J Gen Virol, 1991 Nov, 72 ( Pt 11), 2739 - 45 Frequent detection of bovine polyomavirus in commercial batches of calf serum by using the polymerase chain reaction; Schuurman R et al.; Twenty commercial batches of calf serum, obtained from several suppliers, were tested for the presence of bovine polyomavirus (BPyV) DNA and antibodies against the virus . Using polymerase chain reaction (PCR) technology, BPyV DNA was detected in 70% of the batches; no BPyV was detected in any of the negative control samples . The specificity of the amplification reactions was proven by hybridization . PCR results were confirmed by virus isolation experiments performed with five PCR-positive and five PCR-negative serum batches . The results indicate that the use of calf serum to supplement tissue culture media involves a serious risk of contaminating cell cultures with BPyV . No correlation was observed between the presence or absence of anti-BPyV immunoglobulins and the detection of BPyV-specific DNA sequences in the serum batches. FEBS Lett, 1991 Oct 21, 291(2), 249 - 52 PDGF suppresses the activation of group II phospholipase A2 gene expression by interleukin 1 and forskolin in mesangial cells; Muhl H et al.; Treatment of rat mesangial cells with interleukin 1 beta (IL-1 beta) and forskolin greatly enhanced the expression of group II phospholipase A2 (PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2, as detected by PLA2 activity measurements and immunoprecipitation of culture media of {35S}methionine-labelled mesangial cells . PDGF-BB dose-dependently suppressed the IL-1 beta- and forskolin-induced elevation of PLA2 mRNA, as well as PLA2 synthesis and secretion . In contrast, PDGF-AA had no inhibitory effect . The tyrosine kinase inhibitor genistein dose-dependently antagonized the inhibitory effect of PDGF-BB on IL-1 beta-stimulated PLA2 secretion, thus suggesting that tyrosine phosphorylation may be required for PDGF-BB inhibition of PLA2 gene expression in mesangial cells. Brain Res Dev Brain Res, 1991 Oct 21, 62(2), 293 - 6 Development of glutamate neurotoxicity in cortical cultures: induction of vulnerability by insulin; Schafer M et al.; The effect of insulin on the development of excitotoxic vulnerability in primary cultures of the rat cerebral cortex was examined . Cells were maintained for two weeks in serum-supplemented culture media, in the presence or absence of increasing insulin concentrations . Excitotoxic cell death was induced by 1 mM glutamate . The vulnerability of cells was evaluated by phase contrast microscopy and by the measurement of lactate dehydrogenase (LDH) release due to cytotoxic injury . In addition to a moderate (less than 50%) stimulation of protein and DNA synthesis, insulin produced more than a twofold increase in the excitotoxic vulnerability of cells . The effect of insulin was specific, concentration-dependent and required an intact molecular structure of insulin . Our findings indicate that insulin induces significant changes in cerebral neurons by increasing the lethal vulnerability of cortical cells to excitatory amino acids (EAAs). Science, 1991 Oct 18, 254(5030), 430 - 2 Enhancement of growth of virulent strains of Escherichia coli by interleukin-1; Porat R et al.; Interleukin-1 (IL-1) is a polypeptide cytokine that mediates many physiological responses to infection and inflammation and is a growth factor for certain mammalian cells . Virulent and avirulent clinical isolates of Escherichia coli were grown in culture media in the presence of human IL-1 . IL-1 beta, but not tumor necrosis factor or IL-4, enhanced the growth of virulent, but not avirulent, E . coli . This enhancement was blocked by the IL-1 receptor antagonist (IL-1ra) . Radiolabeled IL-1 bound to virulent but not avirulent E . coli in a specific and saturable fashion; IL-1ra inhibited this binding . Thus, human IL-1 may recognize a functional IL-1-like receptor structure on virulent E . coli and may be a virulence factor for bacterial pathogenicity. Biochim Biophys Acta, 1991 Oct 15, 1086(1), 72 - 80 Lipoprotein secretion by the human hepatoma cell line Hep G2: differential rates of accumulation of apolipoprotein B and lipoprotein lipids in tissue culture media in response to albumin, glucose and oleate; Arrol S et al.; Serum low-density lipoprotein (LDL) concentration is a major determinant of susceptibility to the development of atherosclerosis . A major component of the protein moiety of LDL and its precursor very-low-density lipoprotein is apolipoprotein B (apo B) . The human hepatoma cell line, Hep G2, was used as a model for the investigation of mechanisms which control hepatic secretion of the apo B and lipid components of lipoproteins . Using a sensitive immunoradiometric assay for apo B developed in this laboratory, we showed that bovine serum albumin inhibited and glucose, and fatty acids enhanced the rate of accumulation of apo B in the culture medium of Hep G2 cells . However, these substances did not necessarily affect LDL lipids in the same way as apo B . This finding appeared to be due to Hep G2 cells expressing lipase activities which led to triacylglycerol and phospholipid hydrolysis and lipid reuptake . Reuptake of apo B also occurred, but its rate of accumulation in the culture medium suggested it was a closer reflection of its true secretory rate. Biochim Biophys Acta, 1991 Oct 11, 1080(1), 1 - 10 Purification and partial characterization of a shed 66 kDa melanoma-associated antigen identified by autologous antibody; Vlock DR et al.; We have previously reported the isolation of a 66 kDa melanoma-associated antigen, identified by autologous antibody, in serum and unfractionated spent tissue culture media by Western blot analysis . The antigen, detected by autologous serum S150, was found to be broadly represented on melanoma, glioma, renal cell carcinoma, neuroblastoma and head and neck carcinoma cell lines . S150 did not react with bladder or colon carcinoma, fetal fibroblasts, pooled platelets, lymphocytes and red blood cells, autologous cultured lymphocytes or fetal calf serum . To further characterize the antigen, spent tissue culture media, obtained from autologous melanoma cell line, Y-Mel 84:420, was separated by an isoelectric focusing column . Unabsorbed control serum S150 was noted to have a maximum titer of 1:2040 against autologous melanoma cells as measured by protein A hemadsorption . Following isoelectric focusing the greatest decrease in autologous antibody titer (30-fold) occurred with fractions having a pI between 2 and 3 . Further resolution of the antigen was accomplished with high-pressure ion-exchange chromatography . One of these fractions showed a significantly higher concentration of antigen and was distinctly resolved from bulk serum albumin . Subsequent Western blot analysis, with autologous antibody, of the isolated antigen-containing fraction, confirmed the presence of a single 66 kDa band . Exposure of the antigen, purified by high-pressure ion-exchange chromatography, to neuraminidase ablated recognition by autologous antibody and suggests that sialic acid is present on the protein and may be part of the antigenic epitope . Binding of antigen, obtained following DEAE anion exchange chromatography, was noted to lectins derived from Triticum vulgaris, Dolichos biflorus and Lycopersicon esculentum . Preparative purification of the antigen was accomplished by anion exchange followed by lectin affinity chromatography with a Dolichos biflorus column . Antigen obtained following lectin affinity chromatography subjected to SDS-PAGE and silver stain revealed a single band at 66 kDa . We conclude that a melanoma-associated antigen detected by autologous antibody in spent tissue culture media is an unusually acidic glycoprotein (pI 2-3). Biochim Biophys Acta, 1991 Oct 8, 1090(2), 216 - 22 Stable expression of human tissue-type plasminogen activator regulated by beta-actin promoter in three human cell lines: HeLa, WI-38 VA13 and KMS-5; Morishita H et al.; A high-level and stable expression system of human tissue-type plasminogen activator (t-PA) was accomplished in human cells by selecting a promoter and a host cell line . First, we have constructed two types of t-PA expression plasmids containing 3 kb of the human beta-actin promoter region or 0.3 kb of SV40 early promoter region and these plasmids were transfected into HeLa cells, respectively, and the resulting transfectants were found to secrete various amounts of t-PA derived from the plasmids to the culture media . Southern blot analysis revealed that the beta-actin promoter was more efficient than the SV40 early promoter with regard to the expression level per single copy of the t-PA gene in the transfected HeLa cells . Next, the t-PA expression plasmid containing the beta-actin promoter was also transfected into WI-38 VA13 cells, a human fibroblastic cell line, and KMS-5 cells, a human lymphoid cell line, in order to compare the expression ability of the promoter among these three cell lines . Some of the transfectants from both cell lines were also found to produce t-PA . It was also found that the expression levels in HeLa and WI-38 VA13 seemed to be more efficient than that in KMS-5. Gynecol Oncol, 1991 Oct, 43(1), 29 - 36 Cell kinetic perturbations after irradiation and caffeine in the BG-1 ovarian carcinoma cell line; Wolloch EH et al.; The treatment of ovarian carcinoma includes maximum surgical removal of the tumor tissue followed by irradiation or chemotherapy . In this study, the effects of caffeine on cell cycle traverse have been studied over a 168-hr period after X irradiation in BG-1 cells, an ovarian carcinoma cell line . The results were obtained with dual-parameter flow cytometric measurements of DNA and nuclear protein, using propidium iodide and fluorescein isothiocyanate . After radiation alone, a dose-related arrest of cells in G2 phase and cell kill were observed . Irradiating BG-1 cells with 5 Gy produced an accumulation of the cells in G2 at 24-72 hr postirradiation . When G2 was divided into low nuclear protein (G2A) and high nuclear protein (G2B) compartments, there was a G2A peak accumulation at 24 hr and a G2B peak accumulation at 48-72 hr . The addition of 1 mM caffeine to the culture media, starting immediately postirradiation, prevented G2 arrest, promoting a rapid traverse of cells through G2A to G2B to G1, which was associated with diminished survival. Br J Haematol, 1991 Oct, 79(2), 239 - 45 The high molecular weight form of endothelial cell von Willebrand factor is released by the regulated pathway; Tsai HM et al.; We have previously reported that two forms of von Willebrand factor (vWf) exist in cultured human umbilical vein endothelial cells: a high molecular weight (HMW) form that is released and can be proteolytically cleaved into a series of plasma-like multimers, and a non-secreted low molecular weight (LMW) form . In this study, the mode of vWf release and the relationship between the two forms were examined . As determined by two-dimensional analysis as well as by immunoreactivity with an antibody to the propolypeptide, the LMW form of endothelial cell vWf consisted of a 260 kD pro-vWf polypeptide, while the HMW form consisted of a 225 kD mature polypeptide . Only the 260 kD polypeptide was susceptible to digestion with endoglycosidase H . Release of the HMW form into the culture media was accompanied by a decrease in cellular vWf . Treatment of endothelial cells with cycloheximide or tunicamycin caused a decrease in the LMW form but did not affect the secretion of the HMW form . These results suggest that two pools of vWf exist in endothelial cells--a LMW form of pro-vWf in the endoplasmic reticulum and a HMW form of mature vWf in the storage compartment . Released vWf derives only from the storage pool. Muscle Nerve, 1991 Oct, 14(10), 1003 - 12 Fibrillation and accelerated AChR degradation in long-term muscle organ culture; Wetzel DM et al.; Evaluation of the precise molecular dynamics of endplate maintenance and reorganization has been limited by the lack of available in vitro preparations . We describe an organ culture preparation of mouse diaphragm muscle which permits long-term maintenance of muscle viability . Spontaneous fibrillations, increased levels of extrajunctional acetylcholine receptors, accelerated rates of junctional acetylcholine receptor turnover and maintenance of fine structure of denervated mouse diaphragm muscle in organ culture was evaluated under different culture conditions . Of several standard tissue culture media tested with and without fetal calf serum, medium 199 plus fetal calf serum was best for maintaining this muscle for greater than 2 weeks . The serum component could be partially eliminated by addition of non-glucose energy substrates such as D-beta-hydroxybutyric acid and L-glutamine . This preparation will permit a more controlled examination of the molecular components of endplate diseases. J Mol Endocrinol, 1991 Oct, 7(2), 105 - 12 Stimulatory effect of oestradiol-17 beta and tamoxifen on gross cystic disease fluid protein 15,000 production and mRNA levels in T47D human breast cancer cells; Dejardin L et al.; Oestradiol-17 beta and tamoxifen regulate the synthesis of a gross cystic disease fluid protein (GCDFP-15) in T47D human breast cancer cells . Dose-response curves of GCDFP-15 mRNA contents and GCDFP-15 levels in culture media and cells versus hormone or antihormone concentration have been established . Production of GCDFP-15 was increased by oestradiol-17 beta, tamoxifen and 4-OH tamoxifen . The effect of tamoxifen and 4-OH tamoxifen was greater than the effect of oestradiol-17 beta . Moreover, oestradiol-17 beta and 4-OH tamoxifen acted synergystically in enhancing GCDFP-15 release . The strong oestrogenic effect of the antioestrogen tamoxifen in regulating GCDFP-15 may reflect an unusual interaction between the tamoxifen-oestrogen receptor complex and the DNA oestrogen-responsive elements . As oestrogen control of GCDFP-15 depends also on the cell line studied, investigation of GCDFP-15 could extend our knowledge of the possible mechanism of action of oestrogens or antioestrogens. Am J Physiol, 1991 Oct, 261(4 Suppl), 92 - 6 Specific proteins synthesized in human lymphocytes during hypoxia; Aldashev AA et al.; Hypoxia is a severe stress factor to which man and most other mammalian species are capable of adapting . However, the cellular mechanisms that enable cells to tolerate decreases in ambient oxygen tension are still unknown . We have previously shown that hypoxia induces the synthesis of unique proteins (molecular mass 38, 52, 74, 76 kDa) in human aortic endothelial cells and lymphocytes . In this study we investigated the specificity of hypoxia on the upregulation of these hypoxic stress proteins (HYP) in human peripheral blood lymphocytes and the role of calcium in this response . 