Tyrosine 67 in the epidermal growth factor-like domain of tissue-type plasminogen activator is important for clearance by a specific hepatic receptor. Human tissue-type plasminogen activator (t-PA) is cleared rapidly from the circulation by hepatic receptors, one of which recognizes a site in the epidermal growth factor-like domain of the molecule. To define this site more precisely, we have used oligonucleotide-mediated mutagenesis to introduce amino acid substitutions at specific positions located in turns that connect antiparallel beta-sheets in the epidermal growth factor-like domain. Mutated t-PA proteins with amino acid substitutions of the tyrosine residue at position 67 showed markedly lower rates of endocytosis and degradation by cultured cells of the rat hepatoma (H4) line that express a specific receptor for t-PA, and their half-life in the circulation of rats was extended significantly because of a reduction in the rate of the rapid alpha-phase of clearance. The enzymatic properties and fibrinolytic activity of these mutants in vitro were not significantly different from those of wild-type t-PA. We conclude that tyrosine 67 comprises a key determinant in the clearance of t-PA by a specific hepatic receptor.


Presence of SH2 domains of phospholipase C gamma 1 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor. src homology region 2 and 3 (SH2 and SH3) domains are conserved noncatalytic regions originally described in cytoplasmic tyrosine kinases and subsequently identified in phospholipase C gamma 1 (PLC gamma 1), GTPase-activating protein of ras, and other signaling proteins. Although numerous studies indicate that SH2 domains promote protein-protein interactions by specific binding to tyrosine phosphorylated proteins, the function of SH3 domains is not known. The SH2 domain of PLC gamma 1 binds to certain tyrosine-phosphorylated growth factor receptors, and following phosphorylation on Tyr783 the enzymatic activity of PLC gamma 1 is enhanced, leading to phosphatidylinositol hydrolysis. To determine the functional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in Escherichia coli PLC gamma 1 constructs encoding the region containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjected to quantitative phosphorylation by the epidermal growth factor receptor (EGFR). Analysis of the kinetics of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanced for substrates containing SH2 domains: from S0.5 (average apparent Km) of 110 microM to S0.5 of 20 microM with the addition of a single SH2 domain and S0.5 of 3-4 microM for mutants containing two SH2 domains. The presence of the SH3 domain did not influence the apparent Km of substrate phosphorylation. These results demonstrate that the presence of the SH2 domain in PLC gamma 1 lowers the apparent Km (increases the affinity) of substrate phosphorylation by the EGFR, thereby facilitating PLC gamma 1 phosphorylation and activation.


A nucleoside transporter is functionally linked to ectonucleotidases in rat liver canalicular membrane. Prevention of nucleoside loss in bile is physiologically desirable because hepatocytes are the main source of nucleosides for animal cells which lack de novo nucleoside biosynthesis. We have demonstrated a Na+ gradient-energized, concentrative nucleoside transport system in canalicular membrane vesicles (CMV) from rat liver by studying [3H]adenosine uptake using a rapid filtration technique. The Na(+)-dependent nucleoside transporter accepts purine, analogues of purine nucleosides and uridine; exhibits high affinity for adenosine (apparent Km, 14 microM); is not inhibited by nitrobenzylthioinosine or dipyridamole, and is present in CMV but not in rat liver sinusoidal membrane vesicles. Adenosine transport in right side-out CMV was substantially greater than with inside-out CMV. CMV also contain abundant ecto-ATPase and ecto-AMPase (5'-nucleotidase). These ectoenzymes were shown to degrade nucleotides into nucleosides which were conserved by the Na(+)-dependent nucleoside transport system.


S100 beta stimulates calcium fluxes in glial and neuronal cells. The glial-derived protein S100 beta can act as a mitogen or a neurotrophic factor, stimulating proliferation of glial cells or differentiation of immature neurons. We report here that dimeric S100 beta evokes increases in intracellular free calcium concentrations ([Ca2+]i) in both glial cells and neuronal cells. The [Ca2+]i increase exhibited a rapid transient component which was not affected by removal of extracellular calcium and a sustained component which appeared to require influx of extracellular calcium through Ni(2+)-sensitive channels. S100 beta also stimulated hydrolysis of phosphoinositides, suggesting a mobilization of calcium from intracellular stores. These data suggest that although the final biological responses of neuronal and glial cells to S100 beta are different, transduction of the S100 beta signal in both cell types involves changes in [Ca2+]i.


Infrared evidence of azide binding to iron, copper, and non-metal sites in heart cytochrome c oxidase. Interactions of azide ion with bovine heart cytochrome c oxidase (CcO) at five redox levels (IV) to (0), obtained by zero to four electron reduction of fully oxidized enzyme CcO(IV), were monitored by infrared and visible/Soret spectra. Partially reduced CcO gave three azide asymmetric stretch band at 2040, 2016, and 2004 cm-1 for CcO(III)N3 and two at 2040 and 2016 cm-1 for CcO(II)N3 and CcO(I)N3. Resting CcO(IV) reacts with N3- to give one band at 2041 cm-1 assigned to CuB2+N3 and another at 2051 cm-1 to N3- that is associated with protein but is not bound to a metal ion. At high azide concentrations the weak association of many azide molecules with non-metal protein sites was observed at all redox levels. These findings provide direct evidence for 1) N3- binding to CuB as well as Fea3 in partially reduced enzyme, but no binding to Fea3 in fully oxidized enzyme and no binding to either metal in fully reduced enzyme; 2) a long range effect of the oxidation state of Fea or CuA on ligand binding at heme a3, but not at CuB; and 3) an insensitivity of either Fea3 or CuB ligand site to changes in ligand or oxidation state at the other site. The observed independence of the Fea3 and CuB sites provides further support for Fea3(3)+ OOH, rather than Fea3(3)+ OOCuB2+, as an intermediate in the reduction of O2 to water by the oxidase.


The geranylgeranyl moiety but not the methyl moiety of the smg-25A/rab3A protein is essential for the interactions with membrane and its inhibitory GDP/GTP exchange protein. The smg-25A/rab3A protein (smg p25A), a member of the small GTP-binding protein superfamily, has a C-terminal structure of Cys-Ala-Cys which is post-translationally processed: both cysteine residues are geranylgeranylated followed by the carboxyl methylation of the C-terminal cysteine residue. We reported previously that this posttranslational processing is essential for the interactions of smg p25A with membrane and its inhibitory GDP/GTP exchange protein, named smg p25A GDP dissociation inhibitor (GDI). In this study, we examined which posttranslational modification of smg p25A is necessary for these interactions. The smg p25A which was not posttranslationally processed was produced in Escherichia coli and purified. This protein was then geranylgeranylated at both of the 2 cysteine residues by use of a bovine brain geranylgeranyltransferase in a cell-free system (recombinant smg p25A-GG). By use of this recombinant smg p25A-GG, its membrane-binding activity and its sensitivity to smg p25A GDI were compared with those of the fully posttranslationally processed form of bovine brain smg p25A (smg p25A-GG-Me) and the posttranslationally unprocessed form of bacterial smg p25A (recombinant smg p25A). The membrane-binding activity and sensitivity to smg p25A GDI were similar between the recombinant smg p25A-GG and smg p25A-GG-Me, although recombinant smg p25A lacked both activities. These results indicate that the geranylgeranyl moiety of smg p25A is essential and sufficient for its interactions with membrane and smg p25A GDI and that the methyl moiety is not essential for these interactions.


