Nephron, 1996, 73(2), 273 - 9 Effect of endothelin 1 on fibrinolysis and plasminogen activator inhibitor 1 synthesis in rat mesangial cells; Iwamoto T et al.; Endothelins (ETs) have been known to have a variety of biological functions such as mitogenic stimulation, natriuresis and the stimulation of the proteolytic activity in addition to vasoconstrictive action, which may participate in the process of glomerular diseases pathophysiologically . In this study, the effects of ET-1 on fibrinolysis, plasminogen activator inhibitor 1 (PAI-1) synthesis and PAI-1 mRNA expression were examined in cultured rat mesangial cells (MCs) . The addition of ET-1 (10(-11) to 10(-7) M) into MC cultures reduced fibrinolytic activities assayed by fibrin autography in a dose-dependent manner . For the assay of PAI-1 synthesis, MC culture media metabolically labeled with 35S-methionine were analyzed by SDS-PAGE with fluorography and immuno-precipitation using rabbit antirat PAI-1 antibody . Exposure to ET-1 for 24 h produced a clear dose-dependent effect on the PAI-1 release from the MCs . PAI-1 mRNA expression was also enhanced in parallel with the concentration of ET-1 in the conditioned media . These findings indicate that ET-1 participates in fibrinolysis and the PAI-1 synthesis by MCs, which may thus regulate the degradation of the extracellular matrix in the glomerular microenvironment. Zhonghua Yi Xue Za Zhi, 1996 Jan, 76(1), 41 - 4 {Effects of insulin and captopril on growth of glomerular mesangial cells in STZ-induced diabetic rats}; Geng J et al.; OBJECTIVE: To observe the effects of insulin and captopril on growth of mesangial cells and on production of interleukin-6 (IL-6) and tumor necrosis factor (TNF) in culture media of glomerular cells . METHOD: Streptozocin induced diabetic rats (DM rats) were compared with normal wistar rats . RESULTS: The CPM value of 3H-TdR incorporated into mesangial cells in DM rats was significantly higher than that in Wistar rats . When the culture media was added by 5 x 10(-4) U/ml, 5 x 10(-3) U/ml and 5 x 10(-1) U/ml of insulin, the CPM value incorporated into mesangial cells and the concentration of IL-6 in the culture media of both DM and wistar rats were significantly higher than those of the control (no insulin) . When captopril (10(-6) mol/L, 10(-4) mol/L, 10(-3) mol/L) was added into the media in different concentration, the cpm value of 3H-TdR and the concentration of TNF in DM rats were significantly lower than those of the control (no captopril) . But the CPM value of 3H-TdR in Wistar rats was lower than that in the control, which was only seen in the concentration of 10(-3)M captopril . CONCLUSION: Ins could promote 3H-TdR in corporation and increase the production of IL-6, and captopril inhibited 3H-TdR in corporation and decrease the production of TNF. Reprod Fertil Dev, 1996, 8(2), 259 - 66 Bovine granulosa cells express extracellular matrix proteins and their regulators during luteinization in culture; Zhao Y et al.; This study investigated the ability of bovine granulosa cells to express and secrete collagen, metalloproteinase (MMP) activity and a tissue inhibitor of metalloproteinase (TIMP-1) during luteinization in vitro . Cells from mature (1-2 mL fluid volume) bovine follicles were cultured over 4 days in serum-free medium . Their luteinization during culture was confirmed by a 10-fold increase in progesterone secretion . Samples of cell extracts, culture media and follicular fluid were subjected to Western blotting to identify secreted proteins and to gelatin zymography to detect enzyme activity . Poly A+ RNA, isolated from cells before and after culture, was probed to detect expression of collagen alpha 1(I), collagen alpha 3(IV) and TIMP-1 . The results revealed that: (1) the collagen alpha 1(I) subunit gene was expressed in cells before culture but with greater intensity by Day 4 culture; collagen I protein, on the other hand, was not detectable in culture medium; (2) the collagen alpha 3(IV) subunit gene was expressed at a low level in uncultured cells and could be detected on Day 4 of culture; low amounts of the protein were detected in medium; (3) a 92-kDa band of gelatinase activity (presumed MMP-9) was present in all medium samples, together with bands of unidentified activity; and (5) the TIMP-1 gene was expressed in uncultured cells but its expression increased markedly up to Day 4 of culture . These results show that granulosa luteinization is associated with an increase in the expression of collagen, collagen-degrading enzymes and TIMP-1 . Collagen protein, however, may be only poorly synthesized in this culture model . The results suggest that granulosa-derived cells are a likely source of components of the extracellular matrix during post-ovulatory remodelling of early luteal tissue. Reprod Fertil Dev, 1996, 8(2), 231 - 4 Significant decrease in parathyroid hormone-related protein concentrations in amniotic fluid with labour at term but not preterm; Mitchell MD et al.; It has been determined whether amniotic fluid concentrations of parathyroid hormone-related protein (PTHrP) change with labour . An evaluation of which cells from intrauterine tissues might produce PTHrP has also been conducted . Amniotic fluid was obtained by amniocentesis from women: (1) at term, not in labour; (2) in normal term labour; (3) in preterm labour, undelivered within one week; (4) in preterm labour, delivered within one week; (5) in preterm labour associated with clinical chorioamnionitis; and (6) who were gestation-matched controls for chorioamnionitis patients-women in this group were similar to those in Group 4 but were different patients . Amnion, chorion, and decidual cells were grown by standard techniques and incubated with interleukin-1 beta (IL-1 beta) . PTHrP was assayed in duplicate samples of amniotic fluid or tissue culture media using an immunoradiometric assay . There was a significant reduction in amniotic fluid concentrations of PTHrP during labour at term . Preterm labour was not associated with significant changes in amniotic fluid concentrations of PTHrP although a trend for reduced concentrations was observed . Amnion and chorion produced measurable quantities of PTHrP and rates of production were increased by treatment with IL-1 beta . Decidual cells did not produce detectable amounts of PTHrP . Hence, labour at term is associated with a decrease in amniotic fluid PTHrP concentrations that may reflect reduced amnion production, which in turn may play a permissive or active role in the mechanism(s) of parturition . These data support the view that the mechanisms that control term and preterm labour may be regulated differently. Brain Res Mol Brain Res, 1996 Jan, 35(1-2), 309 - 13 Antisense oligonucleotide to NOR-1, a novel orphan nuclear receptor, induces migration and neurite extension of cultured forebrain cells; Ohkura N et al.; We previously identified a novel orphan nuclear receptor referred to as NOR-1 from rat forebrain cells . This study examined the role of NOR-1 in primary cultured forebrain cells by selectively inhibiting NOR-1 expression by addition of antisense oligonucleotide to the culture media . Treating cells with the antisense oligomer resulted in the following dramatic morphological changes: (i) cell migration, (ii) extension of processes, and (iii) formation of cellular aggregates . Immunocytochemistry for microtubule-associated protein 2 revealed that the processes were filled with neurites growing from neuronal cells . These findings suggest that NOR-1 may be involved in the molecular mechanisms regulating neural differentiation. Osteoporos Int, 1996, 6(2), 111 - 9 Positive interaction between 17 beta-Estradiol and parathyroid hormone in normal human osteoblasts cultured long term in the presence of dexamethasone; Rao LG et al.; We previously developed two models of human osteoblasts with distinct differentiation stages using cells derived from iliac crest trabecular bone explants cultured long term in the presence (HOB + DEX) and absence (HOB - DEX) of 10 nM dexamethasone (DEX) (Wong et al., J Bone Miner Res 1990;5:803) . Using these models from 36 subjects aged 41-80 years, we examined the effects of 17 beta-estradiol (E2) on cell proliferation, osteocalcin (OC) production, alkaline phosphatase (ALP) and basal and parathyroid hormone (PTH)-stimulated adenylate cyclase activities, as well as the steady-state mRNA levels of ALP, collagen type I(COLL), OC, and receptors for E2 (ER) and PTH (PTHr) . E2 alone had no effect on {3H}thymidine uptake in (HOB - DEX) cells but appeared to stimulate the uptake in (HOB + DEX) cells in a dose-dependent manner, with maximum effect at 10(-10)M (p < 0.05) . However, in the presence of 10(-6)M PTH, E2 inhibited the uptake in (HOB - DEX) cells (ANOVA, KW = 18.95, p < 0.005) but stimulated the uptake in (HOB + DEX) cells (KW = 13.52, p < 0.025) . E2 decreased the amount of osteocalcin in culture media from both (HOB - DEX) and (HOB + DEX) cells (p < 0.05) . PTH alone or E2, alone or in combination with 10(-9)M PTH, had no effect on ALP activity in (HOB - DEX) cells . In contrast, in (HOB + DEX) cells, E2 + PTH but not E2 alone, had biphasic effects on ALP activity, with maximum stimulation observed at 10(-11) and 10(-10)M E2, and a return to basal levels at 10(-9)M E2 . E2 decreased basal adenylate cyclase activities in a dose-dependent manner in (HOB + DEX) but not (HOB - DEX) cells (KW = 13.48, p < 0.05) . In (HOB + DEX) cells, E2 had biphasic effects on PTH-stimulated adenylate cyclase activity, with significant stimulation observed at 10(-10)M (p < 0.05) . While E2 had no significant effect on osteoblastic marker mRNA levels in (HOB - DEX) cells, it decreased osteocalcin and stimulated PTHr mRNA levels in (HOB + DEX) cells . Thus, in our human osteoblastic cell models, estrogen regulated metabolic function largely in the more differentiated cells, by modifying the effects of PTH. Eur J Clin Invest, 1996 Jan, 26(1), 30 - 7 Altered release of endothelin-1,2 and thromboxane B2 from trophoblastic cells in pre-eclampsia; Cervar M et al.; The aim of the study was to investigate whether pre-eclampsia is associated with an altered release of vasoactive substances from trophoblastic cells in vitro . Trophoblastic cells from 15 uncomplicated control pregnancies and 18 pre-eclamptic pregnancies at preterm (weeks 31-36; n = 12) and term (weeks 37-40; n = 21) were cultured for 5 days . The concentrations of angiotensin II (AII), endothelin-1,2 (ET-1,2), thromboxane B2 (TXB2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and leukotriene B4 (LTB4) were measured daily in culture media for 5 days by radioimmunoassay . In pre-eclampsia, concentrations of ET-1,2 were decreased (P < 0.01) at both preterm and term, TXB2 concentrations were increased (P < 0.05) only at preterm and the TXB2-6-keto-PGF1 alpha ratio was increased at both preterm and term (P < 0.01) as compared with the controls . Concentrations of AII, 6-keto-PGF1 alpha and LTB4 were similar to the controls . The data suggest that pre-eclampsia is associated with a decreased release of ET-1 and an increased release of TXB2 from trophoblastic cells in vitro. Appl Biochem Biotechnol, 1996 Spring, 57-58, 413 - 22 Culture of the astaxanthinogenic yeast Phaffia rhodozyma in low-cost media; Fontana JD et al.; Growth of the yeast Phaffia rhodozyma was carried out in a simplified medium based on less expensive nutrient sources, such as diluted sugar cane juice, urea, and sodium phosphate . The usual content of the astaxanthin, an oxygenated pink carotenoid useful for fish flesh staining, was improved along with with good cell yields (respective values of > 1300 micrograms/g cells and > 5 g cells/L were observed) . Yeast invertase and urease must therefore play an important role in the implementation of low-cost culture media. Cell Transplant, 1996 Jan-Feb, 5(1), 1 - 12; discussion 13-7, 19 Survival and function of islets during culture; Clayton HA et al.; Cell and tissue culture techniques have improved considerably since the first attempts to maintain explants of animal tissue in vitro . The two major developments that have allowed these improvements are the ability to produce continuous cell lines, thus allowing reproducible results to be obtained, and the definition of media for different cell types, thereby reducing the need for supplements of serum and other extraneous extracts . The requirements of islets in culture have been more difficult to define, largely because islets do not proliferate in culture and proliferation rate cannot therefore be used to measure the suitability of the medium . Further difficulties arise because islets are highly metabolically active "mini-organelles." Although many studies have been undertaken to try and optimize media for the culture islets of Langerhans, the media most commonly used are commercially available media developed for other cell types . There remains ample scope for further refinement of the composition of islet culture media, with the possibility of different media for islets from different species. Basic Res Cardiol, 1996 Jan-Feb, 91(1), 79 - 85 The ability of heat stress and metabolic preconditioning to protect primary rat cardiac myocytes; Cumming DV et al.; Primary rat cardiocytes were subjected to either thermal "preconditioning" for 30 min at 43 degrees C or 20 min metabolic "preconditioning" (10 mM deoxyglucose, 20 mM lactate, pH 6.5) . Eighteen hours later cells were analysed either for hsp 70i expression or subjected to a subsequent lethal heat stress or simulated ischaemia (10 mM deoxyglucose, 20 mM lactate, 0.75 mM sodium dithionite, 12 mM potassium chloride, pH 6.5) for 2 hours and assessed for survival by trypan blue exclusion . Hsp 70i was induced over 100 fold by thermal "preconditioning" and 30 fold by metabolic "preconditioning" (p < 0.001, p < 0.05), hsp 90 was induced 2.71 fold and 2.24 fold (p < 0.001, p < 0.001) by thermal and metabolic "preconditioning" respectively, while hsp 60 was no induced by either treatment . Preconditioned