35S-methionine pulse-labeling studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis and autoradiography demonstrated that normobaric hypoxia (4% O2-5% CO2-91% N2) enhanced synthesis of HYP, whereas heat-shock protein synthesis was not affected . Heat shock (42 degrees C) and cold stress (4 degrees C) did, however, induce synthesis of heat-shock protein but not HYP . The 38-kDa HYP is the major protein for which synthesis is upregulated by hypoxia . Its isoelectric point (pI) is 3.5-4.0, and it is localized in the cytosol . The 52-kDa HYP has a pI of greater than 6.5, and it is also localized in the cytosol . The 74- and 76-kDa HYPs appear to be membrane bound . In addition to hypoxia, an increase in calcium concentration in the culture media (25-50 mM) enhanced synthesis of HYP . An ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)/Ca2+ binding complex, when added to blood lymphocytes during exposure to hypoxia, significantly inhibited HYP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS) South Med J, 1991 Oct, 84(10), 1197 - 8 Technique for drawing blood for cultures: is changing needles truly necessary? Chapnick EK, Schaffer BC, Gradon JD, Lutwick LI, Krigsman SA, Levi M. After drawing blood for culture, medical students and house officers are often taught to change needles before inoculating the culture media . Data to support this practice have been scarce . We obtained simultaneous blood cultures on 75 hospital patients using two techniques: changing needles (C) or not changing needles (NC) before inoculating the cultures . Positive cultures judged to be contaminants were obtained in 4/75 patients in the NC group (5.3%) and in 1/75 patients in the C group (1.3%) . This difference was not statistically significant . The practice of changing needles after drawing blood for culture may not be necessary, thus reducing the risk of needlestick injury. Exp Cell Res, 1991 Oct, 196(2), 246 - 54 Basic fibroblast growth factor upregulates steady-state levels of laminin B1 and B2 chain mRNA in cultured neuroepithelial cells; Drago J et al.; The growth of purified populations of murine neuroepithelial cells isolated from 10 day embryonic (E10) telencephalon and mesencephalon can be specifically enhanced by supplementing growth culture media with basic fibroblast growth factor (bFGF) . One effect of bFGF on cultured neuroepithelial cells was to enhance the amount of laminin expressed at the protein level as detected by immunofluorescence . This was correlated with significant upregulation of steady-state levels of laminin B1 and B2 chain expression as analyzed at the mRNA level . When E12 neuroepithelial cells were split into precursor neuronal or glial subpopulations on the basis of differential expression of major histocompatibility class-1 antigens, only the glial progenitor fraction was found to be capable of detectable laminin synthesis . It is thus possible that a primary action of FGF is to increase the synthesis and release of extracellular matrix molecules from neural cells which act back in a paracrine manner to stimulate differentiation. Mol Cell Endocrinol, 1991 Oct, 81(1-3), 95 - 104 Effect of epidermal and insulin-like growth factors on vectorial secretion of transferrin by rat Sertoli cells in vitro; Spaliviero JA et al.; Within the seminiferous tubules, the Sertoli cells create an impermeable blood-testis barrier and an unique intratubular microenvironment that fosters the development of spermatozoa . The functional differentiation of spermatozoa therefore requires vectorial secretion by Sertoli cells of substances that cannot cross the blood-testis barrier . We investigated the role of epidermal (EGF) and insulin-like growth factors I and II (IGF-I and IGF-II) in the regulation of vectorial secretion of transferrin by Sertoli cells . In order to study the regulation of vectorial transferrin secretion, we modified culture conditions in the twin chamber culture system to maximise gradients of transferrin secretion . Sertoli cells were plated at high density (3-4 x 10(6) cells/well) into chambers of near equal volume, cultured at 37 degrees C and maintained in simple, fully defined media omitting standard supplements (insulin, EGF, FSH) which affect vectorial transferrin secretion . Using this optimised culture system, maximum gradients of transferrin secretion occurred between days 2 and 3 of culture with preferential secretion (mean ratio 3.7 +/- 0.2) directed towards the apical compartment . The transferrin ratio (ratio of transferrin secreted into the upper over the lower chamber) was decreased by insulin and FSH but not by retinoic acid or testosterone, yet all four stimuli increased total transferrin secretion . IGF-I and IGF-II were effective at physiological concentrations (ED50 = 1 ng/ml) in lowering transferrin ratio and were 100-fold more potent than insulin suggesting that insulin effects on vectorial transferrin secretion by Sertoli cells is mediated through type 1 IGF receptors . EGF also reduced the transferrin ratio (ED50 = 50 ng/ml) as well as stimulating total transferrin secretion . The hormonally mediated reduction in transferrin ratio was consistently due to enhanced secretion of transferrin into the lower chamber . In the first demonstration of a highly polarised response of Sertoli cells to hormonal stimuli, the effects of insulin, FSH and EGF on vectorial transferrin secretion were effected primarily via the basal membrane of the Sertoli cell and operated independent of mechanisms controlling total transferrin secretion . These results establish a potential role for epidermal and insulin-like growth factors in the paracrine regulation of vectorial secretion by the Sertoli cell, in particular the developmental regulation of vectorial transferrin secretion by Sertoli cells . These findings also indicate that previous studies which included insulin and EGF routinely in culture media have systematically underestimated apically directed transferrin secretion. J Bone Miner Res, 1991 Oct, 6(10), 1081 - 90 Tissue and urokinase plasminogen activators in bone tissue and their regulation by parathyroid hormone; Leloup G et al.; The identification of the plasminogen activator (PA) types present in bone and the regulation of their activity by parathyroid hormone (PTH) were investigated in cultures of fetal mouse calvariae with the use of either a chromogenic substrate or a zymographic assay . PA was detected essentially in the tissue extracts of the explanted bones, with only 1-2% of the total activity released in the surrounding culture media . From their electrophoretic behavior compared to PAs of other mouse tissues and from their response to a specific antibody raised against the tissue type PA (tPA), two major molecular species, of 70 and 48 kD were identified as tPA and urokinase (uPA), respectively, a third minor species of 105 kD being likely to correspond to complexes between tPA and an inhibitor; the culture fluids, moreover, contained enzymatically active degradation products of uPA of 42 and 29 kD . The PA activity of the bone extracts was only minimally affected by the addition of fibrinogen fragments to the chromogenic assays . PTH induced bone resorption and stimulated in parallel the accumulation of PA in the tissue; other bone-resorbing agents, 1,25-dihydroxyvitamin D3 and prostaglandin E2, had similar effects . Densitometric scanning of the zymograms of the bone extracts indicated that PTH stimulated only the production of tPA and had no effect on that of uPA . However, PTH also enhanced the release of uPA (both the 48 kD and the 29 kD forms) from the bones into the media . Although inhibiting bone resorption, calcitonin had no effect on the PTH-induced accumulation of PA in bone or on the release of tPA, but it prevented the PTH-induced accumulation of 29 kD uPA in the culture fluids . Thus these studies support the view that tPA and possibly also uPA may have a role in the physiology of bone; the nature of this role remains to be elucidated, however. Avian Dis, 1991 Oct-Dec, 35(4), 910 - 9 Experimental infection of turkeys with Mycoplasma gallisepticum of low virulence, transmissibility, and immunogenicity; Dingfelder RS et al.; Three-week-old turkeys were inoculated intranasally with approximately 10(6) colony-forming units (CFU) of putative variant Mycoplasma gallisepticum (MG) strains M876, M35, or the virulent S6 reference strain . Uninoculated turkeys in each group served as contact sentinels . The hemagglutination-inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) were used to determine serologic responses . MG was isolated from 100% and 92% of S6- and M876-inoculated turkeys, respectively, on day 7 PI . However, culture-positive rates among M876-inoculated turkeys declined more rapidly, transmission to contact sentinels took longer and occurred at lower rates, and serologic responses measured by HI and ELISA were lower than in S6-infected turkeys . Testing sera from inoculated turkeys for antibodies to MG in homologous and heterologous ELISA systems indicated that strain M876 was significantly (P less than 0.05) less immunogenic than S6 (days 62 and 95 PI), and that the homologous ELISA was more sensitive (P less than 0.005) . MG strain M35 failed to infect turkeys in three attempts, even though the inocula used were viable on culture media. Am J Vet Res, 1991 Oct, 52(10), 1622 - 5 Influence of polysulfated glycosaminoglycan on equine articular cartilage in explant culture; Caron JP et al.; Articular cartilage explants from 3 horses were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan on proteoglycan biosynthesis . Cultures were exposed to concentrations of 0, 50, or 200 micrograms of the drug/ml for either 2 days or 6 days, and labeled with 35S, before measuring the content of sulfated proteoglycan in the culture media and in extracts of cartilage . In a second experiment, the explants were incubated with the isotope and subsequently exposed to the same concentrations of the polysulfated glycosaminoglycan for 4 days . Subsequently, the amount of remaining labeled proteoglycan was determined . Gel filtration chromatography was used to compare the hydrodynamic size of proteoglycans from the cartilage explants in each experiment . Polysulfated glycosaminoglycan caused a dose-dependent depression of sulfated proteoglycan synthesis, which was statistically significant after 6 days of exposure . Radioactive proteoglycan content in explants was similar in the experiment involving isotopic labeling prior to exposure to the drug . Proteoglycan monomer size was similar in all treatment groups . It was concluded that polysulfated glycosaminoglycan caused a modest depression in proteoglycan synthesis, had little effect on endogenous proteoglycan degradation, and did not influence the size of sulfated proteoglycans synthesized by normal equine chondrocytes in explant culture. Immunology, 1991 Oct, 74(2), 310 - 6 The role of the macrophage in induction of immunosuppression in Trypanosoma congolense-infected cattle; Flynn JN et al.; Impairment of T-cell function in Boran (Bos indicus) cattle during primary infection with Trypanosoma congolense ILNat 3.1 was found to occur in peripheral blood, spleen and, in particular, the lymph nodes . Lymph node cells from infected cattle failed to proliferate in response to mitogenic stimulus and suppressed proliferation of both normal peripheral blood mononuclear cells and lymph node cells in co-culture assays . The addition of indomethacin, to inhibit prostaglandin synthesis, had no effect on the ability of lymph node cells from infected cattle to suppress the proliferative response of responder cells from uninfected cattle . The supplementation of the culture media with catalase, which degrades hydrogen peroxide, either alone or in combination with indomethacin, also did not result in restoration of proliferation . This suggested the presence of suppressor cells in lymph nodes of infected cattle which exert their effects via a prostaglandin-independent mechanism . By depleting lymph node cells from infected cattle of the monocyte-macrophage population using a cell sorter it was possible to abrogate the previously observed immunosuppression, thus indicating a key role for these macrophages in the induction of trypanosome-associated immunosuppression. Am J Physiol, 1991 Oct, 261(4 Pt 2), F600 - 6 Synthesis and action of nitric oxide in rat glomerular mesangial cells; Shultz PJ et al.; Macrophages and certain tumor cell lines can be induced to synthesize nitric oxide (NO) from L-arginine after stimulation with lipopolysaccharide (LPS) or cytokines . In the present study, we have found that culture