA in the serum samples, organ culture media and corneas of donors tested seropositive for HBsAg or anti-HCV in an attempt to obtain information as to the potential infectivity of this donor material . In this study, 29 corneas of 17 donors seropositive by ELISA for HBsAg and 27 corneas of 14 donors seropositive by ELISA for anti-HCV were evaluated . The organ culture media and the sera were screened for the presence of HBV-DNA or HCV-RNA by PCR . The corneoscleral discs were divided into a central trephinate (7 mm) and the corneoscleral rim . Concerning HBV-DNA both tissues were examined separately by polymerase chain reaction (PCR) . In the case of HCV-RNA, a further, more sensitive nucleotide amplification method (NAT), the transcription mediated amplification (TMA), was used to test media, central corneas and cornealscleral rims . The media were additionally tested by PCR . Viral nucleic acid was detected in the sera from 6 of 17 HBsAg positive donors and from 6 of 14 anti-HCV positive donors . Viral genomes could not be detected in the organ culture media nor in the central corneas or corneoscleral rims by PCR at a detection limit of 1000 and 100 copies/ml . Concerning HCV-RNA, two media were positive in the TMA with 50-100 copies/ml . CONCLUSION: according to our results, the risk of transmitting hepatitis B or C virus by penetrating keratoplasty appears to be low: although hepatitis C virus RNA could be detected in 2 media (from two donors) out of 27 with a low concentration of virus copies between 50-100/ml . It remains open whether such a low virus particle number may cause infection in the recipient. Graefes Arch Clin Exp Ophthalmol, 2001 Oct, 239(10), 778 - 82 Evaluation of potential organ culture media for eye banking using a human corneal endothelial cell growth assay; Moller-Pedersen T et al.; BACKGROUND: To evaluate the ability of different commercially available cell culture media to induce proliferation and morphological changes in primary cultures of human corneal endothelial cells (HCEC) . This screening model was used in an attempt to establish a rational basis for the development of well-defined, serum-free preservation media for long-term organ culture of human donor corneas . METHODS: A total of 11 different culture media enriched with 0%, 2%, 5%, and 10% fetal calf serum (FCS) were compared . The test media were divided into three groups: Group 1: Media based on minimal essential medium (MEM), currently used for long-term corneal organ culture in European eye banks; Group 2: F99-based media, enriched for growth of corneal endothelial cells at serum-reduced conditions; and Group 3: Media designed for growth of special cell types or for short-term corneal organ culture . The growth-promoting capacity of each test medium was quantified using an HCEC proliferation assay, whereas changes in cell morphology were evaluated by phase-contrast microscopy . RESULTS: The morphological characteristics of HCEC were best maintained in the group of F99-based media, which also induced the highest level of cell proliferation under serum-reduced conditions . Specifically, the medium F99-Sr (F99 enriched with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids) induced a two- to three-fold higher HCEC density at both 0% and 2% FCS when compared to all other test media, and it also maintained the most endothelial cell-like morphology . Also, at higher serum concentrations (5% and 10% FCS), the cell growth was most prominent in F99-Sr, as well as in the medium SFM that originally was designed for serum-free growth of vascular endothelial cells . CONCLUSION: This study suggests that the media F99-Sr and SFM should be further tested and refined as potential new storage solutions for long-term corneal organ culture at physiological temperatures. Biosci Biotechnol Biochem, 2001 Oct, 65(10), 2311 - 4 Isolation and identification of antheridiogens in the ferns, Lygodium microphyllum and Lygodium reticulatum; Kurumatani M et al.; Antheridiogens in culture media of 6-week-old prothallia of two species of Schizaeaceous ferns, Lygodium microphyllum and Lygodium reticulatum, were analyzed by gas chromatography-mass spectrometry . In both species, the gibberellin A73 methyl ester (GA73-Me) was identified as the most abundant antheridiogen, and the methyl esters of GA9 and of several monohydroxy-GA73 derivatives were also detected . Since both species produced antheridiogens at a high level, they were classified into high-antheridiogen-producing ferns . The response to GA73-Me of gametophytes of both species is also discussed. Arch Toxicol, 2001 Oct, 75(8), 497 - 504 Effects of all-trans-retinoic acid and all-trans-retinoyl glucuronide in two in vitro systems of distinct biological complexity; Ruhl R et al.; In vitro systems are widely used to evaluate the embryotoxic potential of retinoids . The effective concentrations of these retinoids, however, are not consistent in the various in vitro systems used in evaluating embryotoxicity . This may be explained by the different level of complexity for each individual system, which may lead to different concentrations of the substances in the target tissues . To verify this hypothesis we have compared two in vitro systems of distinct biological complexity: the rat whole embryo culture system, and the mouse limb bud organ culture system . The lipid soluble, teratogenic retinoid all-trans-retinoic acid (ATRA), and all-trans-retinoyl-beta-D-glucuronide (ATRAG), an endogenous, water-soluble and biologically active retinoid with limited placental transfer, were compared with regard to their embryotoxic potential in vitro . In both in vitro systems, ATRAG showed a lower degree of embryotoxicity than ATRA . In the limb bud organ culture, ATRAG revealed only slightly less toxicity than ATRA, whereas the effective concentrations of the two compounds in the whole embryo culture system differed by almost two orders of magnitude . During incubation with ATRAG, ATRA is generated by hydrolysis and is found in culture media and exposed tissues . The presence of membrane barriers around the developing embryo in the whole embryo culture system possibly prevents the transfer of ATRAG to the embryo and, therefore, its exposure to the active hydrolysis product ATRA . From these results we conclude that analysis of retinoid concentrations in the culture media and in the exposed tissues is essential for the interpretation of results obtained from in vitro toxicity testing. Anim Reprod Sci, 2002 Jan 23, 69(1-2), 73 - 89 The effect of stimulators and blockers of adrenergic receptors on LH secretion and cyclic nucleotide (cAMP and cGMP) production by porcine pituitary cells in vitro; Siawrys G et al.; The direct effects of alpha- and beta-adrenergic agents on luteinizing hormone (LH) secretion in vitro by porcine pituitary cells and the participation of secondary messengers, adenosine 3'5'-monophosphate (cAMP) and guanosine 3'5'-monophospate (cGMP), in transduction of signals induced by adrenergic agents and gonadotropin-releasing hormone (GnRH) in these cells have been investigated . Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) 1 month before slaughter . OVX gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2 and 3) estradiol benzoate (EB; 2.5mg/100kg b.w.) at 30-36h (OVX+EB I) or 60-66h (OVX+EB II) before slaughter, respectively; (4) progesterone (P(4); 120mg/100kg b.w.) for 5 consecutive days before slaughter (OVX+P(4)) . Anterior pituitaries were dispersed with trypsin and then pituitary cells were cultured (10(6) per well) in McCoy's 5a medium containing horse serum (10%) and fetal calf serum (2.5%) for 3 days, at 37 degrees C and under the atmosphere of 95% air and 5% CO(2) . On day 4 of the culture, the cells were submitted to 3.5h incubation in the presence of GnRH (a positive control), alpha- and beta-adrenergic agonists (phenylephrine (PHEN) and isoproterenol (ISOP), respectively), and alpha- and beta-adrenergic blockers (phentolamine (PHENT) and propranolol (PROP), respectively) . The culture media were assayed for LH (experiment I) and cyclic nucleotides (experiment II).In experiment I, addition of GnRH (100ng/ml) increased LH secretion by pituitary cells taken from gilts of all experimental groups . The effects of alpha- and beta-adrenergic agents on LH secretion by the cells depended on hormonal status of gilts . The LH secretion by pituitary cells of OVX gilts was potentiated in the presence of PHEN (10, 100nM, and 1microM) and PHENT (1microM), alone or in combination with PHEN (100nM) and by the cells derived from OVX+EB I and OVX+P(4) animals in response to PHEN (100nM) and ISOP (1microM) . ISOP (1microM) also stimulated LH secretion by the cells taken from OVX+EB II gilts . In experiment II, GnRH (100ng/ml) increased cGMP production by pituitary cells obtained from all groups of gilts and cAMP secretion by the cells taken from OVX and OVX+P(4) animals . PHEN (100nM) decreased and PROP (1microM) enhanced cAMP production by pituitary cells derived from OVX+EB I and OVX gilts, respectively . Moreover, PHEN (100nM) reduced, while PHENT (1microM) stimulated the release of cGMP by pituitary cells taken from OVX+EB II animals . In turn, ISOP (100nM) decreased and increased cGMP production by the cells derived from OVX+EB II and OVX+P(4) gilts, respectively . PROP (1microM) potentiated cGMP accumulation by pituitary cells taken from OVX+EB I and OVX+P(4) animals.In conclusion, our results suggest that adrenergic agents can modulate LH release by porcine pituitary cells acting through guanyl and adenylyl cyclase and in a manner dependent on hormonal status of gilts. Aquat Toxicol, 2002 Jan, 56(2), 69 - 79 Tolerance and acclimation to zinc of field-collected Daphnia magna populations; Muyssen BT et al.; The zinc tolerance of two Daphnia magna populations collected at a zinc contaminated site was studied . One clone was isolated from each population in order to determine interclonal variation in zinc tolerance . 48hEC50-values, life table parameters, carapace lengths and cellular energy allocation (CEA) were used as test endpoints and compared with the results obtained with a standard laboratory clone . The natural clones were more tolerant to acute zinc toxicity (up to a factor of 4) and exhibited a higher reproduction rate (factor 2) and carapace length (factor 1.2) . The optimal zinc concentrations for the natural clones ranged from 80 to 200 microg Zn/l . When cultured without zinc, the natural clones gradually lost their zinc tolerance . Therefore, the environmental relevance of using toxicity data obtained with organisms (natural, as well as laboratory clones) acclimated to culture media containing no or very small amounts of zinc can be questioned. Am J Respir Cell Mol Biol, 2002 Jan, 26(1), 144 - 51 Expression and regulation of inducible nitric oxide synthase from human primary airway epithelial cells; Donnelly LE et al.; Elevated levels of exhaled nitric oxide are seen in inflammatory airway diseases such as asthma, but the cellular source remains unknown . This study investigated whether human airway epithelial cells express inducible nitric oxide synthase (iNOS) . Human bronchial epithelial cells stimulated with 50 ng/ml interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma express iNOS mRNA, protein and increased nitrite in the cell culture media, which was inhibited by the selective iNOS inhibitor 1400W . Cells derived from subjects with asthma produced less nitrite than cells from normal subjects (6.59 +/- 0.99 microM nitrite, n = 15 versus 3.89 +/- 0.42 microM nitrite, n = 20; P < 0.05) . This was not attributed to steroid treatment of subjects with asthma because there was no difference in the amount of nitrite released from steroid-naive and steroid-treated cells (3.51 +/- 0.46 versus 4.27 +/- 0.7 microM nitrite, n = 10) . Neither dexamethasone nor budesonide inhibited iNOS mRNA induction, protein expression, or nitrite accumulation . The cells were not steroid insensitive because steroids inhibited GM-CSF release . Therefore, although these cells express iNOS under inflammatory conditions, they do not appear to be regulated directly by glucocorticosteroids. Biochim Biophys Acta, 2001 Nov 30, 1534(1), 45 - 55 Predominance of human versus rat phenotype in the metabolic pathways for bile acid synthesis by hybrid WIF-B9 cells; Monte MJ et al.; The rat hepatoma-human fibroblast hybrid cell line WIF-B9 stably exhibits the