Differential regulation of phosphoinositide and phosphatidylcholine hydrolysis by protein kinase C-beta 1 overexpression. Effects on stimulation by alpha-thrombin, guanosine 5'-O-(thiotriphosphate), and calcium. Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.


Mechanism for ganglioside-mediated modulation of a calmodulin-dependent enzyme. Modulation of calmodulin-dependent cyclic nucleotide phosphodiesterase activity through binding of gangliosides to calmodulin and the enzyme. Gangliosides were recently shown to bind to calmodulin (Higashi, H., Omori, A., and Yamagata, T. (1992) J. Biol. Chem. 267, 9831-9838). This prompted us to investigate the effects of gangliosides on the calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. Several species of gangliosides competitively inhibited calmodulin-stimulated phosphodiesterase activity, with GD1b, GT1b, and GD1a being noted to do so particularly (group 1). GM1, GQ1b, and GM2 (group 2) were less inhibitory, and GM3, GM3(NeuGc), GalCer, sulfatide, GgOse4Cer, and oligosaccharide portions of inhibitory gangliosides showed no inhibition in accordance with the binding specificity of calmodulin to gangliosides. Trypsin-activated phosphodiesterase was inhibited by gangliosides with similar specificity, indicating interactions of gangliosides with the enzyme. Inhibition, however, was less than that of calmodulin-dependent activity by these compounds and, in both cases, was eliminated by excess calmodulin. In the absence of calmodulin, group 1 gangliosides at lower concentrations activated the intact enzyme but inhibited it over a certain range of increase in concentration. Ganglioside-dependent modulation of calmodulin-dependent phosphodiesterase activity is thus shown to be due to interactions of gangliosides with both calmodulin and the enzyme, and consequently, ganglioside-calmodulin binding is likely the mechanism for regulation of the enzyme.


The assembly and secretion of ApoB 100-containing lipoproteins in Hep G2 cells. ApoB 100 is cotranslationally integrated into lipoproteins. The possibility that apoB 100 is cotranslationally translocated to the endoplasmic reticulum lumen and integrated into lipoproteins has been investigated. ApoB 100 nascent polypeptides were shown to be secreted from pulse-labeled Hep G2 cells after treatment with puromycin and chase for 1 or 2 h in the presence of puromycin and cycloheximide. These nascent polypeptides banded during sucrose gradient ultracentrifugation between the position of the high (HDL) and the low (LDL) density lipoproteins, revealing an inverse relationship between the length of the polypeptide and the density of the fraction. ApoB 100 occurred in the position of LDL and very low density lipoproteins (VLDL). Electronmicroscopy studies of the apoB-containing particles from the gradient indicated an increase in size with increasing length of the polypeptide. Furthermore, labeling studies indicated that the triglyceride load increased with the length of the polypeptide. An inverse relationship between the size of C-terminally truncated apoB polypeptides and the density of the assembled lipoproteins was also observed in experiments with transfected minigenes coding for apoB 41, apoB 29, and apoB 23. These proteins appeared on HDL particles. Pulse-chase experiments indicated that 80-200-kDa apoB nascent polypeptides on particles with HDL density, with time, were converted into larger polypeptides on lighter particles, to be fully replaced by apoB 100 on LDL-VLDL particles. The formation of these LDL-VLDL particles could be blocked by cycloheximide. Sixty-five percent of pulse-labeled apoB nascent polypeptides present in the microsomal fraction was released by sodium carbonate treatment, and 77% of these polypeptides could be recovered on the immature particles (banding between HDL and LDL) after sucrose gradient ultracentrifugation. Pulse-chase experiments indicated that these nascent polypeptides, on the immature lipoproteins, had the capacity to be precursors for all the apoB 100-containing LDL and VLDL particles formed in the cell. The obtained results indicate that a major portion of the apoB nascent polypeptides in the cell form lipoproteins cotranslationally during the translocation to the lumen of the endoplasmic reticulum.


Mitogen-induced tyrosine-phosphorylated 41- and 43-kDa proteins are family members of extracellular signal-regulated kinases/microtubule-associated protein 2 kinases. Two antipeptide antibodies, one against the peptide corresponding to residues 307-327 (alpha Y91) and one against the peptide corresponding to the C-terminal portion (alpha C92) of the deduced amino acid sequence of the extracellular signal-regulated kinase 1 (ERK1), precipitated two 41-kDa and/or two 43-kDa phospho-proteins from mitogen-stimulated Swiss 3T3 cells. Electrophoretic mobilities on two-dimensional gels of the immunoprecipitated 41- and 43-kDa phosphoproteins were similar to those of the 41- and 43-kDa cytosol proteins, whose increased tyrosine phosphorylation we and others had originally identified in various mitogen-stimulated cells (Cooper, J. A., Sefton, B. M., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37; Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779); phosphopeptide map analysis revealed that they were respectively identical molecules. All those phosphoproteins contained phosphotyrosine, and the more acidic forms contained additional phosphothreonine. Immunoprecipitated 41- and 43-kDa phosphoproteins had serine/threonine kinase activity toward myelin basic protein (MBP) and microtuble-associated protein 2 (MAP2). With the combination of two-dimensional gel electrophoresis and the kinase assay in MBP-containing polyacrylamide gels of the alpha Y91 immunoprecipitates, with or without phosphatase 2A treatment, we showed that only their acidic forms were active. These results clearly indicate that 41- and 43-kDa proteins, the increased tyrosine phosphorylation of which is rapidly and commonly induced by mitogen stimulation of fibroblasts, are family members of ERKs/MAP2 kinases and that phosphorylation both on tyrosine and threonine residues is necessary for their activation.


