Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14542 - 7 Epub 2002 Oct 22. Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyl-D-aspartate (NMDA) receptor coagonist D-serine; De Miranda J et al.; High levels of d-serine occur in the brain, challenging the notion that d-amino acids would not be present or play a role in mammals . d-serine levels in the brain are even higher than many l-amino acids, such as asparagine, valine, isoleucine, and tryptophan, among others . d-serine is synthesized by a serine racemase (SR) enzyme, which directly converts l- to d-serine . We now report that SR is a bifunctional enzyme, producing both d-serine and pyruvate in cultured cells and in vitro . Transfection of SR into HEK 293 cells elicits synthesis of d-serine and augmented release of pyruvate to culture media . We identified substances present in HEK 293 and astrocyte cell extracts that strongly stimulate d-serine production by SR and elicit production of pyruvate . Experiments with recombinant enzyme reveal that Mg(2+) and ATP present in the cell extracts are physiological cofactors and increase 5- to 10-fold the rates of racemization and production of pyruvate . As much as three molecules of pyruvate are synthesized for each molecule of d-serine produced by SR . This finding constitutes a previously undescribed mechanism underlying d-amino acid synthesis in mammals, different from classical amino acid racemases present in bacteria . Our data link the production of d-serine to the energy metabolism, with implications for the metabolic and transmitter crosstalk between glia and neurons. Di Yi Jun Yi Da Xue Xue Bao, 2002 May, 22(5), 388 - 92 Role of p38 mitogen-activated protein kinase in lipopolysaccharide-induced expression of inducible nitric oxide synthase in human endothelial cells; Kan WH et al.; OBJECTIVE: To understand the role of p38 mitogen-activated protein kinase (p38MAPK) in the expression of inducible nitric oxide synthase (iNOS) and NO production in human endothelial cells under the stimulation by lipopolysaccharide (LPS) . METHODS: NO level in the supernatant of the cell culture media was measured with Griess method, and iNOS protein and mRNA expressions by the cells were detected with immunofluorescence analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively . Immunoprecipitation assay was employed to examine p38 MAPK activity . RESULTS: It was shown that in comparison with the basal level of iNOS expression in cultured endothelial cells line ECV304, iNOS mRNA and protein expressions were significantly increased in the cells after LPS stimulation . In response to LPS treatment, obvious enhancement of p38 MAPK activity in ECV304 took place after the stimulation, with the peak level occurring at 15 min that maintained for approximately 45 min before gradual decline . When treated with SB203580 {4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole}, a highly specific inhibitor of p38 MAPK, significant inhibition of LPS-induced iNOS protein and mRNA expressions was observed . CONCLUSIONS: p38 MAPK plays an important role in iNOS expression and NO production in ECV304 cells, and, inhibition of the signal transduction pathway may consequently be an effective approach to reduce the production of iNOS and other cytokines for the treatment of septic shock. Neuropharmacology, 2002 Oct, 43(5), 877 - 88 Thiolic antioxidants protect from nitric oxide-induced toxicity in fetal midbrain cultures; Rodriguez-Martin E et al.; Nitric oxide (NO) may act as a neuroprotector or neurotoxic agent in dopamine neurons, depending on cell redox status . We have investigated the effect of several thiolic antioxidants, glutathione (GSH), its cell permeable analog GSH ethyl ester (GSHEE), and the GSH synthesis precursor L-N-acetyl cysteine (L-NAC), as well as non-thiolic antioxidants like ascorbic acid (AA) and uric acid, on NO-induced toxicity in fetal midbrain cultures . The cultures were treated for 8-24 h with neurotoxic doses of the NO donor diethylamine/nitric oxide complex sodium DEA/NO (200-400 micro M) and/or antioxidants . Thiolic antioxidants, at equimolar concentrations, added at the same time or previous to DEA/NO, protected from cell death, from tyrosine hydroxylase (TH) positive cell number decrease and from intracellular GSH depletion, induced by DEA/NO, without increasing intracellular GSH content . In these conditions, S-nitrosothiol compound formation was detected in the culture media . Protection disappeared when antioxidants were supplied 30 min after NO treatment . Nevertheless, non-thiolic antioxidants, AA and uric acid, with similar peroxynitrite scavenging activity to thiolic antioxidants, and free radical-scavenging enzymes as catalase and Cu/Zn-superoxide dismutase, which prevent extracellular peroxynitrite ion formation, and 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), which prevents intracellular peroxynitrite ion formation, did not rescue cell cultures from neurotoxicity induced by NO . In addition, AA exacerbated DEA/NO-induced toxicity in a dose-dependent manner from 200 micro M AA . The present results suggest that only antioxidants with thiol group exert neuroprotection from NO-induced toxicity in fetal midbrain cultures, probably by direct interaction of NO and thiol groups, resulting in NO blocking . On the other hand, some classical antioxidants, like AA, exacerbate neurotoxicity due to NO. J Orthop Res, 2002 Sep, 20(5), 927 - 33 Expression and enzymatic activity of MMP-2 during healing process of the acute supraspinatus tendon tear in rabbits; Choi HR et al.; We investigated the spontaneous healing process of a surgically created supraspinatus tendon tear in rabbits with specific reference to the expression of matrix metalloproteinase-2 (MMP-2) and its time-course change in enzymatic activity along with the expression of tissue inhibitors of metalloproteinases (TIMPs) . A transverse, full thickness tear of the supraspinatus tendon was created and examined . Immunohistochemical analysis revealed that MMP-2 positive cells were mainly localized at both cutting ends of the tendon, and reparative tissue encroached into the gap from the bursal side . The expression of TIMP-1 was induced in the cells at not only the tendon edges but also the reparative tissue during the healing process . TIMP-2 was constitutively expressed in both the tendon and the reparative tissue . Gelatin zymography using tissue culture media demonstrated latent and active forms of MMP-2 and characteristic time-linked changes of the enzymatic activity . Western blotting confirmed the bands as the latent form of MMP-2 . These results suggest that MMP-2 is expressed and activated during the healing process of acute supraspinatus tendon tear and can play an important role in the remodeling process. Zhonghua Zheng Xing Wai Ke Za Zhi, 2002 Jul, 18(4), 234 - 6 {Substance P up-regulates the TGF-beta 1 mRNA expression of human dermal fibroblasts in vitro}; Hu D et al.; OBJECTIVE: To investigate the role of substance P in the formation of hypertrophic scar . METHODS: Dermal fibroblasts derived from human normal skin were cultured with substance P alone or together with selective non-peptide NK-1 tachykinin antagonist, L-703, 606 oxalate salt . The effect of substance P on proliferation of fibroblasts was measured by MTT assay . Furthermore, the TGF-beta 1 mRNA expression in the fibroblasts was determined by in situ hybridization and image analysis . RESULTS: Substance P enhanced fibroblast proliferation dose-dependently, which showed the maximum rate when the concentration of substance P was 25 ng/ml or higher in the culture media . By 48 hours cultured with 25 ng/ml of substance P, the fibroblasts expressed TGF-beta 1 mRNA more significantly than the fibroblasts without substance P . The effects of substance P on both fibroblast proliferation and TGF-beta 1 mRNA expression could be antagonized by L-703, 606 oxalate salt . CONCLUSION: The results suggest that substance P may play an important role in phenotype changes of fibroblasts in skin scarring. Anal Biochem, 2002 Oct 1, 309(1), 79 - 84 How to make tetracycline-regulated transgene expression go on and off; Rennel E et al.; Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals . While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline . Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines . Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media . The released doxycycline reached sufficiently high levels to completely suppress transgene expression . The effect was not dependent on cell type or the nature of the transgene . However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3h after the initial removal of doxycycline . The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results . These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems. J Matern Fetal Neonatal Med, 2002 Jan, 11(1), 11 - 7 Thrombin-enhanced matrix metalloproteinase-1 expression: a mechanism linking placental abruption with premature rupture of the membranes; Rosen T et al.; OBJECTIVE: Given the strong clinical association between the decidual hemorrhage of placental abruption and subsequent preterm premature rupture of the membranes, we assessed the effects of thrombin on the expression of the potent interstitial collagenase, matrix metalloproteinase-1 (MMP-1), in cultured endometrial stromal and decidual cells . STUDY DESIGN: Stromal cells derived from predecidualized cycling endometrium and decidual cells from term decidua were cultured in a defined medium containing estradiol, to mimic the hormonal milieu of the non-pregnant proliferative phase, or estradiol plus medroxyprogesterone acetate (MPA), to mimic the hormonal milieu of pregnancy, in the presence and absence of thrombin . Culture media were examined for MMP-1 protein levels and cell lysates were examined for steady-state MMP-1 mRNA levels . RESULTS: MPA strongly inhibited MMP-1 levels in endometrial stromal and term decidual cells . However, thrombin overcame this suppression, producing MMP-1 levels that were several-fold higher than control levels . CONCLUSION: Extrapolation of thrombin-enhanced MMP-1 expression in cultured endometrial stromal and decidual cells to the in vivo pregnant state provides an explanation for the strong association between placental abruption and preterm membrane rupture. Biochem Biophys Res Commun, 2002 Oct 18, 298(1), 10 - 16 A double-strand decoy DNA oligomer for NF-kappaB inhibits TNFalpha-induced ICAM-1 expression in sinusoidal endothelial cells; Shibuya T et al.; Altered gene expression of liver sinusoidal endothelial cells (SECs) is associated with impaired immune response . Here we report that the decoy technique effectively suppresses TNFalpha-induced ICAM-1 expression in SEC . An NF-kappaB decoy (NF-kappaB31: 5(')-TGGGGACTTTCCAGTTTCTGGAAAGTCCCCA-3), which contains a consensus sequence for NF-kappaB, was complexed to PLL-g-HA {hyaluronate-grafted poly(L-lysine) copolymer} that permits transfer of exogenous DNA selectively to the SEC . The PLL-g-HA/NF-kappaB31 complex was added to the culture media of LSE cells, a human SEC-derived cell line . Then, cells were stimulated with TNFalpha (5ng/mL) . PLL-g-HA/NF-kappaB31, but not control oligodeoxynucleotides having a reverse or scrambled sequence, inhibited the intranuclear localization of NF-kappaB induced by TNFalpha, with almost complete inhibition at 2.5microg/mL as DNA . NF-kappaB31 attenuated the increase in ICAM-1 mRNA as well as protein levels in LSE cells . The decoy technique in combination with PLL-g-HA may provide a novel strategy for manipulation of SEC functions. Di Yi Jun Yi Da Xue Xue Bao, 2002 Jul, 22(7), 624 - 5 In vitro culture and study of the biological characteristics of rabbit keratocytes; Huo XK et al.; OBJECTIVE: To improve the technique for culturing rabbit keratocytes in vitro and investigate the biological characteristics of these cells . METHODS: Fresh rabbit corneas were obtained and the epithelial and endothelial cells were removed after digestion with trypsin . The stroma was rinsed, minced, and incubated in DMEM/F12 medium supplemented with 10% fetal bovine serum and the biological characteristics of the keratocytes were observed with MTT assay and compared with those of the cells in serum-free culture media . RESULT: On about the third day of incubation, some keratocytes germinated from the stromal tissues and migrated onto the flask surface presenting fibroblast-like arrangement with spindle-shaped appearance . The keratocytes became confluent after 10 day's incubation and the peak of cell proliferation occurred on day 8 in the presence of serum in the media, while in the absence of serum, the peak took place on day 10 . CONCLUSION: The method improved for in vitro keratocyte culture is convenient and effective, and the presence of serum in the media may to some degree affect the growth of the keratocytes. Clin Chest Med, 2002 Sep, 23(3), 623 - 32, vii Pulmonary disease caused by rapidly growing mycobacteria; Daley CL et al.; The rapidly growing mycobacteria (RGM) differ from slow-growing mycobacteria such as Mycobacterium tuberculosis by virtue of their more rapid growth in culture media and their in vitro resistance to standard antituberculosis drugs . The RGM can produce numerous infections including chronic lung disease . The most common causes of pulmonary disease are Mycobacterium abscessus and Mycobacterium fortuitum . This article reviews the management of