cultures had improved survival against subsequent lethal heat stress or simulated ischaemia: Thermal "preconditioning" reduced death from 69.22% to 52.46% upon subsequent "lethal" heat stress and from 49.13% to 36.66% upon subsequent "lethal" simulated ischaemia . Metabolic "preconditioning" reduced cell death from 51.29% to 33.8% against subsequent "lethal" heat stress, and from 69.09% to 55.61% upon subsequent "lethal" simulated ischaemia . A second marker of cell death, the release of lactate dehydrogenase activity into the culture media, was reduced to 65% and 60% of control values for thermally preconditioned cells subjected to "lethal" heat or "lethal" simulated ischaemia respectively . Metabolically "preconditioned" cells demonstrated lactate dehydrogenase activity of 59% and 51% that of control values, when subjected to "lethal" heat or "lethal" simulated "ischaemia" respectively. Comp Immunol Microbiol Infect Dis, 1996 Jan, 19(1), 55 - 63 Brucella abortus differs in the multiplication within bovine chorioallantoic membrane explants from early and late gestation; Samartino LE et al.; The ability of Brucella to infect and grow within extraplacentomal chorioallantoic explants (CAMs) derived from early and late gestational cattle was compared . Following inoculation of CAMs with equal numbers of strain 2308 B . abortus, the infectivity was approximately the same in CAMs from both ages, however, bacterial replication was significantly greater in late gestational CAMs than in early gestational CAMs . Co-culture of both early and late gestation CAMs or culture of both types of CAMs in the presence of tissue culture media collected from either early or late B . abortus inoculated CAMs failed to alter B . abortus growth rates and/or cytopathic effects. Life Sci, 1996, 58(1), 75 - 82 Opioids inhibit dopamine secretion from PC12 rat pheochromocytoma cells in a naloxone-reversible manner; Venihaki M et al.; Opioids inhibit the release of catecholamines in the nervous system . Normal adrenal chromaffin cells produce delta opioids and they respond to them by suppressing the release of their catecholamines . Chromaffin cell tumors, the pheochromocytomas, produce mainly kappa opioids . The aim of this work was: (a) to test if pheochromocytomas retain the response of normal chromaffin cell catecholamines to delta opioids and to naloxone (a general opioid antagonist), and (b) to test if kappa opioids exert any specific effect on catecholamine release from these tumors . Since we have previously shown that, in common with human pheochromocytomas, the PC12 rat pheochromocytoma cells express the prodynorphin gene and secret its kappa opioid products, we used these cells to examine the effect of several opioid agonists and of naloxone on basal, nicotine-, and KCl-induced dopamine release . Dopamine is the main PC12 catecholamine . We have found that the specific kappa opioid agonist U-69593 inhibited the release of dopamine in a dose-dependent manner (IC50=0.5 x 10(-8)M) . Under basal conditions the mean concentration of dopamine in the culture media was 11.25 +/- 0.57 ng/mg of total cellular protein (n=13) . A 30 min exposure to U-69593 at 10(-6) M suppressed basal dopamine release to 58 +/- 2% (n=7) of controls . A 12 hr pre-incubation with U-69593 caused the same degree of suppression . The effect of the synthetic kappa opioid agonist dynorphin A was indistinguishable from that of U-69593 . DADLE (a mu and delta synthetic opioid agonist) was significantly less effective in suppressing dopamine release (IC50=10(-7)M) . The concentration of dopamine following exposure to 10-6 M of DADLE for 30 min was 74 +/- 5% of the controls (n=4) . The mu opioid agonist DAGO was ineffective . The suppressive effect of all opioid agonists was blocked by naloxone suggesting that conventional opioid receptors were involved. Jpn J Cancer Res, 1996 Jan, 87(1), 91 - 7 Evaluation of the relative cytotoxic effects of anticancer agents in serum-supplemented versus serum-free media using a tetrazolium colorimetric assay; Tsai CM et al.; Most cell culture and in vitro drug sensitivity assays utilize serum-supplemented media (SSM) . However, fully defined serum-free media (SFM) offer several advantages and are being used increasingly for initiation and maintenance of cell cultures . Because serum inhibits the in vitro cytotoxicity of certain antineoplastic agents, we investigated the inter-relationships between medium type, cell proliferation and cytotoxic effect . Twenty-four human lung cancer cell lines were tested with nine anticancer agents in both media types . A semi-automated tetrazolium (MTT) colorimetric assay was used for assaying cell survival . Cell lines initiated and maintained in SFM preferentially proliferated in that medium type or proliferated equally well in both media types . In contrast, cell lines established in SSM varied considerably in their medium of preference . There appeared to be a direct correlation or trend between cell proliferative rate and cytotoxicity of all drugs with the possible exceptions of methotrexate and carmustine . In general, the cell lines were more sensitive to anticancer agents when they were exposed in the culture medium in which they preferentially proliferated . Therefore, to determine the influence of culture media on cytotoxicity, we analyzed the data only from lines that replicated equally efficiently in both media . After correction for cell proliferative rate, SSM had a negative effect on the cytotoxic action of some drugs (especially methotrexate, 5-fluorouracil and, to a lesser extent, mitomycin-C) . Our results demonstrate that fully defined SFM may be suitable for initiating cell lines and for use in in vitro cytotoxicity assays for selection of individualized therapy or for screening of new anti-neoplastic agents, and thus may increase the number of antineoplastic agents that can be tested satisfactorily. Circ Res, 1996 Jan, 78(1), 44 - 9 Regulation of matrix metalloproteinases and plasminogen activator inhibitor-1 synthesis by plasminogen in cultured human vascular smooth muscle cells; Lee E et al.; Plasmin and matrix metalloproteinases (MMPs) both participate in extracellular matrix remodeling . This study examined the effects of tumor necrosis factor-alpha (TNF-alpha) and plasminogen on collagenase, stromelysin, and plasminogen activator inhibitor-1 (PAI-1) synthesis of collagenase and stromelysin, which remained predominantly in proenzyme forms, as determined by Western analysis of culture media . In contrast, plasminogen and plasmin not only increased secretion of MMPs but also induced cleavage to their active forms . The serine protease inhibitor aprotinin inhibited this activation of MMPs by plasminogen and plasmin . TNF-alpha reduced plasminogen-induced activation of MMPs, suggesting induction of an inhibitor or plasmin generation, such as PAI-1 . Enzyme-linked immunosorbent assay of culture media showed that TNF-alpha (10 ng/mL) increased PAI-1 secretion by 4.2 fold compared with control (105.5 +/- 9.6) versus 24.9 +/- 1.7 ng/mL, n = 3) . Surprisingly plasminogen also increased PAI-1 secretion by vascular SMCs (3.6-fold over control) . These results demonstrate coordination of cytokines and serine proteases in regulating MMP secretion and activation . In addition, the induction of PAI-1 by TNF-alpha and plasminogen suggests a negative feedback mechanisms limit both plasmin-mediated and MMP-mediated matrix degradation. J Surg Res, 1996 Jan, 60(1), 156 - 62 Differential expression of IGFBPs by normal and hypertrophic scar fibroblasts; Hathaway CL et al.; Insulin-like growth factor-I (IGF-I) is a potent fibroblast mitogen which influences wound healing . IGF action is regulated by a family of six IGF-binding proteins (IGFBPs) . The purpose of this study was to determine if expression of IGFBPs is altered in hypertrophic scarring, a wound-healing condition commonly associated with deep dermal injury . Fibroblast populations from the superficial and deep dermal layers of normal human skin (SN and DN, respectively) and from superficial and deep layers of hypertrophic scars (SSc and DSc, respectively) were established and cultured in serum-free medium with or without several growth factors known to modulate wound healing, including basic fibroblast growth factor, the BB isoform of platelet-derived growth factor, IGF-I, and transforming growth factor-beta (TGF-beta) . IGFBP release was analyzed by radioligand blot assays of culture media . Two main forms of IGFBPs were released, IGFBP-3 and a 24-kDa form which comigrated with serum IGFBP-4 . DSc fibroblasts accumulated significantly more IGFBP-3 into serum-free culture medium than did SN, DN, or SSc fibroblasts in all conditions except TGF-beta treatment and confluence . Additionally, comparisons of IGFBP-3 release by each cell type with and without TGF-beta revealed TGF-beta stimulated IGFBP-3 accumulation by SN and DN fibroblasts but not by SSc or DSc fibroblasts . DN and DSc fibroblasts accumulated significantly more of the 24-kDa IGFBP species than SN or SSc fibroblasts in all conditions except TGF-beta or IGF-I treatment . These findings indicate that superficial and deep dermal fibroblasts are heterogeneous with respect to IGFBP release, and suggest that hypertrophic scar fibroblasts may represent a population of cells with regulatory properties distinct from those of normal dermal fibroblasts. Arch Biochem Biophys, 1996 Jan 1, 325(1), 20 - 8 Superoxide scavenging by Mn(II/III) tetrakis (1-methyl-4-pyridyl) porphyrin in mammalian cells; Gardner PR et al.; The superoxide dismutase mimic Mn(II/III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn(II/III)TMPyP) was examined for its superoxide radical (O2.-)-scavenging ability in cultured mammalian cells . Mn(III)TMPyP (< 5 microM) added to culture media relieved growth inhibition and decreased the inactivation of the O2(.-)-sensitive enzyme aconitase in cells exposed to the O2(.-)-generating phenazine pyocyanine . Treatment of cells with Mn(III)TMPyP did not measurably affect cellular O2.- production as revealed by rates of cyanide-resistant respiration with or without added pyocyanine . In contrast, Mn(II/III)TMPyP enhanced O2.- production in cells when the redox-active naphthoquinone menadione was present as measured by both increased cyanide-resistant respiration rates and aconitase inactivation . In vitro, Mn(II/III)TMPyP catalyzed the oxidation of ascorbate, and menadione enhanced this effect . Mn(III)TMPyP did not protect aconitase when O2.- production was elicited in mitochondria by antimycin A and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone . The results support a reductant-O2.-:oxidoreductase mechanism for O2.- scavenging by Mn(II/III)TMPyP in the mammalian cytosol as proposed for its action in Escherichia coli, but also indicate that Mn(II/III)TMPyP can either scavenge or produce O2.- in cells depending upon the prevailing pathways of Mn(II/III)TMPyP oxidation-reduction. J Bacteriol, 1996 Jan, 178(1), 54 - 60 The ornithine decarboxylase gene odc is required for alcaligin siderophore biosynthesis in Bordetella spp.: putrescine is a precursor of alcaligin; Brickman TJ et al.; Chromosomal insertions defining Bordetella bronchiseptica siderophore phenotypic complementation group III mutants BRM3 and BRM5 were found to reside approximately 200 to 300 bp apart by restriction mapping of cloned genomic regions associated with the insertion markers . DNA hybridization analysis using B . bronchiseptica genomic DNA sequences flanking the cloned BRM3 insertion marker identified homologous Bordetella pertussis UT25 cosmids that complemented the siderophore biosynthesis defect of the group III B . bronchiseptica mutants . Subcloning and complementation analysis localized the complementing activity to a 2.8-kb B . pertussis genomic DNA region . Nucleotide sequencing identified an open reading frame predicted to encode a polypeptide exhibiting strong similarity at the primary amino acid level with several pyridoxal phosphate-dependent amino acid decarboxylases . Alcaligin production was fully restored to group III mutants by supplementation of iron-depleted culture media with putrescine (1,4-diaminobutane), consistent with defects in an ornithine decarboxylase activity required for alcaligin siderophore biosynthesis . Concordantly, the alcaligin biosynthesis defect of BRM3 was functionally complemented by the heterologous Escherichia coli speC gene encoding an ornithine decarboxylase activity . Enzyme assays confirmed that group III B . bronchiseptica siderophore-deficient mutants lack an ornithine decarboxylase activity required for the biosynthesis of alcaligin . Siderophore production by an analogous mutant of B . pertussis constructed by allelic exchange was undetectable . We propose the designation odc for the gene defined by these mutations that abrogate alcaligin siderophore production . Putrescine is an essential precursor of alcaligin in Bordetella spp. Postgrad Med, 1996 Jan, 99(1), 123 - 8, 131-2 Atypical pneumonia . Extrapulmonary clues guide the way to diagnosis; Cunha BA et al.; In atypical pneumonia, causative organisms are difficult to isolate, so careful clinical assessment is essential in arriving at a working diagnosis . Definitive diagnosis through serologic testing is usually retrospective . Either a high initial titer or a fourfold or greater rise between the acute and convalescent titer is considered diagnostic in a patient with compatible illness . Legionella and mycoplasma organisms may be cultured from respiratory secretions if plated on appropriate culture media . Using a syndromic approach, physicians can almost always differentiate typical from atypical community-acquired pneumonia and narrow diagnostic possibilities among the atypical pathogens, making possible institution of early, possibly lifesaving, empirical therapy. Circulation, 1995 Dec 15, 92(12), 3513 - 9 Exposure to shear stress alters endothelial adhesiveness . Role of nitric oxide; Tsao PS et al.; BACKGROUND: Shear stress