medium collected after 24 h from unstimulated rat mesangial cells (MC) contains 6.3 +/- 1.2 microM of NO3-/NO2- (the degradation products of NO) . These levels were significantly increased when MC were incubated with LPS (10 micrograms/ml) for 24 h (23.9 +/- 4.1, P less than 0.05) . The specific inhibitor of NO synthesis, NG-monomethyl-L-arginine (L-NMMA) completely inhibited LPS-stimulated production of NO3-/NO2-, confirming that the NO3-/NO2- was derived from NO within the MC . Recent studies suggest that endothelium-derived relaxing factor (EDRF) produced by vascular endothelium is also NO, and we have previously shown that both EDRF and NO stimulate increases in MC guanosine 3',5'-cyclic monophosphate (cGMP) . Thus we sought to determine whether NO synthesized by the MC could affect cGMP levels within the same cells . After 24-h incubation with LPS (10 micrograms/ml), intracellular cGMP level within the MC was 706.3 +/- 197 (SE) compared with 40.5 +/- 7 fmol/micrograms protein in control MC incubated in media alone (P less than 0.01) . The changes in cGMP in response to LPS were inhibited by greater than 90% by L-NMMA . Similar to LPS, incubation of MC with the cytokine gamma-interferon also increased NO3-/NO2- in the culture media and increased cGMP levels within MC . The induction of NO synthesis within MC and the concomitant stimulation of MC cGMP may be important in the modulation of the effects of endotoxemia, as well as inflammation, within the glomerulus. Mol Cell Endocrinol, 1991 Oct, 81(1-3), 195 - 203 Phenol red: an inhibitor of thyroglobulin iodination in cultured porcine thyroid cells; Gruffat D et al.; Phenol red, commonly used as a pH indicator in tissue culture media, is known to possess estrogenic properties . We investigated the effect of phenol red on the process of thyroglobulin iodination which occurs only at the apical surface of porcine thyroid cells when cultured in porous bottom chambers . When phenol red was added simultaneously to both compartments (apical and basolateral), separated by the polarized monolayer, thyroglobulin iodination was inhibited by about 86% without any effect on thyroglobulin secretion and apical iodine concentration . When phenol red was added separately to either the apical or basal media, inhibition was 68% and 43%, respectively . A large amount of phenol red which was introduced into the basal medium crossed through the monolayer . Thus, inhibition was dependent upon the concentration of phenol red present in the apical compartment . A maximal inhibition was observed from 30 microM apical concentration . Phenol red acts as a substrate for thyroperoxidase in the iodination reaction. Zhonghua Yi Xue Za Zhi, 1991 Oct, 71(10), 572 - 4, 40 {Protective effects of apolipoprotein CII on endothelial cell injury induced by low density lipoprotein}; Liu Q; Human umbilical vein endothelial cells (EC) were cultured . The protective effects of high density lipoprotein (HDL) and apolipoprotein CII (apoCII) on the morphology and function of EC injured by low density lipoprotein (LDL) were studied by observing the morphological changes with phase-contrast and transmission electron microscopy, measuring the release of lactate dehydrogenase (LDH) and secretion of 6-Keto-prostaglandin F1 alpha . The EC injured by LDL showed cell contraction, membrane damage, increased release of LDH and decreased secretion of PGI2 . When HDL or apoCII was added to culture media before LDL addition, the EC showed normal morphology, normal LDH release and PGI2 synthesis . The results indicated that both HDL and apoCII can resist the injurious effect of LDL on cultured EC and that apoCII as well as HDL may play an important role in combating atherogenesis. J Biol Chem, 1991 Sep 25, 266(27), 17770 - 7 Bile acid synthesis in cultured human hepatoblastoma cells; Axelson M et al.; Biosynthetic pathways to bile acids have been studied in HepG2 cells, a well-differentiated human hepatoblastoma cell line . Cholesterol metabolites, in total 29, were isolated from culture media and cells by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry . The production rates/concentrations of cholic acid (CA) and chenodeoxycholic acid (CDCA) in media from control cells were 71 and 74 ng/10(7) cells/h, respectively . Major bile acid precursors were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA), 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid, 7 alpha-hydroxy-3-oxo-4-cholenoic acid, and 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-cholanoic acid, their concentrations being 60, 30, 23, and 10 ng/10(7) cells/h, respectively . These and nine other isolated intermediates formed essentially complete metabolic sequences from cholesterol to CA and CDCA . The remaining steroids were metabolites of the intermediates or autooxidation products of cholesterol . These findings and the observed effect of dexamethasone on production rates suggest that in HepG2 cells the major biosynthetic pathways to primary bile acids start with 7 alpha-hydroxylation of cholesterol and oxidation to 7 alpha-hydroxy-4-cholesten-3-one followed by hydroxylation at either the 26 or 12 alpha position . CDCA is formed by the sequence of 26-hydroxylation, oxidation, and degradation of the side chain and A-ring reduction . CA is formed by the sequence of 12 alpha-hydroxylation, 26-hydroxylation, oxidation, and degradation of the side chain and reduction of the A-ring . An alternative pathway to CA included A-ring reduction of the intermediate 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholestenoic acid to form THCA prior to side chain cleavage . These pathways are not limited to HepG2 cells but may also be important in humans. J Immunol Methods, 1991 Sep 20, 143(1), 1 - 9 In vitro measurement of antigen-specific cell-mediated immune responses using recombinant HIV-1 proteins adsorbed to latex microspheres; Wu JY et al.; Recombinant proteins representing full-length and truncated forms of the human immunodeficiency virus type 1 envelope protein gp160 were produced in E . coli and sf9 insect cells . These proteins were denatured and reduced as a function of purification . We adsorbed these proteins onto latex microspheres and used the protein-coated particles as a vehicle to present the antigen in vitro to splenic mononuclear cells from immune mice . Recombinant proteins presented on the latex particles induced antigen-specific proliferative responses that were dependent on the antigen concentration . The proliferative responses were similar to those produced against an identical protein used in soluble form and equivalent protein concentrations . Latex microspheres coated with recombinant proteins could also induce precursor cytotoxic T lymphocytes to mature to functional effector cells in vitro . The use of the latex microspheres to present recombinant proteins as antigens allowed for the use of denatured proteins in our assay that were not soluble in aqueous solutions, such as cell culture media . This system of delivering recombinant proteins in vitro should greatly facilitate the use of recombinant proteins in assays involving live cells. J Biol Chem, 1991 Sep 5, 266(25), 16948 - 53 Hepsin, a cell membrane-associated protease . Characterization, tissue distribution, and gene localization; Tsuji A et al.; Hepsin, a putative membrane-bound serine protease, was originally identified as a human liver cDNA clone (Leytus, S.P., Loeb, K.R., Hagen, F.S., Kurachi, K., and Davie, E.W . (1988) Biochemistry 27, 1067-1074) . In the present study the human hepsin gene was localized to chromosome 19 at q11-13.2 . The messenger RNA of hepsin is 1.85 kilobases in size and present in most tissues, with the highest level in liver . Hepsin is synthesized as a single polypeptide chain, and its mature form of 51 kDa was found in various mammalian cells including HepG2 cells and baby hamster kidney cells . It is present in the plasma-membrane in a molecular orientation of type II membrane-associated proteins, with its catalytic subunit (carboxyl-terminal half) at the cell surface, and its amino terminus facing the cytosol . Hepsin is found neither in cytosol nor in culture media . The results obtained suggest that hepsin has an important role(s) in cell growth and function. J Biol Chem, 1991 Sep 5, 266(25), 16722 - 9 30-kDa heparin-binding protein of brain (amphoterin) involved in neurite outgrowth . Amino acid sequence and localization in the filopodia of the advancing plasma membrane; Merenmies J et al.; A cDNA library constructed from mRNA of rat brain was used to clone the cDNA that encodes the 30-kDa heparin-binding protein (amphoterin) that is developmentally regulated in brain and enhances neurite outgrowth in cerebral neurons . cDNA and peptide sequencing identified a dipolar sequence that has been previously found in studies of high mobility group 1 protein: the 184-amino acid cationic region is followed by a cluster of 30 anionic residues . The mRNA encoding amphoterin is also developmentally regulated; it is strongly reduced in quantity after the rapid perinatal growth phase of the rat brain . Anti-synthetic peptide antibodies raised according to the sequence of amphoterin were shown to bind specifically to the protein isolated from brain, and were used to detect amphoterin in subcellular fractions and in immunostaining of cells . Amphoterin was found in the cytoplasm of the cell soma, in the cell processes, and the substrate-attached material . In cells that are at an active stage of spreading and extending their cytoplasmic processes amphoterin was especially associated with plasma membrane filopodia . The distinct localization to the filopodia of the advancing plasma membrane suggests that endogenous amphoterin has a role in the extension of neurite-type cytoplasmic processes in developing cells . This inference is further supported by the finding that both anti-amphoterin and the anti-synthetic peptide antibodies in the culture media strongly inhibit the outgrowth of cytoplasmic processes. J Biol Chem, 1991 Sep 5, 266(25), 16380 - 6 Characterization of DNA damage induced by 3,4-estrone-o-quinone in human cells; Nutter LM et al.; The DNA damage induced in a human breast cancer cell line treated with 1,5 (10)-estradiene-3,4,17-trione (3,4-estrone-o-quinone; 3,4-EQ) has been measured qualitatively and quantitatively . Single-strand (ss) but not double-strand (ds) DNA breaks were formed in MCF-7 cells treated with 3,4-EQ . The ss DNA breaks formed in MCF-7 cells were partially repaired after incubation of cells in 3,4-EQ-free media for 2 and 4 h (i.e . 33 and 23% repair, respectively, as compared to the ss DNA breaks in cells after a 1-h exposure to 3,4-EQ without a recovery period) . The formation of interstrand DNA cross-links was demonstrated in MCF-7 cells exposed to the bifunctional alkylating agent, mitomycin C, but not in those exposed to 3,4-EQ . Protein-linked DNA breaks were detected in MCF-7 cells after exposure to camptothecin and etoposide but not 3,4-EQ, suggesting that the ss DNA breaks induced by 3,4-EQ are unlikely to be mediated via topoisomerases . The induction of ss DNA breaks was detected in the estrogen receptor-negative cell line, BT-20, after exposure to 3,4-EQ . Furthermore, excess estradiol in culture media did not prevent 3,4-EQ-induced ss DNA breaks, suggesting that the DNA damage was not mediated via the estrogen receptor . Evaluation of the newly synthesized quinone analogue, 5,6,7,8-tetrahydro-1-2-naphthoquinone, in the ss DNA breakage assay revealed that the A and B ring moiety of 3,4-EQ is sufficient to produce ss DNA breaks in MCF-7 cells. Orv Hetil, 1991 Sep 1, 132(35), 1907 - 12 {A new drug in a new role: dipyridamole in the treatment of HIV-1 infections?