Polysialic acid is associated with sodium channels and the neural cell adhesion molecule N-CAM in adult rat brain. We have studied alpha 2,8-linked polysialic acid (polySia) and the neural cell adhesion molecule (N-CAM) in the adult rat brain by immunohistochemistry and Western blot analysis. Both molecules were widely distributed but not ubiquitous. Various brain regions showed colocalization of polySia and N-CAM. Strong immunoreactivity for polySia was seen in regions which were negative for N-CAM, such as the main and accessory olfactory bulbs. Immunohistochemical evidence for the heterogeneity of polySia expression in different brain regions was confirmed by immunoblotting. We present evidence that N-CAM is not the only polySia bearing protein in adult rat brain. Specifically, immunoprecipitation using the polySia-specific monoclonal antibody mAb 735 precipitated not only N-CAM isoforms carrying polySia, but also the sodium channel alpha subunit. Immunoblotting using sodium channel alpha subunit antibody (SP20) revealed a smear from 250 kDa upwards. PolySia removal using an endoneuraminidase specific for alpha 2,8-linked polysialic acid of 8 or more residues long, reduced this smear to a single band at 250 kDa. Thus both N-CAM and sodium channels carry homopolymers of alpha 2,8-linked polysialic acid in adult rat brain.


Role of protein kinase C and the Na+/H+ antiporter in suppression of apoptosis by granulocyte macrophage colony-stimulating factor and interleukin-3. Granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) suppress apoptosis in hemopoietic cells, a process of active cell death characterized by the degradation of genomic DNA into oligonucleosomic fragments. The present study was therefore initiated with the view that the two growth factors may trigger the same early events in the cell, leading to suppression of apoptosis. We provide evidence here for a role of protein kinase C and of the Na+/H+ antiporter in the signal transduction pathways activated by binding of GM-CSF or IL-3 to their respective receptors, resulting in suppression of apoptosis in target cells. First, kinetic studies indicate that the process is irreversible after two hours of deprivation. The suppression of apoptosis by GM-CSF and IL-3 is dose-dependent, with half-efficient concentrations that are in the range of the dissociation constants of the high affinity GM-CSF or IL-3 receptor, respectively. Second, the use of three inhibitors of protein kinase C (PKC), H7, staurosporine, and sphingosine, in concentrations that are below their toxicity limits, revert the suppression of apoptosis by IL-3 and GM-CSF. Conversely, the use of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, allows a bypass of receptor activation in suppression of apoptosis. Western blotting of cytosolic and membrane proteins indicate that exposure of the cells to GM-CSF, IL-3, or TPA results in translocation of PKC to the cell membrane. Our data, therefore, indicate that the activation of PKC is important in suppression of apoptosis by GM-CSF and IL-3. Third, the two amiloride derivatives 5-(N,N-hexamethylene) and 5-(N-ethyl-N-isopropyl)amiloride that specifically block the function of the Na+/H+ antiport also revert the protective effect of GM-CSF, IL-3, and TPA on MO7-E cells. Further, exposure of the cells to GM-CSF, IL-3, or TPA results in sustained pHi alkalinizatio, which is abrogated when the cells are preincubated with 5-(N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the antiport. Preincubation of the cells with staurosporine, a PKC inhibitor, also significantly reduces the effect of GM-CSF or IL-3 on pHi. Taken together, our data indicate that a functional antiport is required in suppression of apoptosis by GM-CSF, IL-3, or TPA. Furthermore, our results are consistent with the view that GM-CSF or IL-3 receptor activation initiates the sequential activation of PKC and of the Na+/H+ antiporter, resulting in suppression of apoptosis in target cells.


Tensile strength of the interface between hydroxyapatite and bone. Tensile strength of the interface between hydroxyapatite (HA) and bone was tested. Scanning electron microscopy was used to observe the tensile failure mode and the morphological change of hydroxyapatite ceramic surface in bone. The porosity of hydroxyapatite is 14% and pore size less than 2 microns. After 2 weeks of implantation, the tensile strength of the interface is 0.72 MPa. After 4, 8, and 16 weeks, the average tensile strength stayed at 1.5 MPa. SEM showed that tensile failure occurred at the HA-bone interface at the second week, but after 4 weeks, the failure occurred between HA particles within the bulk, and not at the HA-bone interface. Calcified tissue was directly deposited on the HA ceramic surface and exits also in the micropores. Near the interface, sintered necks among HA ceramic particles were subjected to biodegradation.


Participation of poly(ADP-ribose) polymerase in the drug sensitivity in human lung cancer cell lines. Poly(ADP-ribose) polymerase has been generally assumed to be involved in DNA repair. The level of the enzyme in various lung cancer cell lines was examined to determine if it is involved in drug resistance. Among nine cell lines of lung cancer tested, small-cell lung cancer lines, which showed higher sensitivity to cisplatin and etoposide, were unexpectedly found to contain significantly higher poly(ADP-ribose) polymerase activity than five non-small-cell lung cancer cell lines. This activity inversely correlated with IC50 values of lung cancer cell lines to etoposide, an inhibitor of topoisomerase II. The polymerase activity was also examined in several cisplatin-resistant variants of the cell lines. However, no difference was observed between parental and cisplatin-resistant cells. There was no significant relation between poly(ADP-ribose) polymerase activity and IC50 values for cisplatin and carboplatin. Although this enzyme was considered to play some role in the resistance to specific drugs, it might not be a critical factor in cisplatin-induced cytotoxicity.


Type I and type II insulin-like growth factor receptors and their function in human Ewing's sarcoma cells. Binding studies using recombinant human 125I-labelled insulin-like growth factor I ([125I]IGF-I) revealed IGF-I receptors in three Ewing's sarcoma cell lines with Kd ranging from 74 x 10(-12) M to 100 x 10(-12) M and Bmax = 36-63 fmol/mg cell protein. [125I]IGF-I binding was displaced by IGF-I, IGF-II and insulin with IC50 values of 1.5 nM, 6.3 nM and 0.7 microM respectively. Recombinant human [125I]IGF-II radioligand-binding assays in the cell lines disclosed specific binding sites for IGF-II with Kd = (110-175) x 10(-12) M and Bmax varying from 21 fmol/mg to 72 fmol/mg cell protein. Neither IGF-I nor insulin displaced [125I]IGF-II binding. IGF-I was found to increase basal glucose transport by maximally 1.5 times with EC50 = 0.9 nM IGF-I. The efficacy and potency of IGF-II on glucose uptake were comparable to those of IGF-I whereas insulin was ineffective. IGF-I and IGF-II also provoked stimulation of glycogen synthesis in Ewing's sarcoma cells. The maximal glycogenic response was reached at 0.01 microM IGF-I and 0.1 microM IGF-II, the EC50 value being approximately 1 nM IGF-I and 2 nM IGF-II. Insulin did not significantly influence glycogen formation. IGF-I and IGF-II but not insulin increased DNA synthesis in Ewing's sarcoma cells. The maximal mitogenic response was obtained with 10 nM IGF-I or IGF-II with an EC50 value of about 0.7 nM for both peptides. alpha-IR-3, a monoclonal antibody specific for the IGF type I receptor, effectively blocked IGF-I- and IGF-II-mediated metabolic responses. In conclusion, the data show that IGF-I and IGF-II induce rapid and long-term biological responses in Ewing's sarcoma cells exclusively through interaction with IGF type I receptors.