increases the release of nitric oxide (NO) by endothelial cells (ECs) . We and others have provided evidence that endothelium-derived NO inhibits monocyte adhesion to the vessel wall . We therefore hypothesized that previous exposure to shear stress would inhibit endothelial adhesiveness for monocytes by virtue of its effect to increase NO release . METHODS AND RESULTS: Confluent monolayers of bovine aortic endothelial cells, human aortic endothelial cells, or human venous endothelial cells were exposed to laminar fluid flow . Culture media were collected for measurement of NO (by chemiluminescence) and the prostacyclin metabolite 6-keto-prostaglandin F1 alpha . NOx and 6-keto-prostaglandin F1 alpha accumulated in the conditioned medium during laminar fluid flow from 30 minutes to 24 hours in a time-dependent fashion . In another set of studies, ECs previously exposed to flow or to static conditions were washed with Hanks' buffer and exposed to THP-1 cells for 30 minutes . Adherent cells were counted by microscopy . Previous exposure to flow reduced endothelial adhesiveness for monocytes by 50% (P < .05) . The effect of flow on endothelial adhesiveness occurred within 30 minutes . This effect was abrogated by nitro-L-arginine (an antagonist of NO synthesis), as well as by tetraethylammonium ion (an antagonist of the flow-activated potassium channel); the effects of these inhibitors were reversed by the NO donor SPM-5185 . Although the cyclo-oxygenase inhibitor indomethacin totally inhibited the flow-induced production of prostacyclin by ECs, it minimally affected adherence of THP-1 cells . The early effect of flow on endothelial adhesiveness was not mediated by alterations in the expression of the endothelial adhesion molecules VCAM-1 or ICAM-1 as assessed by fluorescent activated cell sorting . CONCLUSIONS: Shear stress alters endothelial adhesiveness for monocytes; at early time points, this effect is largely due to flow-stimulated release of NO and, to a lesser extent, prostacyclin . This effect of flow occurs within 30 minutes and is probably due to alterations in the signal transduction or activation state (rather than the expression) of endothelial adhesion molecules. Southeast Asian J Trop Med Public Health, 1995 Dec, 26(4), 673 - 6 Anti-dengue IgG detection by an indirect ELISA; Tio PH et al.; Protein-free culture media were originally developed for hybridomas to simplify downstream processing and purification . For the same reasons, we have used these protein-free media for passaging dengue 2 virus in C6/36 cells . This provided us with an infected supernatant (DenPF) which could then be used as coating antigens for an indirect enzyme-linked immunosorbent assay (ELISA) to determine dengue IgG levels . Using this preparation, the main immunogenic band as seen by immunoblot appeared to be viral envelope protein (E) . Without the high concentrations of "competing" proteins from fetal calf serum (FCS), the Den2PF could be directly coated onto 96-well ELISA plates . The amount of viral proteins in Den2PF appeared to be sufficient so that there was no need for further purification steps, eg polyethylene glycol (PEG) precipitation, which made this preparation cost effective . It compared favorably with the dengue dot enzyme immunoassay (DEIA; sensitivity of 95.7% and specificity of 95.2%). Virus Res, 1995 Dec, 39(2-3), 165 - 79 Characterization of revertants of a Sindbis virus 6K gene mutant that affects proteolytic processing and virus assembly; Ivanova L et al.; Alphaviruses of the Togaviridae encode a small hydrophobic polypeptide of 55 amino acids, noted as the 6K protein, that is synthesized as part of a polyprotein containing the sequences of the two major transmembranal viral structural glycoproteins . Mutations, insertions and deletions in the 6K appear to selectively interfere with the final stages of virus assembly and budding, producing aberrant, multi-cored infectious viruses . In addition, some of these mutations were pleiotropic and much more inhibitory to virus formation . One of the latter, a substitution of alanine in the wild-type Sindbis virus 6K gene by arginine, has been studied further and shown to interfere with normal proteolytic processing of the polyprotein . Cells infected with this mutant but not the wild-type virus also displayed viral antigens in nuclear membranes and released fragments of membranes into the cell culture media . A revertant, obtained by enrichment for a faster growing strain, 'suppressed' these defects and genetic mapping showed that the arginine codon had been modified to encode a methionine . However, the sequence of the 6K protein in this revertant was not wild-type and the revertant was still defective in assembly as demonstrated by formation of aberrant particles . A complete restoration of wild-type particle formation for this revertant could be effected by modifying the E2 glycoprotein sequence. In Vitro Cell Dev Biol Anim, 1995 Dec, 31(11), 876 - 9 Production of ethanol by cultured insect cells; Takahashi M et al.; Proton Nuclear Magnetic Resonance (1H-NMR) Analysis of insect cell culture media used for cultivating insect cell lines derived from the fleshfly Sarcophaga peregrina, swallowtail butterfly Papilio xuthus, and cabbage armyworm Mamestra brassicae revealed that ethanol appeared in the medium as the cultures aged . By incorporating {13C-1}-glucose into the media, we pursued 13C-NMR spectrograms to show that the ethanol was derived from glucose . Thus, it became evident that the insect cells cultured in vitro produce ethanol from glucose as a metabolite. Hum Reprod, 1995 Dec, 10(12), 3248 - 54 Zinc is a possible toxic contaminant of silicone oil in microdrop cultures of preimplantation mouse embryos; Erbach GT et al.; A batch of silicone oil (dimethylpolysiloxane) is described which had differential effects on the development of 1- and 2-cell preimplantation mouse embryos in vitro when used as a microdrop overlay over two culture media: CZB and KSOM . A high rate of development into blastocysts was observed when using CZB medium; in contrast, development was strongly inhibited when KSOM was used . Other batches of silicone oil or paraffin oil permitted development from the zygote to the blastocyst of an outbred strain of mouse without arrest at the 2-cell stage . Our results show that the higher concentrations of ethylenediaminetetraacetic acid (EDTA) and bovine serum albumin (BSA) in CZB medium, in comparison with KSOM, protect against the toxic component in the oil . Observations also gave circumstantial evidence that the toxic component in the oil is zinc . The beneficial effect of including EDTA in a medium is usually attributed to its chelating toxic metals introduced as impurities in other components of the medium . Our results now show that EDTA also protects against impurities in the oil overlay. Hum Cell, 1995 Dec, 8(4), 195 - 201 {Establishment of a new human endometrial adenocarcinoma cell line, Watanabe cells, containing estrogen receptor}; Satoh T et al.; A new human endometrial adenocarcinoma cell line, Watanabe cells, was established from the ascitic fluid of a relapsed endometrial adenocarcinoma obtained from a 58-year-old woman; this cell line has been maintained in vitro for more than 3 years and 8 months . The cells formed a monolayer in a mosaic fashion and tended to pile up and formed a hemicyst . The population boubling time was 60.0 hours at the 10th generation . The modal chromosomal number of the cells was in the diploid range . The histology of tumors induced by this cell line in athymic nude mice showed poorly differentiated adenocarcinoma, while the initial tumor was a well differentiated adenocarcinoma . Estrogen and progesterone receptors (ER, PR) were demonstrated in the original tumor, whereas ER but not PR were present in the tumors induced in nude mice . CA125, CA19-9 and other tumor markers were positive in culture media of this cell line . The cells showed intrinsic cisplatin-resistance (50% inhibition concentration: > 10 micrograms/ml) at 120 hours of exposure by MTT assay . We believe this cell line will be useful for investigating the mechanisms of progesterone therapy, the biological behaviors of the tumor markers and mechanisms of chemotherapeutic resistance in endometrial carcinoma. J Vet Med Sci, 1995 Dec, 57(6), 1125 - 8 Protective effect of the combined vaccine prepared from cell-free-antigen of Actinobacillus pleuropneumoniae serotypes 1, 2 and 5 in pigs; Oishi E et al.; Cell-free-antigens prepared from a concentrated culture supernatant of Actinobacillus pleuropneumoniae (A . pleuropneumoniae) serotypes 1, 2 and 5 were mixed and emulsified with oil adjuvant . The combined vaccine of these 3 serotypes of A . pleuropneumoniae was tested for its ability to confer protection . Pigs immunized with the combined vaccine survived and showed no clinical signs against an intratracheal challenge with A . pleuropneumoniae . In contrast, control pigs inoculated with concentrated culture media emulsified with oil adjuvant developed typical symptoms of pleuropneumonia after challenge inoculation. P R Health Sci J, 1995 Dec, 14(4), 293 - 6 Inhibition of hematopoiesis by a plasma factor in a case of aplastic anemia associated with systemic lupus erythematosus; Marques JA et al.; Systemic Lupus Erythematosus (SLE) may be associated with inhibition of hematopoiesis mediated by antibodies, T-cells or both . A 41-year-old woman with a five-year history of SLE treated with prednisone was admitted to Cabrini Medical Center in New York . The patient complained of fever, chills, arthralgias, general malaise, weakness and dyspnea on exertion, and showed malar rash, pallor, and a systolic ejection murmur along the left sternal border . Admission work up included a CBC with evidence of moderate pancytopenia, a normal EKG, and a normal chest X-ray . The patient's anemia was symptomatic and required a transfusion of packed red blood cells (PRBC's) . Bone marrow biopsy and aspiration revealed an aplastic marrow with few hypoplastic islands of hematopoietic elements . The patient was treated with plasmapheresis, achieving immediate progress towards recovery . Bone marrow culture studies (erythroid BFU-E, and myeloid CFU-GM) were done by incubating various titers of the patient's acute phase plasma with normal bone marrow cells . This was done to determine if the patient's plasma contained any hematopoietic inhibitory activity, as has been reported in other cases . Our experiments demonstrated marked inhibition of erymathropoiesis and myelopoiesis in vitro, when various titers of the patient's plasma were included in the culture media . Control plasma produced no inhibition . These studies support the hypothesis that a circulating antibody which inhibits hematopoiesis may be produced in SLE patients with aplastic anemia, and be responsible for it. Am J Reprod Immunol, 1995 Dec, 34(6), 381 - 5 IL-1 beta, TNF-alpha, and IL-2 in peritoneal fluid and macrophage-conditioned media of women with endometriosis; Keenan JA et al.; PROBLEM: The presence of various cytokines in human peritoneal fluid has been incompletely evaluated . Changes in cytokine levels may be related to the development of endometriosis, infertility, and activation of peritoneal macrophages . This study assesses levels of IL-1 beta, IL-2 and TNF- alpha in peritoneal fluid and macrophage conditioned media of women with endometriosis . METHOD: Peritoneal fluid was collected from 51 women at the time of diagnostic or operative laparoscopy for benign gynecologic disease . Peritoneal macrophages were isolated, cultured for 24 h, and the culture media collected . IL-1 beta, IL-2, and TNF- alpha levels were determined by commercial ELISA kits . RESULTS: The mean concentration of IL-1 beta and TNF- alpha was significantly higher in macrophage conditioned media of patients with endometriosis (P < 0.02) . However, there were no significant changes in peritoneal fluid cytokine levels . Peritoneal macrophage concentrations were also higher in patients with endometriosis . CONCLUSION: This study supports the concept that endometriosis is associated with activation of peritoneal macrophages, and a higher concentration of these cells . This activation is reflected by the increased levels of cytokines found in macrophage conditioned media . The absence of significant changes in peritoneal fluid cytokine levels would seen to indicate that the above derangements are not responsible for the development or progression of endometriosis. Insect Biochem Mol Biol, 1995 Dec, 25(10), 1115 - 9 Developmental studies on Drosophila melanogaster glutathione S-transferase and its induction by oxadiazolone; Hunaiti AA et al.; Glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene was detected in various developmental stages of Drosophila melanogaster . The specific activity of the enzyme was 110, 35, 25 and 15 nmol/min/mg protein in crude extracts prepared from eggs, larvae, pupae and adult stages respectively . The enzymes from larval, pupal and adult stages were purified and compared . Incorporation of the widely used herbicide oxadiazolone at concentrations of 375 and 563 part/million into the culture media caused 4- and 2.5-fold increase in the enzyme activity in pupal and adult stages respectively. Rinsho Byori, 1995 Dec, 43(12), 1251 - 5 {Detection of Mycoplasma pneumoniae by using polymerase chain reaction and nonradioactive DNA probes}; Yamashita K et al.; Mycoplasma pneumoniae is the causative agent of primary atypical pneumoniae, and two distinct groups (I and II) have been established . Serological tests are relatively insensitive and the diagnosis by culture is time-consuming . This study was therefore undertaken to detect and to identify M . pneumoniae on culture media and in throat swab specimens by using polymerase chain reaction (PCR) and hybridization probes conjugated to alkaline phosphatase (Alp) . Primer pairs were selected for amplification of DNA fragments in the C to D, F, G and I to J regions of the M . pneumoniae