patients with lung disease caused by RGM. Antibiot Khimioter, 2002, 47(4), 3 - 6 {Concentration on Diapak C 16 capsules of lovastatin, mevinolinic acid and other inhibitors of biosynthesis of sterins produced by Penicillium citrinum 89}; Baranova NA et al.; The method of lovastatine and mevinolinic acid known as competitive inhibitors of HMG-CoA-reductase and produced by micromycetes was elaborated . The inhibitors from diluted water solutions were fully absorbed on Diapak C16 patrons . The rate of inhibitors elution from the patrones was more than 95 per cent . Patrons may be used for concentration of lovastatine group inhibitors from the culture media . Inhibitors synthesis by the Penicillium citrinum 89 was investigated in dynamics with the use of Diapak C16 patrones . It was shown that UV-spectrum of inhibitor produced by P . citrinum 89 was identical with compactin spectrum and had absorbance maximum at 230, 237 and 247 nm. Exp Dermatol, 2002 Oct, 11(5), 413 - 20 Organotypic keratinocyte coculture using normal human serum: an immunomorphological study at light and electron microscopic levels; Hinterhuber G et al.; Organotypic human skin equivalents of keratinocytes and fibroblasts embedded in collagen matrix have been the subject of studies dealing with various culture conditions . Development of standardized living skin equivalents using defined culture media containing respective supplements can provide important instruments of investigation in skin biology . In addition, tissue engineering has created human skin substitutes for treatment of acute and chronic wounds . In our study, we generate a modified organotypic human skin equivalent using normal human serum instead of fetal calf serum (FCS) . This living skin equivalent shows regular stratification of the epidermis and the dermal-epidermal junction zone at the light and electron microscopic level after 1 and 3 weeks of coculture . Indirect immunofluorescence reveals regular expression of differentiation antigens and the major structural proteins collagen IV, laminin 5 and the integrin chains alpha 6 and beta 4 at the dermo-epidermal junction zone . Immunoelectron microscopy demonstrates expression of collagen IV, alpha 6 and beta 4 integrin after 1 and 3 weeks of coculture . This organotypic skin model could be the basis for autologous skin grafting for acute or chronic wounds using autologous serum as well as patients' keratinocytes and fibroblasts, thus minimizing the risk of transmitting infectious agents. J Clin Endocrinol Metab, 2002 Oct, 87(10), 4775 - 81 Exendin 4 up-regulates expression of PDX 1 and hastens differentiation and maturation of human fetal pancreatic cells; Movassat J et al.; In addition to stimulating insulin secretion, glucagon-like peptide and its long-acting analog exendin 4 have been reported to increase beta-cell mass by both differentiation/neogenesis of precursor cells and enhanced replication of existing beta-cells . Here, we investigated the effect of exendin 4 in the growth and differentiation of beta-cells from undifferentiated precursors in islet-like cell clusters (ICCs) derived from human fetal pancreases . Our results show that the addition of exendin 4 to the culture media stimulates PDX 1 expression in ICCs as shown by immunofluorescence staining . The up-regulation of PDX 1 was not accompanied by changes in insulin expression because we did not find a significant difference in the number of insulin-positive cells in the exendin 4-treated ICCs, compared with controls . We also tested the effects of exendin 4 in the glucose-induced insulin secretion of human ICCs transplanted under the kidney capsule of athymic rats . In the exendin 4-treated rats (given ip during 10 d) 8 wk after the beginning of the treatment, insulin was released in response to glucose as detected by the measurement of circulating human C-peptide . In control (saline-treated) rats, the basal levels of human C-peptide did not change significantly after glucose stimulation . Thus, exendin 4 induces functional maturation of fetal beta-cells in response to glucose . In these rats, serial sections of the kidney-bearing grafts were examined histologically for insulin containing cells . We found a significant increase in beta-cell number, compared with the control rats . Overall, these results show that in vivo exendin 4 causes growth and differentiation of human fetal beta-cells from undifferentiated precursor cells . It also accelerates the functional maturation of fetal beta-cells as evidenced by their glucose-stimulated insulin secretion. Thromb Haemost, 2002 Oct, 88(4), 632 - 8 Protein C deficiency caused by homozygosity for a novel PROC D180G mutation--in vitro expression and structural analysis of the mutation; Lind B et al.; Homozygosity for a novel D180G mutation in the protease domain of protein C, associated with plasma protein C activity and antigen levels of 8% of normal was identified in a thrombosis prone family . Transient expression of protein C in HK-293 cells and analysis of protein C antigen in culture media and cell lysates showed that the secretion of mutant protein as compared with wild-type protein was reduced by 79% while the intracellular contents were similar . Computer analysis of the X-ray structure of activated protein C and of a theoretical model of the zymogen predicts that the mutation destabilises the molecule locally . Our results are compatible with a relatively unstable mutant molecule that could be trapped inside the cell and degraded . However, if secreted the mutant molecule could have a relatively normal catalytic activity and structure consistent with the plasma levels of protein C activity and the late onset of thrombosis. Lett Appl Microbiol, 2002, 35(4), 272 - 5 The effect of culture preservation techniques on patulin and citrinin production by Penicillium expansum Link; Santos IM et al.; AIMS: To study the influence of culture preservation methods and culture conditions on the production of the mycotoxins patulin and citrinin by Penicillium expansum . METHODS AND RESULTS: Ten strains of Penicillium expansum were preserved using subculture and maintenance at 4 degrees C, mineral oil, drying on silica gel and freeze-drying . Patulin and citrinin production was assessed on yeast extract sucrose agar (YES) and grape juice agar (GJ), using TLC before and after 0.5, 2-3, 6 and 12 months preservation . Citrinin was detected in all cultures for all preservation techniques on YES . The patulin profiles obtained differed with strain and culture media used . CONCLUSIONS: Citrinin production seems to be a stable character for the tested strains . There is a tendency for patulin detection with time apparently more consistent for silica gel storage and freeze-drying, especially when the strains are grown on GJ . SIGNIFICANCE AND IMPACT OF THE STUDY: Variability in the profiles of the mycotoxins tested seems to be more strain-specific than dependent on the preservation technique used. Mol Biotechnol, 2002 Sep, 22(1), 87 - 98 Downstream processing in the biotechnology industry; Kalyanpur M; The biotechnology industry today employs recombinant bacteria, mammalian cells, and transgenic animals for the production of high-value therapeutic proteins . This article reviews the techniques employed in this industry for the recovery of these products . The methods reviewed extend from the centrifugation and membrane filtration for the initial clarification of crude culture media to the final purification of the products by a variety of membrane-based and chromatographic methods . The subject of process validation including validation of the removal of bacterial and viral contaminants from the final products is also discussed with special reference to the latest regulatory guidelines. Res Reprod . 1977 Sep;9(5):4. Albumin and the acrosome change in mammalian spermatozoa; Carbon Catabolite Repression Regulates Glyoxylate Cycle Gene Expression in Cucumber; Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United KingdomWe have previously proposed that metabolic status is important in the regulation of cucumber malate synthase (MS) and isocitrate lyase (ICL) gene expression during plant development . In this article, we used a cell culture system to demonstrate that intracellular metabolic status does influence expression of both of these genes . Starvation of cucumber cell cultures resulted in the coordinate induction of the expression of MS and ICL genes, and this effect was reversed when sucrose was returned to the culture media . The induction of gene expression was closely correlated with a drop in intracellular sucrose, glucose, and fructose below threshold concentrations, but it was not correlated with a decrease in respiration rate . Glucose, fructose, or raffinose in the culture media also resulted in repression of MS and ICL . Both 2-deoxyglucose and mannose, which are phosphorylated by hexokinase but not further metabolized, specifically repressed MS and ICL gene expression relative to a third glyoxylate cycle gene, malate dehydrogenase . However, the addition of 3-methylglucose, an analog of glucose that is not phosphorylated, did not result in repression of either MS or ICL . It is proposed that the signal giving rise to a change in gene expression originates from the intracellular concentration of hexose sugars or the flux of hexose sugars into glycolysis. J Biol Chem, 2002 Dec 20, 277(51), 49831 - 40 Epub 2002 Sep 18. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression; Langley B et al.; Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts . In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3 . In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media . Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation . The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation . Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression . Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin . Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes . Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts. Microsc Res Tech, 2002 Sep 15, 58(6), 488 - 95 Melanization stimulating factor (MSF) and melanization inhibiting factor (MIF) in the integument of fish; Zuasti A; The present studies were directed to demonstrate that adult fish skin contains putative factors that affect chromatophore and/or chromatoblast function . This hypothesis is based upon the possibility that hypo and/or hyperpigmented areas of the skin are so pigmented because of the localized expression of intrinsic factors that are either stimulatory or inhibitory to the differentiation of specific pigment cell types . In all the morphological and biochemical experiments carried out, we used culture media conditioned by dorsal (DCM) or ventral (VCM) skin from different species of fish . Both DCM and VCM were capable of stimulating differentiation of melanophores in neural crest explants . While the stimulation of melanization is an activity present in both dorsal and ventral skin, an inhibitory activity is also present in ventral skin at such a concentration that it overrides the stimulatory activity afforded by DCM . With biochemical assays, we demonstrated that the three important sequential enzymatic steps in melanogenesis are all stimulated by the conditioned media in a dose-dependent manner and this results in an increase in the amount of melanin present in cultured cells . The results of our investigations provide strong evidence that there are intrinsic pigment cell regulatory factors in the integument of fish, the inhibitory activity being stronger in the ventrum, and that those factors strongly influence, perhaps even determine, the pigment patterns of fish . Theriogenology, 2002 Oct 1, 58(6), 1081 - 95 The suppression of fragmentation by stabilization of actin filament in porcine enucleated oocytes; Kawahara M et al.; A thorough understanding of the mechanism underlying fragmentation would contribute to the improvement of the developmental ability of reconstructed embryos after nuclear transfer . We conducted the present study to elucidate the influence of the nuclear transfer method on fragmentation of enucleated oocytes and the relationship between change in actin filament distribution and fragmentation . In Experiment 1, we examined activation rates of in vitro matured oocytes . These were 12.9% in maturation alone, 75.7% in electrical stimulation, and 57.9% in ethanol/cycloheximide treatment . In Experiment 2, we observed a higher rate of fragmentation (P < 0.05) in cultured oocytes that had been enucleated and electrically stimulated than in oocytes subjected to the other treatments (maturation alone, enucleation alone and enucleation plus ethanol/cycloheximide activation) . In Experiment 3, we stained enucleated and electrically stimulated oocytes with rhodamine/phalloidin dye to show discontinuous distributions in the ooplasm of treated oocytes; oocytes in the other treatment groups showed homogenous distributions of actin filaments (AFs) . In Experiment 4, we added cytochalasin B, an inhibitor of AF polymerization, to the culture medium, which prevented fragmentation of enucleated plus electrically stimulated oocytes (cytochalasin B, {+} 0.0%, {-} 60.7% at 24 h after treatment, P < 0.05) . In Experiment 5, we investigated the relationship between fragmentation and alteration in AF distribution in enucleated plus electrically stimulated oocytes . At 0 h of culture, enucleated plus electrically stimulated