}; Szebeni J; Dipyridamole (DP) is a widely used coronary vasodilator and antithrombotic drug . The presented experiments demonstrate that DP inhibits the replication of HIV-1 and markedly potentiates the anti-HIV activity of azidothymidine (AZT) and dideoxycytidine in CD4-positive cells (monocyte-macrophages and T-lymphocytes) . At the same time DP does not potentiate the bone marrow toxicity of AZT, and, in CEM-ss lymphoblastoid cells, it significantly suppresses the cytotoxicity of AZT . Studies on the mechanism of these effects suggest that DP inhibits the intracellular phosphorylation of physiological nucleosides, whereas it does not affect phosphorylation of AZT and other antiviral dideoxynucleoside drugs . This may lead to relative enhancement of the metabolic activation of dideoxynucleoside drugs and the inhibitory action of their active, triphosphate form on HIV reverse transcriptase . Studies comparing the binding of DP to proteins in tissue culture media and in human plasma suggest that in order to achieve significant antiviral potentiation in vivo, high doses of DP will be required. J Protozool, 1991 Sep-Oct, 38(5), 465 - 71 Characterization of the extracellular ribonuclease of Tetrahymena pyriformis W; McKee T et al.; Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media . The extracellular ribonuclease from strain W has been purified and partially characterized . The molecular weight was determined by gel filtration to be 26,500 . The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated . The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity . The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid . The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP . No inhibition of the hydrolysis of Torula yeast RNA was detected . The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes. In Vitro Cell Dev Biol, 1991 Sep, 27A(9), 689 - 97 Extracellular matrix permits the expression of von Willebrand's factor, uptake of di-I-acetylated low density lipoprotein and secretion of prostacyclin in cultures of endothelial cells from rat brain microvessels; Doron DA et al.; Microvascular endothelial cells from the adult rat brain were cultured on Matrigel and found to express many differentiated properties including secretion of prostacyclin (PGI2) and von Willebrand's factor (vWF) . Brain microvascular endothelial cells (BMECs) were purified by dextran and percoll gradients after enzymatic treatment and cultured under various conditions . BMECs that were plated on Matrigel stained positively for factor VIII-related antigen and incorporated Di-I-acetylated low density lipoprotein, whereas BMEC plated on fibronectin, gelatin, or uncoated dishes did not express any of the above properties which are characteristic of endothelial cells . vWF was measured by a sensitive ELISA in the culture media of BMECs plated on different types of matrices . Specificity of the anti-human vWF antibodies for the rat vWF was verified by immunoabsorption on a solid phase, sodium dodecyl sulfate, and Western blot analysis . BMECs also secreted vWF into the culture media only when the cells were plated on Matrigel, and this secretion was augmented after a 6 h incubation with an interleukin-1 tumor necrosis factor-alpha mixture, but not by lipopolysaccharide . From different matrices tested, only Matrigel permitted the secretion of PGI2 by BMECs . Cells also proved to be sensitive to mechanical stimulation and became refractory to secretagogue if the mechanical stimulation was serially repeated . Under the best conditions, stimulation of the cells with bradykinin (1 microM) substantially increased PGI2 secretion . These data indicate that growth of BMECs on Matrigel in vitro permits the expression of classical endothelial cell markers in a manner similar to the behavior of these cells in situ. Biochem J, 1991 Sep 1, 278 ( Pt 2), 369 - 73 Phorbol-ester-mediated expression of the collagen type I pro-alpha 2 gene in mouse 3T3-L1 cells and its absence in a phorbol 12-myristate 13-acetate-non-responsive variant; Stuiver I et al.; We have previously isolated a phorbol 12-myristate 13-acetate (PMA)-non-responsive variant line (VT-1) from mouse 3T3-L1 cells {Shimizu, Fujiki, Sugimura & Shimizu (1986) Cancer Res . 46, 4027-4031} . Differential hybridization of cDNAs obtained from PMA-treated 3T3-L1 and VT-1 cells resulted in the isolation of a number of unique cDNA clones, including one with a high degree of sequence similarity to the type I pro-alpha 2 collagen gene (COL1A2) {Amagai, Inokuchi, Nishikawa, Shimizu & Shimizu (1989) Somat . Cell Mol . Genet . 15, 153-158} . Here we examined the expression of type I collagen pro-alpha 2 {pro-alpha 2(I)} mRNA and production of type I collagen in these two cell lines . In quiescent cells, the pro-alpha 2(I) steady-state mRNA levels were four times greater in 3T3-L1 cells than in VT-1 cells . PMA addition caused the steady-state levels of pro-alpha 2(I) mRNA to be six times greater in 3T3-L1 cells than in VT-1 cells, with a maximum at 30-60 min . The pro-alpha 2(I) protein levels in the extracellular matrix or culture media of 3T3-L1 cells were substantially elevated by PMA treatment, but no significant increase was detected in VT-1 cells . The correlation of collagen expression with a PMA-mediated mitogenic response suggests a new role for collagen as an early component of mitogenic signal transduction. Cancer Res, 1991 Sep 1, 51(17), 4516 - 22 Advantages of adoptive chemoimmunotherapy with polyethylene glycol-cultured, antigen-activated, tumor-infiltrated spleen cells for the complete eradication of lethal MOPC-315 plasmacytomas; Laude M et al.; The incorporation of polyethylene glycol-6000 (PEG) into the culture media of tumor-infiltrated spleen cells (TISpC) and MOPC-315 stimulator tumor cells at a responder to stimulator cell ratio of 30/1 had been shown to lead to the appearance of CD8+ T-cells that were effective in adoptive chemoimmunotherapy (ACIT) of mice bearing a barely palpable MOPC-315 tumor (J . A . Wise, M . B . Mokyr, and S . Dray, Cancer Res., 49:3613-3619, 1989) . Here we show that in the presence of substantially fewer added stimulator tumor cells (responder to stimulator cell ratio, 100/1), the inclusion of PEG in the cultures of TISpC also enhanced the appearance of cells that were highly effective in curing such mice by ACIT . Moreover, these PEG-cultured TISpC were more effective in ACIT than TISpC cultured in the presence of an optimal concentration of recombinant interleukin-2 (60 IU/ml) . The potency of the tumor-eradicating activity of the PEG-cultured TISpC in ACIT was further illustrated by their ability to cause the complete regression of a large (20-22 mm) s.c . MOPC-315 tumor in conjunction with a dose of drug that by itself did not cause tumor regression . PEG-cultured TISpC that were effective against MOPC-315 tumor cells in an antigen-specific manner . In fact, PEG-cultured TISpC were more effective than recombinant interleukin-2-cultured TISpC, not only in ACIT, but also in their ability to lyse MOPC-315 tumor cells in vitro . Thus, a direct specific lytic activity against the tumor by cytotoxic T-lymphocytes is the apparent mechanism through which the complete regression of the large tumor burden is brought about by the PEG-cultured TISpC . Finally, we suggest that the incorporation of PEG to render ineffective lymphoid cells effective in ACIT may offer some advantages compared with the incorporation of recombinant interleukin-2 and may be suitable for protocols to generate human cytotoxic cells for cancer therapy when there are relatively low numbers of available tumor cells. J Virol, 1991 Sep, 65(9), 4902 - 9 Replication of an acutely lethal simian immunodeficiency virus activates and induces proliferation of lymphocytes; Fultz PN; A variant of simian immunodeficiency virus from sooty mangabey monkeys (SIVsmm), termed SIVsmmPBj14, was previously identified and shown to induce acute disease and death within 1 to 2 weeks of inoculation of pig-tailed macaques and mangabey monkeys (P . N . Fultz, H . M . McClure, D . C . Anderson, and W . M . Switzer, AIDS Res . Hum . Retroviruses 5:397-409, 1989) . SIVsmmPBj14 differed from its parent virus, SIVsmm9, not only in pathogenicity but also in multiple in vitro properties . As a first approach to understanding the biological and molecular mechanisms responsible for the acute disease and death induced by this variant, virus-host cell interactions of SIVsmmPBj14 and SIVsmm9 were studied . Initial rates of replication of the two viruses were identical in primary peripheral blood mononuclear cells (PBMC) from normal pig-tailed macaques and mangabey monkeys, but SIVsmmPBj14 infection always resulted in higher yields of virus than did SIVsmm9 infection, as assessed by levels of reverse transcriptase activity in culture supernatants . Surprisingly, despite its cytopathicity for macaque and mangabey CD4+ cells, replication of SIVsmmPBj14 was accompanied by up to 10-fold increases in number of viable cells compared with cell numbers in uninfected or SIVsmm9-infected cultures . Furthermore, SIVsmmPBj14 was shown to infect and replicate in resting PBMC just as efficiently as in mitogen-stimulated PBMC, irrespective of whether exogenous interleukin-2 (IL-2) or antibodies that neutralized IL-2 were added to culture media . Accumulation of virus in culture supernatants of resting PBMC preceded by several days the appearance of activated cells which expressed the IL-2 receptor alpha subunit (CD25), suggesting that activation of cells was not essential for replication . The ability to activate and to induce simian PBMC to proliferate appeared specific for the acutely lethal variant because incorporation of {3H}thymidine by PBMC from naive animals was observed only upon incubation with concentrated, heat-inactivated SIVsmmPBj14 and not with other viruses . Both CD4(+)- and CD8(+)-enriched cell populations proliferated in response to SIVsmmPBj14 . These results are consistent with in vivo observations and suggest that the abilities both to replicate in resting cells and to induce lymphocytes to proliferate may contribute to the extreme virulence of SIVsmmPBj14. Agents Actions, 1991 Sep, 34(1-2), 285 - 8 Synovial activation of chondrocytes: evidence for complex cytokine interactions; Bandara G et al.; Synoviocytes secrete factors which induce the synthesis of neutral metalloproteinases (NMP) and prostaglandin E2 (PGE2) by chondrocytes in a response called "chondrocyte activation" . We analyzed synovial chondrocyte activating factors (CAF) for the presence of cytokines which modulated the NMP production by articular chondrocytes . These studies suggested the presence of several other cytokines in addition to interleukin-1 (IL-1) . Both resting and activated synoviocytes contained mRNA for basic fibroblast growth factor (bFGF) which is a synergist for IL-1 induced NMP production, and secreted bFGF into their culture media . They also expressed mRNA for transforming growth factor beta (TGF beta) which inhibits IL-1 induced NMP production . These cells also produce tumor necrosis factor alpha (TNF alpha) and trace amounts of interleukin-6 (IL-6) . In addition to these there is evidence for a synovial activator of chondrocytes which is distinct from IL-1 . Since a number of recombinant cytokines including TNF alpha, IL-6 and bFGF failed to activate chondrocytes, this could be a novel cytokine. Peptides, 1991 Sep-Oct, 12(5), 1077 - 83 Human pheochromocytoma cells studied in culture contain large amounts of DSIP-like material; Nilsson O et al.; Delta sleep-inducing peptide (DSIP)-like immunoreactive (LI) material has been detected in nine different human pheochromocytoma tumors by immunocytochemistry . In primary tumors subjected to indirect immunofluorescence a variable number of tumor cells (25-75%) showed positive cytoplasmic labeling after incubation with DSIP antiserum . Tumor cells grown in culture were strongly labeled by the DSIP antiserum with DSIP-LI concentrated to cell bodies . Electron microscopic immunocytochemistry (immunogold labeling) of pheochromocytoma cells demonstrated DSIP-LI over the dense core of secretory granules . The presence of DSIP-LI in several HPLC fractions from conditioned culture media indicates secretion of DSIP-LI from cultured pheochromocytoma cells . The observations suggest that DSIP-LI is synthesized and stored in secretory granules before release . The different HPLC profiles from each of the tumors may reflect differences in processing or turnover of DSIP-LI in pheochromocytoma cells. Neuropeptides, 1991 Sep, 20(1), 63 - 72 Bombesin-like peptides in alveolar macrophage: increased release in pulmonary inflammation and fibrosis; Lemaire I et al.; Rat bronchoalveolar cells (99% alveolar macrophages (AM} were obtained by bronchoalveolar lavage and examined for their content of bombesin-like immunoreactivity (BLI) by radioimmunoassay (RIA), immunocytochemistry and high performance liquid chromatography (HPLC) analysis . Rat AM contained and released in their culture media significant levels of BLI, the major molecular form corresponding to gastrin-releasing peptide (GRP) . Release of BLI by AM was not affected by in vitro activation of AM with lipopolysaccharide and muramyl dipeptide, but was enhanced following in vivo treatment with inflammatory agents . AM from animals with inflammation and fibrosis released higher levels of BLI than controls at 3 and 6 weeks after treatment . These changes were correlated with a significant increase in the proportion of low density mature AM as determined by Percoll density gradient fractionation . Together, our data indicate that increased release of BLI by AM may be related to AM maturation and support a role for bombesin-like peptides as modulator(s) of inflammatory reactions. Xenobiotica, 1991 Sep, 21(9), 1091 - 106 Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes; Jauregui HO et al.; 1 . The long-term maintenance of metabolism of representative drugs and steroid hormone substrates by cytochromes P-450, and their inducibility, was investigated in primary cultures of adult rat hepatocytes . Collagenase-isolated cells were seeded on collagen-coated tissue culture dishes and cultured in Chee's essential media in the presence or absence of phenobarbital (PB, 0.75 mM, 96 h or continuously) and 3-methylcholanthrene (MC, 5 microM, 48 h) for up to 45 days . 2 . Hepatic P-450-dependent metabolism of diazepam to its primary oxidized metabolite was inducible by PB both in vivo (monitored in isolated liver microsomes) and in cultured cells (up to 100% and 400% increases in the formation of temazepam and nordiazepam, respectively, after 25 days in culture) . Hepatocyte microsomal androstenedione 16 beta-hydroxylase activity was also induced by PB treatment of the hepatocytes (350-650% increase in 20-day-old cells) . 3 . Western blot analysis revealed that immunoreactive P-450 form PB-4 (IIB1), which catalysed the N-demethylation of diazepam to yield nordiazepam as well as androstenedione 16 beta-hydroxylation when assayed in a purified enzyme system, was substantially elevated following PB treatment of the cultured cells . Similarly, MC induced 7-ethoxycoumarin O-deethylase activity (up to 2000% increase from 5 to 45 days) as well as immunoreactive P-450c (IA1) in the hepatocyte cultures . 