Effect of tobacco smoking on various histological types of lung cancer. In a population-based case/control study the differential lung cancer risk patterns due to tobacco smoking habits of various histological types have been investigated. The cases were 1432 deaths from lung cancer in the years 1980-1987, of which the histological type was known for 627 individuals. There was 54% squamous cell carcinoma, 24% small-cell carcinoma and 17% adenocarcinoma. Controls were 1343 deaths from other causes. Next-of-kin interviews were performed. The results of the study confirmed that cigarette smoking is associated with all histological types of lung cancer; however, the dose/response relationship between smoking and adenocarcinoma differed clearly from that observed in squamous and small-cell carcinomas. In the latter histological types the gradient of risk was much stronger as the number of cigarettes smoked or duration of smoking increased. The overall relative risk for smoking in small-cell and squamous cell carcinoma was 15.4 and 13.5 respectively, whereas that for adenocarcinoma was weaker (relative risk = 3.1). An interesting difference between squamous and small-cell carcinomas was found also for patients who gave up smoking. The effect of stopping was more pronounced in squamous cell carcinoma. The attributable risks for smoking in squamous and small-cell carcinoma were much higher (90% and 88% respectively) than for adenocarcinoma (64%). The data suggest that adenocarcinoma is likely to be related to other factors than tobacco smoking to a greater extent than are squamous or small cell carcinoma. Possible sources of bias, such as missing histological diagnoses, are discussed in detail.


Occurrence of epidermal growth factor receptors in benign and malignant ovarian tumors and normal ovarian tissues: an immunohistochemical study. Epidermal growth factor receptor (EGF-R) was studied with monoclonal antibody 2E9 on 50 ovarian tumors of various histological types and 10 non-tumorous ovarian tissues by immunohistochemistry. Enhanced expression was observed in 26/50 (52%) of the tumors. Only 25 out of 46 epithelial tumors (54%) showed positivity in epithelial tumor cells. Staining was cytoplasmic in all cases. No correlation was established between EGF-R expression and the histological type of the epithelial tumor. Apart from EGF-R expression in tumor cells, low immunoreactivity was also observed in stromal and endothelial cells in both normal and tumorous ovarian tissues. Furthermore in 8/9 specimens containing necrotic areas, EGF-R was noticed in these areas as well. Both of the latter observations may have impact on the evaluation of the prognostic value of EGF-R activity in tumors, when based on EGF-R measurements using biochemical binding studies. We therefore recommend that EGF-R is measured with both methods in studies regarding its clinical value.


A soluble form of the F3 neuronal cell adhesion molecule promotes neurite outgrowth. The F3 molecule is a member of the immunoglobulin superfamily anchored to membranes by a glycane-phosphatidylinositol, and is predominantly expressed on subsets of axons of the central and peripheral nervous system. In a previous paper (Gennarini, G., P. Durbec, A. Boned, G. Rougon, and C. Goridis. 1991. Neuron. 6:595-606), we have established that F3 fulfills the operational definition of a cell adhesion molecule and that it stimulates neurite outgrowth when presented to sensory neurons as a surface component of transfected CHO cells. In the present study the question as to whether soluble forms of F3 would be functionally active was addressed in vitro on cultures of mouse dorsal root ganglion neurons. We observed that preparations enriched in soluble F3 had no effect on neuron attachment but enhanced neurite initiation and neurite outgrowth in a dose-dependent manner. By contrast, soluble NCAM-120 does not have any measurable effect on these phenomena. Addition of anti-F3 monovalent antibodies reduced the number of process-bearing neurons and the neuritic output per neuron to control values. Addition of cerebrospinal fluid, a natural source of soluble F3, also stimulated neurite extension, and this effect was partially blocked by anti-F3 antibodies. Our results suggest that the soluble forms of adhesive proteins with neurite outgrowth-promoting properties could act at a distance from their site of release in a way reminiscent of growth and trophic factors.


Expression of a human hepatocyte growth factor/scatter factor cDNA in MDCK epithelial cells influences cell morphology, motility, and anchorage-independent growth. The addition of exogenous hepatocyte growth factor (HGF)/scatter factor (SF) to MDCK epithelial cells results in fibroblastic morphology and cell motility. We generated HGF/SF producing MDCK cells by transfection with an expression plasmid containing human HGF/SF cDNA. Production of HGF/SF by these cells induced a change from an epithelial to a fibroblastic morphology and increased cell motility. In addition, the HGF/SF producing cells acquired efficient anchorage-independent growth in soft agar but did not form tumors in nude mice. The morphological change and the stimulation of the anchorage-independent growth were prevented by anti-HGF/SF antibody, suggesting that the factor is secreted and then exerts its effects through cell surface receptors.


Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein. Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro. We purified capping protein from Saccharomyces cervisiae. One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J. F., J. F. Cannon, K. Tatchell, C. Hug, and J. A. Cooper. 1990. Nature (Lond.) 344:352-354). Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed-end capping activity. Like chicken capping protein, yeast capping protein is inhibited by PIP2. By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos. In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis.


Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase. We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.


Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast. Topoisomerase II (topoII) and RAP1 (Repressor Activator Protein 1) are two abundant nuclear proteins with proposed structural roles in the higher-order organization of chromosomes. Both proteins co-fractionate as components of nuclear scaffolds from vegetatively growing yeast cells, and both proteins are present as components of pachytene chromosome, co-fractionating with an insoluble subfraction of meiotic nuclei. Immunolocalization using antibodies specific for topoII shows staining of an axial core of the yeast meiotic chromosome, extending the length of the synaptonemal complex. RAP1, on the other hand, is located at the ends of the paired bivalent chromosomes, consistent with its ability to bind telomeric sequences in vitro. In interphase nuclei, again in contrast to anti-topoII, anti-RAP1 gives a distinctly punctate staining that is located primarily at the nuclear periphery. Approximately 16 brightly staining foci can be identified in a diploid nucleus stained with anti-RAP1 antibodies, suggesting that telomeres are grouped together, perhaps through interaction with the nuclear envelope.