oocytes showed discontinuous distributions of AFs, while nontreated oocytes showed homogenous AF distributions . At 24 and 48 h of culture, fragmentation proceeded in enucleated plus electrically stimulated oocytes and the discontinuous AF distribution diminished with time . In Experiment 6, we added hyaluronic acid (HA) to the culture medium, which suppressed fragmentation of enucleated plus electrically stimulated oocytes (HA, {+} 28.5%, {-} 66.4% at 24 h after treatment, P < 0.05) . The results suggest that electrical stimulation induces a change in the AF distribution of oocytes, resulting in fragmentation, and that the addition of HA to the culture media is effective for the suppression of fragmentation. Skin Pharmacol Appl Skin Physiol, 2002 Sep-Oct, 15(5), 335 - 41 Trioxsalen in the presence of UVA is able to induce nuclear factor kappa B binding activity in HaCaT keratinocytes; Sanchez Ruderisch H et al.; It has been described that treatment of cells with high dose psoralen and UVA induce the production of reactive oxygen species (ROS) leading to DNA damage . Transcription factor nuclear factor kappa B (NFkappaB) plays a crucial role in regulating not only cell growth but also cell differentiation, and ROS seem to be partly involved in these mechanisms . The aim of this research was to find out the effect of a combined treatment with trioxsalen (TMP)/UVA on NFkappaB binding activity in HaCaT keratinocytes . HaCaT keratinocytes were treated with 27 microg/l TMP . This concentration did not affect the proliferation rates, nor was it toxic, as shown by cytotoxicity assays . After treatment with TMP with or without UVA (1 J/cm(2)), NFkappaB binding activity in nuclear protein extracts was measured by electrophoretic mobility shift assays . The effect on cytokines and cytokine receptor genes was investigated using cDNA expression arrays . An inhibitory effect on NFkappaB binding activity was found between 30 and 60 min after TMP supplementation of the culture media . UVA irradiation induced a 2-fold increase in NFkappaB binding activity in TMP supplemented HaCaT keratinocytes compared with the non-irradiated control . In addition, NFkappaB binding activity was higher after UVA irradiation with TMP than in UVA irradiated cells in the absence of TMP . TGF-alpha, IL-1R, IL-2Ralpha, IL-12beta and PDGF expression was induced by UVA . However, all of them except PDGF were inhibited by combined TMP/UVA treatment . Using an inhibitor of NFkappaB activation, we found out that under these conditions, these cytokines or cytokine receptor genes are apparently not regulated by NFkappaB . Our results indicate that a combined TMP/UVA treatment of HaCaT keratinocytes induces NFkappaB binding activity, and that this is a synergistic effect . The investigated cytokines, and cytokine receptor genes do not seem to be NFkappaB regulated; however, TMP shows anti-inflammatory capacities in vitro . J Neurosci Res, 2002 Oct 1, 70(1), 36 - 45 Inhibition of insulin-like growth factor I activity contributes to the premature apoptosis of cerebellar granule neuron in weaver mutant mice: in vitro analysis; Zhong J et al.; Evidence from transgenic mice and cultured cerebellar neurons supports an important role for insulin-like growth factor I (IGF-I) in the formation of cerebellar cytoarchitecture . To understand IGF-I's function during cerebellar development, we examined the involvement of IGF-I in the premature apoptosis of granule neurons derived from the cerebella of weaver (wv) mutant mice . Before their demise, wv granule neurons increased the expression and secretion of IGFBP5 in a gene dose-dependent manner . Because IGFBP5 may interfere with the interaction of IGF-I and its receptor, the abnormally high IGFBP5 levels in wv granule neurons suggest that a lack of IGF-I activation may contribute to their premature apoptosis . This hypothesis is supported by a gene dose-dependent decrease in IGF-I receptor (IGF-IR) phosphorylation . More importantly, there is a parallel gene dose-dependent decrease in Akt activity, which was inversely correlated with the activity levels of caspase 3 . On the other hand, adding IGFBP5 antibody into culture media increased the survival of wv granule neurons, whereas adding IGFBP5 decreased the survival of wild-type granule neurons . To delineate the interaction between IGF-I and IGFBP5 on wv granule neurons, we examined neuronal survival after treating with IGF-I, des(1-3) IGF-I, or IGFBP5 antibody . At the same concentration, des(1-3) IGF-I was more effective than IGF-I in promoting survival, in increasing Akt activity, and in decreasing caspase 3 activity . These results indicate that IGF-I's actions on wv granule neurons are normally inhibited by excess IGFBP5, and sufficient IGF-I receptor activation rescues wv granule neurons via stimulating the Akt signaling pathway . Inflamm Res, 2002 Aug, 51(8), 427 - 33 Differential effects of nonsteroidal antiinflammatory drugs on the IL-1 altered expression of plasminogen activators and plasminogen activator inhibitor-1 by articular chondrocytes; Sadowski T et al.; OBJECTIVE: To determine the in vitro effects of several nonsteroidal antiinflammatory drugs on the IL-1 altered expression and activity of tPA, uPA and PAI-1 by articular chondrocytes . METHODS: Bovine chondrocytes were cultured in alginate gel beads . Cells were treated with IL-1alpha in the presence or absence of drugs at various concentrations . Expression of mRNA for the plasminogen activators (uPA and tPA) and their inhibitor (PAI-1) were analyzed by RT-PCR-ELISA . The protein content of PAI-1 in culture media was deter mined by ELISA . PA activity was measured by a functional assay . RESULTS: All tested NSAIDs dose dependently inhibited the IL-1 induced mRNA expression of tPA, whereas only indomethacin and tiaprofenic acid were also able to reduce the expression of uPA . Expression of PAI-1 was elevated by IL-1 without an accompanying increase in secreted amounts of the inhibitor . Indomethacin, naproxen and tiaprofenic acid stimulated the release of PAI-1 into culture media, whereas meloxicam also induced expression of PAI-1 above IL-1 stimulated levels . CONCLUSION: In conclusion, our studies indicate that NSAIDs preferentially inhibit tPA expression by bovine articular chondrocytes . By increasing the production of PAI-1 at therapeutical concentrations meloxicam could reduce PA activity, whereas the other NSAIDs tested mainly enhanced the release of this inhibitor from the extracellular matrix . In how far this would affect the enzyme-inhibitor balance within cartilage has to be determined in further studies. Vet Rec, 2002 Aug 31, 151(9), 260 - 5 Effect of insulin, transferrin and selenium and epidermal growth factor on development of buffalo oocytes to the blastocyst stage in vitro in serum-free, semidefined media; Raghu HM et al.; The in vitro development of buffalo oocytes up to the blastocyst stage was studied in serum-free, semidefined media containing bovine serum albumin, follicle-stimulating hormone (FSH), insulin, transferrin and selenium (ITS) and epidermal growth factor (EGF) . In experiment 1, oocytes aspirated from abattoir-derived ovaries were cultured in eight serum-free, semidefined culture media containing different combinations of these four factors . In experiment 2, the maturation of buffalo oocytes and the development of the embryos were compared in a complex co-culture system and in the serum-free, semidefined media . Supplementation with FSH and EGF significantly (P < 0.05) increased the maturation rates of buffalo oocytes, and the yield of blastocysts was higher (P < 0.05) in media containing EGF and ITS . The yield of blastocysts was lower in the serum-free semidefined media (P < 0.05) than in the complex co-culture system. Neurotoxicology, 2002 Jul, 23(2), 147 - 57 Mechanisms of manganese-induced rat pheochromocytoma (PC12) cell death and cell differentiation; Roth JA et al.; Mn is a neurotoxin that leads to a syndrome resembling Parkinson's disease after prolonged exposure to high concentrations . Our laboratory has been investigating the mechanism by which Mn induces neuronal cell death . To accomplish this, we have utilized rat pheochromocytoma (PC12) cells as a model since they possess much of the biochemical machinery associated with dopaminergic neurons . Mn, like nerve growth factor (NGF), can induce neuronal differentiation of PC12 cells but Mn-induced cell differentiation is dependent on its interaction with the cell surface integrin receptors and basement membrane proteins, vitronectin or fibronectin . Similar to NGF, Mn-induced neurite outgrowth is dependent on the phosphorylation and activation of the MAP kinases, ERK1 and 2 (p44/42) . Unlike NGF, Mn is also cytotoxic having an IC50 value of approximately 600 microM . Although many apoptotic signals are turned on by Mn, cell death is caused ultimately by disruption of mitochondrial function leading to loss of ATP . RT-PCR and immunoblotting studies suggest that some uptake of Mn into PC12 cells depends on the divalent metal transporter 1 (DMT1) . DMT1 exists in two isoforms resulting from alternate splicing of a single gene product with one of the two mRNA species containing an iron response element (IRE) motif downstream from the stop codon . The presence of the IRE provides a binding site for the iron response proteins (IRP1 and 2); binding of either of these proteins could stabilize DMT1 mRNA and would increase expression of the +IRE form of the transporter . Iron and Mn compete for transport into PC12 cells via DMT1, so removal of iron from the culture media enhances Mn toxicity . The two isoforms of DMT1 (+/-IRE) are distributed in different subcellular compartments with the -IRE species selectively present in the nucleus of neuronal and neuronal-like cells. Ultrason Sonochem, 2002 Sep, 9(4), 197 - 203 Effects of dissolved gases and an echo contrast agent on ultrasound mediated in vitro gene transfection; Ogawa R et al.; The effects of acoustic cavitation on in vitro transfection by ultrasound were investigated . HeLa cells were exposed to 1.0 MHz continuous ultrasound in culture media containing the luciferase gene . Transfection efficiency was elevated when an echo contrast agent, Levovist was added or air was dissolved in the medium . When cells were sonicated in medium saturated with Ar, N2 or N2O which have different gamma values (Cp/Cv), or were saturated with He, Ar or Ne with different thermal conductivities, the effectiveness for the dissolved gases in the ultrasound mediated transfection was Ar > N2 > N2O or Ar > Ne > He, respectively . When free radical formation in water by ultrasound was monitored as a measure of inertial cavitation, it was similarly affected by dissolved gases . These results indicate that the efficiency of ultrasound mediated transfection was significantly affected either by occurrence of or by modification of inertial cavitation due to various gases. Anal Bioanal Chem, 2002 Sep, 374(1), 64 - 8 Epub 2002 Jul 23. Direct analysis of nine pharmaceuticals in culture media by use of cartridge separation with electrospray mass spectrometric detection; Li XF et al.; A 2-cm cartridge has been used for separation before electrospray mass spectrometric analysis of pharmaceutical compounds in cell culture media, alleviating the need for sample extraction and desalting procedures . Nine representative pharmaceuticals listed in the biopharmaceutical classification system (BCS) were chosen as the candidate compounds and Hank's balanced salt solution with Hepes buffer (HBSS-Hepes buffer) was used as the cell-culture medium in an effort to study permeability of chemicals through cell monolayers . Effects of several conditions, e.g . pH and buffer concentration in the mobile phase, flow rate, and temperature on separation efficiency were examined . The nine pharmaceuticals were separated within 2 min by use of a 2-cm C(8) cartridge . Relative standard deviations (RSD) from repeated analysis within the same day or over five days were 0.03-0.2% for retention times and 0.6-5.3% for peak areas; antipyrine was used as internal standard . Calibration curves based on peak-area measurements were linear over the range 0.1-20 micro mol L(-1) . The HBSS-Hepes buffer did not interfere with separation and detection; identical separation and peak intensity were obtained when the samples were separately prepared in distilled water or in the culture medium. Domest Anim Endocrinol, 2002 Oct, 23(3), 383 - 96 Regulation of endometrial granulocyte macrophage-colony stimulating factor (GM-CSF) in the ewe; McGuire WJ et al.; Granulocyte macrophage-colony stimulating factor (GM-CSF) increases ovine interferon-tau (oIFNtau) secretion by ovine conceptuses, but endometrial production of GM-CSF has not been characterized . Endometrial GM-CSF expression was evaluated in ovariectomized ewes implanted with estradiol-17beta (E(2)) and/or progesterone (P(4)) for 14 days, in day 14 cyclic and day 14 pregnant ewes . Relative levels of endometrial GM-CSF mRNA were 3-fold higher in E(2)- and E(2)/P(4)-treated ewes than that of control or P(4)-treated ovariectomized ewes . Levels of endometrial GM-CSF mRNA for cyclic ewes were similar to E(2)- and E(2)/P(4)-treated ewes, but amounts of GM-CSF mRNA in pregnant ewes were 2-fold higher . GM-CSF concentrations in endometrial culture media, determined by GM-CSF bioassay, for cyclic and E(2)/P(4)-treated ovariectomized ewes were 3-fold higher than those