4 . These studies demonstrate that cytochrome P-450 activities can be maintained, and also induced, after extended periods of time in hepatocytes cultured using a simple collagen mixture as substrate and a commercially available tissue culture media . This culture system should provide an important tool for further studies of P-450-dependent xenobiotic metabolism in a well-defined, liver-derived cellular system. J Dairy Sci, 1991 Sep, 74(9), 2946 - 51 Enzyme-linked immunosorbent assays for bovine alpha-lactalbumin and beta-lactoglobulin in serum and tissue culture media; Mao FC et al.; Enzyme-linked immunosorbent assays for bovine alpha-lactalbumin and beta-lactoglobulin have been developed for measurements of serum and tissue culture samples . Either alpha-lactalbumin or beta-lactoglobulin antiserum was coated on ELISA plates . Biotinylated proteins were used in competition with unknown amount of proteins in samples . After unbound proteins were washed off, ExtrAvidin-peroxidase and tetramethylbenzidine were then used as a detection system . Crossreactivity of caseins or bovine serum albumin was less than .0001% in either alpha-lactalbumin or beta-lactoglobulin ELISA . Parallel curves from serial dilutions were obtained in serum and media samples . The additivity of alpha-lactalbumin and beta-lactoglobulin ELISA was validated in either serum or medium samples . The intraassay and interassay coefficients of variation for alpha-lactalbumin and beta-lactoglobulin ELISA were below 10% over 51 and 47 assays . The ELISA are useful in mammary gland biology studies for measuring milk whey protein in serum or culture media. Zhong Xi Yi Jie He Za Zhi, 1991 Sep, 11(9), 547 - 9, 518 {Effects of Cordyceps sinensis, rhubarb and serum renotropin on tubular epithelial cell growth}; Tian J et al.; Primary cultured rat tubular epithelium was utilized to investigate the effect of Cordyceps sinensis (CS) on cellular proliferation and metabolism . Judging from incorporation rate of 3H-TdR, it was found that the addition of serum containing CS metabolites into the culture media could promote the DNA synthesis of tubular cells profoundly (P less than 0.001) . In association with its beneficial effects on gentamycin nephrotoxity in vivo study, it is indicated that CS could enhance the regeneration of injured tubular cells . In addition, sera obtained from 5/6 nephrectomized rats (5/6 NT) and rhubarb treated rats were studied to see their effects on tubular cells growth . The results showed that the serum of 5/6 NT rats could promote the DNA synthesis of tubular epithelium, while the presence of experimental serum of rhubarb in culture median markedly inhibited the DNA synthesis of cells. Can J Physiol Pharmacol, 1991 Sep, 69(9), 1288 - 93 Effects of the antiandrogen hydroxyflutamide on progesterone secretion by preovulatory rat follicles in vivo and in vitro; Chandrasekhar Y et al.; Serum and ovarian progesterone levels and in vitro production of progesterone by preovulatory follicles were measured on proestrus in pregnant mare's serum gonadotropin (PMSG) primed immature rats in which the luteinizing hormone (LH) surge and ovulation were blocked by administration of the antiandrogen hydroxyflutamide . Serum progesterone levels observed at 12:00 on proestrus were significantly elevated, twofold above those observed in vehicle-treated controls, by in vivo administration of 5 mg hydroxyflutamide 4 h earlier . In control rats, proestrous progesterone did not increase until 16:00, in parallel with rising LH levels of the LH surge . No LH surge occurred in the hydroxyflutamide-treated rats, ovulation was blocked, and serum progesterone declined throughout the afternoon of proestrus, from the elevated levels present at 12:00 . Administration of human chorionic gonadotropin (hCG) at 11:00 advanced the elevation of serum progesterone by 2 h in vehicle-treated controls and prevented the decline in progesterone levels in hydroxyflutamide-treated rats . The patterns of change in ovarian tissue concentrations with time and treatment were essentially similar to those observed for serum progesterone . In in vitro experiments, progesterone secretion during 24 h culture of preovulatory follicles obtained on PMSG-induced proestrus was significantly increased, sixfold, by addition to the culture media of 370 microM but not of 37 microM hydroxyflutamide . Testosterone (50 nM) and hCG (20 mIU/mL) caused 26- and 14-fold increases, respectively, in progesterone secretion by cultured follicles . Hydroxyflutamide significantly reduced the stimulatory effect of testosterone but not of hCG on progesterone secretion in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Parasitol, 1991 Sep, 21(5), 589 - 96 A nuclear magnetic resonance study of the glucose metabolism of Hymenolepis diminuta exposed to histamine and serotonin in vitro; Novak M et al.; The direct effects of the inflammatory mediators, histamine (HI) and serotonin (SE), on the glucose metabolism of Hymenolepis diminuta in vitro were studied by analyzing the excretory products from culture media, containing D-1-13C-glucose and various concentrations of HI and/or SE, by 1H-nuclear magnetic resonance (n.m.r.) spectroscopy . The results revealed that HI markedly accelerated the glycolysis process by increasing the amount of lactate production . The increased glycolytic activity was reflected in a concentration-dependent increase in glucose uptake . Excretion of acetate was also stimulated by HI . A low concentration of SE significantly increased succinate, acetate and lactate excretions, whereas a high concentration had little effect on lactate production and significantly decreased succinate and acetate excretions . A combination of HI and SE treatment at a low concentration had no significant effect, but at a high concentration showed an additive effect, with an increase in lactate production, a decrease in succinate production and an increase in glucose uptake . Thus this work confirms that HI and SE directly influence, albeit differently, energy metabolism of the tapeworm H . diminuta. Andrologia, 1991 Sep-Oct, 23(5), 325 - 7 Substance P-induced inhibition of Leydig cell steroidogenesis in primary culture; Angelova PA et al.; The effect of substance P (SP) on hamster Leydig cell steroidogenesis in primary culture was investigated . Purified Leydig cells were cultured with or without SP for 24 h . The levels of testosterone and progesterone in the culture media were 174 +/- 20 pg ml-1 and 105 +/- 12 pg ml-1, respectively . In the presence of SP (10(-7) mol l-1), testosterone concentration significantly decreased to 123 +/- 19 pg ml-1, e.g . with 29.3% . By contrast, at the concentration used, SP had no effect on progesterone secretion . The possible molecular mechanism of the SP action on Leydig cell function was discussed . The reported results indicate that SP can modulate Leydig cell steroidogenesis in culture. Gynecol Oncol, 1991 Sep, 42(3), 265 - 72 Effect of differentiation agents on expression of CA 125, alkaline phosphatase, and cytokeratins in human ovarian adenocarcinoma cells (OVCA 433); Brooks SE et al.; A number of chemical agents have been found to influence the proliferation, morphology, enzymatic activity, and antigen expression of neoplastic cells toward a more differentiated phenotype . We studied the effects of differentiating agents retinoic acid, sodium butyrate, and dibutyryl cyclic AMP on the expression of the tumor-associated antigen CA 125 and several biochemical markers of differentiation in cultured OVCA 433 ovarian cancer cells . Treatment of OVCA 433 cells with these agents for 96 hr reduced cellular proliferation and altered cellular morphology . Quantitation of cell surface CA 125 using flow cytometry revealed that CA 125 expression was reduced by 35-50% . The amount of CA 125 antigen shed into the culture media was reduced to a similar degree . In addition, differentiation inducers markedly enhanced cellular alkaline phosphatase activity and induced the expression of a 65-67-kDa cytokeratin . These findings provide support for the induction of a more differentiated phenotype by these agents. Br J Haematol, 1991 Sep, 79(1), 14 - 21 Induction of granulocyte and granulocyte-macrophage colony-stimulating factors from human monocytes stimulated by Fc fragments of human IgG; Ishiguro A et al.; The effect of human IgG on human haemopoiesis has been studied in vitro . Dialysed purified IgG stimulated haemopoietic colony growth by bone marrow mononuclear cells (MNC) but not by monocyte-depleted MNC . Culture media, conditioned by IgG-stimulated peripheral blood MNC, augmented formation of neutrophil-macrophage, eosinophil, and megakaryocyte colonies by monocyte-depleted marrow MNC . Serum-free IgG-conditioned media also contained colony-stimulating activity (CSA) . IgG Fc fragments and heat-aggregated IgG promoted the secretion of CSA, but F(ab')2 fragments, Fab fragments or ultracentrifuged IgG did not . In the cell-selection studies, CSA was produced by highly enriched monocytes following stimulation with Fc fragments . The antiserum against human granulocyte colony-stimulating factor (G-CSF) and/or granulocyte-macrophage CSF (GM-CSF) neutralized the CSA produced by Fc fragment-activated monocytes . Enzyme immunoassays showed G-CSF and GM-CSF in media conditioned by monocytes stimulated with the Fc fragments, heat-aggregated IgG and anti-D-sensitized red blood cells (RBC) . Northern hybridization analysis showed mRNA encoding G-CSF and GM-CSF in RNA extracted from MNC and monocytes cultured with the Fc fragments, but not in the RNA from unstimulated cells or monocyte-depleted MNC . These results indicate that IgG Fc fragments, aggregated IgG and antigen-antibody complexes induce monocytes to produce G-CSF and GM-CSF in vitro . The CSFs release induced by IgG may be involved in the in vivo regulatory network in haemopoiesis. Am J Gastroenterol, 1991 Sep, 86(9), 1150 - 3 The role of epidermal growth factor in the pathogenesis of peptic ulcer disease; Zandomeneghi R et al.; Duodenal biopsies obtained from seven normal subjects and six ulcerous patients were cultured in vitro for 30 min at 37 degrees C under various experimental conditions . Epidermal growth factor (EGF) and somatostatin released in the culture medium were determined by radioimmunoassay . Under basal conditions, EGF and somatostatin levels were significantly higher in normal subjects (11.49 +/- 3.07 ng/mg protein and 3.06 +/- 0.8 ng/mg protein, respectively) than in ulcerous patients (6.9 +/- 1.98 ng/mg protein and 1.75 +/- 1.23 ng/mg protein, respectively) . However, when antibodies to somatostatin and vasoactive intestinal polypeptide (VIP) were added together to the culture media, in ulcerous patients, EGF levels also were lower as absolute values, but were higher as a percentage of variation than controls (p less than 0.05) . The fall of EGF secretion from tissue cultures of ulcerous patients could be the consequence of endocrine cellular loss or damage, rather than the cause of ulceration . Moreover, the EGF-producing cells around the lesion in ulcerous patients seems to be hyperactive, and this hyperfunction of EGF-producing cells might contribute to the in vivo repair of tissue damage. Ann Rheum Dis, 1991 Sep, 50(9), 637 - 41 In vitro effects of methotrexate on peripheral blood monocytes: modulation by folinic acid and S-adenosylmethionine; Nesher G et al.; The mechanism of action of low dose methotrexate in rheumatoid arthritis has not been established . It has been shown to have an anti-inflammatory effect and to inhibit neutrophil chemotaxis, but the effect on monocytes has not been widely studied . Normal donor peripheral blood monocytes were incubated with methotrexate in vitro and their superoxide production, chemotaxis, and phagocytosis subsequently assessed . Additionally, the influence of different culture media, and of folinic acid, and the methyl donor S-adenosylmethionine, and spermidine on the methotrexate mediated effects were evaluated . It was found that methotrexate in low concentrations inhibited in vitro monocyte chemotaxis and superoxide production but only after prolonged incubation . This inhibition was augmented by incubation in medium containing a low methionine concentration and was abolished by folinic acid and S-adenosylmethionine, suggesting that methotrexate may interfere with specific methylation reactions. J Cell Biol, 1991 Sep, 114(6), 1285 - 94 Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10; Bradham DM et al.; Human umbilical vein endothelial (HUVE) cells have been previously reported to express the genes for the A and B chains of PDGF and to secrete PDGF-related factors into culture media . Antihuman PDGF IgG affinity chromatography was used to purify PDGF-related activity from HUVE cell-conditioned media . Immunoblot analysis of the affinity-purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD and that less than 10% of the purified biologically active molecules are PDGF A or B chain peptides . Screening of an HUVE cell cDNA library in the expression vector lambda gtl 1 with the anti-PDGF antibody resulted in the cloning and sequencing of a cDNA with an open reading frame encoding a 38-kD cysteine-rich secreted protein which we show to be the major PDGF-related mitogen secreted by human vascular endothelial cells . The protein has a 45% overall homology to the translation product of the v-src-induced CEF-10 mRNA from chick embryo fibroblasts . We have termed this new mitogen connective tissue growth factor. J Immunol Methods, 1991 Aug 28, 142(1), 83 - 94 Practical limitations of estimation of protein adsorption to polymer surfaces; Underwood PA et al.; The estimation of protein adsorption to polymeric surfaces is a prerequisite for analysing the biological activity of a coated protein both in the evaluation of polymeric biomaterials and in the standardisation of ELISA assays . Direct quantitative methods utilise either measurements of the physical characteristics of the coated surface (equipment necessary for this is not always available), or measurements of radiolabelled protein . We demonstrate that proteins labelled with 125I using chloramine-T contain a significant proportion