cytadhesin P1 genes . Amplified DNA fragments were visualized by staining with ethidium bromide after 2% agarose gel electrophoresis and by Southern hybridization with Alp-labeled probes . No amplification of the P1 genes was seen with any of five related Mycoplasma species, the others from M . pneumoniae . In all of 30 clinical isolates on PPLO medium, M . pneumoniae was detected with the F and G primer pairs, giving 100% of sensitivity . Of 69 throat swab specimens, 25 were positive with the F primer pairs, and 23 positive with the Gen Probe test . From these results, we conclude that the PCR with F or G primer pairs can be adapted as a practical method for the rapid diagnosis of M . pneumoniae infections. Biol Reprod, 1995 Dec, 53(6), 1517 - 26 An estrogen-dependent sheep oviductal glycoprotein has glycan linkages typical of sialomucins and does not contain chitinase activity; DeSouza MM et al.; The deduced amino acid sequence of an estrogen-dependent sheep oviductal glycoprotein (M(r) 90,000-116,000) revealed the presence of several potential sites for glycan substitution on a protein backbone of M(r) approximately 66,500, and identity with chitinases . In order to further define the nature of the secreted glycoprotein, the objectives of the present study were 1) to devise a method to significantly enrich for the glycoprotein from oviductal secretions, 2) to biochemically characterize the glycoprotein by use of lectin blotting and enzymatic and chemical digestion, and 3) to determine whether unfractionated and enriched fractions containing the glycoprotein have chitinase activity . Oviducts were obtained from ovariectomized ewes treated with estradiol for 6 days and explant-cultured for 24 h . The oviductal glycoprotein was enriched approximately 80-85% from explant culture media by Maackia amurensis agglutinin (MAA) lectin affinity chromatography . Enriched fractions containing the oviductal protein were separated on SDS gels, transferred to polyvinyl difluoride, and probed with digoxigenin-labeled lectins . Lectin blotting revealed that the glycoprotein contained the carbohydrate moieties N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, and sialic acid both in alpha(2,3) and alpha(2,6) linkages, typical of sialomucins . Enzymatic digestion with neuraminidase and N-glycanase indicated that approximately 20% and approximately 6% of the molecular weight of the oviductal glycoprotein can be accounted for by sialic acid and N-linked glycans, respectively . The oviductal glycoprotein was resistant to digestion with O-glycanase alone and chondroitinase ABC, with the latter indicating that it was not a proteoglycan . Treatment with trifluoromethanesulfonic acid resulted in a deglycosylated product of M(r) approximately 66,000 immunoreactive with antibodies to the oviductal glycoprotein . No chitinase activity could be detected for unfractionated culture medium proteins or enriched fractions containing the M(r) 90,000-116,000 oviductal glycoprotein when the substrate methylumbelliferyl chitotriose was used . These data show that 1) MAA lectin chromatography can significantly enrich for the M(r) 90,000-116,000 glycoprotein from oviductal secretions, 2) the secreted glycoprotein contains saccharide residues typical of sialomucins, and 3) despite primary amino acid sequence identity, the oviductal glycoprotein does not share an enzymatic relationship with chitinases. Biol Reprod, 1995 Dec, 53(6), 1345 - 52 Evidence for germ cell control of Sertoli cell function in three models of germ cell depletion in adult rat; Boujrad N et al.; In order to clarify the role of germ cells in the regulation of Sertoli cell secretions, three experimental models of germ cell depletion were used: hypodactyl rat mutation (testis weight {TW}: 55% less than controls), in utero busulfan treatment (TW: 88% less than controls), and neonatal experimental cryptorchidism (TW: 72% less than controls) . The aim of this work was to compare the numbers of Leydig and Sertoli cells and the production of germ cells in each experimental model to the in vitro secretions of Leydig and Sertoli cells in conditioned media and to the hormonal serum profiles of the same animal in vivo . In the three models, serum levels of hypophyseal and testosterone hormones were significantly increased and decreased, respectively . In the absence of germ cells, the total length of seminiferous tubules, the total numbers of Sertoli and Leydig cells, and the daily production of germ cells were significantly diminished . The addition of both control and damaged seminiferous tubule culture media (STM: media conditioned by 10 cm of seminiferous tubules) to 10(6) control or damaged Leydig cells led to a further increase of testosterone production after ovine LH stimulation . However, expressed per Sertoli cell, testosterone production by control Leydig cells was reduced by addition of damaged STM as compared to addition of control STM, and similarly, the addition of control STM to damaged Leydig cells enhanced testosterone production more than did the addition of damaged STM . Secretions of transferrin per Sertoli cell in STM were reduced as compared to controls by the absence of germ cells but to a lesser extent than was production of spermatocytes and of spermatids . In conclusion, secretions by Sertoli cells of the paracrine factor involved in the control of testosterone production by Leydig cells and of transferrin are modified by germ cells. Parasitology, 1995 Dec, 111 ( Pt 5), 569 - 74 Spontaneous release of cysteine proteinases but not of pore-forming peptides by viable Entamoeba histolytica; Leippe M et al.; Invasive properties of pathogenic Entamoeba histolytica have been postulated to depend on the secretion or release of cysteine proteinases and pore-forming peptides (amoebapores) by trophozoites . To establish whether such toxic molecules are released by viable trophozoites or upon cellular disintegration, amoebae were maintained in various culture media, and activities in supernatants were monitored over time in correlation to cellular integrity . By measuring the release of the cytoplasmic marker enzyme NADP(+)-alcohol dehydrogenase, it became apparent that release of amoebapore was accompanied by cellular disintegration . In contrast, considerable quantities of cysteine proteinases were found to be present in culture supernatants also when amoebae remained intact . Treatment of amoebae with concanavalin A, bacterial lipopolysaccharides or the calcium ionophore A23187 did not result in amoebapore secretion suggesting that here target cell contact is required as an essential stimulus. Lab Invest, 1995 Dec, 73(6), 899 - 911 Production of matrix metalloproteinases at the bone-implant interface in loose total hip replacements; Yokohama Y et al.; BACKGROUND: Incidence of aseptic loosening of hip prostheses is increasing in recent years . Previous studies suggested involvement of proteinases and cytokines in the accelerated bone lysis associated with loosening . EXPERIMENTAL DESIGN: To investigate the role of matrix metalloproteinases (MMPs) in the loosening we immunolocalized MMP-1 (tissue collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-9 (gelatinase B) and their common inhibitors, tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), in the bone-prosthesis interface membranes . In situ hybridization was performed for the detection of MMP-9 mRNA in the membranes . The amounts of these MMPs and TIMPs in the tissue were measured by the sandwich enzyme immunoassays and enzyme activities assayed using radiolabeled collagen, gelatin, and carboxymethylated transferrin substrates . We also examined the ability of the cells from interface membranes to resorb mouse calvaria bone . RESULTS: The membranes obtained from the loose bone-implant interface were composed of fibrous granulation tissue containing numerous multinucleated giant cells with high density polyethylene debris . Immunohistochemical examination revealed that the giant cells were strongly positive for MMP-9 and weakly for MMP-1 . Expression of MMP-9 mRNA in the cells was demonstrated by in situ hybridization . MMP-2 and TIMP-2 were immunolocalized mainly in the fibroblasts . TIMP-1 was localized in the endothelial cells of the blood vessels and weakly in fibroblasts . However, MMP-3 was almost negative in the membrane tissue . Sandwich enzyme immunoassays showed that MMP-9 levels are significantly higher in both homogenates and culture media of the cup and stem interface membranes than the control pseudocapsule . Gelatinolytic activity was also remarkably higher in the membrane samples than the control . The cells isolated from the membranes had no ability to resorb calvaria bone . CONCLUSIONS: These data demonstrate that MMP-9 is produced by the multinucleated giant cells appeared by the reaction to polyethylene debris in the interface membranes . This proteinase may play a role in degradation of the extracellular matrix macromolecules present around and on the surface of the bone trabeculae, facilitating the osteoclastic bone resorption. J Endocrinol, 1995 Dec, 147(3), 449 - 61 Different molecular and messenger ribonucleic acid forms of insulin-like growth factor-binding protein-3 in the pregnant baboon (Papio anubis); Gargosky SE et al.; The ratio of the serum concentrations of insulin-like growth factors (IGF) to IGF-binding protein (IGFBP)-3 is highly correlated (Baxter & Martin 1986) . During pregnancy in the baboon, this ratio is perturbed; serum IGFBP-3 concentrations increase 10-fold, yet IGF-I levels are unaltered and IGF-II is increased only 2-fold (Giudice et al . 1993) . The aims of this study were to determine the molecular distribution of IGFBP-3 and to identify the tissue source and form(s) of IGFBP-3 during pregnancy in the baboon . Serum of non-pregnant and pregnant baboons, and conditioned media of decidua and placental explant cultures were characterized using neutral size-exclusion chromatography in combination with Western ligand blot, Western immunoblot, an IGFBP-3 radioimmunoassay (RIA) and an IGFBP-3 protease assay . Localization of immunoreactive IGFBP-3 was determined by immunocytochemistry, and expression of IGFBP-3 mRNA in the placental and decidual explants was examined by Northern blot analysis . RIA confirmed that immunoreactive IGFBP-3 is increased 10-fold in pregnancy serum compared with non-pregnancy serum . Size-exclusion chromatography combined with an IGFBP-3 RIA revealed that, unlike non-pregnancy serum where 70% of the immunoreactive IGFBP-3 elutes in the 150 kDa ternary complex, equal amounts of immunoreactive IGFBP-3 were measured in pregnancy serum in the < or = 150 and 60 kDa IGFBP regions . Western analysis revealed that non-pregnancy serum contained predominantly a 45-40 kDa IGFBP-3 doublet and a 28 kDa immunoreactive form of IGFBP-3, while in pregnancy serum IGFBP-3 existed as a 45-40 kDa doublet, as well as 26-28 kDa and 18 kDa immunoreactive forms . These alternative forms of IGFBP-3 were not attributable to detectable IGFBP-3 protease activity . To identify the source of the increased serum levels of IGFBP-3 during pregnancy, we examined explant culture media of baboon decidua and placenta . Size-exclusion chromatography combined with RIA and Western analysis revealed that: (1) more immunoreactive IGFBP-3 was produced by decidual cultures than by placental explants, but less 45-40 kDa IGFBP-3 was present in decidua; (2) the immunoreactive forms of IGFBP-3 detected in decidua were similar to those found in maternal serum; (3) placental explants secreted only 45-40 kDa IGFBP-3 in culture . IGFBP-3 was immunohistochemically localized to the cells of placental villi, and to the perinuclear region of the decidual cells and staining for IGFBP-3 was more intense in the decidua than in the placenta . Northern analysis of the explant cultures revealed two IGFBP-3 mRNA transcripts of 2.4 and 1.7 kb in both decidua and placenta which may account for the different immunoreactive forms of IGFBP-3 detected in the baboon . However analysis of non-pregnancy liver also revealed two IGFBP-3 transcripts of 2.4 and 1.7 kb . These data suggest that the two transcripts are not solely pregnancy-associated and levels of protein may be the reason for detection of multiple immunoreactive IGFBP-3 fragments in pregnancy. J Clin Endocrinol Metab, 1995 Dec, 80(12), 3784 - 7 Partial purification and amino acid sequence analysis of endometriosis protein-II (ENDO-II) reveals homology with tissue inhibitor of metalloproteinases-1 (TIMP-1); Sharpe-Timms KL et al.; De novo synthesized endometriosis protein-II (ENDO-II; M(r) 28,000 to 32,000; pI 7.0 to 9.0) was partially purified from rat endometriotic tissue culture media using affinity chromatography and two-dimensional SDS-PAGE . The protein was electrophoretically transferred to polyvinyl difluoride membranes which were stained with Coomassie blue R-250 . The stained protein corresponding to ENDO-II (M(r) 28,000 to 32,000; pI 7.0 to 9.0) was cut from the membranes for amino acid sequencing . Partial amino acid sequence was determined by automated Edman degradation using a gas phase sequencer and phenylthiohydantoin analyzer . Sequence analysis of ENDO-II yielded 25 residues: C S C A P T H P Q T A F C N S D L V I R K F M G . Comparison to sequence databanks demonstrated significant homology with rat (100%) and human (84%) tissue inhibitor of metalloproteinases-1 (TIMP-1) . Western blot analysis using a TIMP-1 antibody confirmed amino acid sequence analysis . In conclusion, ENDO-II shares sequence homology with TIMP-1 and cross-reactivity with TIMP-1 antibody and subsequently identifies production of TIMP-1 by endometriotic tissues . The synthesis and secretion of TIMP-1 by endometriosis may derange the normal proteolytic milieu of the peritoneal cavity and contribute to the etiology and underlying physiological sequelae associated with the disease endometriosis. J Cell Physiol, 1995 Dec, 165(3), 639 - 46 Ornithine decarboxylase activity is associated with proliferation but not