of control, E(2)- and P(4)-treated ovariectomized ewes; however, amounts of GM-CSF in pregnant ewes were 2-fold higher . Immunoreactive GM-CSF, examined by western blot, was detected in the culture medium from E(2)/P(4)-treated ovariectomized, cyclic and pregnant ewes . Luminal and glandular epithelia and stromal regions were determined to be sites of GM-CSF expression by immunohistochemistry and in situ hybridization techniques . Data indicate that combined E(2) and P(4) treatment of ovariectomized ewes is sufficient to restore GM-CSF expression to the level found in cyclic ewes; however, GM-CSF mRNA and protein in pregnant ewes is 2-fold greater than in ovariectomized or cyclic ewes . These data suggest that the conceptus, in addition to steroids, may play a role in the regulation of endometrial production of GM-CSF. Environ Toxicol Chem, 2002 Sep, 21(9), 1788 - 95 Use of anodic stripping voltammetry in predicting toxicity of copper in river water; Wang Z et al.; The labile concentration and toxicity of Cu as influenced by alkalinity and different concentrations of ethylenediaminetetraacetic acid (EDTA) and naturally derived fulvic acid (FA) were determined by bioassays carried out in the culture media for Daphnia magna (D . magna) . The labile concentration of Cu was obtained by differential pulse anodic stripping voltammetry with a double-acidification method (DAM-DPASV) . Changes in water alkalinity did not affect the labile concentration of Cu, but increase in alkalinity did reduce the mortality of D . magna . In the presence of EDTA and FA, both labile concentration of Cu and mortality were reduced . By excluding Cu-carbonate complexes from the labile concentration, a bioavailable concentration of Cu ({Cu*}) was obtained and was used to predict the acute toxicity of Cu on D . magna . For natural waters, the labile concentration of Cu was measured by DAM-DPASV, and {Cu*} was calculated using MINTEQ A2 software (developed by the U.S . Environmental Protection Agency) based on the anion composition of waters . This procedure was tested for waters and sediment elutriates sampled from the Le An River (Jiangxi Province, China) that were severely polluted by the discharges from a copper mine . The results showed that {Cu*} was a good indicator for Cu toxicity and could be used under field conditions. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1997, 29(3), 303 - 309 Expression of a Ligninase by a New Hybrid Baculovirus with Expanded Host Range; Lin QY et al.; We have generated and purified a new recombinant baculovirus with expanded host range . AcNPV DNA and BamHI-digested BmNPV DNA were co-transfected into Spodoptera frugiperda SF21 cells . Progeny viruses were used to infect BmN cells, which are normally resistant to AcNPV infection, in order to screen for recombinant viruses with cross-infectivity . A recombinant virus was isolated by plaque purification and analyzed . This isolate was able to infect, replicate and produce polyhedrin in both the SF21 and BmN cells . DNA restriction endonuclease analysis showed that it was a hybrid (HyNPV) of AcNPV and BmNPV . Co-transfection of this HyNPV virus with a transfer vector containing a ligninase H8 gene insert into SF21 cells yielded a recombinant baculovirus expressing the ligninase . When this recombinant baculovirus was used to infect either SF21 or BmN cells, ligninase proteins could be found in the lysed cells as well as in the cell culture media . We have thus demonstrated that this hybrid virus can be utilized in the generation of recombinant baculovirus expressing foreign proteins in two host cells which normally can not be infected by AcNPV nor BmNPV. Anesthesiology, 2002 Sep, 97(3), 630 - 5 Growth cone collapsing effect of lidocaine on DRG neurons is partially reversed by several neurotrophic factors; Radwan IA et al.; BACKGROUND: Local anesthetics were suggested to have a potential for neurotoxicity in both clinical reports and laboratory experiments . Growing neurons have been shown to be susceptible to the toxic effects of local anesthetics in culture . These findings have generated the interest in factors that would rescue the neurons affected by the neurotoxicity of local anesthetics . METHODS: Primary cultured dorsal root ganglia were isolated from age-matched chick embryos and exposed to lidocaine . After 60 min of exposure, the culture media were replaced to wash out the lidocaine . Neurotrophic factors (NTFs)-brain-derived neurotrophic factor, glial-derived neurotrophic factor, or neurotrophin 3-were added to the replacement media to examine the capacity of these NTFs to support the reversibility of the lidocaine-induced growth cone collapse . The growth cone collapse assay was applied a quantitative method of assessment . RESULTS: When any of the three NTFs was added to the replacement media at a minimum concentration of 10 ng/ml, significantly high reversibility of the lidocaine-induced growth cone collapse was observed, especially at 48 h after washout (P < 0.05) . At that time point, there was no significant difference between the values of growth cone collapse percentage in the cells that were exposed to lidocaine and supported by the NTFs after the washout, and the control cells (not exposed to lidocaine) (P > 0.05) . CONCLUSION: The NTFs-brain-derived neurotrophic factor, glial-derived neurotrophic factor, and neurotrophin 3-were demonstrated to support the reversibility of lidocaine-induced growth cone collapse in primary cultured sensory neurons, an effect that was concentration- and time-dependent . Because similar effects were observed after tetracaine washout, the supporting effects of NTFs may not be specific to lidocaine. Hua Xi Yi Ke Da Xue Xue Bao, 1999 Sep, 30(3), 340 - 2 {The influential factors in using cadmium reduction method for measurement of the stable products of NO--nitrates and nitrites}; Gu L et al.; To establish an efficient assay method for detecting nitric oxide indirectly, we compared the effects of cadmium filing, 5 mmol/L and 80 mmol/L copperized cadmium filing on the measurement of nitrates and nitrites (the stable products of NO) using the cadmium reduction method . The results demonstrated that cadmium filing was the most efficient cadmium preparations (P < 0.001) . It's mean reduction rate was 96%, and it showed stronger reduction effectiveness in deproteinization cell culture media . The results indicated that cadmium filing has strong anti-interference capacity in biological fluid . We recommend the cadmium filing reduction method because it is simple, practical, inexpensive, highly efficient and can be performed in ordinary laboratories. Hua Xi Yi Ke Da Xue Xue Bao, 1999 Sep, 30(3), 233 - 5 {Effects of insulin on synthesis and secretion of apolipoproteins A I, A II, B100, C III and E by cultured HepG2 cells}; Wang H et al.; This study was intended to observe the effects of insulin on synthesis and secretion of apolipoproteins by hepatic cells . The HepG2 cells were cultured in RPMI 1640 media with bovine insulin (1 microgram/ml media and 10 micrograms/ml media) for 72 hours, and the apolipoproteins contents in cultured media were measured by radioimmunodiffusion assay (RID) kits developed by authors' research laboratory . 40 fold lyophilizely condensed culture media were used for the assays . The results showed that the relative amounts of apo A I, A II, B100, C III and E secreted by HepG2 cells detected by this method were 824 +/- 27, 813 +/- 24, 4875 +/- 82, 597 +/- 74 and 97 +/- 4 ng/mg cell protein, respectively . When 1.0 microgram/ml medium of bovine insulin was added into the culture media, apo A I and apo E secretion remained unchanged . When compared with the control, while apo A II tended to decrease, apo C III tended to increase, and apo B100 secretion decreased by 21.2%; when 10.0 micrograms/ml medium of insulin was added, apo A I, A II and B100 secretion decreased by 6.9% (P < 0.05), 44.3% (P < 0.01) and 29.3% (P < 0.01) respectively, while apo C III secretion increased by 51.8% (P < 0.01) . These results support certain parts of the hypothesis of pathogenesis of Chinese endogenous hypertriglyceridemia proposed by Bing-Wen Liu. Poult Sci, 2002 Aug, 81(8), 1191 - 8 The effect of hepatocyte growth factor on turkey satellite cell proliferation and differentiation; Zeng C et al.; The effect of hepatocyte growth factor (HGF) on turkey satellite cell proliferation and differentiation was examined in cell culture . Satellite cell clones were established from one muscle of an individual turkey . The results showed that HGF is a potent activator and mitogen of turkey satellite cells and embryonic myoblasts with maximal stimulation at 1 ng/mL . HGF is also an inhibitor of differentiation of turkey satellite cells . Heterogeneity in the responsiveness to HGF in the turkey satellite cell population was observed between clones selected for fast (Early) or slow (Late) rates of proliferation . However, two other Early clones exhibited responses similar to those of two other Late clones . When combined with insulin-like growth factor (IGF) and fibroblast growth factor (FGF), singularly or in combination, HGF did not exert any additive or synergistic effects on Early or Late clone proliferation . Whereas when combined with IGF, FGF, and platelet-derived growth factor (PDGF), HGF significantly stimulated proliferation of the Late clone but not the Early clone . Addition of anti-HGF antibody to culture media diminished proliferation and provided evidence of autocrine production of HGF by turkey satellite cell cultures . Heterogeneity also exists in the turkey satellite cell population with respect to autocrine production of HGF. Bioessays, 2002 Sep, 24(9), 845 - 9 Quiet please, do not disturb: a hypothesis of embryo metabolism and viability; Leese HJ; This review uses nutritional markers of normal and impaired development to address the question; what makes a viable mammalian preimplantation embryo? Resolution of this question is important to ensure the long-term safety of embryo-based biotechnologies in man and domestic animals, the optimisation of embryo production and culture conditions and the development of methods to select viable embryos for replacement . After considering the nutrition of embryos and somatic cells, and the phenomenon of caloric restriction, it is concluded that preimplantation embryo survival is best served by a relatively low level of metabolism; a situation achieved by reducing the concentrations of nutrients in culture media and encouraging the use endogenous resources . Biotechnol Bioeng, 2002 Oct 5, 80(1), 73 - 83 Determination of oxygen gradients in engineered tissue using a fluorescent sensor; Kellner K et al.; Nutrient and oxygen supply of cells are crucial to tissue engineering in general . If a sufficient supply cannot be maintained, the development of the tissue will slow down or even fail completely . Previous studies on oxygen supply have focused on measurement of oxygen partial pressures (pO(2)) in culture media or described the use of invasive techniques with spatially limited resolution . The experimental setup described here allows for continuous, noninvasive, high-resolution pO(2) measurements over the cross-section of cultivated tissues . Applying a recently developed technique for time-resolved pO(2) sensing using optical sensor foils, containing luminescent O(2)-sensitive indicator dyes, we were able to monitor and analyze gradients in the oxygen supply in a tissue over a 3-week culture period . Cylindrical tissue samples were immobilized on top of the sensors . By measuring the luminescence decay time, two-dimensional pO(2) distributions across the tissue section in contact with the foil surface were determined . We applied this technique to cartilage explants and to tissue-engineered cartilage . For both tissue types, changes were detected in monotonously decreasing gradients of pO(2) from the surface with high pO(2) to minimum pO(2) values in the center of the samples . Nearly anoxic conditions were observed in tissue constructs ( approximately 0 Torr) but not in excised cartilage discs ( approximately 20 Torr) after 1 day . Furthermore, the oxygen supply seemed to strongly depend on cell density and cell function . Additionally, histological analysis revealed a maximum depth of approximately 1.3 mm of regular cartilage development in constructs grown under the applied culture conditions . Correlating analytical and histological analysis with the oxygen distributions, we found that pO(2) values below 11 Torr might impair proper tissue development in the center . The results illustrate that the method developed is an ideal one to precisely assess the oxygen demand of cartilage cultures . Parasitol Res, 2002 Oct, 88(10), 946 - 9 Epub 2002 Jun 14. Acid phosphatase activity in excretion/secretion products from Heligmosomoides polygyrus adults: an indicator of the physiological status of the worms; Martinez-Grueiro MM; Acid phosphatase (AP) activity was detected in 24 h culture media from adult Heligmosomoides polygyrus . Female and male excretion/secretion products showed similar specific activity . For both, the AP had a pH optimum of 4.0 and was inhibited by sodium fluoride, tartaric acid, and sodium orthovanadate . The release of AP by adult worms was significantly inhibited by adverse incubation conditions (temperatures of 20 degrees C and 4 degrees C), known physiological perturbers ( t-butylhydroperoxide and sodium azide), and broad