of 125I label which is unable to adsorb to polyvinyl microtitre wells . This non-bindable label is not separable from protein by fractionation on Sephadex G-25 but does dissociate upon SDS gel electrophoresis . The proportion of non-adsorbable label increases with storage time at -70 degrees C, the effect being accelerated at higher temperatures . A method is described for estimating protein adsorption which is both independent of the non adsorbable fraction but can also be used to estimate it . The use of biotin as a protein label does not apparently result in deterioration of adsorption with short storage times . The problem of subsequent desorption in ELISA buffers, tissue culture media, or body fluids is discussed. Anal Biochem, 1991 Aug 15, 197(1), 225 - 30 Adaptation of the plasma renin radioimmunoassay for use with HIV-1 protease; Hyland LJ et al.; We have demonstrated the use of a radioimmunoassay to quantitate the peptidolytic activity of human immunodeficiency virus, type 1 (HIV-1) protease using a tetradecapeptide substrate of porcine renin, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser . HIV-1 protease catalyzes cleavage of this substrate at the same Leu-Leu bond as does porcine renin, resulting in the formation of authentic angiotensin-I . The angiotensin-I product is then detected by use of a commercially available renin plasma assay kit, which constitutes the basis of the RIA . The radioimmunoassay provides detection of the protease-catalyzed formation of angiotensin-I at picomolar concentrations in vitro . We demonstrate the use of this assay in determining IC50 values for two HIV-1 protease inhibitors present in cell culture media and in standard assay buffer . An example of the potential development of this assay for the quantitation of these inhibitors present in ex vivo plasma samples is also presented. Biochem Biophys Res Commun, 1991 Aug 15, 178(3), 1429 - 36 Characterization of mucin-type oligosaccharides with the sialyl-Le(a) structure from human colorectal adenocarcinoma cells; Kitagawa H et al.; Oligosaccharides with the sialyl-Le(a) structure have been isolated on an affinity column of a monoclonal antibody, MSW 113, from mucin-type glycoproteins derived from the surfaces of SW 1116 and LS 180 cells, and their secretions . The oligosaccharides were polydisperse with respect to molecular size, the oligosaccharides derived from glycoproteins in culture media being larger than those in cell lysates, as assessed by gel filtration . Some of the oligosaccharides were susceptible to degradation by endo-beta-galactosidase (E . freundii), as judged from the change in the gel filtration pattern . These results indicate that oligosaccharides with the sialyl-Le(a) structure derived from mucin-type glycoproteins produced by human colonic cancer cells are extremely large in size and complex in structure, and that some of them contain the poly-N-acetyllactosamine structure. J Biol Chem, 1991 Aug 5, 266(22), 14418 - 24 Catalytic triad residue mutation (Asp156----Gly) causing familial lipoprotein lipase deficiency . Co-inheritance with a nonsense mutation (Ser447----Ter) in a Turkish family; Faustinella F et al.; We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity . We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors . Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues . Both patients were homozygous for both mutations . Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations . In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector . Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant . Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly . Immunoreactive LPLs were produced in all cases . The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients . The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity . Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity . We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance . Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS) Zh Mikrobiol Epidemiol Immunobiol, 1991 Aug, (8), 21 - 3 {The standardization of the in-vitro tetanus toxin neutralization test on Chinese hamster ovary cells}; Khardina AA et al.; A test for the titration of B . pertussis toxin with antisera on Chinese hamster ovary (CHO) cells has been worked out . B . pertussis protective antigenic cell-free complex containing 48-54% of B . pertussis toxin has been used as antigen . The specificity of the effect of this complex on CHO cells has been confirmed in the toxicity neutralization test with antisera . CHO cells have been adapted to reagents and culture media made in the USSR . The titration of B . pertussis toxin and antisera on CHO cells did not require the use of highly purified antigen. Vet Immunol Immunopathol, 1991 Aug, 29(1-2), 183 - 8 Optimal conditions for in vitro mitogen-induced proliferation of peripheral blood lymphocytes in breeding foxes; Kostro K et al.; The proliferative response of fox peripheral blood lymphocytes to nonspecific mitogens: leucoagglutinin (LA), concanavalin A (Con A) and pokeweed mitogen (PWM) was studied . Microcultures were kept at 39 degrees C in a humidified atmosphere containing 5% CO2 . The highest 3H-thymidine incorporation was observed, when Con A was used, while LA and PWM showed weaker but significant stimulatory action . Optimal doses of mitogens were: 5 micrograms/ml for Con A, 5 micrograms/ml for LA and a dilution of 1:100 for PWM . The maximal stimulation index for Con A was about 240 and up to 100 for LA or PWM . The maximal lymphocyte proliferation was observed when culture media were supplemented with 10% serum . When proliferation kinetics were studied, the peak response was observed on Day 2. Parasitology, 1991 Aug, 103 Pt 1, 85 - 95 In vitro cultivation of adult Litomosoides carinii: evaluation of basic culture media, gas phases and supplements; Mossinger J; Adult Litomosoides carinii, recovered from cotton rats (Sigmodon hispidus) 4-5 months post-infection (p.i.), were cultivated in vitro with emphasis on investigations into the development of intra-uterine embryonic stages . Baseline values for the embryonic status of female worms were established immediately after recovery from the hosts . In such females, on average, 16% of the intra-uterine stages were fully formed microfilariae while the remainder belonged to the early embryonic classes that were characterized . For the evaluation of culture success, apart from survival of worms in vitro, the rate of microfilariae development (mf rate) served as a major parameter . Of the five basic culture media RPMI 1640, F12, L15, NCTC 135, and IMDM, tested without supplementation, RPMI 1640 yielded by far the best results (survival = 14 days; mf rate = 41%), and was therefore chosen as the routine medium . In comparison with 5% CO2 in nitrogen, a gas phase of 5% CO2 in air was superior, although the resulting oxygen tension of 138 mmHg in the medium was not physiological . Addition of 10% newborn or foetal bovine serum to the medium in some cases distinctly influenced results . Effects of different batches of sera varied from 'filaricidal' to 'very promoting' . Co-cultivation of worms and Sigmodon cells, or rhesus monkey LLCMK2 cells, only marginally improved results . Of the serum substitutes Ultroser G, BMS, and Clex, the latter had a moderately promoting effect . The protein supplements bovine serum albumin, transferrin and haemoglobin significantly improved results, and could replace certain batches of serum . Supplementation with the haemin moiety alone was less effective than with haemoglobin . The anti-oxidants glutathione plus ascorbic acid proved beneficial in combination with a serum supplement only . Results from other experiments with multiple supplementation also suggest that various supplements may act only in a synergistic manner . The longest average time that adult L . carinii survived in vitro was 3-4 weeks . The highest mf rate was 78%, which indicated that all normal embryonic stages present in the uteri of a female at the start of culture completed their development to microfilariae, however, oogenesis ceased in vitro . The parameters for embryonic development employed proved to be highly sensitive for the judgment of various culture conditions. J Rheumatol, 1991 Aug, 18(8), 1243 - 6 Disseminated Nocardia brasiliensis infection: an unusual complication of immunosuppressive treatment for childhood dermatomyositis; Klein-Gitelman MS et al.; The use of steroids combined with cytotoxic drugs has increased in the last decade . The concomitant increase of opportunistic infections has contributed significantly to morbidity and mortality of patients treated with immunosuppressive agents . We describe a child with dermatomyositis who developed disseminated Nocardia brasiliensis infection while receiving steroids and methotrexate . Infectious etiology was established by gram stain . The patient was treated successfully . Disseminated Nocardia brasiliensis infection is rare with a high reported mortality . Diagnosis may be delayed secondary to insidious onset, similarity of clinical manifestations to other pathogens and slow growth in routine culture media . Nocardia should be considered early in the evaluation of infection in patients treated with immunosuppressive agents. Neuropeptides, 1991 Aug, 19(4), 237 - 42 Differential secretion of enkephalin-like peptides from bovine adrenal chromaffin cells; Cherdchu C et al.; Stimulation of chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) or depolarizing concentrations of K+ resulted in a significant secretion of high and low molecular weight enkephalin-like peptides (ELPs) into the culture medium . BioGel P-10 column chromatography was used to characterize the ELPs in chromaffin cell extracts and in culture media before and after stimulation with either DMPP or K+ . DMPP (50 microM) stimulation produced a significant secretion of primarily low molecular weight (less than 3 kDa) ELPs whereas 56 mM K+ caused a secretion of both high and low molecular weights ELPs . The expected decrease in cellular content of low molecular weight peptides was not observed regardless of stimulation type . Our results support the hypothesis that the precursor/product ratio of secreted ELPs is dependent upon the nature of the chromaffin cell stimulus . Moreover the cellular content of low molecular weight ELPs is not depleted with either type of stimulation. Lab Invest, 1991 Aug, 65(2), 250 - 7 Carbon stripping extracts serum free fatty acids: implications for media supplementation of cultured type II pneumocytes; Viscardi RM et al.; Carbon stripping is a process that is widely used to remove hormones from serum . Because addition of serum to culture media also provides exogenous fatty acids that influence lipid metabolism of cultured cells, we investigated the effects of carbon stripping on the composition of the phospholipid and free fatty acid fractions in fetal bovine serum and the effects of these changes on phosphatidylcholine synthesis by cultured adult alveolar type II cells . Carbon stripping resulted in quantitative and qualitative changes in serum free fatty acids . The process effectively extracted greater than or equal to 99% free fatty acids and, to a lesser extent, phospholipids . There were also qualitative changes in the relative composition of the remaining free fatty acids with a selective loss of oleic and linoleic free fatty acids . However, the relative composition of the serum phospholipid fatty acid fraction was unaffected . Type II cells isolated from adult male rat lung and cultured in Dulbecco's modified Eagle's medium supplemented with 10% carbon-stripped fetal bovine serum (FBS-CS) incorporated {3H}choline into phosphatidylcholine at a rate 36% less than the rate of control cells cultured with unstripped FBS . Addition of oleic acid to FBS-CS supplemented media increased total phosphatidylcholine synthesis by adult type II cells by 67-71% . In contrast, addition of palmitic acid inhibited PC synthesis 51-67% . The combination of oleic and palmitic acids resulted in a rate of {3H}choline incorporation into phosphatidylcholine similar to the rate for control cells cultured in FBS-CS-supplemented media alone . Although synthesis of disaturated phosphatidylcholine was unaffected by exogenous fatty acids, addition of fatty acids altered the proportion of disaturated phosphatidylcholine synthesis relative to total phosphatidylcholine synthesis . The presence or absence of the hormones, dexamethasone and triiodothyronine, did not explain the difference in rate of phospholipid synthesis by type II cells cultured in untreated versus carbon-stripped serum supplemented media . These results suggest that the removal of serum free fatty acids by carbon stripping can influence phospholipid metabolism of cultured type II cells . Because serum free fatty acids influence cellular lipid composition and potentially cell metabolic functions, carbon-stripped serum may not be the optimal choice for media supplementation of cultured cells. Gan To Kagaku Ryoho, 1991 Aug, 18(11), 1812 - 6 {Antitumor Effector mechanism of plant alkaloid preparation, cepharanthin, by intratumoral administration}; Ebina T et al.; The antitumor effect of biscoclaurine alkaloids, at a distant site was examined in the double grafted tumor system, in which mice received simultaneous intradermal inoculations of Meth-A in both right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with 0.5 mg of CR in the right tumor on days 3, 4 and 5 . CR inhibited the growth of not only the right but also the left, non-treated tumor . In order to examine the role of lymph nodes in the antitumor activity of CR, regional (axillary and inguinal) lymph nodes were resected . Since in resected mice the