Local expression and exocytosis of viral glycoproteins in multinucleated muscle cells. We have analyzed the distribution of enveloped viral infections in multinucleated L6 muscle cells. A temperature-sensitive vesicular stomatitis virus (mutant VSV ts045) was utilized at the nonpermissive temperature (39 degrees C). As expected, the glycoprotein (G protein) of this mutant was restricted to the ER when the multinucleated cells were maintained at 39 degrees C. We demonstrate that this G protein remained localized when the infection was performed at low dose. By 4 h after infection the G protein patches spanned an average of 220 microns. The localization was independent of nuclear positions, showing that the ER was a peripheric structure. Thus, the infection did not recognize nuclear domains characteristic of nuclearly encoded proteins. After release of the 39 degrees C block, transport through a perinuclear compartment into a restricted surface domain lying above the internal G protein patch occurred. Accordingly, the transport pathway was locally restricted. After a 16-h infection the G protein spanned 420 microns, while the matrix protein occupied 700-800 microns of the myotube length. Double infection of multinucleated L6 muscle cells with Semliki Forest virus and VSV at high multiplicities showed that the glycoprotein of each virus occupied intracellular domains which were devoid of the other respective glycoprotein. Taken together, these findings indicate that the viral glycoproteins did not range far from their site of synthesis within the ER or other intracellular membrane compartments in these large cells. This result also suggests that relocation of viral RNA synthesis occurred slowly.


Ligand-induced formation of the leukotriene B4 receptor-G protein complex of human polymorphonuclear leukocytes. The components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4 (LTB4) were examined by Sephacryl S-300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and filtered on Sephacryl S-300 prior to addition of [3H] LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4 bound to PMNL membranes prior to solubilization was recovered predominantly with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4 and subsequent solubilization, radioactivity was recovered predominantly with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP-PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4 either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4 receptor protein-G protein complex. That the maximum specific binding of [35S] guanosine-5'-0-3-thiotriphosphate (GTP-gammaS) to LTB4-binding proteins in the Sephacryl S-300 effluent corresponded to the 140 kD protein supported the presence of a G protein in the LTB4 receptor complex.


Dihydropyridine calcium antagonist modulates cholesterol metabolism and eicosanoid biosynthesis in vascular cells. Recent clinical studies have shown that calcium channel blockers can retard and possibly reduce the angiographic progression of coronary artery disease. Calcium channel blockers also inhibit dietary-induced atherosclerosis in animal models of this disease. In this study, we delineate potential cellular and molecular mechanisms by which nicardipine, a dihydropyridine calcium antagonist, may alter lipoprotein and cholesterol trafficking, affect the regulatory signal transduction pathways involved in accelerating cholesteryl ester (CE) catabolism in vascular smooth muscle cells, and modulate cell-cell interactions of vascular and inflammatory cells. We demonstrate in arterial smooth muscle cells that nicardipine increases 1) LDL binding, uptake, and degradation, 2) RNA transcript levels for the LDL receptor, 3) CE catabolic activity, 4) PGI2 release, and 5) RNA transcript levels for cyclooxygenase. Furthermore, nicardipine blocked cytokine-induced monocyte adhesion to endothelial cells and smooth muscle cells. Taken together, these findings support the hypothesis that nicardipine may function as an anti-atherosclerotic agent by promoting CE catabolism and cholesterol clearance and by reducing monocyte adhesion to the activated endothelium.


Regulation of aromatase activity in FSH-primed rat granulosa cells in vitro by follicle-stimulating hormone and various amounts of human chorionic gonadotrophin. The influence of various amounts of human chorionic gonadotrophin (HCG), with or without follicle-stimulating hormone (FSH), on aromatase activity, progesterone- and cAMP-accumulation in rat granulosa cells was investigated. Cells were isolated from immature diethylstilbestrol-treated rats and primed for 2 days in vitro with FSH (50 mIU/ml) to give a maximum response of the cells with respect to the induction of aromatase activity. After FSH priming, small amounts of HCG (1-5 mIU/ml) increased aromatase activity during an additional culture period of 24 h, with a maximum of 4- to 5-fold at 5-8 mIU/ml HCG. At higher concentrations of HCG, aromatase activity declined reaching a plateau (1.5-fold stimulation) at 20 mIU/ml HCG. When HCG was combined with FSH (50 mIU/ml) following the priming period, aromatase activity was stimulated in an additive way, reaching a maximum at 4-5 mIU/ml HCG followed by a decline, similar to that seen in the absence of FSH. HCG induced a dose-dependent increase in cAMP with a maximum at 20 mIU/ml. This increase in cAMP was higher when HCG was combined with FSH. Aromatase activity was inhibited by 3-isobutyl-methyl-xanthine and forskolin, both of which induce elevated cAMP levels in the cell. HCG, and HCG in combination with FSH, increased the production of progesterone in a dose-dependent manner until a plateau was reached. In the presence of FSH, this plateau was reached at lower HCG concentrations. Both R5020 and progesterone showed dose-dependent inhibition of aromatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Differential sperm performance as judged by the zona-free hamster egg penetration test relative to differing sperm penetration techniques. A prospective study on 61 unselected semen samples from infertile patients was conducted to evaluate the effects of sperm preparation techniques on the outcomes of the zona-free hamster egg penetration test (HEPT) to assess the in-vitro fertilizing capacity of spermatozoa. Each semen sample was divided into two equal portions before separation of the spermatozoa from seminal plasma either by the single-tube swim-up method, or using a two-layer discontinuous Percoll gradient. Spermatozoa were incubated overnight for initiation of capacitation after which HEPT was performed. The swim-up spermatozoa were further divided into two subgroups before HEPT as follows: with or without (control) treatment for 20 min with 50% (v/v) pooled, human follicular fluid (hFF) which had not been heat-inactivated. It was demonstrated (P less than 0.05) that the Percoll-separated spermatozoa exhibited higher penetration scores (percentage penetration rate and penetration index) than the control or the hFF-treated swim-up spermatozoa. A short exposure (20 min) to hFF significantly increased the penetration scores in HEPT for swim-up spermatozoa (P less than 0.05) but the average results were still significantly lower (P less than 0.05) than those of the Percoll separated spermatozoa, which had received no hFF treatment. Based on these findings, we conclude that the Percoll separation technique is better than the centrifugal pelleting and single-tube swim-up technique for reducing the false-negative results in HEPT. In addition, the use of hFF can significantly improve the performance of the swim-up sperm samples in HEPT.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples. Two methods of density gradient centrifugation, Percoll (P) and mini-Percoll (MP), were compared with the swim-up technique for preparing spermatozoa from each of 40 abnormal semen samples. P and MP produced similar results with a mean recovery of spermatozoa with progressive motility which was significantly higher (18-19%) than that achieved with swim-up (5%). However, the swim-up method resulted in the recovery of spermatozoa with a higher mean motility (89 versus 58%), velocity (69 versus 56 microns/s), percentage with normal morphology (22 versus 16%) and intact acrosomes (61 versus 36%) than P and MP. The mean amplitude of lateral head displacement was the only characteristic of spermatozoa in semen which correlated with the recoveries of motile spermatozoa. Combining MP and swim-up methods for 10 samples produced a higher recovery (11 versus 6.9%) of spermatozoa with significantly better mean motility (94 versus 87%) than did swim-up alone. Although P and MP resulted in greater yields of motile spermatozoa than the swim-up preparation, the latter procedure selected higher proportions of spermatozoa with improved characteristics (velocity, intact acrosomes and normal morphology) which correlate with fertilization rates in vitro. It is concluded that P and MP are not superior to swim-up. However, sequential MP and swim-up preparation improves yields of high quality spermatozoa from some abnormal semen samples and therefore has potential for improving fertilization rates.