spectrum anthelmintics (albendazole, levamisole, morantel, and ivermectin) . These results indicate that the AP activity level in the culture medium may be an indicator of the physiological status of the worms. J Neurophysiol, 2002 Sep, 88(3), 1475 - 90 Differential involvement of Ca(2+) channels in survival and neurite outgrowth of cultured embryonic cockroach brain neurons; Benquet P et al.; The contribution of voltage-gated calcium channels (VGCC) to the development of cultured embryonic cockroach brain neurons was assessed using pharmacological agents . VGCC currents were recorded using the patch-clamp technique and were found to be blocked dose-dependently by micromolar concentrations of mibefradil . The activation and inactivation properties of the calcium channels enable a sizeable calcium current to flow at rest (about -30 and -20 mV in high-potassium culture media) . As expected, the cytoplasmic-free calcium concentration was found to rise when the extracellular potassium concentration was raised from 3 to 15 and 30 mM . The effects of VGCC blockers and calcium chelators were different in fresh and in mature cultures in which the neurons were connected to each other to form a defined network . In fresh cultures, the two non-selective VGCC blockers (verapamil and mibefradil) induced a dose-dependent cell death that was proportional to their blocking effect on I(Ba) . This effect could not be prevented by addition of fetal calf serum to the culture medium . A similar effect was obtained using intra- or extracellular calcium chelating agents (10 microM BAPTA-AM or 10 mM EGTA) . Quite unexpectedly, blockade of the P/Q-like (omega-Aga WA-sensitive) component of the calcium current by 500 nM of omega-AgaTx IVA had no lethal effect, suggesting that the corresponding channels are not involved in the survival mechanism . As expected from their lack of effect on I(Ba), isradipine, nifedipine, and omega-CgTx GVIA did not induce cell death . When the neurons started growing neurites, their sensitivity to calcium channel blockade by mibefradil decreased, indicating a correlation between neurite outgrowth and resistance to calcium depletion . In mature cultures, the neurons became resistant to mibefradil, verapamil, and BAPTA-AM . However, these agents, as well as omega-AgaTx IVA, had a significant inhibitory effect on the increase in diameter of the connectives that linked adjacent clusters of neurons . This effect has been shown to result, in the case of mibefradil, from an inhibition of neurite outgrowth characterized by a significant reduction of the number of primary neurites and secondary branchings but not to a significant modification of the diameter of individual neurites . These results support the view that, as in vertebrates, calcium influx through VGCC plays an important role in survival and neurite outgrowth of cultured embryonic insect neurons . The differential contribution of the P/Q-like and R-like (omega-Aga WA-sensitive) calcium channels in these processes is discussed. J Biochem (Tokyo), 2002 Sep, 132(3), 493 - 9 Mutational analysis of residues in two consensus motifs in the active sites of cathepsin E; Liu J et al.; Cathepsin E, an intracellular aspartic proteinase of the pepsin family, is composed of two homologous domains, each containing the catalytic Asp residue in a consensus DTG motif . Here we examine the significance of residues in the motifs of rat cathepsin E by substitution of Asp98, Asp283, and Thr284 with other residues using site-directed mutagenesis . Each of the mutant proenzymes, as well as the wild-type protein, was found in culture media and cell extracts when heterologously expressed in human embryonic kidney 293T cells . The single mutants D98A, D283A, and D283E, and the double mutants D98A/D283A and D98E/D283E showed neither autocatalytic processing nor enzymatic activities under acidic conditions . However, the D98E and T284S mutants retained the ability to transform into the mature forms, although they exhibited only about 13 and 40% of the activity of the wild-type enzyme, respectively . The K(m) values of these two mutants were similar to those of the wild-type enzyme, but their k(cat) values were greatly decreased . The K(i) values for pepstatin and the Ascaris pepsin inhibitor of the mutants and the wild-type enzyme were almost the same . The circular dichroism spectra of the two mutants were essentially the same as those of the wild-type enzyme at various pH values . These results indicate that (i) Asp98, Asp283, and Thr284 are indeed critical for catalysis, and (ii) the decrease in the catalytic activity of D98E and T284S mutants is brought about by an effect on the kinetic process from the enzyme-substrate complex to the release of the product. J Clin Microbiol, 2002 Sep, 40(9), 3499 - 501 Early detection of negative BACTEC MGIT 960 cultures by PCR-reverse cross-blot hybridization assay; Romano L et al.; We evaluated the efficacy of a PCR-reverse cross-blot hybridization assay, a test which permits identification of mycobacteria by means of species-specific probes and a Mycobacterium-specific probe, for early detection of negative BACTEC MGIT 960 mycobacterial cultures . Aliquots of 549 cultures were collected 7 days after the culture media were inoculated with various clinical specimens and tested with the molecular assay . PCR results were compared to those obtained at the end times with the BACTEC MGIT 960 system . Of the 549 specimens analyzed, 484 were found to be negative and 64 were found positive by both methods; one specimen, found to be positive by the BACTEC MGIT 960 system, was identified as negative by the molecular assay . In view of its high negative predictive value (99.8%), the PCR-reverse cross-blot hybridization assay appears to be a valid tool for early detection of negative BACTEC MGIT 960 cultures. Life Sci Space Res, 1965, 3, 139 - 41 Bacterial ecologies in limonite; Vishniac W; Limonite (Fe2O3 . nH2O) may be a constituent of the Martian surface . We have prepared culture media with ferric hydroxide as an electron acceptor . One medium contained ethanol, another gaseous hydrogen and carbon dioxide . Bacterial growth without light and oxygen suggests that ferric iron serves as a terminal respiratory electron acceptor . The oxidation of ferrous hydroxide may be carried out by photosynthetic bacteria . A ferrous-ferric couple may thus support bacterial respiration and photosynthesis in the absence of oxygen . This cycle may account for the dark markings of Mars. Med Lav, 2002 May-Jun, 93(3), 267 - 78 {In vitro study of the nephrotoxic mechanism of mercuric chloride}; Aleo MF et al.; OBJECTIVES: Mercury (Hg), one of the most diffused and hazardous organ-specific environmental contaminants, exists in a wide variety of physical and chemical states, each of which with unique characteristics of target organ specificity . Exposure to Hg vapour and to organic mercurials specifically affects the CNS, while the kidney is the target organ for inorganic Hg compounds . Despite the increasing number of studies, the molecular bases of the nephrotoxic potential of Hg has not, up to now, been clarified, even if there is evidence suggesting that the ability of the metal to interact with proteins (thiol groups) or to generate oxygen radicals may play a major role . Within this context, the aim of the present study was to investigate, in vitro, the mechanism(s) of the early nephrotoxic potential of mercury chloride (HgCl2), one of the most diffused and biologically active mercury (Hg2+) compounds . For this purpose, two kidney-derived in vitro systems (the MDCK and the LLC-PK1 cell lines) were tested for their sensitivity to the salt, and MDCK was chosen as the most suitable in vitro model for our study . As possible biological markers of the organ-specific toxicity of the metal we analysed: i) critical biochemical parameters related to oxidative stress conditions (effect of Hg2+ on the anti-oxidant status of the cell), and ii) gap-junctional function (GJIC) . METHODS: Classical toxicity tests (MTT and NR) were used for assessing the sensitivity (IC50) of LLP-CK1 and MDCK cell lines to the mercuric salt . Complete solubilisation of the salt in the culture media was verified by inductively coupled plasma mass spectrometry (ICP-MS) . The influence of the metal on cell growth rate and viability were evaluated by conventional proliferation assays . For the following mechanistic studies, cells were exposed for different time periods (4 to 72 hours) to non-cytotoxic (0.1-50 microM) HgCl2 concentrations . The biochemical analysis of the pro-oxidant properties of the mercuric compound was performed by the measurement of anti-oxidant cellular defences against H2O2 {catalase (Cat), glutathione peroxidase (Gpx), and total glutathione (GSH)} . The influence of the metal on the GJIC capacity of MDCK cells was assessed by the "microinjection/dye-coupling" assay . RESULTS: Among the two kidney-derived in vitro systems, MDCK cell line was the most specifically sensitive to the toxic effect of HgCl2: it was, consequently, chosen as a "tubular cell model" for the following experimental steps . Tested for various time periods at increasing concentrations, the HgCl2 effect on MDCK cell proliferation and viability was found to be time- and dose-related . For concentrations < or = 50 microM, HgCl2 inhibits MDCK cell growth rate, being this effect significant (> 50% in respect to untreated controls) from the 24th from the beginning of the treatment, while, for concentrations > 50 microM, the metal causes cell death . Concerning the influence of HgCl2 on MDCK anti-oxidant defences, the most interesting results were obtained by analysing the influence of the mercury salt on the GSH cell content and Gpx activity . Both were, in fact, significantly affected by the presence of the mercury ion . HgCl2 also induced a rapid, dose- and time-related inhibitory effect on the GJIC capacity of the cells . CONCLUSIONS: Even if further investigations are needed to better clarify the possible causal relationship between our findings, they indicate that: a) MDCK cells represent a suitable in vitro model for the study of Hg nephrotoxicity; b) GJIC function is, among those considered in our study, one of the most sensitive biological endpoints for investigating the mechanism(s) of Hg2+ specific toxicity. Diabetes, 2002 Sep, 51(9), 2734 - 41 Androgens decrease plasma adiponectin, an insulin-sensitizing adipocyte-derived protein; Nishizawa H et al.; Adiponectin, an adipose-specific secretory protein, exhibits antidiabetic and antiatherogenic properties . In the present study, we examined the effects of sex hormones on the regulation of adiponectin production . Plasma adiponectin concentrations were significantly lower in 442 men (age, 52.6 +/- 11.9 years {mean +/- SD}) than in 137 women (53.2 +/- 12.0 years) but not different between pre- and postmenopausal women . In mice, ovariectomy did not alter plasma adiponectin levels . In contrast, high levels of plasma adiponectin were found in castrated mice . Testosterone treatment reduced plasma adiponectin concentration in both sham-operated and castrated mice . In 3T3-L1 adipocytes, testosterone reduced adiponectin secretion into the culture media, using pulse-chase study . Castration-induced increase in plasma adiponectin was associated with a significant improvement of insulin sensitivity . Our results indicate that androgens decrease plasma adiponectin and that androgen-induced hypoadiponectinemia may be related to the high risks of insulin resistance and atherosclerosis in men. Clin Sci (Lond), 2002 Aug, 103 Suppl 48, 94S - 97S Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a; Kulathila R et al.; In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence . The recombinant baculovirus was then used to infect High Five(TM) insect cells in suspension culture . Both the monomeric (85 kDa) and dimeric (170 kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography . Typically, approximately 11 mg of ECE-1a monomer and 6 mg of dimer were obtained from l litre of culture medium . No interconversion of the two forms was detected after purification . Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500 mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA . However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the K(m) and k(cat) values for the monomer were 2.2 microM and 1.6 min(-1) respectively, while those of the dimer were 1.4 microM and 4.9 min(-1) respectively . These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1 . The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization. Clin Sci (Lond), 2002 Aug, 103 Suppl 48, 35S - 38S Three vasoactive peptides, endothelin-1, adrenomedullin and urotensin-II, in human tumour cell lines of different origin: expression and effects on proliferation; Takahashi K et al.; Evidence has accumulated showing that vasoactive peptides, such as endothelin-1, adrenomedullin and urotensin-II, are expressed in various kinds of tumour cells . In the present study, the expression of endothelin-1 and endothelin receptors was studied in eight human tumour cell lines: T98G (glioblastoma), IMR-32 and NB69 (neuroblastoma), BeWo (choriocarcinoma), SW-13 (adrenocortical carcinoma), DLD-1 (colonic carcinoma), HeLa (cervical carcinoma) and VMRC-RCW (renal carcinoma) . Reverse transcriptase-PCR showed expression of endothelin-1 mRNA in seven out of the eight cell lines, the exception being BeWo cells . ET(A) receptor mRNA was expressed in T98G, IMR-32 and NB69 cells, but weakly in the other cells . ET(B) receptor mRNA was expressed in IMR-32, NB69 and BeWo cells, but only weakly in T98G and HeLa cells . Immunoreactive endothelin was detected in the culture media of six out of the eight cell lines, but not in that of IMR-32 or BeWo cells . Treatment of T98G cells with an anti-endothelin-1 antibody or an anti-adrenomedullin antibody for 24 h decreased cell numbers to approx . 