with T3-induced differentiation of Caco-2 cells; Jumarie C et al.; Ornithine decarboxylase (ODC) activity and polyamine (putrescine, spermidine, spermine) concentrations were measured in parallel in enterocyte-like Caco-2 cells maintained under various culture conditions . ODC activity was maximal at the beginning of the exponential growth phase, decreasing dramatically thereafter to a negligible level at confluency (day 9) . Kinetic studies performed on day 3 revealed the presence of a single enzyme with a Km around 200 microM and a Vmax of about 2 nmol CO2 released/h/mg protein . Similar values were obtained in both serum-supplemented and transferrin/selenium (TS)-defined culture media, indicating that ODC kinetic parameters are not affected by any factors present in serum . Polyamine concentrations were maximal on day 5 . By day 9, they returned to initial levels and remained at these fairly high values until day 21 . Since we have previously shown (Jumarie and Malo, 1994, in Vitro Cell . Dev . Biol., 30A:753-760) that triiodothyronine (T3) stimulates differentiation but not proliferation of Caco-2 cells maintained in TS-defined medium, we investigated if it induces differentiation by a polyamine-dependent mechanism . Short- and long-term measurements revealed similar ODC activity and polyamine levels whether T3 was present or not in the culture medium . These results clearly demonstrate that polyamine synthesis is more likely to be associated with Caco-2 cell proliferation, and that the T3 effect on Caco-2 cell differentiation does not involve polyamine biosynthesis . Moreover, our data show that ODC activity is not solely regulated by intracellular polyamine concentration. Brain Res Dev Brain Res, 1995 Nov 21, 89(2), 270 - 80 Perturbation of target-directed neurite outgrowth in embryonic CNS co-cultures grown in the presence of ethanol; Heaton MB et al.; Studies were conducted to determine the influence of ethanol on target-directed fiber outgrowth in culture, using embryonic chick spinal cord-muscle, and fetal rat septal-hippocampal co-cultured explants . Process extension from the spinal cord and septal explants in control cultures was selectively oriented toward the appropriate target tissue . Ethanol in the culture medium (500 mg/dl) eliminated this target-oriented outgrowth in both systems, although the overall extent of neurite outgrowth was not affected . In an effort to further characterize the source of this disruption, target explants were grown alone, with and without ethanol, and the target-conditioned culture media was subsequently harvested and placed on newly plated spinal cord or septal explants, to determine whether ethanol decreased the target production of soluble substances . To determine whether deposition of substrate-bound materials by the target tissue was affected by ethanol, spinal cord or septal explants were plated in wells which had previously been occupied by the appropriate target tissue . These studies revealed that ethanol significantly inhibited production of soluble and substrate-bound materials by muscle explants, but not by hippocampal explants . It was concluded that the ethanol-induced loss of target-directed neurite outgrowth in the spinal cord explants could be accounted for primarily by the attenuated production of neurotropic/neurotropic substances by the muscle tissue . The loss of target-directionality in the septal explants appeared to be due to other factors, possibly related to ethanol-induced compromise of the capacity of the septal neurons to respond appropriately to target-derived neurotrophic/neurotropic substances . The implications of these results for the fetal alcohol syndrome are considered. Spine, 1995 Nov 15, 20(22), 2373 - 8 Herniated cervical intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2; Kang JD et al.; STUDY DESIGN . Herniated cervical disc specimens were obtained from patients undergoing surgical discectomy for persistent radiculopathy and cultured in vitro to determine whether various biochemical agents were being produced . OBJECTIVES . Our hypothesis is that biochemical mediators of inflammation and tissue degradation play a role in cervical intervertebral disc degeneration and in the pathophysiology of cervical radiculopathy . SUMMARY OF BACKGROUND DATA . Neck pain with or without radiculopathy is a common clinical problem, but the etiology of neck pain and the exact pathophysiology of radiculopathy remain uncertain . We have previously reported the production of various biochemical agents by herniated lumbar disc specimens in vitro . Because of a lack of such studies in the literature with respect to the cervical spine, the purpose of this study was to determine whether similar biochemical agents of inflammation and tissue degradation were being produced by herniated cervical disc specimens . METHODS . Eighteen herniated cervical discs were obtained from 15 patients undergoing anterior disc surgery . The specimens were cultured and incubated for 72 hours, and the media were subsequently collected for biochemical analysis . Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E2, and a variety of cytokines were performed . As a control group, six cervical discs specimens were obtained from three patients undergoing anterior surgery for traumatic burst fractures, and similar biochemical analyses were performed . RESULTS . The culture media from the herniated cervical disc specimens showed increased levels of matrix metalloproteinase activity compared with the control discs . Similarly, the levels of nitric oxide, prostaglandin E2, and interleukin-6 were significantly higher in the herniated disc specimens compared with the control discs . Interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of the herniated or control discs . CONCLUSIONS . Herniated cervical disc specimens were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6 . These results were similar to those obtained in herniated lumbar disc specimens that we have previously reported . These products may be intimately involved in the biochemistry of disc degeneration and the pathophysiology of radiculopathy. Cell Adhes Commun, 1995 Nov, 3(4), 273 - 81 Growth stimulation of human skin fibroblasts by elastin-derived peptides; Kamoun A et al.; Elastin-derived peptides (kappa-elastin: kappa E, mean molecular mass: 75 kDa), either coated onto plastic dishes or added to culture media (0.26 to 1.33 nM) stimulated the growth of human skin fibroblasts (HSF) strains obtained from different donors and tested at different cell passages (4 to 12) . Coated 44.4 micrograms/cm2 insoluble elastin (iE) exhibited the same action; coated iE or kappa E significantly modifies the HSF morphology: after 5-6 days of culture, HSF are more elongated, and at preconfluence state, formation of HSF clusters surrounding iE were observed . Increased 3H thymidine incorporation and proliferative effect of HSF by kappa E (1.3 to 2.2 fold as compared to control cells) was observed after a lag phase period which raised with initial HSF density . Optimal proliferative effect was obtained at kappa E 8.5 10(-10) M, a value close to the dissociation constant (kD = 2.7 10(-10) M) of kappa E to HSF . Valine-glycine-valine-alanine-proline-glycine (VGVAPG), but not valine-glycine-valine (VGV) or Valine-glycine-valine-valine-glycine-alanine (VGVVGA) also significantly stimulated, optimally at 7.0 10(-10) M, HSF proliferation . It was concluded that the stimulatory influence of elastin derived peptides on HSF proliferation was mediated through a binding to plasmalemmal receptor of HSF. Ophthalmic Surg Lasers, 1995 Nov-Dec, 26(6), 568 - 71 Complement-derived anaphylatoxins in human donor corneas treated with excimer laser; Gardner BP et al.; BACKGROUND AND OBJECTIVE: An inflammatory response produced by excimer laser photorefractive keratectomy (PRK) may be associated with the subsequent corneal haze and regressions in refractive error observed after treatment . Complement-derived anaphylatoxins, potent mediators of inflammation, may have a role in postoperative healing . MATERIALS AND METHODS: Twenty right human donor corneas underwent a 6-D excimer laser PRK treatment . The corresponding left donor corneas served as the controls . After incubation in tissue culture media for 6 hours and elution in phosphate-buffered saline with EDTA for 24 hours, complement-derived anaphylatoxins C3a, C4a, and C5a were measured in corneal eluates by radioimmunoassay . RESULTS: Compared with control corneas, the excimer PRK corneas failed to demonstrate a significant increase in C3a, C4a, or C5a levels (P > .05) . CONCLUSIONS: These results suggest that the excimer laser at this dose does not activate significant complement in the cornea. Infection, 1995 Nov-Dec, 23(6), 378 - 9 Comparison of BacT/Alert blood culture bottles with lytic media for culture of peritoneal dialysis fluid; Wust J et al.; A comparison was made between the Septi-Chek "Release" system containing saponins, standard BacT/Alert blood culture bottles and FAN BacT/Alert blood culture bottles for culturing CAPD fluids . Seventy-seven CAPD effluent specimens were tested . No differences could be found . Therefore, lytic agents are not necessary when dialysates are cultured in blood culture media. J Bone Miner Res, 1995 Nov, 10(11), 1660 - 5 Effects of extracellular calcium on insulin-like growth factor II in human bone cells; Honda Y et al.; Extracellular calcium concentration is critically important for normal function of the body . Recently, reports have shown that cells derived from parathyroid glands contain an extracellular calcium receptor that is responsive to changes in extracellular calcium . Bone is intimately involved in calcium homeostasis; therefore, we sought to test the hypothesis that extracellular calcium has direct effects on bone cells . Extracellular calcium was increased by the addition of varying concentrations of CaCl2 (0.4-2.0 mM) to the control medium . An increase in extracellular calcium increased cell proliferation, as assessed by 3H-thymidine incorporation, in a number of cell types including normal human bone cells derived from vertebrae (HBV155) and a number of human osteosarcoma cell lines . The increase in cell proliferation by elevated CaCl2 was dose dependent, whereas MgCl2 was not effective at the doses tested (up to 2 mM added MgCl2) . To test the hypothesis that the mitogenic activity of elevated extracellular calcium involved a growth factor, levels of insulin-like growth factor II (IGF-II) were measured in the conditioned medium of HBV155 cells by radioimmunoassay after removal of binding proteins by size exclusion chromatography . The effects of an increase in extracellular calcium by 1 mM were: 1) increased culture media levels of IGF-II within 1 h of treatment, 2) the increase in IGF-II levels reached a maximum after 8 h of treatment, and 3) IGF-II levels were still elevated after 24 h of treatment . Furthermore, a blocking monoclonal antibody against IGF-II abolished the increased cell proliferation in HBV155 cells following elevation of extracellular calcium . Taken together, these findings suggest that an increase in extracellular calcium results in an increase in IGF-II which is required for the subsequent increase in cell proliferation. Chin Med J (Engl), 1995 Nov, 108(11), 820 - 4 Production of transforming growth factor-beta by cultured rat mesangial cells; Yao J et al.; The purpose of this study was to examine whether transforming growth factor-beta (TGF beta) acts as an autocrine cytokine in cultured mesangial cells . Measangial cell conditioned media (CM) were prepared and tested for their effect on mesangial cell proliferation . CM showed a concentration dependent inhibition on mesangial cell proliferation and the activity was enhanced by treating conditioned media with acid . Gel filtration analysis showed peak inhibitory activity to reside in fractions with an estimated molecular weight range of 16-30 KD . The activity was partially blocked by anti-TGF beta antibody, but not nonimmune control IgG . The presence of TGF beta was confirmed using the mink lung epithelial cell assay . Furthermore, the addition of anti-TGF beta antibody directly into culture media significantly enhanced mesangial cell proliferation . These results demonstrate that measangial cell produce both active and latent forms of TGF beta, which functions as an autocrine growth inhibitor for mesangial cells. J Neurol Sci, 1995 Nov, 133(1-2), 24 - 30 Deficiency of a neuronal growth-sustaining factor in fibroblasts of patients with Alzheimer's disease; Plioplys AV; A previous report has shown a deficiency of a cholinergic differentiating factor in spent culture media in which Alzheimer's disease (AD) patient fibroblasts were grown (Kessler, 1987) . We used a similar approach to investigate whether AD fibroblast-conditioned medium demonstrated central nervous system (CNS) neuronal growth sustaining properties . For these investigations we used cultured fetal murine telencephalic vesicle neurons . Control fibroblast-conditioned medium produced statistically significant neuronal survival as compared to AD fibroblast-conditioned medium or to nonconditioned medium . There was no statistically significant difference between AD fibroblast-conditioned medium and nonconditioned medium results . These results suggest that there may be a deficiency in AD of a CNS neuronal growth-sustaining factor. J Reprod Fertil, 1995 Nov, 105(2), 331 - 8 Trophic effects of myeloid leukaemia inhibitory factor (LIF) on mouse embryos; Lavranos TC et al.; Myeloid leukaemia inhibitory factor (LIF) is expressed at highest concentrations in the maternal endometrial glands at about the stage of blastocyst implantation . LIF is also expressed by the extraembryonic membranes of the early mouse embryo . Embryos of different ages were cultured with, or without, LIF, and embryo growth in vivo and in vitro was examined to determine whether LIF is important for embryo development . Supplementing embryo culture media with 1000 