structural and functional characteristics of normal differentiated hepatocytes . The abilities of these cells to synthesize bile acids and amidate them with glycine and taurine were investigated . The release of bile acids into the culture media over 72 h was assessed by gas chromatography-mass spectrometry . WIF-B9 cells were able to synthesize bile acids (1.10+/-0.17 nmol/mg protein) but less efficiently than rat hepatocytes in primary culture (2.19+/-0.19 nmol/mg protein; P<0.01) . The patterns of major bile acid species produced by both types of cells were also different . Cholic acid (CA; 72%) and beta-muricholic acid (19%) were the major bile acids produced by rat hepatocytes, while chenodeoxycholic acid (CDCA) accounted for only 4.5% of total bile acids . In contrast, muricholic acids were absent, while CA (62%) and CDCA (34%) were the most abundant bile acids synthesized by WIF-B9 cells . Using reverse transcription-polymerase chain reaction and gene- and species-specific primers for key enzymes involved in bile acid synthesis, the expression of human, but not rat, orthologues of CYP7A1, CYP27, CYP8B and CYP7B1 was found in WIF-B9 cells . Induction of cell stress by serum deprivation did not change the amount of total bile acids synthesized by these cells, but an inversion of the CA-to-CDCA ratio from 1.8 to 0.3 together with a marked increase in the proportion of intermediate metabolites related to the acidic pathway was found . Using 500 microM radiolabeled CA and 2 mM of taurine or glycine, the ability to amidate CA over 48 h was determined by high performance liquid chromatography . Rat hepatocytes conjugated more than 90% CA with either amino acid, whereas this ability was very poor (< 2%) in WIF-B9 cells . Regarding the expression of enzymes and the products of bile acid synthesis, it may be concluded that the human phenotype predominates over that of the rat in WIF-B9 cells . Moreover, these cells are almost completely unable to further conjugate primary bile acids, which facilitates the manipulation of these steroids in analytical procedures . These characteristics make WIF-B9 cells a suitable in vitro model to carry out studies on bile acid synthesis by 'human-like' metabolic pathways. Exp Clin Endocrinol Diabetes, 2001, 109(8), 416 - 8 Polichlorinated biphenyls (PCB126 and PCB 153) action on proliferation and progesterone secretion by cultured in vitro porcine luteal cells; Augustowska K et al.; Summary:To characterise PCBs action on luteal cell steroidogenesis and cell viability two PCB congeners were selected as model substances . PCB 126 because of its dioxin-like configuration and high toxicity while 153 because it is one of the most commonly detected congeners in breast milk . Luteal cells collected from mature corpora lutea were cultured in M199 medium at 37 degrees C . Control cultures were maintained in that medium alone, while other cultures were supplemented with either PCB 126 (5, 10, 50 and 100 pg/ml) or PCB 153 (5, 10, 50 and 100 ng/ml) . After 24 h, 48 h and 72 h of culture media were collected for P4 content analysis . Cell viability was measured using LDH cytotoxicity test . Exposure of luteal cells to all doses of PCB 126 for 24 h had no effect on progesterone secretion while longer, 48 h and 72 h exposure decreased progesterone secretion in a statistically significant manner . Concentration dependent decrease in progesterone secretion by luteal cells was seen after 24 h and 48 h exposure to PCB153 while concentration dependent increase in progesterone secretion was noted after 72 h exposition to this congener . The toxic effect of both congeners was observed only after 72 h exposition to 50 pg/ml and 100 pg/ml in the case of PCB 126 and 50 ng/ml and 100 ng/ml in the case of PCB 153 . In conclusion, these results suggest that there are differences in PCB 153 and 126 action in luteal cells . Since information concerning mechanism of PCBs action on luteal cells is scarce, these preliminary experiments are of pioneering character. Mol Reprod Dev, 2001 Dec, 60(4), 457 - 62 Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos; Wei Z et al.; The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR) . Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF) . Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment . In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5 . In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA . In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively . RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts . The PCR products were subjected to direct DNA sequencing . There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition . They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA . However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA . With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group . When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition . When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml . EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing . Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin) . However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation . Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media . J Cell Biochem, 2001, 81(S36), 232 - 246 Estrogen suppression of EGFR expression in breast cancer cells: A possible mechanism to modulate growth; Yarden RI et al.; Epidermal growth factor receptor (EGFR) is a transmembrane receptor whose overexpression in breast cancer predicts for poor prognosis and is inversely correlated with expression of estrogen receptor (ER) . This study was designed to investigate whether estrogen plays an active role in suppression of EGFR expression in estrogen-responsive breast cancer cell lines expressing low levels of EGFR . Upon withdrawal of estrogen, EGFR mRNA and protein increased 3-6 fold in MCF-7, T47D, and BT474 ER+ breast cancer cells . This was reversible upon addition of estradiol back to the culture media, but only after prolonged treatment . Nuclear run-on assays and studies with the transcription inhibitor actinomycin D demonstrated that regulation is at the transcriptional level . These results indicate that in the presence of estrogen, ER+ breast cancer cells possess active mechanisms to suppress EGFR expression . Up-regulation of EGFR in response to estrogen depletion and growth inhibition could represent an attempt to rescue cell growth by utilizing an alternative pathway . Indeed, we found that estrogen-depleted breast cancer cells are more sensitive to the mitogenic effects of EGF and TGF-alpha, and simultaneous blockade of both estrogen and EGFR signaling pathways induced cell death . J . Cell . Biochem . Suppl . 36: 232-246, 2001 . Am J Obstet Gynecol, 2001 Dec, 185(6), 1314 - 7 Effect of carbon dioxide on human ovarian carcinoma cell growth; Smidt VJ et al.; OBJECTIVE: Laparoscopy may be associated with increased risk of ovarian carcinoma wound metastases . This study was designed to determine whether carbon dioxide exposure increases the growth of human ovarian cancer cells in vitro . STUDY DESIGN: Immortalized ovarian epithelial carcinoma cell (SKOV-3 cell line) cultures were exposed to carbon dioxide, nitrous oxide, or culture media with decreased pH for up to 3 hours . Cell growth was determined with the use of a spectrophotometric assay, and the results were compared with control cells by paired t tests and linear regressions analysis . RESULTS: Carbon dioxide exposure increased SKOV-3 cell growth by 52% after 4 days in culture . The increased cell growth had a linear relationship to the length of carbon dioxide exposure . Cells that were exposed to either nitrous oxide or media with pH 6.3 showed a trend toward decreased growth . CONCLUSION: Carbon dioxide exposure increases the in vitro growth of human ovarian carcinoma cells by an effect that is independent of the carbon dioxide-related decrease in the culture media pH. Neurobiol Dis, 2001 Dec, 8(6), 1057 - 68 Generation and characterization of immortalized human microglial cell lines: expression of cytokines and chemokines; Nagai A et al.; Microglia are a major glial component of the central nervous system (CNS), play a critical role as resident immunocompetent and phagocytic cells in the CNS, and serve as scavenger cells in the event of infection, inflammation, trauma, ischemia, and neurodegeneration in the CNS . Studies of human microglia have been hampered by the difficulty of obtaining sufficient numbers of human microglia . One way to circumvent this difficulty is to establish permanent cell lines of human microglia . In the present study we report the generation of immortalized human microglial cell line, HMO6, from human embryonic telencephalon tissue using a retroviral vector encoding myc oncogene . The HMO6 cells exhibited cell type-specific antigens for microglia-macrophage lineage cells including CD11b (Mac-1), CD68, CD86 (B7-2), HLA-ABC, HLA-DR, and ricinus communis agglutinin lectin-1 (RCA), and actively phagocytosed latex beads . In addition, HMO6 cells showed ATP-induced responses similar to human primary microglia in Ca2+ influx spectroscopy . Both human primary microglia and HMO6 cells showed the similar cytokine gene expression in IL-1beta, IL-6, IL-8, IL-10, IL-12, IL-15, and TNF-alpha . Using HMO6 cells, we investigated whether activation was induced by Amyloid-beta fragments or lipopolysaccharide (LPS) . Treatment of HMO6 cells with Amyloid-beta 25-35 fragment (Abeta(25-35)) or Amyloid-beta 1-42 fragment (Abeta(1-42)) led to increased expression of mRNA levels of cytokine/chemokine IL-8, IL-10, IL-12, MIP-1beta MIP-1, and MCP-1, and treatment with LPS produced same results . Expression of TNF-alpha and MIP1-alpha was not detected in unstimulated HMO6 cells, but their expression was later induced by long-term exposure to Abeta(25-35) or Abeta(1-42.) ELISA assays of spent culture media showed increased protein levels of TNF-alpha and IL-8 in HMO6 cells following treatment with Abeta(25-35) or LPS . Taken together, our results demonstrate that treatment of human primary microglia and HMO6 immortalized human microglia cell line with Abeta(25-35), Abeta(1-42) and LPS upregulate gene expression and protein production of proinflammatory cytokines and chemokines in these cells . The human microglial cell line HMO6 exhibits similar properties to those documented in human microglia and should have considerable utility as an in vitro model for the studies of human microglia in health and disease. J Endocrinol, 2001 Dec, 171(3), 435 - 43 Human chorionic gonadotropin (hCG) beta-core fragment is produced by degradation of hCG or free hCG beta in gestational trophoblastic tumors: a possible marker for early detection of persistent postmolar gestational trophoblastic disease; Okamoto T et al.; The present study was undertaken to investigate whether human chorionic gonadotropin (hCG) beta-core fragment (hCG beta cf) was directly produced by gestational trophoblastic tumors . Immunoreactivity of hCG beta cf was demonstrated in the extracts as well as in the culture media of hydatidiform mole tissues . It was also present in the extracts of choriocarcinoma tissues, and its molar concentration exceeded that of intact hCG . The presence of hCG beta cf was then confirmed by gel chromatography and Western blot analysis . Immunohistochemistry showed localization of hCG beta cf immunoreactivity to the syncytiotrophoblasts and scattered cells in the stroma of mole tissue, and to syncytiotrophoblastic cells in choriocarcinoma . Immunoreactivity of hCG beta cf was also detected in the sera of the patients with gestational trophoblastic disease, although the hCG beta cf/hCG ratio was less than one hundredth of that in the tissue extracts . Serial measurement of serum hCG beta cf levels after mole evacuation showed that they declined much more rapidly than those of hCG and became undetectable in the patients with subsequent spontaneous resolution, while hCG beta cf remained or became detectable before the rise of hCG was observed in the patients with subsequent persistent trophoblastic disease . Taken together, these results suggest that hCG beta cf is directly produced by gestational trophoblastic tumors, and monitoring of hCG beta cf in the serum after mole evacuation may be useful for early prediction of subsequent development of postmolar persistent trophoblastic disease. J Reprod Immunol, 2002 Jan, 53(1-2), 289 - 303 Human malignant cell death by apoptosis-inducing nucleosides from the decidua derived CD57(+)HLA-DR(bright) natural suppressor cell line; Mori T et al.; The CD57(+)HLA-DR(bright) natural suppressor (57.DR-NS) cell line derived from human decidual tissue mediated apoptosis of human leukemia Molt4 and carcinoma BeWo/GCIY cells but not human fibroblast WI-38 cells, and apoptosis-inducing nucleosides (AINs) appeared to be involved . Six AINs were released into 57.DR-NS cell culture media and were isolated by the combination of physicochemical procedures of C18 preparative column, thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) . Subsequently, we demonstrated that AINs could induce apoptosis in the human malignant Molt4/BeWo/GCIY cell line but not human normal WI-38 fibroblasts . Apoptosis was characterized by DNA strand breaks and activation of the caspase cascade, especially caspase-3 . The administration of AINs into GCIY tumor bearing SCID mice culminated in suppression of tumor growth due to apoptosis of tumor cells. Int Arch Allergy Immunol, 2001 Oct, 126(2), 140 - 6 Roles of superoxide dismutase in rat mast cell granules; Fukuishi N et al.; BACKGROUND: It has been suggested that in the granules of rat mast cells there is some kind of superoxide dismutase (SOD), but details of this SOD in mast cells remain unclear . In the present study, we studied the mode of existence of SOD in mast cells and its releasing mechanism from the granules . In addition, we discussed the physiological role of SOD in allergic events . METHODS: Purified rat mast cells were disrupted with a sonic disrupter and granules (sample I) were separated from supernatant (sample II) by centrifugation . The granules were treated with 1 mM Ca(2+), and the supernatant (sample III) was separated from the pellet (sample IV) . Sample III was applied to a heparin column and the eluate was used as sample V . SOD activity was measured in these samples . RESULTS: SOD existed in mast cell granules as a heparin-binding and an inactive form . However, when granules were released and exposed to high Ca(2+) concentration, SOD was discharged from heparin and shifted to the active form . The expression of macrophage inflammatory protein-1 alpha mRNA was enhanced when hydrogen peroxide (H(2)O(2)) or sample III with the xanthine-xanthine oxidase system were added to the culture media . CONCLUSIONS: These findings suggest that in stimulated rat mast cells, the released SOD may transform the generated superoxide anion into H(2)O(2), and the generated H(2)O(2) may enhance the expression of chemokine mRNA in the mast cells . Gastroenterology, 2001 Dec, 121(6), 1354 - 71 Prostanoid production via COX-2 as a causative mechanism of rodent postoperative ileus; Schwarz NT et al.; BACKGROUND & AIMS: This study demonstrates a significant role for cyclooxygenase (COX)-2 and prostanoid production as mechanisms for surgically induced postoperative ileus . METHODS: Rats, COX-2+/+, and COX-2-/- mice underwent simple intestinal manipulation . Reverse-transcription polymerase chain reaction and immunohistochemistry were used to detect and localize COX-2 expression . Prostaglandin levels were measured from serum, peritoneal lavage fluid, and muscularis culture media . Jejunal circular muscle contractions were measured in an organ bath, and gastrointestinal transit was measured in vivo . RESULTS: The data show that intestinal manipulation induces COX-2 messenger RNA and protein within resident muscularis macrophages, a discrete subpopulation of myenteric neurons and recruited monocytes . The manipulation-induced increase in COX-2 expression resulted in significantly elevated prostaglandin levels within the circulation and peritoneal cavity . The source of these prostanoids could be directly attributed to their release from the inflamed muscularis externa . As a consequence of the molecular up-regulation of COX-2, we observed a decrease in in vitro jejunal circular muscle contractility and gastrointestinal transit, both of which could be alleviated pharmacologically with selective COX-2 inhibition . These studies were corroborated with the use of COX-2-/- mice . CONCLUSIONS: Prostaglandins, through the induction of COX-2, are major participants in rodent postoperative ileus induced by intestinal manipulation. Early Pregnancy, 2000 Jul, 4(3), 176 - 90 Investigation of an in vitro model of trophoblast invasion; Trew AJ et al.; OBJECTIVES: To re-investigate methods for visualising cytotrophoblast invasion using the Matrigel invasion model . STUDY DESIGN: Cytotrophoblast cells were isolated from pooled first trimester placentae and cultured on Matrigel-coated transwells in media supplemented with either 5 ng/ml vascular endothelial growth factor (VEGF) or 10 ng/ml epidermal growth factor (EGF) . Invasion was visualised using standard and confocal fluorescent microscopy . RESULTS: The purity of the enriched cytotrophoblast cells was 84-100% . Immunofluorescent cytokeratin staining examined with an inverted fluorescent microscope suggested that cytokeratin-positive cells were present on the underside of the membrane, having invaded through the Matrigel on the upper surface . However, confocal microscopy indicated that these cells did not invade through the Matrigel, and no viable cells were identified in the culture media below the membrane . CONCLUSION: These results suggest that cytokeratin-positive staining on Matrigel-coated transwells is not necessarily indicative of cell invasion, and that similar studies should be interpreted with caution depending on the method of quantification. Eur J Med Res, 2001 Nov 20, 6(11), 465 - 72 Differential effects of radiocontrast agents on human umbilical vein endothelial cells: cytotoxicity and modulators of thrombogenicity; Fauser C et al.; The endothelium plays a central role in the regulation of blood flow and coagulation . The impact of radiocontrast agents on endothelial cells is therefore potentially clinically important, particularly in percutaneous interventions for acute coronary thrombosis . The effects of radiocontrast agents on endothelial cell viability and determinants of thrombogenicity were studied in human umbilical vein endothelial cells (HUVECs) in vitro . Intercellular tight junctions were assessed using immunofluorescence microscopy and measurement of the transmonolayer electrical resistance (TMR) . The concentrations of endothelin-1 (E), von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1) and thrombomodulin (T) were measured in the cell culture media . The ionic, high osmolal radiocontrast agent diatrizoate induced concentration-dependent cell death and an opening of tight junctions with the attendant abolition of the TMR . The concentration of E decreased, vWF increased in the cell culture media, the concentration of PAI-1 and T was not significantly changed by diatrizoate . Radiocontrast agents with reduced osmolality (ioxaglate: ionic; iopamidol: non-ionic) induced an increase in PAI-1 and vWF, but E and T were not significantly changed . CONCLUSIONS: Radiocontrast agents have differential effects on endothelial cells in vitro including the secretion of modulators of thrombogenesis . The effects are most pronounced in the markedly hyperosmolal compound diatrizoate suggesting a contributory role of hypertonicity . Ioxaglate and iopamidol both increased the prothrombotic factors vWF and PAI-1 to the same degree indicating a similar risk of thrombogenicity between low-osmolal ionic and non-ionic radiocontrast agents in this in vitro model. J Biochem (Tokyo), 2001 Dec, 130(6), 721 - 6 Clearance of extracellular and cell-associated amyloid beta peptide through viral expression of neprilysin in primary neurons; Hama E et al.; Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite constantly anabolized and catabolized in the brain . We previously demonstrated that neprilysin is the major Abeta-degrading enzyme in vivo . To investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating Abeta levels, we expressed neprilysin in primary cortical neurons using a Sindbis viral vector and examined the effect on Abeta metabolism . The corresponding recombinant protein, expressed in the cell bodies and processes, exhibited thiorphan-sensitive endopeptidase activity, whereas a mutant neprilysin with an amino acid substitution in the active site did not show any such activity . Expression of the wild-type neprilysin, but not the mutant, led to significant decreases in both the Abeta40 and 42 levels in the culture media in a dose-dependent manner . Moreover, neprilysin expression also resulted in reducing cell-associated Abeta, which could be more neurotoxic than extracellular Abeta . These results indicate that the manipulation of neprilysin activity in neurons, the major source of Abeta in the brain, would be a relevant strategy for controlling the Abeta levels and thus the Abeta-associated pathology in brain tissues. Anticancer Res, 2001 Jul-Aug, 21(4A), 2281 - 6 Taxotere and vincristine inhibit the secretion of the angiogenesis inducing vascular endothelial growth factor (VEGF) by wild-type and drug-resistant human leukemia T-cell lines; Avramis IA et al.; Recent studies have shown that angiogenesis, which is induced by VEGF, may be involved in the pathogenesis of hematopoietic malignancies . A human leukemia model consisting of T-lymphoblastic CEM/0, 7 monoclonal refractory clones resistant to both cytosine arabinoside (ara-C) and L-asparaginase (ASNase), Jurkat/E6-1 and U937, representing the leukemic blasts from relapsed patients with leukemias was investigated for secretion of VEGF before and after treatment with various agents . The T-lymphoblastic cell line, Jurkat/E6-1, was used as the negative control, which has been characterized as not expressing mRNA nor the VEGF protein, and did not secrete VEGF . With no treatment, U937, the positive control, secreted the highest VEGF concentration of 1612.7 pg/ml . The CEM/O wild type cell line and 5 other drug-resistant clones secreted VEGF at levels ranging from 180.9 to 414.2 pg/ml . Two CEM drug-resistant clones, CEM/ara-C/G/ASNase-0.5-1 and CEM/ara-C/G/ASNase-1-1, lacked VEGF production . Docetaxel (Taxotere, TXR), Vincristine (VCR), ASNase, and the Fit-1/Fc chimera, a specific inhibitor of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, were tested for inhibition of VEGF secretion . Treatment of the leukemic cell lines with 2 microg/ml Flt-1/Fc chimera for 24 hours completely inhibited VEGF secretion to the detection limit of the assay (<10pg/ml) . After 24 hours incubation with Flt-1/Fc chimera, the leukemic cells appeared to be undergoing apoptosis, based on microphotography examination, suggesting that VEGF could be used in an autocrine loop to promote cell survival by the leukemic cells . Treatment with 0.5, 1, and 2 microg/ml Flt-1/FC chimera for 48 hours demonstrated a 15-25% growth inhibition by MTT assay . Strong inhibition of VEGF secretion in the culture media was observed after 10 microM TXR or 0.1 microM VCR for 24 hours in the wild-type and drug-resistant clones, except CEM/ara-C/I, in comparison with controls . In contrast, treatment with 1 IU/ml ASNase, a specific T-cell protein inhibitor, in 5 cell lines for 24 hours demonstrated no inhibition of VEGF in CEM/0 3 drug-resistant clones and the myeloid U937 line . We conclude that the leukemia cell lines actively secrete VEGF, in vitro . TXR and VCR, but not ASNase, strongly inhibit the VEGF production, suggesting that inhibition of this growth factor may be a mechanism of antileukemic activity . Moreover, the leukemic cell lines examined here may constitute a useful model to study antiangiogenic drugs, alone or in combination with established drug regimens used against refractory leukemias. Biochim Biophys Acta, 2001 Dec 1, 1515(2), 144 - 58 Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells; Ishida T et al.; Dioleoylphosphatidylethanolamine (DOPE)-containing liposomes that demonstrated pH-dependent release of their contents were stabilized in the bilayer form through the addition of a cleavable lipid derivative of polyethylene glycol (PEG) in which the PEG was attached to a lipid anchor via a disulfide linkage (mPEG-S-S-DSPE) . Liposomes stabilized with either a non-cleavable PEG (mPEG-DSPE) or mPEG-S-S-DSPE retained an encapsulated dye at pH 5.5, but treatment at pH 5.5 of liposomes stabilized with mPEG-S-S-DSPE with either dithiothreitol or