84% and 90% of control respectively . Treatment with the ET(A) receptor antagonist BQ-610 (1 microM) significantly decreased cell number to about 90% of control, whereas the ET(B) receptor antagonist BQ-788 had no significant effect . On the other hand, exogenously added endothelin-1, adrenomedullin or urotensin-II (0.1 microM) had no significant effects on cell number . These results suggest that endothelin-1 acts as a paracrine or autocrine growth stimulator in tumours . The effect of endothelin-1 on tumour growth appears to be mediated by the ET(A) receptor. J Assist Reprod Genet, 2002 Aug, 19(8), 368 - 75 Objective assessments of temperature maintenance using in vitro culture techniques; Cooke S et al.; PURPOSE: To assess the ability of various facets of embryo culture (microscope stage warmers, volumes of culture media, culture vessel lids, and type of culture incubator) to maintain a constant temperature in vitro . METHODS: Ability to maintain 37.0 degrees C in the microenvironment of gametes was recorded by digital thermocouple in the chosen facets of in vitro culture . RESULTS: Stage warmers are highly variable in their ability to maintain the set temperature (range 33.8 degrees C-37.0 degrees C after 60 s) . Temperature loss in culture media is both volume and vessel dependent, and the direct heat transfer culture incubator (MINC) has superior temperature maintenance compared with a large volume air convection incubator (FORMA), where temperature regain from 35.0 degrees C to 37.0 degrees C took 5.5 min compared to >20 min . CONCLUSIONS: There are large measurable differences in the ability to maintain set temperature that depend on the stage warmer used, volume of media, use of vessel lids, and the type of incubator chosen for IVF culture. J Bone Joint Surg Am, 2002 Aug, 84-A(8), 1405 - 12 Recombinant adeno-associated virus-mediated osteoprotegerin gene therapy inhibits wear debris-induced osteolysis; Ulrich-Vinther M et al.; BACKGROUND: Aseptic loosening of orthopaedic implants secondary to wear debris-induced osteolysis is a serious problem . Osteoprotegerin (OPG) is a natural decoy protein that inhibits osteoclast activation and bone resorption . This study investigated whether gene therapy using a recombinant adeno-associated viral vector that expresses OPG can inhibit wear debris-induced osteolysis . METHODS: A recombinant adeno-associated virus (rAAV) vector co-expressing OPG (rAAV-OPG-IRES-EGFP) was generated . A control vector expressing b-galactosidase (rAAV-LacZ) was also prepared . In vitro validation experiments were performed to determine rAAV-OPG-IRES-EGFP transduction efficiency, OPG expression level and function in bone wafer, and osteoclastic activity . The effect of rAAV-OPG-IRES-EGFP in vivo gene therapy on wear debris-induced osteolysis was then evaluated in a mouse calvarial model in which a single intramuscular injection of the vector was administered prior to the introduction of the wear debris . The effects of the rAAV-OPG-IRES-EGFP gene therapy on wear debris-induced osteoclastogenesis and bone resorption were determined by histomorphometry on day 10 . RESULTS: In vitro experiments revealed that 100% of human embryonic kidney 293 cells were transduced at a multiplicity of infection of 1000 with both rAAV-OPG-IRES-EGFP and rAAV-LacZ . At a rAAV-OPG-IRES-EGFP multiplicity of infection of 1000, an OPG concentration of 135 ng/mL of culture media was achieved after four days . Using a bone-wafer assay for osteoclast activity, we found that treatment with rAAV-OPG-IRES-EGFP reduced resorption sevenfold compared with parathyroid hormone-stimulated controls and elevenfold compared with rAAV-LacZ controls . Furthermore, a seventeenfold decrease in RANKL and macrophage colony-stimulating factor-induced splenocyte osteoclastogenesis was observed in co-cultures containing rAAV-OPG-IRES-EGFP-infected fibroblasts . In vivo administration of rAAV-OPG-IRES-EGFP resulted in detectable transduction of myocytes at the injection site and a significant increase in expression of serum OPG levels by the second day (p < 0.05) . Maximal concentrations were obtained on day 6 and then leveled off throughout the observation period . In contrast, serum OPG could not be detected in the sham-treated, uninfected titanium-stimulated, or rAAV-LacZ-infected mice . In the control mice, titanium implantation resulted in a threefold increase in the mean number of osteoclasts adjacent to the sagittal suture as well as a twofold increase in the mean area of soft tissue in the sagittal suture compared with the sham-treated mice . In contrast, osteoclast numbers remained at basal levels, and the area of soft tissue in the sagittal suture was markedly reduced in titanium-implanted animals that received rAAV-OPG-IRES-EGFP treatment, demonstrating a complete inhibition of osteolysis in response to titanium particles . CONCLUSIONS: A single intramuscular injection of the rAAV-OPG-IRES-EGFP vector can efficiently transduce myocytes to produce high levels of OPG . The OPG effectively inhibits wear debris-induced osteoclastogenesis and osteolysis . Clinical Relevance: Currently, there is no approved drug therapy to prevent or inhibit periprosthetic osteolysis . Although preclinical studies have identified potential drug therapies (i.e., bisphosphonates), there is no evidence that these drugs can effectively treat aseptic loosening in patients . This is the first evidence that in vivo OPG gene therapy can be used to prevent wear debris-induced osteolysis. Anticancer Res, 2002 Jul-Aug, 22(4), 2423 - 7 Hyaluronidase is more elevated in human brain metastases than in primary brain tumours; Delpech B et al.; BACKGROUND: Hyaluronidase is hypothesised to play a role in cancer invasion and metastasis formation . MATERIALS AND METHODS: Hyaluronidase activity was investigated at pH 3.8 in extracts of 30 human brain tumours (17 glioblastomas and 13 brain metastases of carcinomas) and in cancer cell cultures with the ELSA method and zymography . RESULTS: In brain metastases, hyaluronidase activities were significantly higher than in glioma extracts (9.16 +/- 4.48 mU/g vs 4.25 +/- 5.74) which was not explained by serum hyaluronidase contamination . Serum hyaluronidase of tumour patients' sera was within the normal values determined in 28 matched blood donors'sera (33.8 +/- 11 U/l) . The maximum hyaluronidase/albumin (U/g) ratio was 0.9, below which the hyaluronidase content of tumours was below the maximum value calculated from the albumin content of the tumour extract and could not be considered as local production by tumour cells . The hyaluronidase content and hyaluronidase/albumin ratio of metastasis extracts was significantly higher than in glioma extracts and patients' sera, whereas no significant difference was found between the ratios of glioma extracts and sera . The production of hyaluronidase was studied in cell extracts and in culture media of 3 human glioma-derived cell lines and of the brain metastasis-derived cell line SA87 . Hyaluronidase activity of the metastasis-derived cell line SA87 was 100 to 1000-fold that of glioma cell lines . CONCLUSION: These results suggest that hyaluronidase is associated with the more aggressive cancer cells and is directly or indirectly involved in brain metastasis phenotype. Quintessence Int, 2002 Jul-Aug, 33(7), 496 - 502 Root instrumentation with an erbium:yttrium-aluminum-garnet laser: effect on the morphology of fibroblasts; Rossa CJ et al.; OBJECTIVE: The purpose of this study was to evaluate the effect of erbium:yttrium-aluminum-garnet laser instrumentation of root surfaces on the morphology of fibroblasts from continuous lineage . METHOD AND MATERIALS: Dentinal slices with 4 mm2 of surface area were obtained from teeth extracted for severe periodontal involvement . Specimens were assigned to one of three treatment groups: group 1, application of the laser with an energy level of 250 mJ at 103 pulses per second; group 2, application of the laser with an energy level of 80 mJ at 166 pulses per second; and group 3, similar to group 2, but with concomitant water irrigation of the device . The specimens were incubated in multiwell plates containing cell culture media . After 24 hours, the specimens were submitted to routine preparation for scanning electron microscopy . Three independent and blind examiners used photomicrographs to evaluate the morphology of the fibroblasts: 0 = without cells; 1 = flat cells; 2 = round cells; and 3 = combination of round and flat cells . RESULTS: Statistical analysis indicated that there were significant differences among treatment groups and that group 3 was significantly different from groups 1 and 2 . CONCLUSION: There was no difference between groups 1 and 2 in the morphology of fibroblasts . Laser instrumentation with concomitant irrigation impaired the adhesion of fibroblasts to dentinal surfaces. Life Sci, 2002 Apr 21, 70(21), 2521 - 33 Effects of epoxyeicosatrienoic acids on polymorphonuclear leukocyte function; Pratt PF et al.; During periods of ischemia and vascular injury, factors are released which recruit monocytes and polymorphonuclear leukocytes (PMNs) to the site of injury by promoting adherence to the endothelium and transmigration across the endothelial cell (EC) layer . During coronary artery stenosis, we have shown that the endothelium-derived, cytochrome P450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are elevated . Therefore, we examined if the EETs could stimulate PMN adherence to cultured ECs . Pretreatment of ECs with EETs for either 30 min or 4 hr did not alter the adherence of 51Cr-labelled PMNs to ECs while phorbol myristate acetate (PMA) produced a 4-fold increase in PMN adherence . The combination of EETs and PMA did not significantly augment or diminish PMA-induced PMN adherence to ECs . When ECs and 51Cr-labelled PMNs were coincubated, treatment with EETs alone did not alter PMN adherence . However, when EETs and PMA were added together during the coincubation of ECs and 51Cr-labelled PMNs, the EETs produced a concentration-related decrease in PMN adherence . Microscopic analysis of the culture media bathing the cells revealed aggregates of the labeled PMNs . We examined the effects of the EETs on PMN aggregation . 8,9-EET (10, 50, and 100 microM) increased PMN aggregation (7 +/- 3, 35 +/- 10, and 65 +/- 11%) and intracellular calcium by 1.7 +/- 0.5, 4.7 +/- 1.4, and 6.8 +/- 2.3-fold above basal . 5,6-, 11,2- and 14,15-EETs also stimulated aggregation . FMLP stimulated the production of superoxide; however, 8,9-EET did not . These observations indicate that the decrease in PMN adherence observed in the coincubation experiment is the result of EET-induced PMN aggregation . Given the increase in EET production during coronary artery stenosis, these data may provide insight into their potential biological significance during myocardial ischemia and vascular injury. Cancer Biol Ther, 2002 Mar-Apr, 1(2), 168 - 76 Pharmocologic inhibitors of the mitogen activated protein kinase cascade have the potential to interact with ionizing radiation exposure to induce cell death in carcinoma cells by multiple mechanisms; Qiao L et al.; Recent studies have shown that inhibition of stress-induced signaling via the mitogen activated protein kinase (MAPK) pathway can potentiate the toxic effects of chemotherapeutic drugs and ionizing radiation . Because of these observations, we have further investigated the impact upon growth and survival of mammary (MDA-MB-231, MCF7, T47D), prostate (DU145, LNCaP, PC3) and squamous (A431) carcinoma cells following irradiation and combined long-term exposure to MEK1/2 inhibitors . Exposure of carcinoma cells to ionizing radiation resulted in MAPK pathway activation initially (0-4 h) and modestly enhanced MAPK activity at later times (24 h-96 h) . Inhibition of radiation-induced MAPK activation using MEK1/2 inhibitors potentiated radiation-induced apoptosis in two waves, at 21-30 h and 96-144 h after exposure . The potentiation of apoptosis was not observed in MCF7, LNCaP, or PC3 cells . At 24 h, the potentiation of apoptosis was independent of radiation dose whereas at 108 h, apoptosis correlated with increasing dose . Removal of the MEK1/2 inhibitor either 6 h or 12 h after exposure abolished the potentiation of apoptosis at 24 h . At this time, the potentiation of apoptosis correlated with cleavage of pro-caspases -8, -9 and -3, and with release of cytochrome c into the cytosol . Inhibition of caspase function using a pan-caspase inhibitor ZVAD blocked the enhanced apoptotic response at 24 h . Selective inhibition of caspase 9 with LEHD or caspase 8 with IETD partially blunted the apoptotic response in MDA-MB-231, DU145 and A431 cells, whereas inhibition of both caspases reduced the response by > 90% . Removal of the MEK1/2 inhibitor either 24 h or 48 h after exposure abolished the potentiation of apoptosis at 108 h . Incubation of cells with ZVAD for 108 h also abolished the potentiation of apoptosis . In general agreement with the finding that prolonged inhibition of MEK1/2 was required to enhance radiation-induced apoptosis at 108 h, omission of MEK1/2 inhibitor from the culture media during assessment of clonogenic survival resulted in either little or no significant alteration in radiosensitivity . Collectively, our data show that combined exposure to radiation and MEK1/2 inhibitors can reduce survival in some, but not all, tumor cell types . Prolonged blunting of MAPK pathway function following radiation exposure is required for MEK1/2 inhibitors to have any effect on carcinoma cell radiosensitivity. J Vasc Surg, 2002 Aug, 36(2), 263 - 70 Effect of adenoviral titer and instillation pressure on gene transfer efficiency to arterial and venous grafts ex-vivo; Brevetti LS et al.; OBJECTIVE: Adenoviral-mediated gene transfer to arterial and venous grafts has potential in the treatment of a number of vascular diseases . Despite widespread use of these vectors to mediate gene transfer to blood vessel walls, the optimal transduction conditions for each type of vessel has yet to be determined . Our objective was to study the effect of adenoviral titer and instillation pressure on efficiency of gene transfer to arterial and venous grafts ex-vivo . METHODS: Jugular vein and carotid artery segments of 8 cm were harvested from Yorkshire Cross pigs . Tissue culture media or different titers of an adenoviral vector encoding human placental alkaline phosphatase (hpAP) were instilled into venous and arterial grafts at 0 mm Hg or 80 to 100 mm Hg of pressure and bathed externally in the same solution at 37 degrees C for 30 minutes . The grafts were rinsed, opened longitudinally, and incubated in culture media at 37 degrees C for 48 hours . Grafts were fixed and stained for hpAP transgene expression to quantitate percent luminal transduction or homogenized for alkaline phosphatase (AP) activity to determine total transmural transduction . RESULTS: For venous grafts, the percent luminal area stained for hpAP was greatest with 10(8) plaque-forming units/mL at 0 mm Hg (81% +/- 7%) and decreased with increasing titers (53% +/- 9% at 10(9) pfu/mL and 44% +/- 11% at 5 x 10(9) pfu/mL; n = 7; P <.05) . No increase in percent luminal area stain was achieved with an instillation pressure of 80 to 100 mm Hg at any viral titer . The inverse finding was observed in arterial grafts . For arterial grafts, the greatest percent luminal area stained was achieved with 5 x 10(9) pfu/mL at 80 to 100 mm Hg (76% +/- 7%) . An instillation pressure of 80 to 100 mm Hg increased the percent luminal area stained at 10(8) pfu/mL from 31% +/- 9% to 66% +/- 8% (n = 8; P =.01) . For venous grafts, total AP activity peaked with 10(9) pfu/mL at 0 mm Hg and decreased with an instillation pressure of 80 to 100 mm Hg (30.6 +/- 9.7 U/mg versus 10.9 +/- 2.5 U/mg; n = 7; P <.01) . However, for arterial grafts, total AP activity peaked with 5 x 10(9) pfu/mL (0 mm Hg) and increased with an instillation pressure of 80 to 100 mm Hg (32.8 +/- 9.9 U/mg versus 63.4 +/- 20.5 U/mg; n = 8; P <.05) . CONCLUSION: High transduction efficiency can be achieved with adenoviral-mediated gene transfer of arterial and venous grafts . Gene transfer with the vascular graft's physiologic pressure conditions improved transduction efficiency for the artery (80 to 100 mm Hg) and vein (0 mm Hg) . Comprehensive analysis of adenoviral transduction conditions is important to realize the full promise of adenoviral-mediated gene transfer. Clin Exp Immunol, 2002 Aug, 129(2), 379 - 84 Functional changes in rheumatoid fibroblast-like synovial cells through activation of peroxisome proliferator-activated receptor gamma-mediated signalling pathway; Yamasaki S et al.; Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand dependent transcriptional factor known to be a regulator of adipogenesis . Recent studies have also shown that stimulation of PPARgamma inhibits the transcriptional activities of other nuclear factors and down-regulates proinflammatory cytokine synthesis in T cells and monocytes . We examined, in the present study, the functional significance of PPARgamma expressed in fibroblast-like synovial cells (FLS) isolated from patients with rheumatoid arthritis (RA) . Incubation of FLS with a synthetic PPARgamma ligand, troglitazone, inhibited endogenous production of TNF-alpha, IL-6 and IL-8, as well as matrix metalloprotease-3 (MMP-3), without inducing apoptosis of the cells . The gelatinase activity of FLS culture media was also inhibited by troglitazone . Electrophoretic mobility shift assay (EMSA) showed a significant reduction in the DNA binding activity of NF-kappaB in troglitazone-treated FLS in response to TNF-alpha or IL-1beta . Moreover, long-term cultivation of FLS with troglitazone resulted in morphological changes with marked lipid accumulation in these cells . Our results show a negative regulatory function for PPARgamma on cytokine and MMP production together with inhibition of cytokine-mediated inflammatory responses in rheumatoid synovial cells . Our results also suggest that FLS could differentiate into adipocyte-like cells in the presence of proper stimulatory signals including PPARgamma. Biomaterials, 2002 Oct, 23(19), 4001 - 10 An evaluation of accelerated Portland cement as a restorative material; Abdullah D et al.; Biocompatibility of two variants of accelerated Portland cement (APC) were investigated in vitro by observing the cytomorphology of SaOS-2 osteosarcoma cells in the presence of test materials and the effect of these materials on the expression of markers of bone remodelling . Glass ionomer cement (GIC), mineral trioxide aggregate (MTA) and unmodified Portland cement (RC) were used for comparison . A direct contact assay was undertaken in four samples of each test material, collected at 12, 24, 48 and 72 h . Cell morphology was observed using scanning electron microscopy (SEM) and scored . Culture media were collected for cytokine quantification using enzyme-linked immunosorbent assay (ELISA) . On SEM evaluation, healthy SaOS-2 cells were found adhering onto the surfaces of APC variant, RC and MTA . In contrast, rounded and dying cells were observed on GIC . Using ELISA, levels of interleukin (IL)-1beta, IL-6, IL-18 and OC were significantly higher in APC variants compared with controls and GIC (p<0.01), but these levels of cytokines were not statistically significant compared with MTA . The results of this study provide evidence that both APC variants are non-toxic and may have potential to promote bone healing . Further development of APC is indicated to produce a viable dental restorative material and possibly a material for orthopaedic J Clin Endocrinol Metab, 2002 Aug, 87(8), 3774 - 8 Estrogen represses whereas the estrogen-antagonist ICI 182780 stimulates placental CRH gene expression; Ni X et al.; CRH and estrogens, produced by placental trophoblasts, have been suggested to play pivotal roles in the control of human parturition . Estrogen has been shown to affect hypothalamic CRH expression . Therefore, we evaluated 17 beta-estradiol (E2) in the regulation of CRH gene expression in placental cells . E2 inhibited CRH mRNA expression in a dose-dependent manner, which paralleled the decrease in CRH protein levels in culture media . A complete estrogen receptor (ER) antagonist, ICI 182780, not only blocked repression of CRH mRNA levels by E2, but up-regulated CRH mRNA and protein synthesis . An ER alpha-mixed agonist/antagonist and ER beta antagonist, 4-hydroxytamoxifen, also down-regulated CRH gene expression . Using quantitative RT-PCR, we found that placental trophoblasts express predominantly the ER alpha form of the receptor . Transient transfection assays conducted in the choriocarcinoma cell line JEG-3 demonstrated that E2 repressed CRH promoter activity, whereas the antagonist ICI 182780 up-regulated CRH promoter activity when ER alpha was cotransfected . These studies demonstrate that E2 represses placental CRH gene expression through an ER alpha-mediated mechanism . Estrogen may therefore modulate placental CRH production, influencing the rate of rise of maternal plasma CRH concentrations and potentially the length of gestation. Diagn Microbiol Infect Dis, 2002 Aug, 43(4), 297 - 302 Rapid differentiation between clinically relevant mycobacteria in microscopy positive clinical specimens and mycobacterial isolates by line probe assay; Johansen IS et al.; The Inno LiPA Mycobacteria assay, based on PCR amplification of the 16-23S rRNA spacer region of Mycobacterium species, has been designed for identification of mycobacteria grown in culture media and discrimination between Mycobacterium tuberculosis complex, M . avium, M . intracellulare, M . kansasii, M . gordonae, M . xenopi, scrofulaceum and M . chelonae group including M . abscessus . In order to evaluate the system as a fast diagnostic tool, the assay was for the first time used directly on 14 microscopy positive clinical specimens and 71 isolates and the results were compared to those of conventional identification using 16S rDNA analysis and biochemical properties . The assay only misidentified one strain, which was found to be M . avium complex instead of M . intracellulare as found by the conventional tests . The assay allows rapid discrimination of the eight most clinically relevant mycobacteria in microscopy positive clinical specimens and isolates within 6 h in the same procedural run. Tsitologiia, 2002, 44(4), 357 - 63 {Properties of endoribonuclease activity of proteasomes from K562 cells . II . Analysis of specific mRNA nucleolysis by proteasomes}; Mittenberg AG et al.; The ability of 26S proteasomes from the human proerythroleukaemic cell line K562 to degrade high-molecular-weight cytoplasmic RNAs, particularly specific messenger RNA, has been detected . The addition of hemin to K562 cells in the culture media leads to redistribution of proteasomes and their migration mainly to the cytoplasm . The human wild type p53 gene mRNA was shown to be specifically nucleolized by proteasomes . These particles displayed endoribonuclease activity towards mRNA for Renilla sp . luciferase . Proteasomes also specifically degraded Alu-containing mRNAs . A supposition is made about the involvement of proteasomes in stability control of specific RNA groups. Toxicol Appl Pharmacol, 2002 Jul 15, 182(2), 160 - 8 Percutaneous absorption of explosives and related compounds: an empirical model of bioavailability of organic nitro compounds from soil; Reifenrath WG et al.; The percutaneous absorption potentials of (14)C-labeled 2,4,6-trinitrotoluene (TNT), trinitrobenzene, 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene, 2,6-diamino-4-nitrotoluene, N-methyl-N,2,4,6-tetranitrobenzamine, hexahydro-1,3,5-trinitro-1,3,5-triazine, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, and 2,2-thiobis(ethanol) were determined from two soil types, Yolo having 1.9% carbon and Tinker having 9.5% carbon . TNT skin absorption from another low-carbon soil (Umatilla) was also determined . Approximately 10 microg/cm(2) of radiolabeled compound was applied in 5 microl of acetone or 10 mg/cm(2) of soil to excised pigskin mounted in skin penetration-evaporation chambers . Absorption from acetone served as a control . Radiolabel recovered from the dermis and tissue culture media (receptor fluid) was summed to determine the percentage of absorption from the soils . For each compound, percentage absorptions of radiolabel were highest from acetone solution and lowest from Tinker soil, with the results from Yolo soil being intermediate . Skin absorptions of TNT from Yolo and Umatilla soils were similar . For TNT in all vehicles, the penetration rate of radiolabel into the receptor fluid was highest during the 1- to 2-h interval after dosing . HPLC analysis of TNT radiolabel in receptor fluid at maximum flux indicated extensive conversion to monoamino derivatives and other metabolites . For 2,4-DNT and 2,6-DNT applications in Yolo soil, percentage absorptions approached those obtained from acetone applications . After 2,4-DNT and 2,6-DNT applications (acetone and soils), HPLC analysis of radiolabel in receptor fluid during the period of maximum flux revealed no significant metabolites . Skin absorption of the nitro compounds from soils was found to correlate with the compound's water solubility and vapor pressure . These findings formed the basis of an empirical model to predict skin bioavailability. Pancreatology, 2002, 2(4), 402 - 6 Specific amino acid profile in culture media conditioned by human pancreatic cancer cell lines; Wang F et al.; BACKGROUND: In malignant diseases, circulating amino acid profiles correlate with organ sites of malignancy . Direct effects of malignant cells on the extracellular amino acid profile are still uncertain . METHODS: Free amino acids were measured in serum-free culture media (RPMI-1640) conditioned by two human pancreatic cancer cell lines (Panc-1 and HPAF), a hamster pancreatic cancer cell line (PC-1), a human epidermoid carcinoma cell line (A-431), and a human fibroblastic cell line (Ag-1523) . Non-conditioned RPMI-1640 medium was used as control . RESULTS: Amino acid profiles were changed in all the conditioned media, caused by a decrease or increase in the original amino acids and by the appearance of amino acids that were not present in non-conditioned medium . Media conditioned by two human pancreatic cancer cell lines showed similar amino acid profiles, which were characterized by a decrease in glutamine, cysteine and serine, increase in glycine, proline and glutamic acid and appearance of ornithine and alanine . CONCLUSION: Culture media show changed amino acid profiles following incubation with cell lines of pancreatic or non-pancreatic origins . Different human pancreatic cancer cell lines cause similar changes in amino acid profiles of media . J Biol Chem, 2002 Oct 11, 277(41), 38148 - 58 Epub 2002 Jul 22. Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex . Evidence that all strains synthesize glycosylated p-hydroxybenzoic methly esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene; Constant P et al.; Diesters of phthiocerol and phenolphthiocerol are important virulence factors of Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans . They are both long-chain beta-diols, and their biosynthetic pathway is beginning to be elucidated . Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M . tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains . To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M . tuberculosis were analyzed . We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M . tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putative p-hydroxybenzoic acid precursor . We also show that all the strains of M . tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M . leprae, and strains of M . tuberculosis that produce phenolphthiocerol derivatives . Complementation of the H37Rv strain of M . tuberculosis, which is devoid of phenolphthiocerol derivatives, with the fused pks15/1 gene from M . bovis BCG restored phenolphthiocerol glycolipids production . Conversely, disruption of the pks15/1 gene in M . bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid . These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to give p-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives. Anim Reprod Sci, 2002 Aug 15, 72(3-4), 137 - 51 Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers; Reis A et al.; To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCC's) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals . More OCC's were collected during OPU1 than OPU2 (means +/- S.E.M . = 7.2 +/- 0.47 versus 5.7 +/- 0.44; P = 0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55 +/- 3% versus 47 +/- 3%) . The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean +/- S.E.M . = 81 +/- 2% and 71 +/- 7% for OPU1 and OPU2, respectively) . Corresponding blastocyst yields from good quality OCC's (24 +/- 3% and 26 +/- 4%) also did not differ . Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3 +/- 0.28 ng/ml versus 2.5 +/- 0.45 ng/ml; P < 0.05) . In contrast, plasma progesterone concentrations were higher at OPU1 (6.6 +/- 0.48 ng/ml versus 3.9 +/- 0.53 ng/ml; P < 0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1 . Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCC's suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones . Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process . Hypertens Res, 2002 May, 25(3), 455 - 60 Lowering of blood pressure improves endothelial dysfunction by increase of nitric oxide production in hypertensive rats; Hatta T et al.; To investigate the effects of angiotensin converting enzyme inhibitor (ACE-I) and other antihypertensive agents on the nitric oxide (NO) release during hypertension, seven- and fourteen-week-old SHR and deoxycorticosterone acetate (DOCA)-salt rats were treated with hydralazine, manidipine (Ca antagonist) or quinapril (ACE-I) for 3 weeks to lower blood pressure . Systolic blood pressure (SBP) was measured by the tail cuff method once each week . Endothelial cells (ECs) derived from the descending aorta of the treated rats were cultured and NOx levels in culture media were measured with an NO analyzer based on the Griess reaction . In both SHR and DOCA-salt rats, antihypertensive therapy lowered SBP to levels similar to those of control rats . The only exception was quinapril treatment of DOCA-salt rats . Although NOx release by ECs derived from hypertensive rats was improved by antihypertensive therapy, the effect was most pronounced in SHR treated with quinapril . In addition, restoration of NOx release was much more remarkable in younger SHR . NOx release was significantly higher in DOCA-salt rats treated with quinapril than in control rats without reduction of SBP . These results suggest that lowering blood pressure improves release of NO by ECs during hypertension and that the time at which antihypertensive therapy is started is also important to preserve endothelial function . Furthermore, ACE-I is suggested to protect endothelial function by increasing NO production in addition to lowering blood pressure. J Hand Surg {Am}, 2002 Jul, 27(4), 615 - 20 Flexor tendon healing in vitro: effects of TGF-beta on tendon cell collagen production; Klein MB et al.; Flexor tendon healing is complicated by adhesions to the surrounding sheath . Transforming growth factor beta (TGF-beta) is a cytokine with numerous activities related to wound healing . We examined the effects of TGF-beta-1, -2 and -3 on tendon cell proliferation and collagen production . Three separate cell lines--sheath fibroblasts, epitenon and endotenon tenocytes--were isolated from rabbit flexor tendons and cultured separately . Cell culture media was supplemented with 1 or 5 ng/mL of TGF-beta-1, -2, or -3 . Cell number and collagen I and III production were measured and compared with unsupplemented control cultures . The addition of TGF-beta to cell culture media resulted in a decrease in cell number in all 3 lines that did not reach statistical significance . There was a significant increase (p <.05) in collagen I and III production with the addition of all 3 TGF-beta isoforms . Modulation of TGF-beta production may provide a mechanism to modulate adhesion formation clinically. Endocrinology, 2002 Aug, 143(8), 2872 - 9 Antigen presentation by vaginal cells: role of TGFbeta as a mediator of estradiol inhibition of antigen presentation; Wira CR et al.; Estradiol is known to inhibit antigen presentation in the vagina . We report here that TGFbeta mediates the action of estradiol on vaginal antigen presenting cells (APC) . When vaginal APC from ovariectomized rats were incubated with increasing concentrations of TGFbeta1 and TGFbeta2 in the presence of ovalbumin-specific T cells and ovalbumin, both TGFbeta1 and TGFbeta2 inhibited vaginal cell antigen presentation, whereas IL-6, IL-10, and TNFalpha had no consistent effect . In other experiments, estradiol-induced inhibition of antigen presentation by vaginal cells was partially reversed when vaginal APC were incubated with anti-TGFbeta antibody . In contrast, anti-TNFalpha, anti-IL-6, and anti-IL-10 had no effect on antigen presentation . The effect of anti-TGFbeta was seen with vaginal APC from ovariectomized rats treated with estradiol for 1 d as well as 3 d . Finally, analysis of TGFbeta in the culture media of vaginal cells from saline- and estradiol-treated rats indicated that the TGFbeta produced is biologically active . In response to estradiol, vaginal cell production of TGFbeta was significantly greater than that seen with control cells . These studies suggest that estradiol regulation of antigen presentation by vaginal cells is mediated through the local production of TGFbeta by vaginal cells. Int J Radiat Oncol Biol Phys, 2002 Aug 1, 53(5), 1314 - 8 Pronounced radiosensitization of cultured human cancer cells by COX inhibitor under acidic microenvironment; Shah T et al.; PURPOSE: To demonstrate the influence of pH on the cytotoxicity and radiosensitization by COX (cyclooxygenase) -1 and -2 inhibitors using established human cancer cells in culture . METHODS AND MATERIALS: Nonselective COX inhibitor, ibuprofen (IB), and selective COX-2 inhibitor, SC-236, were used to determine the cytotoxicity and radiosensitization at varying pH of culture media . Human colon carcinoma cell line (HT-29) was exposed to the drug alone and in combination with radiation at different pH of the cell culture media . The end point was clonogenic ability of the single-plated cells after the treatment . RESULTS: Cytotoxicity and radiosensitization of IB increased with higher drug concentration and longer exposure time . The most significant radiosensitization was seen with IB (1.5 mM) for 2-h treatment at pH 6.7 before irradiation . The dose-modifying factor as defined by the ratio of radiation doses required to achieve the same effect on cell survival was 1.8 at 10% survival level . In contrast, SC-236 (50 microM for 2-8 h) showed no pH-dependent cytotoxicity . There was modest increase in the cell killing at lower doses of radiation . CONCLUSION: An acidic pH was an important factor affecting the increased cytotoxicity and radiosensitization by ibuprofen . Radiation response was enhanced at shoulder portion of the cell survival curve by selective COX-2 inhibitor. Eur J Vasc Endovasc Surg, 2002 Jul, 24(1), 72 - 80 Enhanced invasive properties exhibited by smooth muscle cells are associated with elevated production of MMP-2 in patients with aortic aneurysms; Goodall S et al.; BACKGROUND: abdominal aortic aneurysms (AAA) are associated with excessive vascular matrix remodelling . Recent findings suggest a systemic overproduction of matrix metalloproteinases-2 (MMP-2) by vascular smooth muscle cells (SMC) may be pivotal aetiologically . SMC migration is facilitated by MMP mediated proteolysis of the basement membrane and extracellular matrix . Our aim was to see if enhanced MMP-2 production by these SMC exhibit increased invasion, in an in vitro model of migration . METHOD: SMC were derived from inferior mesenteric vein (IMV) harvested from patients undergoing aneurysm repair (n=6) or colectomy for diverticulosis (n=6, control) . Using a modified Boyden chamber chemotaxis was measured towards platelet derived growth factor (PDGF) and foetal calf serum (FCS) and invasion through a Matrigel layer . MMP-2 production was quantified by ELISA and gelatin zymography . RESULTS: chemoattractant studies demonstrated no difference in the effect of PDGF or FCS between the two populations of SMC . However, invasive studies demonstrated a significant increase in the number of migrating SMC isolated from IMV of AAA patients . Analysis of culture media extracts revealed that this difference was associated with a significant increase in production of MMP-2 . CONCLUSION: SMC derived from patients with AAA demonstrate increased invasive properties when compared to a control group . Increased migration appears to be due to overproduction of MMP-2 . The enhanced migratory potential of these SMC may lead to extracellular matrix remodelling and subsequent medial disruption demonstrated in the aneurysmal aorta . These data further support evidence of the proteolytic role of MMP-2 in cell migration. J Neuropathol Exp Neurol, 2002 Jul, 61(7), 585 - 96 Galectin-1 modulates human glioblastoma cell migration into the brain through modifications to the actin cytoskeleton and levels of expression of small GTPases; Camby I et al.; We show that high-grade astrocytic tumors with high levels of galectin-1 expression are associated with dismal prognoses . The immunohistochemical analysis of galectin-1 expression of human U87 and U373 glioblastoma xenografts from the brains of nude mice revealed a higher level of galectin-1 expression in invasive areas rather than non-invasive areas of the xenografts . Nude mice intracranially grafted with U87 or U373 cells constitutively expressing low levels of galectin-1 (by stable transfection of an expression vector containing the antisense mRNA of galectin-1) had longer survival periods than those grafted with U87 or U373 cells expressing normal levels of galectin-1 . Galectin-1 added to the culture media markedly and specifically increased cell motility levels in human neoplastic astrocytes . These effects are related to marked modifications in the organization of the actin cytoskeleton and the increase in small GTPase RhoA expression . All the data obtained indicate that galectin-1 enhances the migratory capabilities of tumor astrocytes and, therefore, their biological aggressiveness. J Neurochem, 2002 Jul, 82(2), 240 - 8 An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells; Nunez-Millacura C et al.; Intracellular reactive iron is a source of free radicals and a possible cause of cell damage . In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons . Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool . The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant . When iron in the culture medium increased from 1 to 40 microm, IRP2 activity decreased to nil . In contrast, IRP1 activity decreased when iron increased up to 20 microm, and then, unexpectedly, increased . IRP1 activity at iron concentrations above 20 microm was functional as it correlated with increased (55) Fe uptake . The increase in IRP1 activity was mediated by oxidative-stress as it was largely abolished by N-acetyl-L-cysteine . Culturing cells with iron resulted in proteins and DNA modifications . In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media . As a consequence, iron accumulated and generated oxidative damage . This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death. Biochem Pharmacol, 2002 Jul 15, 64(2), 267 - 76 Novel mechanism of Vitamin E protection against cyclosporine A cytotoxicity in cultured rat hepatocytes; Andres D et al.; Cyclos