Decreased herpes simplex viral immunity and enhanced pathogenesis following stressor administration in mice. Mild electric footshock stress was delivered during the dark portion of a 12:12 h light:dark cycle to C57BL/6 female mice that were infected with herpes simplex virus-type 1 (HSV). The studies were designed to correlate viral titer with both humoral and cell-mediated immune responses to HSV infection. Footshock was observed to result in decreased HSV-specific immunity. The numbers of leukocytes in spleens and draining popliteal lymph nodes of footshocked mice were depressed compared to both apparatus control and home cage control mice. A significant suppression of the HSV-specific cytotoxic T lymphocyte (CTL) response was observed in both the spleen and popliteal lymph nodes of footshocked mice. Serum IgM anti-HSV antibody titers were also depressed in footshocked mice. These changes were shown to be correlated with significantly increased viral titers in footshocked mice compared to control mice. These data demonstrate that administration of a relatively mild stressor is associated with depressed HSV-specific cellular and humoral immunity and is associated with increased pathogenicity.


Mediator release in cerebrospinal fluid of human immunodeficiency virus-positive patients with central nervous system involvement. In this study we evaluated the release of some mediators of inflammatory reactions such as histamine (H), leukotriene B4 (LTB4), leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) in the cerebrospinal fluid (CSF) of 15 patients with acquired immunodeficiency syndrome (AIDS), eight with opportunistic infections of the central nervous system (CNS) and seven without HIV-related neurological pathology, and of 25 HIV-negative control subjects with other neurological diseases. The cerebrospinal LTB4 level was increased in all the AIDS patients (mean 348 pg/ml); the control group revealed normal levels of LTB4 in the CSF (mean 63.2 pg/ml). The PGD2 level in the HIV-positive (mean 264 pg/ml) patients was higher than of the control subjects (mean 50 pg/ml), while low LTC4 levels were found both in the HIV-positive and control groups. We did not find any significant concentration of H in the CSF of either the HIV-positive or the control subjects. These findings may be due to the presence of chronic HIV infection or to the opportunistic infections of the CNS that so often occur in the latest stages of the disease.


Immunomodulatory effect of peripheral benzodiazepine receptor ligands on human mononuclear cells. Immunomodulatory effect of ligands active at the peripheral benzodiazepine receptor (PBR) was examined in human peripheral blood mononuclear cells (PBMC). Ro5-4864, PK11195 and diazepam suppressed phytohemagglutinin (PHA) and concanavalin A (ConA) induced proliferation of PBMC. All three ligands inhibited interleukin-3-like activity (IL-3-LA) secretion, while the production of interleukin-2 (IL-2) was inhibited by Ro5-4864 and diazepam only. The selective central benzodiazepine ligand clonazepam did not affect the cellular immune functions examined. Our results indicate an in-vitro immuno-suppressive activity of peripheral and mixed, but not central type benzodiazepine ligands.


High-frequency transvaginal sonographic examination for the potential malformation assessment of the 9-week to 14-week fetus. A study was undertaken to evaluate the ability of the high-frequency transvaginal scanning method to consistently image first- and early second-trimester fetal structures, such as body contours, long bones, fingers, face, palate, feet, toes, and the four-chamber view. Ninety-seven low-risk pregnancies were scanned from 9 weeks to 14 menstrual weeks inclusive. Accurate dating was ascertained. The results showed that consistent detection of the respective structures was achieved at the following menstrual ages: sagittal contours at 9 weeks to 10 weeks, long bones at 10 weeks to 11 weeks, fingers at 12 weeks, face and palate at 12 weeks, feet and toes at 13 weeks, and the four-chamber view at 14 weeks. The organs and structures examined could be detected at 9 weeks to 14 weeks inclusive. An increasing number of structure were detected consistently with the increasing menstrual age. The study supports the possibility of searching for specific malformations at or after the menstrual ages mentioned, or performing a more comprehensive malformation evaluation after 13 weeks.


Effect of measurement variability on Rossavik growth model specification and prediction of growth outcome at birth. We have studied how variability in second-trimester ultrasound measurements affects the process of fetal growth evaluation based on individual fetal growth curve standards specified by Rossavik growth models. The head and abdominal short axes of two second-trimester scans of a patient with normal fetal growth were increased or decreased by increments ranging from 0.1 cm to values equal to the two standard deviations of the interobserver variability for these measurements--i.e., 0.3 cm for head short axis and 0.4 cm for abdominal short axis. The largest increments affected the Growth Potential Realization Index for weight at birth by 1% to 13% when applied to head short axis, and by 2% when applied to abdominal short axis. In contrast, the same increments had little effect (1%) on the Growth Potential Realization Indices for head and abdominal circumferences. Whereas 0.1-cm increments had no effect on any of the individual growth models or predicted birth characteristics, some combinations of these small errors involving both head and abdominal short axes changed the Growth Potential Realization Index for weight by as much as 15%, and those for head and abdominal circumferences by 3% and 8%, respectively. Under some scenarios, fetal growth status could be falsely classified as normal or abnormal. These results show that the reliability of individualized growth assessment depends to a great extent on excellent ultrasound technique, at least for certain parameters.


Sonographic measurement of penile erectile volume. For more accurate determination of the penile erectile volume in the flaccid and erectile states, and the difference between them, 20 impotent patients from 36 years old to 70 years old were enrolled in the study. Penile Doppler ultrasonography was performed to check the diameter of each corpus cavernosum, and diameter and peak flow velocity of each cavernous artery before and after intracavernous injection with 10 micrograms to 20 micrograms prostaglandin E1 (PGE1). The penile length was measured from base to midglans manually before and after PGE1 injection. The erectile volume in flaccid and erectile states was calculated as pi r2 x length. The summation of erectile volume of left and right corpus cavernosum was taken as the total erectile volume. The patients were classified into good (10 patients) and poor (10 patients) response groups based mainly on the mean peak flow velocity of both cavernous arteries greater or less than 25 cm/s. There was a significant increase of erectile parameters (penile length, diameter of corpus cavernosum, and total erectile volume) after PGE1 injection in each group of patients. For the total of 20 patients, the mean increase of penile length was 4.27 cm (80.0%), the diameter of corpus cavernosum was 0.66 cm (77.9%), and total erectile volume was 27.87 mL (459.0%). A comparison of the increase of penile erectile parameters after PGE1 injection between the good and poor response groups of patients revealed a significantly greater increase of the diameter of corpus cavernosum in the good response patients.