cell-free extracts caused contents release due to cleavage of the PEG chains and concomitant destabilization of the DOPE liposomes . While formulations loaded with doxorubicin (DXR) were stable in culture media, DXR was rapidly released in human plasma . pH-Sensitive liposomes, targeted to the CD19 epitope on B-lymphoma cells, showed enhanced DXR delivery into the nuclei of the target cells and increased cytotoxicity compared to non-pH-sensitive liposomes . Pharmacokinetic studies suggested that mPEG-S-S-DSPE was rapidly cleaved in circulation . In a murine model of B-cell lymphoma, the therapeutic efficacy of an anti-CD19-targeted pH-sensitive formulation was superior to that of a stable long-circulating formulation of targeted liposomes despite the more rapid drug release and clearance of the pH-sensitive formulation . These results suggest that targeted pH-sensitive formulations of drugs may be able to increase the therapeutic efficacy of entrapped drugs. Placenta, 2001 Nov, 22(10), 837 - 45 Secretion of annexin V from cultured cells requires a signal peptide; Wang X et al.; Annexin V is an intracellular protein that lacks a hydrophobic signal peptide . However, there are several studies reporting the extracellular presence of annexin V . In this study, we designed transgenes of annexin V with or without an attached secretory signal peptide and investigated the secretion of the transgene products in COS-7 cells . The signal peptide, targeted annexin V to the endoplasmic reticulum (ER), the Golgi and culture media of transfected cells . In contrast, without the signal peptide, annexin V was present only in the cytoplasm and was not detected in the medium.To confirm our results we also evaluated the presence of extracellular annexin V in two cultured cell lines: BeWo, a choriocarcinoma cell model of placental trophoblasts, and human umbilical vein endothelial cells (HUVEC) . Our results showed that annexin V was immunolocalized on the surfaces of both cells but could not be detected in the culture medium of either cell type . Our results suggest that the secretion of annexin V required the recombinant addition of a hydrophobic signal peptide and that the limited quantities of endogenous cell surface annexin V on BeWo and HUVEC cells is most likely derived from adjacent damaged cells . J Biomater Sci Polym Ed, 2001, 12(8), 911 - 20 Incorporation of sulfonylurea into sugar-carrying polymers and their effects on insulin secretion from MIN6 cells in a solution state; Pa KH et al.; A hypoglycemic sulfonylurea (SU) was incorporated into non-reducing glucose-bearing polystyrene (PS) derivative for enhanced interaction with, and insulin secretion from an insulinoma cell line (MIN6) . SU derivative was copolymerized with styrene having sugar moiety such as maltose or lactose . About an 80% increase in insulin secretion for poly(N-p-vinylbenzyl-D-maltonamide-co-SU) (p(VMA-co-SU)) was observed when the copolymer was added to the culture medium (SU: 25 mol%) . whereas about 50% increase for poly(N-p-vinylbenzyl-D-lactonamide-co-SU) (p(VLA-co-SU)) was observed when compared with unstimulated cells . From the measurement of flow cytometry, the fluorescence intensity of MIN6 cells incubated in culture media containing FITC-labeled SU-incorporated copolymer increased remarkly owing to specific interaction between receptors in the cell membrane and SU ligands in the copolymer . When sugar and/or SU-incorporated copolymer were co-entrapped with the cells in agarose or collagen gel as an extracellular matrix for three-dimensional culture . p(VMA-co-SU) enhanced more insulin secretion from MIN6 cells than other polymers and it was found that collagen gel was more effective in insulin secretion than agarose Neurosci Lett, 2001 Nov 23, 315(1-2), 73 - 6 Long-term natural culture of cochlear sensory epithelia of guinea pigs; Zhao HB; To culture and maintain mammalian cochlear cells in vitro is still a big challenge . Only immortalized cochlear cell lines are available . With refinement of culture media and techniques, cochlear sensory epithelial cells of guinea pigs have been cultured without any genetic manipulation using a modified Keratinocyte medium for more than 6 months . The isolated cell clones by cloning cylinders showed a large, flat, epithelial morphotype and expressed cytokeratin and a tight junction associated protein ZO-1, but did not express vimentin . These cells were also labeled with Brn3.1 and calretinin, which are regarded as early hair cell markers . The immunostaining confirmed the culture cells derived from cochlear sensory epithelia . These non-immortalized natural cochlear cells provide valuable cell sources for molecular and genetic studies of the inner ear. Biomacromolecules, 2000 Summer, 1(2), 208 - 18 A hyaluronic acid-taxol antitumor bioconjugate targeted to cancer cells; Luo Y et al.; A cell-targeted polymeric prodrug prepared from Taxol and chemically modified hyaluronic acid (HA) was evaluated in vitro . Herein we report four results in support of the selective uptake and targeted toxicity of the HA-Taxol prodrug . First, a fluorescently labeled HA-Taxol (FITC-HA-Taxol) was synthesized and used to demonstrate cell-specific binding and uptake using flow cytometry and confocal microscopy . Second, the selective cytotoxicity of FITC-HA-Taxol allowed direct correlation of uptake with selective cytotoxicity . Third, the rapid uptake and selective cytotoxicity of HA-Taxol bioconjugates could be blocked by either excess HA or by an anti-CD44 antibody, but not by chondroitin sulfate (CS) . Finally, the release of free Taxol from HA-Taxol in human plasma or in cell culture media revealed that the free drug was hydrolytically released from the bioconjugate by cleavage of the labile 2' ester linkage . Taken together, these data support the notion that the targeted cytotoxicity of HA-Taxol bioconjugates requires receptor-mediated cellular uptake of the bioconjugate followed by hydrolytic release of free Taxol. Biomacromolecules, 2001 Fall, 2(3), 851 - 5 Cytotoxicity of a unimolecular polymeric micelle and its degradation products; Schmalenberg KE et al.; The cytotoxicity of a unimolecular polymeric micelle (1) and its degradation products was assessed by cell proliferation and viability with L929 mouse areolar/adipose fibroblasts . Polymer 1 and poly(ethylene glycol) (PEG) were diluted to concentrations from 10(-4) to 10(-6) M in culture media, whereas the degradation products were diluted to concentrations (10(-4) to 10(-6) M) that would theoretically occur upon degradation of polymer 1 . The polymer degradation products that were evaluated included mucic acid, hexanoic acid, 1,1,1-tris(4-hydroxyphenyl)ethane (THPE),2,3,4,5-tetrakis-hexanoyloxy-hexanedioic acid or MA(hex), Core(hex), and PEG5, which is PEG of molecular weight 5000 . Cells exposed to polymer 1 proliferated at the same rate as cells grown in polymer-free or PEG5-containing solutions up to 36 h . In both the polymer 1 and PEG5 solutions, cytotoxicity was not observed at any concentration (up to 10(-4) M) as indicated by cell attachment, growth, and morphology . Fibroblasts exposed to the degradation products fared as well as fibroblasts in contact with polymer 1 and PEG5, except for cells exposed to the highest concentration (10(-4) M) of THPE. Int J Clin Oncol, 2001 Apr, 6(2), 84 - 9 Indomethacin increases the cytotoxicity of cis-platinum and 5-fluorouracil in the human uterine cervical cancer cell lines SKG-2 and HKUS by increasing the intracellular uptake of the agents; Ogino M et al.; BACKGROUND: Various approaches have been made to increase the cytotoxicity of cis-platinum (CDDP) and 5-fluorouracil (5FUra), and to reduce their adverse effects . We used two human cancer cell lines of the uterine cervix (SKG-2 and HKUS) and selected indomethacin as a modifying agent to assess its effect on the cytotoxicity of CDDP and 5FUra . METHODS: The effect of indomethacin on the cytotoxicity of CDDP and 5FUra in these tumor cells was evaluated by in-vitro Alamar blue assay, and intracellular uptake of free-CDDP and 5FUra was measured to find out how indomethacin modified the cytotoxicity of CDDP and 5FUra . RESULTS: Indomethacin showed no cytotoxic effect on these tumor cells, but significantly (P < 0.005-0.001) increased the cytotoxicity of both CDDP and 5FUra, compared with single treatment by each agent . The intracellular uptake of both free-CDDP and 5FUra was measured in culture media containing the agents alone (single treatment) or the agents with indomethacin (0.001, 0.01, and 0.1 microgram/ml; combined treatment) . The intracellular contents of both free-CDDP (SKG-2, HKUS; P < 0.001) and 5FUra (SKG-2; P < 0.005, HKUS; P < 0.02) were significantly higher in the combined treatment than in the single treatment . CONCLUSION: This study shows that indomethacin increased the cytotoxicity of both CDDP and 5FUra by increasing the intracellular incorporation of free-CDDP and 5FUra, and suggests the effectiveness of indomethacin in reducing the dosage of CDDP and 5FUra or increasing CDDP and 5FUra cytotoxicity when these agents are given without reducing the dose. Kidney Int, 2001 Nov, 60(5), 1930 - 7 Peritoneal dialysate fluid composition determines heat shock protein expression patterns in human mesothelial cells; Arbeiter K et al.; BACKGROUND: Low biocompatibility of peritoneal dialysis fluids (PDF) contributes to mesothelial injury . We investigated whether the heat shock proteins (HSP)-27, HSP-72, and HSP-90 are differentially induced upon exposure of mesothelial cells to PDF and whether this was affected by selective modulation of the physicochemical properties of PDF . METHODS: Human mesothelial cells (Met5A and primary human mesothelial cells) were exposed to acidic lactate and glucose-monomer based PDF (CAPD2 and CAPD3), to control culture media, or to a neutral lactate and glucose-monomer-based PDF with reduced levels of glucose degradation products (BALANCE) . Expression of HSP-27, HSP-72, and HSP-90 and cellular distribution of HSP-72 were assessed by Western blotting and immunocytochemistry . RESULTS: Mesothelial cells exhibited strong constitutive expression of HSP-27 and to a lesser extent HSP-72 and HSP-90 . Exposure of the cells to CAPD2 and CAPD3 resulted in strong up-regulation of HSP-72 . HSP-27 levels were slightly increased, but HSP-90 levels were unchanged upon exposure to CAPD2 or CAPD3 . In contrast, exposure of the cells to BALANCE did not affect HSP-27 or HSP-72 expression . The acidic pH and glucose degradation products were found to be principal in mediating increased HSP-72 expression upon exposure to PDF . CONCLUSIONS: Analysis of HSP expression represents a novel tool to assess biocompatibility of PDF . Among the HSP investigated, HSP-72 is the most predictive and accurate parameter to assess mesothelial cell injury in the early phase of exposure to PDF. Acta Vet Hung, 2001, 49(3), 319 - 29 Porcine theca cells produce immunoreactive beta-endorphin and change steroidogenesis in response to opioid agonist; Kaminski T et al.; In earlier in vitro experiments opioids affected steroidogenesis in porcine luteal and granulosa cells . The present studies were undertaken to examine the effects of FK 33-824 (opioid agonist) alone or in combination with LH, PRL or naloxone (NAL, opioid antagonist) on steroidogenesis in cultured porcine theca cells . Moreover, we have tested beta-endorphin-like immunoreactivity (beta-END-LI) concentrations in culture media under control conditions and following treatments of theca cells with LH, PRL, progesterone (P4), oestradiol (E2) or testosterone (T) . FK 33-824 and NAL significantly increased P4 release by theca cells and inhibited stimulatory effect of LH on this steroid output . PRL-induced P4 secretion from the cells was blunted only by FK 33-824 . Secretion of androstenedione (A4) and T was essentially elevated in the presence of FK 33-824 and this potentiation of both androgen release was completely abolished by PRL . NAL blocked stimulatory effect of the opioid agonist only in case of T . Secretion of oestradiol and oestrone was completely free from the influence of both the opioid agonist and antagonist . Pig theca cells were able to produce beta-END-LI but none of tested hormones (LH, PRL, P4, E2 and T alone or in combination) significantly affected this production . In conclusion, these data indicate that porcine theca cells may produce beta-END-LI and change their steroidogenesis in response to opioid peptides. Biochem Biophys Res Commun, 2001 Nov 16, 288(5), 1149 - 54 Hypoxia down-regulates endostatin production by human microvascular endothelial cells and pericytes; Wu P et al.; Endostatin is a potent anti-angiogenic factor derived from the C-terminal region of collagen XVIII and is implicated in the regulation of physiological and pathological angiogenesis . In this study, reverse transcription-polymerase chain reaction analysis of poly(A+) RNA demonstrated the presence of mRNA for collagen XVIII in human endothelial cells (EC) and pericytes, the very constituents of microvessels wherein angiogenesis takes place . Enzyme immunoassay revealed that both cell types liberated endostatin into culture media and that the endostatin levels were decreased by hypoxia, the principal cause of angiogenesis . Northern and Western blot analyses revealed that while the collagen XVIII/endostatin mRNA levels were invariant between hypoxic and normoxic conditions, the collagen XVIII protein levels in EC and pericytes decreased by hypoxia . Further, exogenously administered intact endostatin was significantly decreased when it was incubated with hypoxic conditioned media of endothelial cells or pericytes, but not with normoxic media . The results suggest that the reduction of autocrine endostatin may take an active part in hypoxia-driven angiogenesis . J Biol Chem, 2002 Jan 18, 277(3), 2118 - 24 Epub 2001 Nov 06. The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa; Chen M et al.; Type VII collagen is a major component of anchoring fibrils, attachment structures that mediate dermal-epidermal adherence in human skin . Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder caused by mutations in the type VII collagen gene and perturbations in anchoring fibrils . In this study, we produced recombinant human type VII collagen in stably transfected human 293 cell clones and purified large quantities of the recombinant protein from culture media . The recombinant type VII collagen was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation . Purified type VII collagen bound to fibronectin, laminin-5, type I collagen, and type IV collagen and also supported human dermal fibroblast adhesion . In an attempt to establish genotype-phenotype relationships, we generated two individual substitution mutations that have been associated with recessive DEB, R2008G and G2749R, and purified the recombinant mutant proteins . The G2749R mutation resulted in mutant type VII collagen with increased sensitivity to protease degradation and decreased ability to form trimers . The R2008G mutation caused the intracellular accumulation of type VII collagen . We conclude that structural and functional studies of in vitro generated type VII collagen mutant proteins will aid in correlating genetic mutations with the clinical phenotypes of DEB patients. Toxicol In Vitro, 2001 Dec, 15(6), 729 - 40 Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay; Pessina A et al.; This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC) . In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM . In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility . After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified . Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories . Valid tests represented 95% of all tests attempted . The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics . As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant . It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy . On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential. J Parasitol, 2001 Oct, 87(5), 1167 - 8 Axenic culture of Schistosoma mansoni sporocysts in low O2 environments; Bixler LM et al.; Recent successes in culturing intramolluscan larval stages of Schistosoma mansoni have relied on synxenic culture with a cell line (Bge) developed from embryos of a molluscan host Biomphalaria glabrata . To further facilitate progress toward control of schistosomiasis, a system for axenic in vitro culture of the parasite has now been developed . When culture media were preconditioned by Bge cells, sporocysts lived longer in vitro and produced more offspring . Because Bge-derived components could be protecting sporocysts from oxidative stress, axenic sporocysts were cultured at lowered O2 levels . In an hypoxic environment, S . mansoni sporocysts grew well and produced daughter sporocysts continuously under axenic conditions and in a medium completely lacking host molecules . Sporocyst production occurs independently of host influence. J Nutr, 2001 Nov, 131(11), 2943 - 50 Fatty acids enhance GRO/CINC-1 and interleukin-6 production in rat intestinal epithelial cells; Yoshida H et al.; Intestinal mucosal immunity is modulated by cytokine release from intestinal cells, but little is known about the relation between nutrient absorption and cytokine release . In this study, we examined how exposure to fatty acids affects the production of growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) and interleukin (IL)-6 in rat intestinal epithelial cells (IEC) . The long-chain fatty acids, oleic, linoleic and arachidonic acids, and the middle-chain fatty acid octanoic acid were administered to subconfluent cultures of IEC-6 cells alone, or in combination with IL-1beta and transforming growth factor (TGF)-beta . The GRO/CINC-1 and IL-6 concentrations in culture media were determined by sandwich enzyme immunoassay . In epithelial cells, GRO/CINC-1 and IL-6 mRNA expression were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and mitogen-activated protein kinase (MAPK) activities determined by immunoblotting . Administration of long-chain fatty acids significantly increased the GRO/CINC-1 and IL-6 secretion into culture media, and this secretion was markedly increased (P < 0.05) in the presence of IL-1beta or TGF-beta . Octanoic acid had no effect on GRO/CINC-1 or IL-6 production . Furthermore, treatment with long-chain fatty acids significantly enhanced the GRO/CINC-1 and IL-6 expression that was induced by IL-1beta or TGF-beta . MAPK activity was significantly enhanced by treatment with long-chain fatty acids . Inhibitors of phospholipase C, protein kinase C or MAPK significantly reduced the fatty acid-induced increase in GRO/CINC-1 secretion, whereas a calcium/calmodulin inhibitor did not attenuate the secretion . These results suggest that long-chain fatty acids enhance cytokine release under conditions of inflammatory stimulation in the intestinal mucosa. Biochim Biophys Acta, 2001 Oct 18, 1549(2), 161 - 73 Delineation of the minimal catalytic domain of human Galbeta1-3GalNAc alpha2,3-sialyltransferase (hST3Gal I); Vallejo-Ruiz V et al.; The CMP-Neu5Ac:Galbeta1-3GalNAc alpha2,3-sialyltransferase (ST3Gal I, EC 2.4.99.4) is a Golgi membrane-bound type II glycoprotein that catalyses the transfer of sialic acid residues to Galbeta1-3GalNAc disaccharide structures found on O-glycans and glycolipids . In order to gain further insight into the structure/function of this sialyltransferase, we studied protein expression, N-glycan processing and enzymatic activity upon transient expression in the COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence . The expressed soluble polypeptides were detected within the cell and in the cell culture media using a specific hST3Gal I monoclonal antibody . The soluble forms of the protein consisting of amino acids 26-340 (hST3-Delta25) and 57-340 (hST3-Delta56) were efficiently secreted and active . In contrast, further deletion of the N-terminal region leading to hST3-Delta76 and hST3-Delta105 gave also rise to various polypeptides that were not active within the transfected cells and not secreted in the cell culture media . The kinetic parameters of the active secreted forms were determined and shown to be in close agreement with those of the recombinant enzyme already described (H . Kitagawa, J.C . Paulson, J . Biol . Chem . 269 (1994)) . In addition, the present study demonstrates that the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites. Reproduction, 2001 Nov, 122(5), 687 - 93 Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR; Natale DR et al.; The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development . These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos . Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting . We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos . Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine embryos . These studies have demonstrated that culture conditions influence the amount of marker gene transcripts and downregulate or induce the expression of novel genes during early development . Optimized defined culture media maintain embryonic gene expression patterns closely resembling those displayed by embryos derived in vivo . Preimplantation mammalian embryos display an impressive capacity to respond to the pressures that suboptimal culture environments place upon them . However, this plasticity operates within a defined range of tolerances . Continued research using molecular techniques will lead to increased understanding of developmental mechanisms causing culture-related phenotypic abnormalities in post-implantation embryos. Osteoarthritis Cartilage, 2001, 9 Suppl A, S55 - 63 Contacts with fibrils containing collagen I, but not collagens II, IX, and XI, can destabilize the cartilage phenotype of chondrocytes; Farjanel J et al.; OBJECTIVE: Cell-matrix interactions are important regulators of cellular functions, including matrix synthesis, proliferation and differentiation . This is well exemplified by the characteristically labile phenotype of chondrocytes that is lost in monolayer culture but is stabilized in suspension under appropriate conditions . We were interested in the role of collagen suprastructures in maintaining or destabilizing the cartilage phenotype of chondrocytes . DESIGN: Primary sternal chondrocytes from 17-day-old chick embryos were cultured in gels of fibrils reconstituted from soluble collagen I from various sources . The culture media either contained or lacked FBS . Cells were cultured for up to 28 days and the evolution of the phenotype of the cells was assessed by their collagen expression (collagens II and X for differentiated chondrocytes and hypertrophic chodrocytes, repectively; collagen I for phenotypically modulated cells), or by their secretion of alkaline phosphatase (hypertrophic cartilage phenotype) . RESULTS: The cells often retained their differentiated phenotype only if cultured with serum . Under serum-free conditions, cartilage characteristics were lost . The cells acquired a fibroblast-like shape and, later, synthesized collagen I instead of cartilage collagens . Shape changes were influenced by beta1-integrin-activity, whereas other matrix receptors were important for alterations of collagen patterns . Heterotypic fibrils reconstituted from collagens II, IX, and XI did not provoke this phenotypic instability . CONCLUSIONS: Chondrocytes sensitively recognize the suprastructures of collagen fibrils in their environment . Cellular interactions with fibrils with appropriate molecular organizations, such as that in cartilage fibrils, result in the maintenance of the differentiated cartilage phenotype . However, other suprastructures, e.g . in reconstituted fibrils mainly containing collagen I, lead to cell-matrix interactions incompatible with the cartilage phenotype . The maintenance of the differentiated traits of chondrocytes is pivotal for the normal function of, e.g., articular cartilage . If pathologically altered matrix suprastructures lead to a dysregulation of collagen production also in vivo compromised cartilage functions inevitably will be propagated further. Semin Reprod Med, 2001 Sep, 19(3), 259 - 68 Blastocyst versus day 2 or 3 transfer; Schoolcraft WB et al.; The formulation of new sequential culture media, capable of supporting the development of viable human blastocysts, has reopened the discussion regarding the best day for embryo transfer following in vitro fertilization (IVF) . Although several laboratories have reported overall increases in implantation rate and IVF efficiency following the transfer of blastocysts, others have failed to observe any benefit from extended culture . While culture conditions for the mammalian embryo undoubtedly have improved significantly over the past few years, relatively little