antitumor activity of CR against the left tumor was weakened, the regional lymph nodes have a very important role in the antitumor effect of intratumoral administration of CR at a distant site . Spleen cells prepared from CR treated mice were examined for Lyt-1, Lyt-2 and L3T4 phenotypes . The number of Lyt-1 positive lymphocytes increased in the spleen after intratumoral administration of CR . Isolated tumor cells obtained from the right tumor treated with CR and the left side tumor on day 6 were cultured for 24 h . The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophil or macrophage . Significant neutrophil chemotactic factor (NCF) activity was detected in the culture media from CR-treated right tumor tissue, and macrophage chemotactic factor (MCF) activity was detected in the culture media from left tumor tissue . CR-induced NCF was partially neutralized by treatment with anti-human IL-8 IgG, and might be murine IL-8 factor . These results suggest that intratumoral administration of CR first induces neutrophils in the right tumor, then Lyt-1 positive cells in the spleen, and subsequently induces cytotoxic macrophages in the left, non-treated tumor, thus bringing about the regression of distant tumors. Invest Ophthalmol Vis Sci, 1991 Aug, 32(9), 2610 - 21 Influence of culture conditions on the androgen control of secretory component production by acinar cells from the rat lacrimal gland; Hann LE et al.; Research has shown that androgens regulate the production of secretory component (SC), the IgA antibody receptor, by lacrimal gland acinar cells in vivo . This study was designed to establish an optimal culture system to permit analysis of this endocrine-acinar cell interrelationship in vitro . Acinar cells were isolated from male rat lacrimal glands and cultured on Matrigel (Collaborative Research, Bedford, MA) in serum-free Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 media that contained a variety of supplements . Under these conditions, acinar cells responded to dihydrotestosterone (DHT) exposure with a significant increase in SC output . Replacement of the DMEM/Ham's F12 media base with either Modified Eagle's Medium (MEM) or low-calcium MEM inhibited this hormone response and dramatically reduced cell recovery after 4 days of culture . Similarly, decreased concentrations or deletions of selected media supplements, including insulin, which binds to acinar cells, and dexamethasone, led to a significant diminution in the extent of androgen action, as well as to a decline in cell maintenance . In contrast, removal of high-density lipoprotein from culture media or the addition of fetal bovine serum (FBS) or cholera toxin significantly enhanced basal and DHT-associated SC production by acinar cells . With regard to extracellular matrices, Matrigel proved to be superior to collagen type I, laminin, fibronectin, or the Primaria (Falcon, Oxnard, CA) plastic surface in providing support for acinar cell association or hormone-related function . In summary, our results show that the media formulation, supplement profile, and extracellular matrix composition are important for maximal expression of androgen-induced effects by lacrimal gland acinar cells in vitro. Fertil Steril, 1991 Aug, 56(2), 332 - 9 Absence of a direct effect of recombinant tumor necrosis factor-alpha on human sperm function and murine preimplantation development; Wincek TJ et al.; OBJECTIVE: This study was designed to test the in vitro effects of human recombinant tumor necrosis factor (rTNF) on sperm motility, fertilization, and preimplantation development . DESIGN: A sensitive enzyme immunoassay was used to determine half-lives of rTNF and confirm concentrations of cytokine throughout experimental conditions . Effect of rTNF on human sperm survival was measured by computer-assisted methodology, and effect on human sperm penetration was assessed by hamster ova penetration . Cytokine effect on murine gamete interaction was determined by in vitro fertilization (IVF) . Murine preimplantation development was assessed by in vitro development of cryopreserved-thawed one-cell zygotes . RESULTS: The half-life of rTNF was reduced by the addition of sperm to culture media (P less than 0.001) . Sperm motility (P = 0.245) and hamster ova penetration (P = 0.62) were not altered by incubations in the presence of concentrations of rTNF up to 10,000 U/mL . Mouse IVF (P = 0.60) and preimplantation development (P = 0.56) were not altered by rTNF in concentrations up to 5,000 U/mL . CONCLUSIONS: These results demonstrate rTNF by itself does not interfere with gamete function or early embryo development. Biol Reprod, 1991 Aug, 45(2), 245 - 51 Overcoming the 2-cell block by modifying standard components in a mouse embryo culture medium; Lawitts JA et al.; The 2-cell block may be caused by inappropriate concentrations of commonly used constituents of embryo culture media . Almost all zygotes obtained by fertilizing CF1 ova with hybrid B6D2F1/CrlBR sperm did not develop beyond the 2-cell stage when cultured in Whittingham's medium M16 . This 2-cell block was overcome by lowering the concentrations of NaCl, KCl, KH2PO4, glucose, and pyruvate, either individually or in combination . The effects of changing the concentration of either NaCl or KCl depend on the concentration of NaHCO3 in the medium . Although a high percentage of embryos grew to the 4-cell stage in several media with lowered concentrations of certain components, the media are not optimal for complete preimplantation embryo development since the yield of blastocysts is low. Cancer Biochem Biophys, 1991 Aug, 12(2), 95 - 101 Electric field stimulation of human osteosarcoma-derived cells: a dose-response study; Naegele RJ et al.; In vitro electrical stimulation of human osteosarcoma-derived cells resulted in increased cell adherence and current directed cell migration . We have developed an electrical exposure system in which two steel electrodes imbedded in media-based agar, poured in a standard culture dish, are used to apply electric field signals to cells in culture without ion contamination from the electrodes . The cells were exposed to a 100 Hz pulsed DC electric signal at peak field strengths of 1, 10, 100, and 625 mV/cm in the culture media . The data showed no change in cell adherence at 1 and 10 mV/cm, an increase in adherence at 100 mV/cm, and a decrease in both adherence and cell proliferation at 625 mV/cm . Electric field stimulation in vivo has been found useful in accelerating the healing of fractures and non-unions, and the repair of surgical and cancer-related skeletal defects. Early Hum Dev, 1991 Aug-Sep, 26(2), 93 - 9 Spontaneous chromosome fragility in chorionic villus cells; Miguez L et al.; Human fragile sites are only very rarely expressed spontaneously . In this paper we report the presence of non-random spontaneous chromosome lesions (CL) in chorionic villus samples and their coincidence with fragile site (FS) bands . The average number of CL was about 9% both in RPMI-1640 and in Chang media . To determine any possible influence of external factors other than culture media, the results were grouped according to age of gestation . No differences were observed among the different groups . A total of 101 chromosome lesions could be precisely identified by sequential Leishman Staining/Wright G-banding; 76.2% of them coincided with FS-bands . The most affected region was at 1q12-1q21.1 (15.8% of total CL); other FS with a clustering of breakpoints in our study were 1p36, 1q44, 2q37, 3p24, 3q27, 10q22 and 16q23 . These results suggest that spontaneous expression of some FS could be a characteristic of embryonic tissues. J Virol Methods, 1991 Aug, 33(3), 345 - 53 Immunoaffinity purification of two major proteins of bovine leukemia virus (gp51 and p24) and their use for discrimination between vaccinated and infected animals; Merza M et al.; The outer envelope glycoprotein gp51 and the core protein p24 of bovine leukemia virus (BLV), were purified from culture media of FLK-BLV cells by a single-step procedure, using immunoaffinity chromatography based on monoclonal antibodies to the respective proteins . About 90% of the envelope glycoprotein in the culture medium was recovered as a highly purified product . Both purified protein (gp51 and p24) preparations, were found to be highly specific antigens by ELISA, and did not cross-react with sera raised against the other antigen . The conformational epitopes on the purified gp51 were preserved as judged by their reactions with the corresponding monoclonal antibodies . The p24 ELISA reacted only with sera from naturally infected animals and not with sera from animals immunized with an experimental gp51-iscom vaccine . The p24 antigen is therefore useful for discriminating between BLV-infected animals and those immunized with a gp51 subunit vaccine. Endocrinology, 1991 Aug, 129(2), 848 - 58 Acute phosphate depletion dissociates hormonal stimulated second messengers in osteoblast-like cells; Green J et al.; The acute effect (24 h) of either phosphate depletion or phosphate surfeit on hormonal stimulated signal transduction systems was studied in the osteoblastic cell line UMR-106 . Elevation of intracellular Ca2+ ({Ca2+}in), induced by different calciotropic hormones (PTH, prostaglandin E2, endothelin) was blunted by acute phosphate depletion, whereas at high inorganic phosphate (Pi) concentrations the rise in {Ca2+}in was augmented . Basal {Ca2+}in was not altered by either Pi depletion or Pi excess . The effect of acute phosphate depletion on hormonal mediated {Ca2+}in rise was not observed in the absence of extracellular Ca2+ suggesting that under these conditions, the release of Ca2+ from intracellular stores, is not affected . Also, nonhormonal calcium entry pathways such as depolarization-activated calcium channels or protein kinase C-activated Ca2+ channels were not affected by acute phosphate depletion . cAMP accumulation in the cells, either through receptor or nonreceptor-mediated mechanisms, increased under low Pi conditions and decreased as Pi concentration in the culture media was progressively increased from 0 to 2 mM during 24 h of incubation . Changes in Pi concentration had no effect on basal cAMP generation by the cells . The facilitative effect of acute Pi depletion on agonist-induced cAMP accumulation could be demonstrated in both the presence and absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM) . PTH receptor binding assessed with {Nle8 Nle18 Tyr34} bovine PTH (1-34) NH2 was not altered by phosphate depletion . We conclude that exposure of osteoblasts to different Pi environments modulates the second messenger responses to hormones in a reciprocal fashion so that acute phosphate depletion down-regulates {Ca2+}in signals while augmenting cAMP generation and vice versa . Inasmuch as bone resorption processes can be modulated by Ca2+ and cAMP the data presented herein suggest that the altered bone resorptive response to calciotropic hormones (e.g . PTH), under surfeit or deficit of phosphate, is mediated by changes in {Ca2+}in and cAMP. Endocrinology, 1991 Aug, 129(2), 1059 - 65 Effects of substance-P and neuropeptide-Y on in vitro steroid release by porcine granulosa and luteal cells; Pitzel L et al.; The presence of substance-P (SP)- and neuropeptide-Y (NPY)-like immunoreactivity was recently shown in nerves that innervate the ovary . In the present in vitro study we demonstrate that both peptides have direct effects on ovarian steroidogenesis . In cultured porcine granulosa (G-) cells, neither peptide affected progesterone (P) production under basal conditions, but they both inhibited gonadotropin-stimulated P secretion . In luteal (L-) cell cultures, basal as well as hCG-stimulated P release were dose-dependently inhibited by NPY (ED50, 4 x 10(-9) M; identical for both, basal and stimulated release), while SP had only a moderate inhibitory effect (ED50, 6 x 10(-8) M) . In the presence of AP13, a specific SP antagonist, the inhibitory effect of SP on P release was abolished, which suggests a receptor-mediated effect . In addition, we determined androstenedione (A) and estradiol (E2) release into G- and L-cell culture media . While E2 production in G-cell cultures was not influenced by SP and NPY, both peptides had a dose-dependent stimulatory effect on E2 secretion by L-cells . In contrast to E2 release, A secretion by G- as well as L-cell cultures was increased by gonadotropins . Both SP and NPY decreased gonadotropin-stimulated A secretion by G- and L-cells under basal as well as hCG-stimulated conditions . Furthermore, we demonstrate SP immunoreactivity in media of G- and L-cell cultures with a HPLC retention time identical to that of synthetic SP . This may suggest ovarian synthesis, in which case the peptide exerts auto- and/or paracrine effects on ovarian steroidogenesis . From these in vitro results we suggest that SP and NPY have a modulatory effect on ovarian function in pigs not only by their well known regulatory effects of blood supply, but also by a direct effect on ovarian steroidogenesis. J Biochem (Tokyo), 1991 Aug, 110(2), 189 - 95 Efficient purification of TIMP-2 from culture medium conditioned by human hepatoma cell line, and its inhibitory effects on metalloproteinases and in vitro tumor invasion; Umenishi F et al.; Metalloproteinase inhibitors were surveyed with the culture media of 19 kinds of human tumor cell lines, using transin (rat stromelysin) as the target enzyme . This survey showed that most of the cell lines more or less secreted inhibitor activity, and that a human hepatoma cell line, HLE, secreted an extremely high inhibitor activity into the culture medium . Two kinds of metalloproteinase inhibitors were purified from the serum-free conditioned