Accuracy of transvaginal ultrasonography for detection of hematosalpinx in ectopic pregnancy. The transvaginal approach has significantly improved the accuracy of ultrasonography for the detection of ectopic pregnancy. However, there has been limited emphasis given to determining the sensitivity of ultrasonography when a hematosalpinx was used as a specific finding to identify an ectopic pregnancy. The sensitivity of transvaginal ultrasonography was evaluated for the detection of a hematosalpinx defined as an "echogenic homogeneous or inhomogeneous, rounded or elongated structure" in a group of patients with surgically proven ectopic pregnancy. Retrospectively, transvaginal ultrasonography showed a hematosalpinx in 16 out of 18 (88.8%) tubal pregnancies. In 6/6 (100%) patients with a ruptured tube and 10/12 (83.3%) patients with an unruptured tube, a hematosalpinx was detected sonographically. A gestational sac with a live embryo was seen in 26.3% of these patients. The significance of identifying a hematosalpinx, predictability of rupture and implication in the treatment of ectopic pregnancy are discussed.


Doppler echocardiographic measurement of cardiac output in man using mitral annulus method. The mitral inflow method of measuring cardiac output with pulsed Doppler two-dimensional echocardiography was developed and validated against the thermodilution technique in 42 patients. A mitral inflow method combined the velocity of left ventricular inflow at the mitral annulus (apical long-axis view) with the cross-sectional area of the annulus calculated from its diameter (parasternal long-axis view). A good correlation was observed between thermodilution and Doppler measurements of cardiac output (r = 0.93). The ratio of Doppler measurements to thermodilution measurements will be between 0.76 and 1.15 for about 95% of cases. There was a good correlation between percentage change in thermal cardiac output and those in Doppler cardiac output (r = 0.95); the limits of agreement were -11.5% to 10.5%, suggesting that this method can be most useful for assessing relative changes in cardiac output.


Detection of human papillomavirus in matched cervical smears and biopsy specimens by non-isotopic in situ hybridisation. To determine the relative diagnostic sensitivity of non-isotopic in situ hybridisation (NISH) for the diagnosis of human papillomavirus (HPV) on matched smears and biopsy specimens; to compare the NISH signal type in the two samples; and to correlate the NISH data with the morphological diagnosis. HPV samples were assayed individually by NISH with digoxigenin labelled probes (HPV6, 11, 16, 18, and 33) on routinely collected paraffin wax embedded cervical biopsy specimens and for high risk HPVs with a cocktail of similarly labelled probes (HPV16, 18, 33) on matched smears. These were taken at the same colposcopic examination from 32 patients investigated for an abnormal cervical Papanicolaou (PAP) stained smear. An HPV signal was present in 18 (56%) biopsy specimens and in 14 (44%) smears. There was higher concordance of sets of data in the presence of cytopathic wart virus changes. The superiority of biopsy over smear in detecting HPV was mainly the result of examining the entire cervical biopsy specimen rather than cells scraped from the cervical surface. The NISH signal type in both biopsy specimen and smear was similar; it has been shown that NISH type 1 signal correlates with episomal viral replication and type 2 and 3 signals with viral integration. These data show that NISH on cervical smears is a worthwhile primary screen for HPV infection. The NISH signal types in cervical smears are similar to those previously described in cervical biopsy specimens.


Alterations in monoamine receptors in the brain of suicide victims. Suicide has been linked to alterations in the serotonergic and noradrenergic systems. Using in vitro quantitative receptor autoradiography, we compared the binding of 125I-lysergic acid diethylamide (5-hydroxytryptamine-2 sites) and 125I-pindolol (beta-adrenergic sites) with slide-mounted sections from the prefrontal and the temporal cortex of a group of suicide victims, matched to control specimens on postmortem interval, age, sex, and race. A specific laminar distribution of 5-hydroxytryptamine-2 binding was found in both groups, with layers III and IV having the highest level of binding, but beta-adrenergic binding did not differ across cortical layers. Binding to both 125I-lysergic acid diethylamide and 125I-pindolol was increased in the prefrontal cortex of the suicide victims, whereas only beta-adrenergic binding was increased in the temporal cortex of the suicide group.


Digestion kinetics of fiber: influence of in vitro buffer pH varied within observed physiological range. In vitro buffer pH reflective of the diurnal variation in ruminal pH was evaluated for its impact on digestion kinetics of NDF from three forage sources. Alfalfa hay, bromegrass hay, and corn silage were incubated in phosphate-bicarbonate buffer solution adjusted to pH 6.8, 6.5, 6.2, 6.0, 5.8, or 5.5 using 1 M citric acid. Ash-free NDF was measured after 0, 6, 12, 18, 24, 48, 72, and 96 h of fermentation. The experiment was replicated three times; kinetic parameters of fiber digestion were estimated by logarithmic transformation, and by linear and nonlinear regression procedures. Lag in NDF digestion increased as pH fell from 6.8 to 6.5 and again when pH decreased below 6.0. Decreasing buffer pH below 6.2 dramatically reduced NDF digestion rate for alfalfa hay and corn silage but had no significant effect on NDF digestion rate of bromegrass hay. Lag in NDF digestion increased below pH 6.0 for both alfalfa and corn silage but increased only when pH fell below 6.2 for bromegrass. Results suggest that lowered pH exerts its negative effect on NDF digestion between pH 6.2 and 5.8, as evidenced by increased lag and decreased rate, and that critical pH and specific, affected component of digestion varied among forages.


Ruminal buffers: temporal effects on buffering capacity and pH of ruminal fluid from cows fed a high concentrate diet. In vitro characteristics of several buffers and alkalinizing agents commonly utilized to reduce ruminal acid load were evaluated. Ruminal fluid was collected from five cows consuming a diet containing concentrate and sorghum silage in a 68:32 ratio (DM basis). This fluid was incubated with either NaHCO3, a natural sodium sesquicarbonate, a multielement buffer or MgO (7.1 g/L of ruminal fluid), or no buffer for 48 h; flasks were removed and analyzed for pH, buffering capacity, and buffer value index every 12 h during the 48-h incubation. The buffer value index accounts simultaneously for alterations in pH and buffering capacity. Compared with the unbuffered control, all buffering compounds increased ruminal fluid buffer value index. However, the buffer value index separated these buffering compounds into two categories. The NaH-CO3 and sodium sesquicarbonate exhibited similar buffer value indexes; both were markedly higher than those for the multielement buffer and MgO. Although NaHCO3 and sodium sesquicarbonate each increased both ruminal fluid pH and buffering capacity sharply, the multielement buffer only increased pH and buffering capacity moderately. The increase in buffer value index for MgO primarily was due to an increase in pH. Both NaHCO3 and sodium sesquicarbonate were fully active within the first 12 h of incubation; activity of multielement buffer and MgO reached a plateau at 24 h. Compared with the multielement buffer and MgO, NaHCO3 and sodium sesquicarbonate should be more beneficial in preventing short-term postprandial increases in ruminal fluid hydrogen ion concentration; because of their slower release rates, the multielement buffer and MgO should help stabilize ruminal acid-base status, but efficacy might be reduced because of passage out of the rumen.