attention has been paid to the quality of oocytes derived from ovarian hyperstimulation or the quality and receptivity of the endometrium following such hormonal regimes . It appears that differences in controlled ovarian hyperstimulation are among the major factors determining embryo quality and subsequent implantation . This therefore has confounded comparisons between different laboratories . In spite of this there are a growing number of reports demonstrating that the advantages of extended culture and blastocyst transfer, such as increased implantation rates, are not limited to specific groups of patients or specific etiologies . Rather, blastocyst transfer may be of benefit to the majority if not all patients attending for IVF. Clin Exp Immunol, 2001 Oct, 126(1), 131 - 6 Expression of membrane-type 1 matrix metalloproteinase in rheumatoid synovial cells; Honda S et al.; Membrane-type 1 matrix metalloproteinase (MT1-MMP) is thought to be a putative regulator of pro-gelatinase A (MMP-2) in the rheumatoid synovium . In this study, we examined the effects of IL-1beta, one of the inflammatory cytokines, on the expression of MT1-MMP and the activation of pro-MMP-2 using rheumatoid synovial cells . We also studied the effects of KE-298 (2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid), a new disease-modifying anti-rheumatic drug (DMARD), on MT1-MMP expression of rheumatoid synovial cells . Type B synovial cells (fibroblast-like synovial cells) were cultured with KE-298 (25-100 microg/ml) in the presence of IL-1beta for 48 h . Activation of pro-MMP-2 secreted from synovial cells was analysed by gelatin zymography . Reverse transcription-polymerase chain reaction (RT-PCR) methods were used to detect MT1-MMP mRNA . MT1-MMP protein expression on synovial cells was examined by anti-MT1-MMP immunoblot . An active form of MMP-2 was demonstrated in the culture media conditioned by IL-1beta-stimulated synovial cells . In addition, MT1-MMP mRNA and protein expression of rheumatoid synovial cells were increased by IL-1beta treatment . KE-298 blocked this IL-1beta-induced pro-MMP-2 activation and MT1-MMP expression, but did not affect IL-1beta-induced tissue inhibitor of metalloproteinase-2 (TIMP-2) secretion from rheumatoid synovial cells . These findings indicate that activation of rheumatoid synovial cells by IL-1beta results in the induction of MT1-MMP expression . Given that MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokine may contribute to articular destruction in rheumatoid arthritis (RA) . KE-298 may prevent this process by down-regulating MT1-MMP expression. Parasitology, 2001 Oct, 123(Pt 4), 357 - 63 Addition of hypoxanthine to culture media allows in vitro cultivation of Babesia bovis and B . bigemina at reduced serum concentrations; Neves L et al.; The microaerophilous stationary phase system (MASP) was introduced in 1980 and successfully used as a standard technique for Babesia bovis and B . bigemina in vitro culture . The percentage of serum in the medium and the dependence on specific serum donors have been recognized as important constraints both for immunochemical studies and for the logistics of culture routine . In the present study the supplementation of RPMI 1640 with hypoxanthine at a concentration between 50 and 200 microM has enabled patterns of growth of B . bovis and B . bigemina to be achieved comparable to the standard technique with a simultaneous reduction of serum concentration from 40% to 5% . With hypoxanthine-supplemented medium it was possible to either replace the bovine serum from a specific donor with horse serum or use commercial adult bovine serum or foetal calf serum at 10% . When the serum replacement media Albumax II and GF21 were used, the growth of both B . bovis and B . bigemina markedly decreased after 3 x 72 h cycles . However, when these species were cultivated in culture flasks previously coated with cells from a murine peritoneal lavage, continuous parasite growth was achieved. Biorheology, 2001, 38(4), 347 - 53 Fluid shear stress suppresses interleukin 8 production by vascular endothelial cells; Kato H et al.; The effects of shear stress on interleukin 8 (IL-8) production by human umbilical vein endothelial cells (HUVEC) were studied by subjecting the HUVEC to a steady flow laminar shear stress of up to 0.7 N/m(2) in a parallel plate flow chamber . Shear stress decreased IL-8 mRNA expression in a dose and time-dependent fashion . High glucose concentrations increased IL-8 mRNA levels in a MAPK-p38-dependent manner, which was suppressed by shear stress . Measurement of IL-8 protein in HUVEC culture media by ELISA demonstrated that IL-8 secretion was also increased by high glucose and suppressed by shear stress . These results suggest that the anti-atherogenic effect of shear stress arises partly from the suppression of the production of IL-8 which has been shown to trigger the adhesion of monocytes to a vascular endothelium and also acts as a mitogen and chemoattractant for vascular smooth muscle cells. Theriogenology, 2001 Sep 15, 56(5), 955 - 67 Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa; Bogliolo L et al.; The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity . The first objective of this study was to compare the effect of two different culture media and two different incubation times on in vitro maturation (IVM) of domestic cat oocytes . The second objective was to determine the developmental competence of in vitro matured cat oocytes after intracytoplasmic sperm injection (ICSI) with cat spermatozoa . Oocytes recovered from ovaries of ovariectomized cats were cultured either in TCM 199 medium or in synthetic oviductal fluid (SOF), both of which were supplemented with cysteamine, BSA, FSH, LH . Nuclear maturation was assessed after 24 h and 40 h of incubation . Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 40 h of incubation were significantly higher (P<0.001) after culture with SOF (88/110, 80% and 159/192, 82.8%) than TCM 199 (86/129, 66.7% and 58/90, 64.4%) . Oocytes (n = 231) matured in vitro in SOF for 24 h were fertilized by ICSI with frozen-thawed epididymal cat spermatozoa . After ICSI, one group of oocytes (n = 129) was activated with ethanol, and a second group (n = 102) was not activated . The developmental competence of all ICSI oocytes was examined after 7 days of in vitro culture . After 28 h of culture, the cleavage frequency of ICSI-activated oocytes was significantly higher (P<0.001) than that of IC J Assist Reprod Genet, 2001 Sep, 18(9), 519 - 25 Effect of essential amino acids on mouse embryo viability and ammonium production; Lane M et al.; PURPOSE: To examine the effect of essential amino acids concentrations on mouse embryo development . METHODS: Mouse embryos were cultured in medium with different concentrations of essential amino acids and development to the blastocyst stage and viability assessed . Ammonium production resulting from medium breakdown and amino acid metabolism by embryos were also assessed . RESULTS: Reducing the essential amino acid concentration significantly increased blastocyst development and cell numbers . Lowering the essential amino acid concentration decreased ammonium production in the medium . CONCLUSIONS: Culture media for the development of preimplantation embryos should have a reduced essential amino acid concentration to facilitate embryo development. J Biol Chem, 2001 Dec 14, 276(50), 47608 - 14 Epub 2001 Oct 17. Ca2+ pools and cell growth . Evidence for sarcoendoplasmic Ca2+-ATPases 2B involvement in human prostate cancer cell growth control; Legrand G et al.; The present study demonstrates for the first time that intracellular calcium-ATPases and calcium pool content are closely associated with prostate cancer LNCaP cell growth . Cell growth was modulated by changing the amount of epidermal growth factor, serum, and androgene in culture media . Using the microspectrofluorimetric method with Fura-2 and Mag Fura-2 as probes, we show that in these cells, the growth rate is correlated with intracellular calcium pool content . Indeed, an increased growth rate is correlated with an increase in the calcium pool filling state, whereas growth-inhibited cells show a reduced calcium pool load . Using Western blotting and immunocytochemistry, we show that endoplasmic reticulum calcium pump expression is closely linked to LNCaP cell growth, and are a common target of physiological stimuli that control cell growth . Moreover, we clearly demonstrate that inhibition of these pumps, using thapsigargin, inhibits LNCaP cell growth and prevents growth factor from stimulating cell proliferation . Our results thus provide evidence for the essential role of functional endoplasmic reticulum calcium pumps and calcium pool in control of prostate cancer LNCaP cell growth, raising the prospect of new targets for the treatment of prostate cancer. Endocrinology, 2001 Nov, 142(11), 4930 - 6 Effect of adrenal and ovarian androgens on type 4 follicles unresponsive to FSH in immature mice; Wang H et al.; The present study investigates the physiological significance of dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, androstenedione (Delta(4)), dihydrotestosterone (DHT), estrone (E1), and E2 on recombinant human FSH- (rhFSH) resistant type 4 follicles obtained from immature mice . Type 4 follicles of a diameter of 100-120 microm with one or two granulosa cell layers around the oocyte and an intact basal lamina with theca cells were isolated from the ovaries of 11-d-old BDF-1 mice and cultured with medium alone (control) or with dehydroepiandrosterone, dehydroepiandrosterone sulfate, T, Delta(4), DHT, E1, or E2 at concentrations ranging from 1 x 10(-11) to 1 x 10(-7) M for 4 d . We examined the mean diameters of type 4 follicles, levels of immunoreactive (IR)-inhibin, and E2 and progesterone in the culture media on day 4 . In addition, we evaluated follicular cell proliferation by immunofluorescence staining with 5-bromo-2'-deoxyuridine . All tested androgens significantly increased the diameter of type 4 follicles in a dose-dependent manner without the production of IR-inhibin and E2 . The nuclei of granulosa cells in type 4 follicles cultured with all tested androgens exhibited intense 5-bromo-2'-deoxyuridine-positive staining, compared with those of controls . In contrast, neither E1 nor E2 had any stimulatory effects . The stimulatory effects of T, Delta(4), or DHT were inhibited by an AR antagonist in a dose-related fashion but not by an aromatase inhibitor . Furthermore, all tested androgens had a synergistic effect with rhFSH on follicular growth and the production of IR-inhibin and E2 . These results demonstrated that neither adrenal nor ovarian androgens are arteriogenic but that they stimulate type 4 follicles unresponsive to rhFSH and augment the responsiveness of these follicles to rhFSH. Int J Oncol, 2001 Nov, 19(5), 1049 - 55 Effect of platelet activating factor and butyrate on the expression of interleukin-2 receptor alpha in nasopharyngeal carcinoma cells; Tsai MH et al.; Serum level of soluble interleukin-2 receptor alpha (sIL-2Ralpha) has been shown to correlate with disease progression and prognosis of cancer patients . However, the available information about the source and the pathophysiological regulation of IL-2Ralpha in cancer cells is limited . This study addressed the questions of prognostic value and the source of sIL-2Ralpha in patients with nasopharyngeal carcinoma (NPC) . Biological regulation of IL-2Ralpha was characterized in NPC cell lines . Serum sIL-2Ralpha levels of 113 NPC patients were measured by enzyme-linked immunosorbent assay (ELISA) . Levels of sIL-2Ralpha in NPC patients were significantly higher than that in the healthy controls, and sIL-2Ralpha levels were correlated with disease progression and patient survival . IL-2Ralpha was identified in cancer cells by immunocytochemistry . In vitro, IL-2Ralpha expression was markedly increased following treatment with platelet activating factor and/or n-sodium butyrate . Increased secretion of IL-2Ralpha was also detected in the culture media . The secreted IL-2Ralpha could functionally bind IL-2 . These results indicate that elevated sIL-2Ralpha was often detected in patients with advanced NPC . The elevated sIL-2Ralpha could be shed from NPC cells by a yet to be determined mechanism and IL-2Ralpha expression in NPC cells could be upregulated by platelet activating