medium of HLE cells . The major inhibitor, which showed a single protein band with a molecular weight (Mr) of 21,000 (21k) (nonreduced) or 24k (reduced) on SDS-polyacrylamide gel electrophoresis, was identified as TIMP-2 (tissue inhibitor of metalloproteinases-2) by the analysis of its N-terminal amino acid sequence . The other was immunologically identified as TIMP . Purified TIMP-2 inhibited the activities of transin, matrin (pump-1), Mr 72k gelatinase, and interstitial collagenase with 1:1 stoichiometry . When the latent precursor form (Mr 57k) of transin was incubated with p-aminophenylmercuric acetate as an activating reagent, TIMP-2 inhibited the conversion of the intermediate form (Mr 45k) into the mature enzyme (Mr 42k) . This indicated that TIMP-2 regulates not only the activity of the mature enzyme but also the autolytic processing of the proenzyme . TIMP-2 also inhibited in vitro tumor invasion through reconstituted basement membrane (matrigel) in chemotaxis chambers, showing that the metalloproteinase inhibitors as well as the extracellular matrix metalloproteinases are involved in tumor invasion through basement membrane and other extracellular matrices. Mol Cell Endocrinol, 1991 Aug, 79(1-3), 85 - 92 Affinity labeling of endothelin receptors in bovine and rat lung membranes by N epsilon 9-azidobenzoyl-125I-endothelin-1; Kundu GC et al.; Endothelin-1 (ET-1) is a potent, vasoconstrictive peptide isolated from culture media of vascular endothelial cells . The binding of ET-1 to membrane preparations from rat and bovine lung was studied using radioiodinated ET-1 (125I-ET-1) . With both membrane preparations, 125I-ET-1 showed saturable binding to a single class of high affinity sites . Scatchard analysis of the binding data gave dissociation constants (Kd) for ET-1 of 0.22 nM and 0.15 nM, and receptor densities (Bmax) of 6.1 pmol/mg and 2.7 pmol/mg for rat and bovine lung membranes, respectively . Photo-reactive radioiodinated ET-1, N epsilon 9-azidobenzoyl-125I-ET-1, was synthesized and purified as a mono-reactive affinity labeling reagent . This reagent was used for affinity labeling of ET-1 receptor in bovine and rat lung membranes . Photoaffinity labeling followed by sodium dodecyl sulfate gel electrophoresis and autoradiography gave a radiolabeled protein band with an apparent Mr of 34,000 in both membrane preparations . The labeling of this protein band was inhibited by cold ET-1 in a concentration-dependent manner . Labeling was not abolished by unrelated peptides such as angiotensin II and {Arg8}-vasopressin, or by structurally related bee venom apamin . These results indicate that the ET-1 receptor or its ligand binding subunit consists of a 34,000 Da polypeptide. Exp Cell Res, 1991 Aug, 195(2), 284 - 94 The biochemical and structural maturation of human skeletal muscle cells in culture: the effect of the serum substitute Ultroser G; Benders AA et al.; On the basis of the percentage creatine kinase-MM, human skeletal muscle cells cultured on growth and differentiation media containing the serum substitute Ultroser G reach a significantly higher maturation grade after 7 days of differentiation than cells cultured on serum-containing media . They also remain viable for longer periods . The myotubes are much longer, their nuclei are often localized in rows on the periphery, and they show cross-striation more frequently . The activities of creatine kinase, citrate synthase, cytochrome c oxidase, AMP deaminase, and phosphorylase are significantly higher . Extending the differentiation period to 3 weeks increases the maturation grade of the cultures and the activities of all the enzymes mentioned before, except phosphorylase . A correlation exists between the enzyme activities and the maturation grade of the muscle cells . The content of fatty acid-binding protein also increases significantly with the maturation grade in contrast to the palmitate oxidation rate . The AMP deaminase and creatine kinase activity and the percentage MM-type remain lower in cultured cells than in adult muscle and the hexokinase activity remains higher, but the other enzyme activities become comparable after 20 days of differentiation . The myotubes, derived from Ultroser G-containing culture media, show spontaneous contractions after 12 days and cross-striation after 20 days when immunostained for the M-subunit of creatine kinase . These cells possess clusters of acetylcholine receptors, but aggregation of desmin at the site of the clusters was never detectable . The possibility of cultivating muscle cells with a predictable maturation grade allows the study of muscle development and muscular diseases caused by differentiation defects or by deficiency of a maturation-dependent (iso)enzyme. Biochem Biophys Res Commun, 1991 Jul 31, 178(2), 625 - 33 Purification and characterization of human recombinant insulin-like growth factor binding protein 3 expressed in Chinese hamster ovary cells; Tressel TJ et al.; Recombinant human insulin-like growth factor binding protein 3 (hIGFBP-3) stably expressed in chinese hamster ovary cells (CHO cells) has been purified to homogeneity from serum-free culture media . The purified protein migrates as a doublet (45/43 kDa) upon SDS-PAGE . The purified recombinant hIGFBP-3 is fully active and binds one mole of IGF-I per mole of recombinant binding protein . When the transfected CHO cells are treated with tunicamycin a single 29 kDa hIGFBP-3 protein is observed . This expressed hIGFBP-3 protein maintains its ability to bind IGF-I . N-Glycanase treatment of the purified hIGFBP-3 protein results in a protein that migrates similar to E . coli-derived IGFBP-3 upon SDS-PAGE under reducing conditions (30 kDa) . Carboxymethylation of hIGFBP-3 suggests that all 18 cysteines are involved in disulfide linkages . These results represent the first purification and characterization of recombinant hIGFBP-3 expressed in CHO cells. J Immunol, 1991 Jul 1, 147(1), 144 - 8 Nitric oxide production is required for murine resident peritoneal macrophages to suppress mitogen-stimulated T cell proliferation . Role of IFN-gamma in the induction of the nitric oxide-synthesizing pathway; Albina JE et al.; Lymphocyte proliferation in Con A- or LPS-stimulated murine splenic cell (SC) cultures was suppressed by the addition of excess macrophages . In Con A-stimulated cultures, suppression was associated with the expression of nitric oxide-synthesizing pathway (NOSP) activity as demonstrated by the accumulation of nitrite, a degradation product of nitric oxide (NO), in the culture supernatants . That NO, a cytotoxic and anti-proliferative metabolite of l-arginine, or other reactive nitrogen intermediates generated through the NOSP mediated the suppressive effect was suggested by the reversal of suppression brought about by the addition of a specific inhibitor of the NOSP (NG-monomethyl-l-arginine acetate) to the culture media . No NOSP activity was detectable in LPS-stimulated SC/macrophage cocultures . The role of T cell-derived IFN-gamma in the induction of the NOSP was investigated by the use of anti-IFN-gamma-mAb . Antibody-treated Con A supernatants failed to induce the NOSP in macrophages, and the addition of the mAb to Con A-stimulated SC/macrophage cocultures obviated the suppressive effects . Indomethacin and catalase only partially restored proliferation in Con A-stimulated SC/macrophage cocultures but were remarkably efficient in preventing macrophage-dependent suppression when LPS was used as the mitogenic stimulus . These results demonstrate a regulatory system of potential relevance in sites of predominant macrophage infiltration by which T cell-derived IFN-gamma activates the production of the mediator, NO, that suppresses T cell proliferation . In addition, these data demonstrate that, although the suppressive effects of excess macrophages appear to be expressed nonspecifically toward both T and B cells, suppression is mediated through a different mechanism in each case. J Surg Res, 1991 Jul, 51(1), 33 - 9 Comparison of immediate seeding of endothelial cells with culture lining of small diameter ePTFE carotid interposition grafts; Hussain SM et al.; This study is the first to compare chronic healing characteristics of immediately seeded grafts with those of grafts lined by autogenous venous endothelial cells in tissue culture prior to implantation . Ten mongrel dogs had a segment of external jugular vein excised for enzymatic harvest of endothelial cells . After approximately 21 days growth in tissue culture, 4 X 10(6) cells/ml were inoculated into a 6-cm length of 4 mm i.d . ePTFE for formation of a confluent lining in culture media . The remaining external jugular vein had its endothelial cells enzymatically harvested for immediate seeding of an identical length of preclotted ePTFE . Both grafts were implanted end-to-end in the carotid position and excised after 30 days . In 6 of the 10 dogs, grafts were patent bilaterally; all others were occluded . Planimetric measurements on patent grafts with immediate seeding showed a thrombus-free surface area of 56 +/- 39% compared to 86 +/- 15% for culture-lined grafts (P = 0.046) . Endothelial coverage was 70 +/- 24% for immediately seeded grafts and 29 +/- 21% for culture-lined grafts (P = 0.016) . We conclude that immediate seeding and culture lining of autogenous endothelial cells in small diameter ePTFE grafts produce equivalent short-term patency . While culture-lined grafts have an initially less thrombogenic luminal surface, subsequent development of a confluent endothelial lining is slower than that with an immediate seeding preparation, and thus would appear to offer no significant clinical benefit, especially in light of the complexity culture lining adds to the procedure. Indian J Med Res, 1991 Jul, 93, 225 - 31 Fine structure of Calymmatobacterium granulomatis with particular reference to the surface structure; Chandra M et al.; Ultrastructural study of C . granulomatis, the causative organism of donovanosis (granuloma inguinale), in human tissue revealed the presence of a complex cell envelope . The cytoplasm of these organisms showed presence of electron dense polar material, in addition to regular bacterial structures like mesosome, ribosomes, and nuclear material . Surface appendages i.e., fimbriae and blebs were studied in detail . Origin of these structures was clearly endogenous to the cell wall . Morphology of fimbrium at the site of its attachment to the cell membrane has been described . A distinct layer of homogenous material of varying density surrounding the organism indicated the possibility of it being a capsulePIP: The morphological structure of Calymmatobacterium granulomatis, the organism responsible for donovanosis, was investigated in biopsy material from 15 patients . The use of human material was necessary because this organism is difficult to grow in artificial culture media . 1-10 organisms per cell from 1.5-2.5 mcm in size were observed singly or in clusters, most frequently in the vacuolated cytoplasm of macrophages in the upper portion of the dermis . Most notable was the limitation of these organisms by a complex cell envelope comprised of at least 2 layers--the corrugated cell wall and the cytoplasmic membrane . In most cases, the protoplasm was enclosed by more than 2 trilaminar membranes . Cytoplasm at the periphery contained ribosomes and fine membranes, while the nuclear materials tended to be located in the cell's center . The organism's surface was covered with multiple filamentous processes (fimbriae) and round vesicles (blebs) . Unique to this study was the finding of electron dense polar bodies at poles . The basal portion of the fimbriae was more electron dense than the remaining portion . Also notable was the observation that fimbriae are attached to the inner membrane and do not arise from the cell wall . The blebs also appeared to be endogenous to the cell wall and could be seen detaching and moving away from the parent organism . Finally, a distinct layer of homogenous material was observed on light microscopy to surround the organism and is postulated to represent capsular material . Vet Immunol Immunopathol, 1991 Jul, 28(3-4), 317 - 26 Spontaneous murine thymocyte comitogenic activity consistent with interleukin-1 in cattle naturally infected with Mycobacterium paratuberculosis; Kreeger JM et al.; The production of comitogenic activity consistent with interleukin-1 (IL-1) activity by blood monocytes from cattle with naturally acquired paratuberculosis was examined by murine thymocyte proliferation . In addition, IL-1-like activity in response to homologous and heterologous antigens was determined . Activity was determined in nine cattle naturally infected with Mycobacterium paratuberculosis and six non-infected cattle . Comitogenic properties were measured in response to M . paratuberculosis antigen (johnin), bacterial lipopolysaccharide (LPS) as a positive control, and culture media as a negative control . Monocytes from infected cattle spontaneously released high levels of IL-1-like activity in the absence of stimuli and significantly (P less than 0.05) increased activity in response to LPS . With johnin, M . bovis PPD and KLH stimulation, comitogenic activity was similar to spontaneous levels . Non-infected cattle had significantly (P less than 0.05) increased comitogenic activity when blood monocytes were stimulated with KLH, M . bovis PPD, johnin, and lipopolysaccharide when compared with non-stimulated cells in that group . Johnin produced the greatest response in non-infected animals . The data suggest that blood monocytes in infected cattle are non-specifically activated with respect to IL-1 production . Alternatively, a defective regulatory mechanism for IL-1 may be operat