U recombinant human LIF ml-1 increased the number of eight-cell mouse embryos developing beyond the hatched blastocyst stage in vitro from 62.1% to 85.1% (P < 0.05) . LIF significantly increased the number of embryos hatching (33.8% versus 7.65% for controls 96 h after hCG injection, P < 0.001), completely hatching (85.1% versus 62.1%, P < 0.05), and exhibiting trophoblast outgrowth (13.5% versus 0% 120 h after hCG treatment, 85.1% versus 47.0% 144 h after hCG treatment, P < 0.001) in vitro . LIF-treated embryos also displayed a significantly greater area of trophoblast outgrowth than did controls as early as day 5 in culture (P < 0.005) . These data show that LIF enhances mouse eight-cell embryo development in vitro, as seen by the accelerated rate of embryo hatching and trophoblast outgrowth . In addition, enhanced embryo survival in vivo is shown, following the transfer of LIF-treated embryos into a pseudopregnant recipient female . Expression of mRNA encoding LIF was detected in endometrial cells cultured in monolayer from uteri of day 3 pregnant females, explaining the known embryotrophic effects of endometrial coculture . This expression was not enhanced significantly by treatment with oestradiol (3.7 x 10(-5) mol l-1) or progesterone (3.2 x 10(-6) mol l-1) or both hormones . These results indicate that LIF could have a dual action in early embryogenesis as an embryotrophin and as a factor required for embryo implantation . Multiple roles for LIF are consistent with the expression of this factor at embryonic, extraembryonic and maternal sites during early embryogenesis. J Neurobiol, 1995 Nov, 28(3), 363 - 80 Conditions for the primary culture of eye imaginal discs from Drosophila melanogaster; Li C et al.; We have established a primary culture system for Drosophila eye imaginal discs . With this system, we were able to obtain neurite outgrowth from intact eye discs, eye disc fragments, and dissociated eye imaginal disc cells . Immunoreactivity to antibody 24B10 indicates that these extending neurites are photoreceptor axons . Three culture media were tested for their ability to support the survival of and neurite extension from eye disc fragments in vitro at 23 degrees C . These, with supplements, were: five parts of Schneider's Drosophila medium with four parts of basal Eagle's medium ("4 + 5"); Leibovitz's L-15 medium (L-15); and Shields and Sang's M3 modified medium (MM3) . We obtained the best results with MM3 supplemented with 2% fetal bovine serum (FBS) . Eye disc fragments survived in this medium for at least 20 days . Pigmentation in the nonphotoreceptor pigment cells in cultures from the prepupa required the presence of 20-hydroxyecdysone (20-HE) (1 micrograms/ml), whereas neurite outgrowth was seen in the absence of 20-HE . Donor animals had to fall within a range of ages to obtain appropriate eye disc differentiation in vitro . Eye disc from 5-h pupae (P + 5) or older commenced ommachrome synthesis in vitro in a temporal sequence close to that found in vivo, whereas the in vitro synthesis of this pigment was delayed in eye discs from younger flies . Average neurite length was not affected by age among pupae younger than P + 5; but neurite outgrowth from P + 24 was scarce, probably because by this time photoreceptor axons had already grown in vivo and were severed and unable to regenerate in vitro . Eye discs taken from third instar larvae or white prepupae continued their mitotic activity in vitro . Together with the advance of the morphogenetic furrow at the leading edge of retinal development, this observation is consistent with the evidence that pattern formation continues in vitro . Morphogenetic changes were manifested in cultures . Viability tests with calcein AM and ethidium bromide revealed few dead cells in living cultures. Biol Reprod, 1995 Nov, 53(5), 1038 - 50 Isolation and characterization of a 30-kDa endometrial glycoprotein synthesized during the estrous cycle and early pregnancy of the pig; Geisert RD et al.; Endometrial polypeptide synthesis, which is regulated through ovarian steroid secretion and steroid production by the developing conceptus, not only provides the necessary secretory components vital to conceptus development but also presents the adhesive changes in the epithelial surface essential for conceptus attachment . In the present study, a 30-kDa, basic endometrial glycoprotein (pGP30) was isolated and characterized during the estrous cycle and early pregnancy of the pig . Uterine flushings and endometrial culture media were obtained from gilts on Days 0, 5, 10, 12, 15, and 18 of the estrous cycle and Days 10, 12, 15, and 18 of pregnancy . A polyclonal antibody was generated to pGP30 after isolation of medium from Day 15 pregnant endometrial cultures separated by gel filtration and PAGE . Western blot analysis indicated that the antiserum reacted with isoforms of pGP30 and cross-reacted with a 90-kDa component in serum that was not removed after cleavage of the oligosaccharide chains from the 90-kDa glycoprotein . Antiserum did not detect a 30-kDa band in media from cultures of kidney, fat, heart, muscle, liver, or serum; however, heart and muscle did contain bands of different molecular masses that cross-reacted with the antiserum . Multiple bands of higher molecular mass (35-40 kDa) were detected in the endometrial cultures from gilts on Days 0 through 10 of the estrous cycle . Treatment of ovariectomized gilts with estradiol-17 beta stimulated a similar response . During the mid- to late luteal phase of the estrous cycle (Days 12-18), the 30-kDa band as well as an additional 32-kDa band was present on Western blots . Administration of progesterone for 14 days stimulated the synthesis of both the 30- and 32-kDa products in ovariectomized gilts . However, only the pGP30 was detected on Days 12-18 of pregnancy . Immunocytochemical localization with antiserum to pGP30 indicated that the glycoprotein is present in the endometrial epithelium, with the surface epithelium demonstrating the strongest reaction product . Discrete changes in staining and cellular localization were observed during the early stages of the estrous cycle (Days 0-5) and the midluteal (Day 10) phase . A similar response was achieved with administration of steroids to ovariectomized gilts . Data indicate that discrete changes in epithelial synthesis of the endometrial glycoprotein occur at the time of conceptus trophoblastic elongation and placental attachment in the pig. J Clin Endocrinol Metab, 1995 Nov, 80(11), 3273 - 8 Opposing actions of transforming growth factor-beta and glucocorticoids in the regulation of fibronectin expression in the human placenta; Guller S et al.; Alterations in the expression of extracellular matrix (ECM) proteins in the placenta and fetal membranes have been linked to parturition whether occurring before or at term . In the present study, we examined the individual and combined effects of transforming growth factor (TGF)-beta and dexamethasone (DEX) on the expression of oncofetal fibronectin (onfFN), i.e . a major ECM protein synthesized by placenta, in cytotrophoblasts isolated from human term placentas to establish a model system from which to evaluate the actions of positive and negative regulators of ECM protein expression in the human placenta . Cytotrophoblasts were maintained for 21=62 h in medium supplemented with 4% charcoal-stripped calf serum in the presence or absence of TGF-beta (2 ng/mL) and DEX (10(-7) mol/L) . Levels of onfFN in culture media were determined by immunoassay . TGF-beta treatment alone induced approximately a 150% increase in media levels of onfFN after 21 and 45 h of culture when compared with control, whereas DEX treatment alone reduced levels of onfFN to 15% of control levels . Media levels of onfFN in cells treated with both TGF-beta and DEX were 40-90% of control levels . Similarly, treatment of cells with TGF-beta alone promoted a 100-250% increase in rates of FN synthesis and levels of FN messenger ribonucleic acid, whereas DEX treatment alone reduced these indices of FN expression to approximately 10% of control levels . In cells treated with TGF-beta and DEX, levels of ECM protein synthesis and FN messenger ribonucleic acid were between 30 and 100% of control values . Similar patterns of regulation of FN expression by TGF-beta and DEX were observed when experiments were carried out in serum-free medium . Our results suggest that during pregnancy, TGF-beta and glucocorticoids may be important opposing physiological regulators of placental ECM protein expression. Int Arch Allergy Immunol, 1995 Nov, 108(3), 211 - 23 Blood monocytes in rheumatoid arthritis are highly adherent to cultured endothelium; Mazure G et al.; Monocytes from 17 patients with rheumatoid arthritis (RA) were more adherent than monocytes from 17 control patients to monolayers of pig aortic endothelium irrespective of whether sera was included (median 27-34% increase; p = 0.002) or omitted (median 27% increase; p = 0.022) from the culture media . When human umbilical vein endothelial cells were used as the adherence substrate, rheumatoid monocytes from an additional 21 patients demonstrated a median 31% (p = 0.004) and 20% increase (p = 0.004) in adhesion when compared with monocytes from 21 normal healthy subjects in the absence and presence of autologous sera, respectively . Activation of control monocytes with muramyl dipeptide or treatment with RA sera increased their attachment to endothelium (mean 34 +/- 14% increase; p < 0.001) . The expression of the adhesion molecules CD11b (p < 0.005), CD18 (p < 0.005), CD62L (p = 0.01) was enhanced on rheumatoid monocytes, but antibody-blocking studies suggested that CD18 and CD62L were not responsible for the augmented binding of the rheumatoid cells . A subpopulation of rheumatoid monocytes possessed a very low net negative surface charge, a property that favours binding to vessel walls . We propose that many rheumatoid monocytes are predisposed for sheer-resistant adhesion to vascular endothelium. Am J Respir Cell Mol Biol, 1995 Nov, 13(5), 563 - 9 Expression of calcitonin gene-related peptide by cultured rat alveolar type II cells; Hastings RH et al.; Calcitonin gene-related peptide (CGRP) immunoreactivity is found in the airways in terminals of primary sensory afferents, in neuroendocrine cells, and in tracheal serous cells . This study shows that rat alveolar epithelial cells express immunoreactive CGRP also . Freshly isolated cells contained 34 +/- 23 fmol CGRP/10(7) cells (n = 4) . Cultured type II cells secreted CGRP at a stable rate for 3 days after cell isolation, averaging 206 +/- 14 fmol CGRP/well/day (750,000 cells plated/well with approximately 30% efficiency) . The extracellular CGRP immunoreactivity eluted in the same fraction as rat CGRP-beta on high performance liquid chromatography . Secretion of CGRP from type II cells was reversibly blocked by monensin, an inhibitor of secretory protein transport . CGRP secretion was stimulated in a concentration-dependent fashion by phorbol myristate acetate, but it was not affected by forskolin, capsaicin, bradykinin, or nicotine . CGRP was not detected in culture media from alveolar macrophages or fibroblasts, potential contaminants of primary type II cell cultures . Calcitonin is expressed by neuroendocrine cells, but it was not detected in conditioned media from type II cell cultures . Thus, type II alveolar epithelial cells express and secrete CGRP . Secretion occurs constitutively and is regulated by a protein kinase C-dependent pathway . Secretion is unaffected by increases in cyclic adenosine monophosphate or by treatments that induce release of CGRP from sensory afferent nerve terminals in the airways. Am J Physiol, 1995 Nov, 269(5 Pt 1), L631 - 6 Tachykinins induce gelatinase production by guinea pig alveolar macrophages: involvement of NK2 receptors; D'Ortho MP et al.; To determine whether tachykinins induce gelatinase production by guinea pig alveolar macrophages (AM), and to characterize the mechanism involved, we incubated AM with substance P (SP), neurokinin A (NKA), or the NH2-terminal fragment of SP, SP(1-7) . The effects of increasing concentrations of selective NK1 and NK2 agonists on tachykinin-induced gelatinase production were also evaluated, as were the effects of a selective NK2 antagonist . Gelatinase activity in conditioned culture media (CCM) was assessed by zymography and quantified by image analysis . SP increased 92-kDa gelatinase activity in CCM of AM in a concentration-dependent manner, with a maximum increase at 10(-4) M . NKA, the NH2-terminal fragment of SP, and an NK1-selective agonist had no effect . In contrast, a selective NK2 agonist induced a concentration-dependent increase in gelatinase activity . The increase in this activity induced by SP and the selective NK2 agonist was inhibited by a selective NK2 antagonist . We conclude that SP induces gelatinase production by AM through NK2 receptor activation . The release of gelatinase may constitute one mechanism through which SP contributes to the epithelial lesions observed in bronchial hyperreactivity and asthma. Am J Physiol, 1995 Nov, 269(5 Pt 1), C1311 - 6 Induction of hypertrophic responsiveness to isoproterenol by TGF-beta in adult rat cardiomyocytes; Schluter KD et al.; In a previous publication we reported that hypertrophic responsiveness to beta-adrenoceptor stimulation can be induced in isolated cardiomyocytes when these are cultured for 6 days in presence of fetal calf serum (FCS; Pinson et al., J . Mol . Cell . Cardiol. . 