factor and butyrate. Hybridoma, 2001 Aug, 20(4), 265 - 72 Characterization of monoclonal antibodies against carcinoembryonic antigen (CEA) and expression in E . coli; Kim SH et al.; Eight monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) were characterized . Five clones are IgG(1), two clones are IgM and one clone is IgG(2b); all have kappa light chain . The affinities are in the range of 1.1 x 10(-7) approximately 2.4 x 10(-9) M; the affinities of two IgM clones could not be estimated because of their low enzyme-linked immunoadsorbent assay (ELISA) signal . Each clone was constructed as single-chain Fv (scFv) and expression was performed in E . coli . Four clones out of 8 could express scFv soluble to culture media and the expression was confirmed further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting . The nucleotide and amino acid sequences of V(H) and V(L) of four scFvs were deduced and their family and subgroup were analyzed . We found that the clones that do not express the scFv have aberrant kappa chain (incorrect V/J recombination or stop codon); in contrast, their heavy chain sequences proved correct . The E . coli-expressed scFvs showed 1.5 x 3.4-fold lower affinities (2.8 x 10(-8) approximately 3.6 x 10(-9) M) than those of hybridoma-derived parental antibodies except the one clone (C5), which exhibited approximately 10(-6) M of affinity. Clin Orthop, 2001 Oct, (391 Suppl), S208 - 18 Human meniscus cell: characterization of the primary culture and use for tissue engineering; Nakata K et al.; Human meniscus cells from 47 surgically excised menisci were grown in primary culture . Cell proliferation and morphologic features were evaluated in three different culture media . Human meniscus cells showed three distinguishable cell types in monolayer culture: elongated fibroblastlike cells, polygonal cells, and small round chondrocytelike cells . These cells proliferated in Dulbecco's modified Eagle's medium, but by Day 7, elongated fibroblastlike cells became predominant . Cells did not proliferate in Ham's nutrient mixture-F-12 . In a mixture of Ham's nutrient mixture-F-12 and Dulbecco's modified Eagle's medium, cells proliferated, maintaining their morphologic features and their ability to express messenger ribonucleic acids for aggrecan and Types I, II, and III collagen . Hyaluronan enhanced cellular proliferation without altering morphologic features or chondroitin sulfate production . Cultured human meniscus cells attached to a porous collagen sponge after cell seeding . Gene transfer was successful and an introduced gene was expressed by the cells, indicating that human meniscus cells can undergo gene manipulation . The finding that cells collected from small surgical specimens of human meniscus could be cultured, propagated, and seeded onto a collagen scaffold holds promise for the development of a cell-based, tissue engineered collagen meniscus. J Eukaryot Microbiol, 2001 Sep-Oct, 48(5), 588 - 94 The sterol composition of Trypanosoma cruzi changes after growth in different culture media and results in different sensitivity to digitonin-permeabilization; Rodrigues CO et al.; Respiration, oxidative phosphorylation . and the corresponding changes in membrane potential (deltapsi) of Trypanosoma cruzi epimastigotes grown either in liver infusion-tryptose (LIT) or brain heart infusion (BHI) culture medium were assayed in situ using digitonin to render their plasma membrane permeable to succinate, ADP, safranine O, and other small molecules . When the cells were permeabilized with 64 microM digitonin, a concentration previously used with epimastigotes, the ability of the cells grown in LIT medium to sustain oxidative phosphorylation was demonstrated by the detection of an oligomycin-sensitive decrease in mitochondrial membrane potential induced by ADP . In contrast, the cells grown in BHI medium were not able to sustain a stable membrane potential and did not respond to ADP addition . Analyses of oxygen consumption by these permeabilized cells indicated that the rate of basal respiration, which was similar in both cell types, was significantly decreased by 64 microM digitonin . Addition of ADP to the permeabilized cells grown in LIT medium promoted an oligomycin-sensitive transition from resting to phosphorylating respiration in contrast to the cells grown in BHI medium, whose respiration decreased steadily and did not respond either to ADP or CCCP . Titration of the cells grown in BHI medium with different digitonin concentrations indicated that their mitochondria have higher sensitivity to digitonin than those grown in LIT medium . Analysis of the sterol composition of epimastigotes grown in the two different media showed a higher percentage of cholesterol in total and mitochondrial extracts of epimastigotes grown in BHI medium as compared to those grown in LIT medium, suggesting the involvement of this sterol in their increased sensitivity to digitonin-permeabilization. Ann N Y Acad Sci, 2001 Sep, 943, 340 - 51 Role of the proteasome in the regulation of fetal fibronectin secretion in human placenta; Yuehong MA et al.; The goal of the current study was to examine the role of the ubiquitin-proteasome system (UPS), a pathway of intracellular degradation, in the regulation of fetal fibronectin (FFN) expression in human placenta . Primary cultures of cytotrophoblasts (CTs) and placental mesenchymal cells (PMCs) were isolated from human term placentas and were maintained in serum-free medium (SFM) in the presence of inhibitors of proteasome-mediated degradation (e.g., MG132) as well as inhibitors of other proteases . Levels of secreted FFN and interleukin (IL)-8 in culture media were quantitated by enzyme-linked immunosorbent assay (ELISA), and cell viability was assessed by trypan blue exclusion . Intracellular levels of FFN and ubiquinated proteins were measured by Western blotting, and levels of fibronectin mRNA were determined following Northern blotting . We found that proteasome inhibitors (MG132, MG262, and PSI) potently suppressed levels of secreted FFN in cultures of CTs, but they not did affect levels of IL-8 . Lysosomal, calpain, and serine protease inhibitors as well as the anti-inflammatory compound sulfasalazine did not markedly affect levels of secreted FFN in CT cultures . Proteasome inhibitors did not compromise cell viability during the initial 16-18 hours of treatment and did not affect intracellular levels of FFN protein or fibronectin mRNA . The efficacy of suppression of FFN in CT culture media by proteasome inhibitors reflected their effects on intracellular accumulation of ubiquinated proteins . By contrast, the presence of proteasome inhibitors did not alter levels of secreted FFN in cultures of PMCs . We conclude that inhibitors of proteasome-mediated degradation potently and specifically suppressed extracellular expression of FFN in CTs through a cell type-specific pathway that did not involve alterations in FFN synthesis . This suggests that accumulation of ubiquinated proteins in the presence of proteasome inhibitors blocks FFN secretion or promotes the extracellular degradation of FFN . This experimental paradigm will be useful for dissecting the role of the UPS in regulating CT function. Environ Toxicol, 2001 Oct, 16(5), 391 - 6 Extraction and purification of the zwitterions cylindrospermopsin and deoxycylindrospermopsin from Cylindrospermopsis raciborskii; Norris RL et al.; The hepatotoxin cylindrospermopsin (CYN) has been isolated from the cyanobacterium Cylindrospermopsis raciborskii (C . raci.) . Efforts to study this toxin have been hampered by the time-consuming requirement to extract it from cultures of the organism . It is usually extracted from lyophilized cells collected from a laboratory culture . Our preliminary work suggested far more of the toxin is available in solution in the culture media than in the cells collected . We have therefore investigated the use of commercially available solid phase extraction sorbents to extract CYN from culture media in which C . raci . has been grown . A range of reverse phase and ion-exchange sorbents were tested across a range of pHs for their ability to retain CYN without success . Subsequently, graphitized carbon cartridges were found to retain CYN strongly . Elution with 5% formic acid in methanol allowed the CYN to be regained for final purification by HPLC . Deoxy-CYN, an analog of CYN can also be extracted using this procedure. Microbiol Immunol, 2001, 45(8), 571 - 7 Chemotaxis of oral treponemes toward sera and albumin of rabbit; Umemoto T et al.; The motility and chemotaxis of human oral spirochetes Treponema denticola ATCC 35404, T . medium ATCC 700293, and T . vincentii ATCC 35580 were examined by a capillary assay method . Of five sera three human oral treponemes were dominantly chemoattractant to the rabbit serum . The checkerboard analysis of chemotaxis toward rabbit serum clearly showed that the motile T . denticola cells swam toward the culture media containing higher concentrations of the rabbit serum . T . denticola chemotaxis to the rabbit serum was clearly reduced by heating serum, and rabbit albumin contributed by 60 to 70% to its chemotaxis to the rabbit serum . Western blotting analysis demonstrated that these treponemes possessed rabbit albumin-binding polypeptides with approximate molecular sizes of 65 kDa and 70 kDa . Immunoelectron microscopy demonstrated that a 65 kDa rabbit albumin-binding polypeptide was located on the outer envelopes, suggesting that the rabbit albumin-binding polypeptide is responsible for chemotaxis toward rabbit serum. Nutr Cancer, 2001, 39(1), 126 - 31 Effect of the molar ratio of branched-chain to aromatic amino acids on growth and albumin mRNA expression of human liver cancer cell lines in a serum-free medium; Saito Y et al.; Supplementation of branched-chain amino acids (BCAAs) is often used for the treatment of hepatic encephalopathy and low albuminemia in Japan . In this scenario, although many cases are complicated with hepatocellular carcinoma in chronic viral infection, the effect of BCAA levels on hepatocellular carcinoma cells remains unclear . We investigated the effect of the molar ratios of BCAAs to aromatic amino acids (AAAs) on the growth and albumin mRNA expression of cultured human liver cancer cell lines, HCC-M, HCC-T, PLC/PRF/5, and Hep G2 . To exclude the effect of fetal serum in culture media on modification of the growth and albumin transcription of cell lines, we used a synthetic serum-free medium . We found that an increase in the molar ratio of BCAAs to AAAs reduced the growth of Hep G2 cells, and it increased albumin mRNA expression in this cell line at a molar ratio of 0.1-10 . These results suggest that the molar ratio of BCAAs to AAAs affect the growth and mRNA expression of some liver cancer cells, and supplementation of BCAAs may at least be beneficial to patients with cirrhosis, even complicated with liver cancer. Invest Ophthalmol Vis Sci, 2001 Oct, 42(11), 2610 - 5 Lovastatin-induced cytoskeletal reorganization in lens epithelial cells: role of Rho GTPases; Maddala RL et al.; PURPOSE: To understand the involvement of isoprenylated small guanosine triphosphatases (GTPases) in lovastatin-induced cataractogenesis, Rho- and Rac-mediated cell adhesion and actin cytoskeletal reorganization were investigated in lovastatin-treated lens epithelial cells . METHODS: The effects of lovastatin on F-actin reorganization (phalloidin staining), focal adhesion formation (paxillin or vinculin), cell-cell adhesions (cadherin and beta-catenin), and protein tyrosine phosphorylation were evaluated in human and porcine lens epithelial cells by immunocytochemical staining with specific antibodies . To explore the involvement of the Rho and Rac GTPases in lovastatin-mediated effects, changes in distribution of Rho and Rac GTPases were analyzed by Western blot analysis, and the effects of C3-exoenzyme on lovastatin-induced cytoskeletal changes were evaluated by immunocytochemical analysis . RESULTS: Lovastatin induced drastic changes in cell shape in both human and porcine lens epithelial cells, including a profound loss of actin stress fibers, focal adhesions, protein phosphotyrosine, and cell-cell adhesions . Lovastatin treatment also led to the accumulation of nonisoprenylated Rho and Rac GTPases in cytosolic fraction . Supplementation of culture media with geranylgeranyl pyrophosphate dramatically reversed the lovastatin-induced morphologic and cytoskeletal changes, whereas farnesyl pyrophosphate was ineffective . Treatment of cells with C3-exoenzyme (a Rho GTPase-specific inhibitor), however, abolished the geranylgeranyl-supplementation-induced recovery from the morphologic and cytoskeletal effects of lovastatin . CONCLUSIONS: This study demonstrates that inhibition of protein prenylation by lovastatin leads to disruption of actin cytoskeletal organization, and to loss of integrin-mediated focal adhesions and cadherin-mediated cell-cell adhesions in lens epithelial cells . Based on isoprenoid supplementation studies, it could be concluded that impairment of geranylgeranylated Rho and Rac GTPase function is most likely responsible for lovastatin-induced cytoskeletal changes in lens epithelial cells. Eur J Endocrinol, 2001 Oct, 145(4), 505 - 11 Effect of cytokines and growth factors on the secretion of inhibin A, activin A and follistatin by term placental villous trophoblasts in culture; Mohan A et al.; OBJECTIVE: Maternal serum inhibin A and activin A are higher in pre-eclampsia than in normal pregnancy . The placenta is a source of these proteins in pregnancy . The aim of this study was to investigate the effect of growth factors and proinflammatory cytokines that are raised in pre-eclampsia on the secretion of dimeric inhibin A, activin A and follistatin by villous cytotrophoblasts in culture . DESIGN AND METHODS: Villous cytotrophoblasts were prepared from term placentae and cultured in serum-free media . Cells were treated with increasing concentrations of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, transforming growth factor (TGF)-beta1, granulocyte and monocyte colony-stimulating factor (GMCSF), inhibin A, activin A and follistatin for 2 days . Culture supernatants were assayed for human chorionic gonadotrophin (hCG), inhibin A, activin A and follistatin as appropriate . Experiments were repeated at least three times with each cytokine or growth factor and the data pooled . RESULTS: Cytotrophoblasts syncytialise and spontaneously secrete hCG, inhibin A and activin A in culture . Follistatin levels were <20 pg/ml in most experiments . Activin A secretion was increased in culture in a dose-dependent manner by IL-1beta (approximately 150%, P<0.05), TNF-alpha (approximately 35%, P=0.02) and GMCSF (approximately 100%, P<0.01) . hCG secretion was inhibited in a dose-dependent manner by TNF-alpha (50%, P<0.05) . Inhibin A was stimulated by IL-1beta ( approximately 30%, P=0.05) . Inhibin A, activin A, follistatin or TGF-beta1 did not have a significant effect on any measured parameters . CONCLUSIONS: These data show that inflammatory cytokines increase the secretion of activin A by trophoblasts in culture . The presence of very low levels, or no follistatin (<20 pg/ml) in the culture media suggests 'free' activin A could have autocrine/paracrine effects on cytotrophoblasts . Inhibin A secretion was stimulated by IL-1beta . However, absence of an effect by the other cytokines investigated on inhibin A in this study suggests that the mechanism(s) involved in increasing maternal circulating levels of inhibin A and activin A in pre-eclampsia are controlled differentially. Anesthesiology, 2001 Sep, 95(3), 726 - 33 Direct neurotoxicity of tetracaine on growth cones and neurites of growing neurons in vitro; Saito S et al.; BACKGROUND: Local anesthetics have direct neurotoxicity on neurons . However, precise morphologic changes induced by the direct application of local anesthetics to neurons have not yet been fully understood . Also, despite the fact that local anesthetics are sometimes applied to the sites where peripheral nerves may be regenerating after injury, the effects of local anesthetics on growing or regenerating neurons have never been studied . METHODS: Three different neuronal tissues (dorsal root ganglion, retinal ganglion cell layer, and sympathetic ganglion chain) were isolated from an age-matched chick embryo and cultured for 20 h . Effects of tetracaine were examined microscopically and by a quantitative morphologic assay, growth cone collapse assay . RESULTS: Tetracaine induced growth cone collapse and neurite destruction . Three neuronal tissues showed significantly different dose-response, both at 60 min and at 24 h after the application of tetracaine (P < 0.01) . The ED50 values (mean +/- SD) at 60 min were 1.53+/-1.05 mM in dorsal root ganglion, 0.15+/-0.05 mM in retinal, and 0.06+/-0.02 mM in sympathetic ganglion chain cultures . The ED50 values at 24 h were 0.43+/-0.15 mM in dorsal root ganglion, 0.07+/-0.03 mM in retinal, and 0.02+/-0.01 mM in sympathetic ganglion chain cultures . Concentration of nerve growth factor in the culture media did not influence the ED50 values . The growth cone collapsing effect was partially reversible in dorsal root ganglion and retinal neurons . However, in the sympathetic ganglion culture, no reversibility was observed after exposure to 1 mM tetracaine for 10 or for 60 min . Bupivacaine had similar neurotoxicity to the three types of growing neurons . (The ED50 values at 60 min were 2.32+/-0.50 mM in dorsal root ganglion, 0.96+/-0.16 mM in retinal, and 0.18+/-0.05 mM in sympathetic ganglion chain cultures . The ED50 values at 24 h were 0.34+/-0.09 mM in dorsal root ganglion, 0.21+/-0.06 mM in retinal, and 0.45+/-0.10 mM in sympathetic ganglion chain cultures.) CONCLUSIONS: Short-term exposure to tetracaine produced irreversible changes in growing neurons . Growth cones were quickly affected, and neurites degenerated subsequently . Sensitivity varied with neuronal type and was not influenced by the concentration of nerve growth factor . Because a similar phenomenon was observed after exposure to bupivacaine, the toxicity to growing neurons may not be unique to tetracaine. J Cell Biochem, 2001 Aug 1-9, 83(2), 200 - 3 Novel role of extracellular carbon dioxide in lymphocyte proliferation in culture; Chakrabarti R et al.; CO(2)/HCO(3)(-) buffering system is indispensable to maintain the pH of culture media for long-term cell culture . Now-a-days, the zwiterionic hydrogen buffer HEPES is widely used as an additional buffer in the commonly used culture media . There are reports on the successful use of HEPES-buffered media, under CO(2)/HCO(3)(-) free conditions, for long-term cell cultures . However, still CO(2)/HCO(3)(-) buffering system is widely used . We aimed at investigating the reason for this . We found that lymphocytes proliferate in response to concanavalin A only in HCO(3)(-)-buffered medium in the presence of 5% CO(2), but not in the HEPES-buffered medium in the absence of CO(2) . However, lymphocyte proliferation was observed in HEPES-buffered medium in the presence of 5% CO(2) and in the absence of HCO(3)(-) . On the other hand, a low level proliferation was observed in HEPES-buffered medium supplemented with HCO(3)(-) in the absence of CO(2) . Supplementation of the culture medium with TCA cycle intermediates and the precursors for the salvage pathway of nucleotide synthesis did not support the lymphocyte proliferation at all . Based on these findings and other reports, we suggest that extracellular CO(2) plays a novel role in cell proliferation . Appl Environ Microbiol, 2001 Oct, 67(10), 4858 - 62 New additive for culture media for rapid identification of aflatoxin-producing Aspergillus strains; Fente CA et al.; A new reliable, fast, and simple method for the detection of aflatoxigenic Aspergillus strains, consisting of the addition of a cyclodextrin (a methylated beta-cyclodextrin derivative) to common media used for testing mycotoxin production ability, was developed . We propose the use of this compound as an additive for fungal culture media to enhance the natural fluorescence of aflatoxins . The production of aflatoxins coincided with the presence of a bright blue or blue-green fluorescent area surrounding colonies when observed under long-wavelength (365-nm) UV light after 3 days of incubation at 28 degrees C . The presence of aflatoxins was confirmed by extracting the medium with chloroform and examining the extracts by high-pressure liquid chromatography with fluorescence detection. Laryngoscope, 2001 Jul, 111(7), 1147 - 55 L-n-acetyl-cysteine protection against cisplatin-induced auditory neuronal and hair cell toxicity; Feghali JG et al.; OBJECTIVES: The aim of this study is to determine the efficacy of L-N-acetyl-cysteine (L-NAC) as a protectant for inner ear auditory sensory cells against the toxic effects of cisplatin . STUDY DESIGN: Prospective laboratory study of the otoprotective effect of L-NAC on auditory neurons and hair cells in vitro . METHODS: The study has two arms . The first arm evaluated the neuroprotective effect of L-NAC on early postpartum auditory ganglion cell cultures . Two culture media were used . The two media differed in that one of them was enhanced by the addition of neurotrophins (neurotrophin type 3 and brain-derived neurotrophic factor) and a growth factor (transforming growth factor-beta1) . Then the survival of cisplatin-treated auditory neurons was studied before and after pretreatment with protective levels of L-NAC . The second arm of the study evaluated the effect of L-NAC on cisplatin damage initiated to auditory hair cells . Early-postpartum organ of Corti explants were grown in culture . Their rate of survival was studied after exposure to toxic levels of cisplatin . Then, survival of cisplatin-damaged hair cells was studied after they were pretreated with L-NAC . RESULTS: Pretreatment of cultures with L-NAC protected both auditory neurons and hair cells from the effects of exposure to toxic levels of cisplatin . This observed otoprotective effect was dose dependent . CONCLUSIONS: Our in vitro studies have demonstrated that L-NAC protected both auditory neurons and hair cells from the toxic effects of cisplatin . Because it protects both of these inner ear structures, L-NAC may be potentially useful in protecting hearing, in general, from cisplatin-induced damage . In addition, L-NAC has low systemic and mucosal toxicity . It also has a low molecular weight that may allow it to readily cross the round window membrane . All these characteristics make it potentially suitable for transtympanic application for the prevention of the ototoxicity of cisplatin in vivo. Exp Biol Med (Maywood), 2001 Oct, 226(9), 847 - 53 Reduced oxygen tension increases atrial natriuretic peptide release from atrial cardiocytes; Klinger JR et al.; To test the hypothesis that reduced oxygen tension stimulates cardiac atrial natriuretic peptide (ANP) secretion, we measured ANP release and expression in neonatal rat atrial and ventricular cardiac myocytes exposed to 45 min and 3, 6, and 24 hr of 3% or 21% oxygen . In atrial cardiocytes, the percentage of increase in culture media ANP concentration from baseline was greater in cells exposed to 3% than in cells exposed to 21% oxygen after 3 hr (814% +/- 52% vs . 567% +/- 33%, P < 0.05) and 6 hr of exposure (1639% +/- 91% vs . 1155% +/- 73%, P < 0.05) . No differences in the percentage of increase in culture media ANP concentration was seen at 45 min (284% +/- 27% vs . 201% +/- 16%, P = NS) or 24 hr (2499% +/- 250% vs . 2426% +/- 195%) . There was a significant increase in cellular ANP content between 3 and 24 hr in atrial cardiocytes exposed to 21% oxygen (105% +/- 40% vs . 296% +/- 60%, P < 0.05), but not in atrial cardiocytes exposed to 3% oxygen (118% +/- 20% vs . 180% +/- 26%, P = NS) . Steady-state ANP mRNA levels in atrial cardiocytes were not affected by oxygen tension . In ventricular cardiocytes, oxygen tension did not affect ANP secretion, cellular ANP content, or steady-state ANP mRNA levels . We conclude that reduced oxygen tension increases release of ANP from atrial, but not ventricular cardiocytes and that this mechanism may contribute to the elevation in plasma ANP seen during acute hypoxia. Anal Biochem, 2001 Oct 1, 297(1), 15 - 24 High-performance liquid chromatographic technique for the simultaneous determination of lactone and hydroxy acid forms of camptothecin and SN-38 in tissue culture media and cancer cells; Boyd G et al.; Analysis of camptothecins in biologic media is hampered by chemical hydrolysis of the parent lactone (form I) to an inactive hydroxy acid (form II) . A solid-phase extraction (SPE) method utilizing C2-bonded silica particles (100 mg, 1 ml) is presented for simultaneous determination of forms I and II