25: 477-490, 1993) . The role of transforming growth factor-beta (TGF-beta) in this induction process has now been investigated . Isolated cardiomyocytes from adult rats were cultured for 6 days in presence of 20% FCS . It was found that induction of hypertrophic responsiveness to beta-adrenoceptor stimulation was abolished when a neutralizing anti-TGF-beta 1 antibody was added to FCS-containing culture medium . In culture media with FCS contents (5%) too low to induce hypertrophic responsiveness to beta-adrenoceptor stimulation, addition of 1 ng/ml TGF-beta 1,2 induces this responsiveness . It was demonstrated that cardiomyocytes already release TGF-beta into culture media on day 1 of culture and that they continue to do so in presence of FCS supplements of > 5% . The results demonstrate that hypertrophic responsiveness to beta-adrenoceptor stimulation is induced in cardiomyocytes by an autocrine mechanism involving TGF-beta 1 as mediator. J Cell Biol, 1995 Nov, 131(4), 1025 - 37 Manganese effectively supports yeast cell-cycle progression in place of calcium; Loukin S et al.; Metal ion requirements for the proliferation of Saccharomyces cerevisiae were investigated . We used bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relatively acid tolerant chelator, to reduce the free metal ion concentrations in culture media . Chelatable metal ions were added back individually and in combination . In addition to a requirement for approximately 10 pM external free Zn2+ we found an interchangeable requirement for either 66 nM free Ca2+ or only 130 pM free Mn2+ . Cells depleted of Mn2+ and Ca2+ arrested as viable cells with 2 N nuclei and tended to have very small minibuds . In the absence of added Mn2+, robust growth required approximately 60 microM total internal Ca2+ . In the presence of added Mn2+, robust growth continued even when internal Ca2+ was < 3% this level . Chelator-free experiments showed that MnCl2 strongly and CaCl2 weakly restored high-temperature growth of cdc1ts strains which similarly arrest as viable cells with 2 N nuclear contents and small buds . Its much greater effectiveness compared with Ca2+ suggests that Mn2+ is likely to be a physiologic mediator of bud and nuclear development in yeast . This stands in marked contrast to a claim that Ca2+ is uniquely required for cell-cycle progression in yeast . We discuss the possibility that Mn2+ may function as an intracellular signal transducer and how this possibility relates to previous claims of Ca2+'s roles in yeast metabolism. Prostate, 1995 Nov, 27(5), 277 - 86 Influence of dihydrotestosterone, epidermal growth factor, and basic fibroblast growth factor on the cell kinetics of the PC3, DU145, and LNCaP prostatic cancer cell lines: relationship with DNA ploidy level; Janssen T et al.; The cell kinetics (percentage of cells in the S+G2 phases of the cell cycle) and the DNA ploidy levels (nuclear DNA content) were determined in 108 samples each of the PC3, DU145, and LNCaP prostate cancer models . This was carried out by means of the digital cell image analysis of Feulgen-stained nuclei . Two to three hundred cell nuclei were analyzed for each of the 324 samples under study . The three cell lines were submitted to experimental conditions including the addition of dihydrotestosterone (DHT), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), either alone or in combination, to the culture media . The results show that under the present culture conditions, the PC3 cell line was DHT-, EGF- and bFGF-insensitive . In contrast to what is generally reported in the literature, the DU145 cell line was DHT- and EGF-sensitive under the present culture conditions, but bFGF-insensitive . The LNCaP cell line was DHT-sensitive, but EGF- and bFGF-insensitive . While mainly tetraploid, the three cell lines nevertheless exhibited a significant level of heterogeneity in their nuclear DNA content distributions . Indeed, the proportions of non-tetraploid (diploid, hyperdiploid, triploid, hypertriploid, hypertetraploid, polymorphic) DNA histograms were 14% in the PC3, 16% in the DU145, and 29% in the LNCaP cell lines . These results suggest that the DNA ploidy level would not influence the hormone sensitivity level in the cell lines since they had significantly distinct hormone sensitivity profiles while remaining mainly tetraploid. Metabolism, 1995 Nov, 44(11), 1469 - 74 Role of cellular ribose-5-phosphate content in the regulation of 5-phosphoribosyl-1-pyrophosphate and de novo purine synthesis in a human hepatoma cell line; Boer P et al.; 5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo purine synthesis . However, the role of ribose-5-phosphate (R5P), the precursor for PRPP, in the regulation of PRPP and de novo purine synthesis has not yet been clarified conclusively . This study was designed to clarify interrelationships between R5P content, PRPP availability, and the rate of de novo purine synthesis in the cultured human hepatoma cell line (HepG2), a plausible model for normal human hepatocytes . Increasing glucose concentration in the culture media from 0 to 10 mmol/L resulted in a 2.9-fold elevation of cellular R5P content (from 107 +/- 31 to 311 +/- 57 nmol/g protein), associated with a correlated increase of 7.14-fold in cellular PRPP availability (from 4.76 +/- 3.4 to 34 +/- 8.4 pmol/mg protein/min) and of 149-fold in the rate of de novo purine synthesis (from 55 to 8,204 dpm/mg protein/h) . Plotting the rate of de novo purine synthesis versus R5P content indicates that at a wide range of R5P content, including that prevailing in hepatocytes under physiological conditions, the rate of purine synthesis depends on R5P content . A similar dependence was also demonstrated for PRPP availability . The rate of de novo purine synthesis exhibited a sigmoidal dependence on PRPP availability . The demonstration in human hepatocytes of dependence of the rate of purine synthesis on R5P content has implications concerning the pathogenesis of purine overproduction associated with several inborn and acquired conditions in man. Mol Cell Endocrinol, 1995 Oct 30, 114(1-2), 35 - 42 Growth hormone inhibits differentiation of avian epiphyseal growth-plate chondrocytes; Monsonego E et al.; The effect of chicken growth hormone (cGH) on the proliferation and differentiation of avian growth-plate chondrocyte was evaluated in culture . In culture, addition of ascorbic acid to the culture media caused cell differentiation . Treatment of proliferating chondrocytes with cGH caused a time-dependent increase in collagen type II gene expression together with a decrease in the appearance of osteopontin (OPN) in the medium . In addition, the ascorbic acid-dependent increase in alkaline phosphatase (AP) activity was inhibited by cGH . IGF-I, on the other hand, caused an increase in AP activity in the ascorbic acid-treated chondrocytes . In the presence of ascorbic acid, cGH did not affect collagen type II gene expression or the appearance of OPN in the medium . Proliferation of avian growth-plate chondrocytes, in contrast to mammalian chondrocytes, was not stimulated by GH alone, although the presence of cGH was essential for chondrocyte survival in long-term culture . cGH in combination with epidermal growth factor (EGF) stimulated cell proliferation . These results suggest that GH inhibits differentiation in avian growth-plate chondrocytes, thereby sustaining their proliferative state and maintaining their sensitivity to growth factors such as EGF. Brain Res, 1995 Oct 30, 697(1-2), 1 - 16 Nitric oxide-mediated death of cultured neonatal retinal ganglion cells: neuroprotective properties of glutamate and chondroitin sulfate proteoglycan; Nichol KA et al.; The release of nitric oxide and stimulation of glutamate receptors by excitatory amino acids has been linked to neuronal degeneration and toxicity . In the rat retina approximately 60% of retinal ganglion cells (RGCs) die during the first postnatal week . In this study we examined the effects of nitric oxide synthase blockers and glutamate on the survival of neonatal RGCs in vitro over a 16 h assay period . Less than 10% of P1 RGCs survived in serum free defined media alone (control), however survival was increased, in a dose-dependent manner, when L-glutamate (10 microM-10 mM) was added to the media; a maximum of 70% of RGCs could be maintained with the addition of 5 mM glutamate . This effect was blocked by the NMDA and non-NMDA receptor blockers APV and DNQX and was age dependent; the survival of RGCs from P5 but not P7 rats was enhanced by the addition of glutamate even in high calcium concentrations (10 mM) . When the nitric oxide synthase blockers L-NAME (5 mM) or haemoglobin (25 microM) were added to the culture media, up to 61% of P1 RGCs survived . The addition of the 480 kDa chondroitin sulfate proteoglycan (SCCP) previously shown to enhance RGC survival in vivo and in vitro, potentiated the action of glutamate and L-NAME and increased RGC survival to over 90% with almost all RGCs expressing a profusion of processes . These results suggest that the release of nitric oxide and glutamate by cells within the retina may contribute to the regulation of RGC numbers in vivo during development. Transplantation, 1995 Oct 27, 60(8), 854 - 60 Functional studies of rat, porcine, and human pancreatic islets cultured in ten commercially available media; Holmes MA et al.; There have been no extensive studies investigating the effect of tissue culture media on the in vitro functional characteristics of rat, porcine and human Islets of Langerhans . We therefore aimed to compare ten commercially available tissue culture media on the basis of their ability to maintain islet viability . Following isolation, islets were cultured free-floating in the ten media (RPMI 1640-11mM glucose (control), RPMI 1640-2.2mM glucose, Dulbecco's MEM, TCM 199, CMRL 1066, Iscove's MEM, Waymouth's MEM, Serum-Free medium, Ex-cell 300, Ham's F-12) and viability was assessed after 24 hr, 3 days, and 7 days on the basis of macroscopic appearance, cell membrane integrity, and insulin secretion in response to glucose stimulation both by dynamic incubation and by perifusion . Each islet species demonstrated physiological insulin release characteristics in all media--however, it was possible to distinguish between the media by comparing the stimulation indices calculated from the insulin release studies . Significantly higher stimulation indices were produced in Iscove's MEM for rat islets, in Ham's F-12 for porcine islets and in CMRL 1066 for human islets . Over the entire culture period a significant deterioration in function was observed in all species cultured in the control media, although this was reversed when islets were cultured in the optimal media . Furthermore, in the case of porcine and human islets a significant improvement in function over the seven-day period was noted in the optimal media . In conclusion, of the commercially available media, the optimal tissue culture medium for rat islets is Iscove's MEM, for porcine islets is Ham's F-12, and for human islets is CMRL 1066. Arch Biochem Biophys, 1995 Oct 20, 323(1), 87 - 96 A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli; Richardson TH et al.; Human cytochrome P450s 2C8, 2C9, 2C18, and 2C19 and rabbit cytochrome P450s 2C1, 2C2, 2C4, 2C5, and 2C16 were expressed from their respective cDNAs in Escherichia coli as chimeric enzymes in which a portion of the N-terminal membrane anchor sequence was replaced with a modified sequence derived from P450 17A . For 2C1 and 2C2 removal of the extraneous 3'-untranslated sequence allowed the successful expression of constructs that were unproductive in its presence . The levels of expression varied from 180 to 1500 nmol/liter of culture and the addition of delta-aminolevulinic acid to the culture media increased the amount of spectrally detectable P450 for several of these enzymes 2- to 10-fold . The catalytic properties of the modified human 2C P450s expressed in E . coli were concordant with previously published data for several marker substrates including (S)-mephenytoin for P450 2C19, tolbutamide and tetrahydrocannabinol (THC) for P450 2C9, and taxol for P450 2C8 . Interestingly, P450 2C19 catalyzed the 21-hydroxylation of progesterone and, to a lesser extent, catalyzed the formation of 16 alpha-hydroxyprogesterone . The rabbit enzyme P450 2C16 catalyzed the formation of 17 alpha- and 16 alpha-hydroxyprogesterone in addition to 21-hydroxylation . P450 2C19 also catalyzed the methylhydroxylation of tolbutamide and the 7-hydroxylation of THC at rates that were similar to or greater than that of P450 2C9 . This work has identified important factors required for the high-level expression of 2C subfamily P450s in E . coli . The availability of these enzymes will facilitate detailed kinetic measurements for known and yet to be identified substrates. J Biol Chem, 1995 Oct 13, 270(41), 24496 - 501 Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in hemoglobin degradation; Becker MM et al.; A cDNA encoding a Schistosoma japonicum aspartic proteinase was cloned, sequenced, and found to encode a zymogen of 380 amino acid residues, and its gene was shown to be present as a single copy in the S . japonicum genome . Identity comparisons showed that the enzyme (Sjpasp) was most closely related to the cathepsin Ds . The deduced amino acid sequence has four potential glycosylation sites, two of which are in identical positions to the two glycosylation sites of human kidney lysosomal cathepsin D . Furthermore, all four disulfide bonds found in mammalian cathepsin D sequences are present in Sjpasp, although the beta-hairpin (loop 3), which is cleaved during maturation of vertebrate cathepsin Ds to yield light and heavy chain subunits, is absent from Sjpasp . While most residues involved in substrate specificity and catalysis of aspartic proteinases are preserved in Sjpasp, several residues in these regions exhibit changes that may result in a novel substrate specificity . Aspartic proteinase activity is present in extracts of adult S . japonicum and Schistosoma mansoni and in culture media in which schistosomes were maintained and was capable of digesting hemoglobin . The schistosome aspartic proteinase may play a pivotal role in the catabolism of hemoglobin obtained from host erythrocytes. Biochem Biophys Res Commun, 1995 Oct 13, 215(2), 721 - 9 Serine and cysteine proteinase inhibitors prevent nitric oxide production by activated macrophages by interfering with transcription of the inducible NO synthase gene; Griscavage JM et al.; The objective of this study was to ascertain the mechanism by which serine and cysteine proteinase inhibitors interfere with production of NO by LPS-activated rat alveolar macrophages . Macrophages were incubated in the presence of LPS+ test agent for 24 hr . Culture media were analyzed for NOX- accumulation, harvested cells were assayed for iNOS activity, and cellular RNA was extracted for determination of iNOS mRNA by Northern blot analysis . TPCK, TLCK, calpain inhibitor 1 (CPI-1) and calpain inhibitor 2 (CPI-2) each inhibited NOX- production and inducible iNOS expression in a concentration-dependent manner at 1-100 microM . TPCK and CPI-1 were about 10-fold more potent than TLCK and CPI-2, respectively . These data suggest that a chymotrypsin-like serine or cysteine proteinase is required for the LPS-inducible expression of the iNOS gene, perhaps by mechanisms involving activation of transcription factor NF-kappa B . Accordingly, a potent inhibitor of NF-kappa B activation whose action is attributed to inhibition of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) was tested . Z-IE(O-t-Bu)A-Leucinal abolished NOX- production and inducible iNOS expression at 1 microM and showed over 50% inhibition at 10 nM . These observations indicate that inhibitors of MPC interfere with iNOS induction and provide strong evidence that MPC functions importantly in iNOS induction in macrophages. Biochem Biophys Res Commun, 1995 Oct 13, 215(2), 691 - 7 Secretion of prostaglandins elicited by lipopolysaccharide and ethanol in cultured rat Kupffer cells; Victorov AV et al.; Prostaglandins (PGs) released by cultured rat Kupffer cells in response to stimulation with lipopolysaccharide (LPS) or ethanol were extracted from culture media, separated by HPLC and measured by radioimmunoassay . LPS (0.5-5 micrograms/ml) enhanced, after a 3-4 hrs lag period, the production of PGE2 (7-10 fold by 24 hrs), thromboxane B2 (2-3 fold) and PGD2 . PG 6-keto-F1 alpha, PGF2 alpha (20-50% each) . This effect was not inhibited by 30 microM aspirin but was reduced by dexamethasone . Ethanol (25-85 mM) gradually increased the release of PGE2 (40-90% by 24 hrs) and other PGs (10-30%), with 30 microM aspirin eliminating this effect . When added together with LPS, ethanol potentiated the endotoxin action . We suggest that LPS causes synthesis of the inducible cyclooxygenase-2 form in Kupffer cells, whereas ethanol exerts its effect via the pre-existing cyclooxygenase-1 mainly by increasing the free arachidonic acid content. J Immunol Methods, 1995 Oct 12, 186(1), 17 - 25 Long-term production of human monoclonal antibodies by human-mouse heterohybridomas; Yoshinari K et al.; Production of monoclonal antibodies (mAbs) by fused somatic cells was first developed by Kohler and Milstein two decades ago, but its utilization for the production of human mAbs, particularly those bearing kappa chains, has been difficult because heterohybridomas formed with mouse myeloma cells are unstable and tend to lose certain of their human chromosomes . We have stabilized two such heterohybridomas over one year period and induced the production of kappa-bearing and lambda-bearing human mAb, respectively . Increased productivity was achieved by adding the Na+K(+)-ATPase inhibitor, ouabain and a cell mitosis inhibitor, cytochalasin B, to the cell culture media. J Immunol Methods, 1995 Oct 12, 186(1), 151 - 4 Variability in the growth sustaining capacity of medium batches; Hugin AW et al.; Medium batches, analysed in various spontaneous and mitogen induced proliferation assays, revealed heterogeneity in their growth promoting activity . This can critically affect test results and suggests that culture media can be a source of variability and problems in cell culture work . The manufacturers of media should broaden their quality control of medium batches. Biochim Biophys Acta, 1995 Oct 4, 1239(1), 74 - 80 The effect of lethal acid stress on Na+/H+ exchanger isoforms in cultured inner medullary collecting duct cells: deletion of NHE-2 and over expression of NHE-1; Singh G et al.; Cultured inner medullary collecting duct (mIMCD-3) cells express Na+/H+ exchanger isoforms NHE-2 and NHE-1 (Soleimani et al . (1994) J . Biol . Chem . 269, 27973-27978) . In the present studies we examined the effect of lethal acid stress on Na+/H+ exchanger activity and isoform expression in mIMCD-3 cells . mIMCD-3 cells were incubated for 10 min with 20 mM ammonium, and exposed to an ammonium-free acidic solution (pH 6.0) for 120 min . Thereafter, cells were recovered and grown in normal culture media . The surviving clones were isolated and subjected to two additional cycles of acid stress . A mutant clone was isolated and characterized for Na+/H+ exchange activity and isoform expression . The mutant mIMCD-3 clone demonstrated significant over-expression of Na+/H+ exchange activity as assessed by acid-stimulated 22Na influx (11.56 nmol/mg protein in mutant vs . 4.06 nmol/mg in parent cells, P < 0.001, n = 4) and sodium-dependent pHi recovery from an acid load (0.55 pH/min in mutant vs . 0.28 pH/min in parent cells, P < 0.01, n = 6) . A dose-response inhibition of the exchanger showed that the mutant cells were very sensitive to dimethylamiloride (IC50 158 nM in mutant vs . 889 nM in parent mIMCD-3 cells, P < 0.001) . To compare the Na+/H+ exchanger isoforms in mutant and parent mIMCD-3 cells, poly(A)+ RNA was isolated from each group and probed with radiolabeled NHE-1 or NHE-2 cDNA . The expression of NHE-1 mRNA was increased by approximately 100% in mutant cells . The NHE-2 mRNA, on the other hand, was found to be absent in mutant mIMCD-3 cells . Examination of the regulatory mechanisms of the Na+/H+ exchanger isoforms in parent mIMCD-3 cells, which express NHE-2 and NHE-1, and mutant mIMCD-3 cells, which only express NHE-1, would be helpful in elucidating the roles of NHE-2 and NHE-1 in inner medullary collecting duct cells. Teratology, 1995 Oct, 52(4), 205 - 14 Diamide-induced alterations of intracellular thiol status and the regulation of glucose metabolism in the developing rat conceptus in vitro; Hiranruengchok R et al.; Direct oxidation of embryonic reduced glutathione (GSH) by a thiol oxidant, diamide, has been demonstrated to result in increased glutathione disulfide (GSSG) and protein-glutathione mixed disulfide (protein-S-SG) formation, which is accompanied by embryotoxicity and reductions in amniotic fluid volume . The altered functions of critical proteins or enzymes caused by the formation of protein-S-SG perturb cellular metabolism and may be involved in the embryotoxicity produced by GSH oxidation . The present study investigates changes in the metabolism of glucose through glycolysis and the pentose phosphate shunt pathways (PPP) and their related enzymes under the oxidative conditions produced by diamide exposure in organogenesis-stage rat conceptus (gestational day 10) in vitro . The metabolism of glucose via the PPP, measured as amounts of CO2 production from D-{1-14C}-glucose, was significantly increased in the conceptus exposed to 100-500 microM diamide to levels 2.5-3-fold those of controls . It was found that these substantial increases in the PPP activity did not correlate well with a moderate activation of glucose 6-phosphate dehydrogenase (G6PD) activity, the key enzyme in the PPP pathway . Changes in glycolysis due to diamide treatment were also determined by measurements of lactate production from D-{U-14C}-glucose . Production of lactate by the conceptus exposed to 250-500 microM diamide for 60 min was reduced (to approximately 54% of control values) concomitantly with a significant inhibition of the glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase (GPD) and phosphofructokinase (PFK), indicating an overall decrease in glycolysis . Diamide was found to produce a differential effect on the enzymatic activities determined in this study, with greater degrees of inhibition seen in the tissue supernatants from the visceral yolk sac (VYS) compared to those from the embryo . Activities of GPD and PFK were decreased to approximately 22% and 43% control values, respectively, when determined in the supernatants from the VYS of the conceptus exposed to 500 microM diamide for 60 min . In addition, more than 90% of the GPD activity in the VYS, but not the embryo, was rapidly inhibited by the thiol alkylating agent N-ethylmaleimide (NEM, 100 microM) within 15 min of the exposure . In contrast to diamide and NEM, no alterations in lactate production were seen in the conceptus treated with the GSH depletor L-buthionine-S,R-sulfoximine (1 mM) for 5 hr in the culture media . Further experiments demonstrated that the activity of the GPD, inhibited by a 30-min incubation with 500 microM diamide, can be reversed after removal of diamide and that this effect was potentiated by subsequent treatment with dithiothreitol (30 mM), a thiol reducing agent . These results indicated the involvement of thiol/disulfide status in regulation of the metabolism of glucose in the developing conceptus and support the hypothesis that GSH oxidation and protein-S-SG formation could be a critical event associated with mechanisms of embryotoxicity elicited by oxidative stress . It was suggested in this study that, under these experimental conditions, embryotoxicity induced by diamide is primarily mediated via altered VYS functions, including disrupted energy production (glycolysis). Mol Membr Biol, 1995 Oct-Dec, 12(4), 331 - 7 An investigation into the role of N-glycosylation in the functional expression of a recombinant heteromeric NMDA receptor; Chazot PL et al.; The effect of N-glycosylation on the assembly of N-methyl-D-aspartate (NMDA) heteromeric cloned receptors was studied . Thus human embryonic kidney (HEK) 293 cells were cotransfected with N-methyl-D-aspartate R1 (NR1) and N-methyl-D-aspartate R2A (NR2A) clones and the cells grown post-transfection in the presence of tunicamycin (TM) . TM treatment resulted in a decrease of the NR1 subunit with M(r) 117 000 with a concomitant increase in a M(r) 97 000 immunoreactive species previously identified as the non-N-glycosylated NR1 subunit . In parallel, TM caused a dose-dependent inhibition of {3H}MK801 binding to the expressed receptor which was a result of an approximate four-fold reduction in the Dissociation Constant (KD) but with no change in the number of binding sites (Bmax) . NMDA receptor cell surface expression was unchanged following TM treatment but it did result in a decrease in the percentage cell death post-transfection compared to control samples . The removal of TM from the cell culture media resulted in a return to the control KD value for {3H}MK801 binding and partial reglycosylation of newly synthesized NR1 subunit . These results demonstrate that N-glycosylation is requisite for the efficient expression of functional NR1/NR2A receptors . Furthermore, they suggest that N-glycosylation may be important for the correct formation of the channel domain of the NR1/NR2A receptor. Can J Microbiol, 1995 Oct, 41(10), 951 - 4 Fetal bovine serum induces changes in fatty acid composition of Trypanosoma cruzi phosphoinositides; Racagni G et al.; Fetal bovine serum (FBS) is a necessary constituent of the culture media employed to foster the growth of Trypanosoma cruzi epimastigote forms . In different laboratories, the serum is used at final concentrations of 5 or 10% . We have normally supplemented the complex medium with 10% FBS . Under this condition we have described the fatty acid composition of the total lipids and of the phosphoinositide fractions . Additionally, we have reported the increase of polyphosphoinositides and phosphatidic acid after cholinergic stimulation . Since further attempts to reproduce these results with 5% FBS in the culture medium were not successful, the effect of the FBS concentration on the fatty acid composition of phospholipids from the T . cruzi epimastigote forms was thoroughly examined . This work showed that when the FBS concentration supplementing the culture medium was reduced from 10 to 5%, the fatty acid composition of the phosphoinositides was altered while the other major phospholipids were not significantly affected . The most relevant result was the decrease in the content of linoleic acid (18:2) and the increase of palmitoleic acid (16:1) in phosphatidylinositol 4,5-bisphosphate . Phosphatidylinositol (PI) and phosphatidylinositol phosphate also exhibited similar changes in the same fatty acids . The C2 fatty acid composition of the phosphoinositides, under the same conditions, is also reported here for the first time. J Assist Reprod Genet, 1995 Oct, 12(9), 590 - 3 Interferon gamma and interleukin 10 levels in preimplantation embryo culture media; Ozornek MH et al.; PURPOSE: The aim of our study is to elucidate whether human oocytes/embryos secrete IFN gamma and/or IL-10 and whether the fertilization process depends on the balance between these cytokines . METHODS: A total of 142 embryo culture media from 24 patients were collected and the cytokine levels were tested with ELISA . RESULTS: IFN gamma and IL-10 were detectable in 40.1% and 29.6% of culture media respectively . The difference of IFN gamma and IL-10 levels in media from fertilized oocytes between day 1 and day 2 are significant (0.46 vs . 1.47 and 34.2 vs . 12.7, respectively) . However there was no significant difference between the IFN gamma levels of the media from fertilized and nonfertilized oocytes 0.46 vs . 0.85 at day 1 and 1.47 vs 1.49 at day 2, as well as IL-10 levels 34.2 vs . 30.9 at day 1 and 12.7 vs . 9.58 at day 2 respectively . CONCLUSIONS: Human preimplantation embryos secrete the cytokines IFN gamma and IL-10 . No effect of these cytokines on fertilization process could be shown. Mol Reprod Dev, 1995 Oct, 42(2), 188 - 99 Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro; McKiernan SH et al.; Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids . This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a compleme