Supplementation of dairy cow diets with calcium salts of long-chain fatty acids and nicotinic acid in early lactation. Forty multiparous Holstein cows were assigned to one of four treatments 15 d postpartum according to milk yield during wk 2 postpartum to examine the effects of supplementing niacin, Ca salts of long-chain fatty acids, and their interaction. Treatments were control, niacin (12 g/d), Ca salts of long-chain fatty acids (3% of dietary DM), or a combination of niacin and Ca salts. On d 99 postpartum, all cows were fed the control treatment for 2 wk to evaluate residual effects. Milk and FCM yields, blood plasma NEFA and beta-hydroxybutyrate concentrations, and apparent total tract hemicellulose digestibility were increased; milk protein percentage, milk SNF percentage, and blood plasma glucose concentrations were reduced by treatments containing the Ca soaps. Niacin supplementation increased milk protein content and yield but reduced blood plasma beta-hydroxybutyrate concentration. During the residual period, in which all cows received the control treatment, milk yield and plasma NEFA concentration remained elevated, milk protein and SNF contents remained depressed, and milk fat content was reduced for cows previously supplemented with Ca salts of long-chain fatty acids. Methionine and phenylalanine uptakes by the mammary gland were enhanced by niacin supplementation. Results indicated that dairy cattle in early lactation yielded more milk when their diets were supplemented with Ca salts of long-chain fatty acids and that niacin supplementation increased milk protein content and yield.


Effect of a diet high in monounsaturated fat from almonds on plasma cholesterol and lipoproteins. The effect of almonds as part of a low saturated fat, low cholesterol, high-fiber diet was studied in 26 adults (13 men, 13 women). The baseline diet was modified in a similar way for all subjects by limiting meat, fatty fish, high-fat milk products, eggs, and saturated fat. Grains, beans, vegetables, fruit, and low-fat milk products were the foundation of the diet. During the almond diet period, raw almonds (100 mg/day) supplied 34 g/day of monounsaturated fatty acid (MUFA), 12 g/day of polyunsaturated fatty acid, and 6 g/day of saturated fatty acid. Almond oil was the only oil allowed for food preparation. There was a rapid and sustained reduction in low-density lipoprotein cholesterol without changes in high-density lipoprotein cholesterol. This was reflected in a total plasma cholesterol decrease from (means +/- SEM) 235 +/- 5.0 at baseline to 215 +/- 5.0 at 3 weeks, and to 214 +/- 5.0 mg/dl at 9 weeks (p less than 0.001). When the consumption of nuts high in MUFA increases the fat content of the diet, reduction rather than elevation of plasma cholesterol has to be expected, possibly due to the MUFA content of these nuts.


Impact of child day care on infectious diseases in adults. The authors review diseases that occur in day care centers and their impact on day care providers and parents. Acute infections of the gastrointestinal and respiratory tracts are the most commonly transmitted illnesses from children in day care centers to adults. The most important infections acquired by adults are those with the potential for significant clinical morbidity, such as hepatitis A, or those that generate concern, such as cytomegalovirus and parvovirus B19, because of their potential to produce congenital infections in pregnant women.


The pharmacological management of hypertension in pregnancy. The management of pregnancy-induced hypertension (PIH) and preeclampsia using antihypertensive drug therapy remains contentious. Conflicts arise due to differences in diagnostic criteria and varying attitudes regarding the value of treating hypertension, which is only one aspect of this systemic disorder. The following review assesses the role of individual agents and their effects upon both maternal and foetal/neonatal wellbeing. Human clinical trials of each of the main antihypertensive drugs used in the management of PIH/preeclampsia are reviewed. The value of randomized, placebo-controlled trials and long-term paediatric follow up is stressed. A number of agents have a favourable benefit-risk profile for use in women with PIH/preeclampsia; these include alpha-methyldopa, beta-blockers, hydralazine, prazosin, calcium channel antagonists and ketanserin. Diazoxide and sodium nitroprusside may also be used for acute severe hypertension. Angiotensin converting enzyme inhibitors are contra-indicated. Low-dose aspirin is presently being investigated in multicentre trials and may play a major role in the prevention of preeclampsia. Decisions regarding the need for antihypertensive treatment during pregnancy and the selection of a specific antihypertensive agent should be based upon an assessment of the relative benefits and risks for the individual patient. In future studies, the effects of antihypertensive agents upon the underlying pathophysiological processes involved in PIH/preeclampsia may guide therapeutic decision making.


Molecular biology of renin. I: Gene and protein structure, synthesis and processing. This two-part review aims to consolidate information on renin structure, synthesis, precursor processing, intracellular shunting and transcriptional control. Such revolutionary advances in the molecular knowledge of renin stem from the cloning of mouse renin complementary DNA in the early 1980s. Amongst many of the key studies such as cloning of human, mouse and rat renin genes, which have been crucial to progress, several highlights covered in this part include: differences in DNA sequences that might explain species differences in tissue-specific expression; identification of inactive renin as pro-renin, the activation site and the molecular explanation for this activation by many proteases; a different processing site for rat pro-renin; and identification of cathepsin B as a likely pro-renin activator in kidney and a kallikrein as the activator of the duplicated mouse renin gene product. Cloning of the renin gene has greatly increased our understanding of the processes that precede secretion of this important enzyme.


Increased nerve growth factor levels in spontaneously hypertensive rats. Increased sympathetic innervation has been reported in spontaneously hypertensive rats (SHR); however, the precise mechanisms involved are not yet clear. Nerve growth factor (NGF), a neurotrophic peptide in peripheral sympathetic neurons, is believed to contribute to this phenomenon. We measured the content of NGF in SHR and control Wistar-Kyoto (WKY) rats during development. Mesenteric artery, spleen, heart and sciatic nerve were isolated and homogenized. NGF content in the supernatant fractions was measured using a highly sensitive and specific two-site enzyme immunoassay. At 3 weeks of age, SHR had a greater NGF content in the spleen, the sciatic nerve and the mesenteric artery than WKY rats. However, these differences disappeared completely at 12 weeks of age. Cardiac NGF content was slightly lower in 3-week-old SHR and, conversely, higher in 12-week-old SHR than in age-matched WKY rats. These findings suggest that, except for the heart, the SHR tissues observed overproduce NGF at a young age, leading to enhancement of peripheral sympathetic nervous system activity and the production of vasoconstrictive catecholamines.


Arginine vasopressin response to hypertonicity in hypertension studied by arginine vasopressin assay in unextracted plasma. To investigate whether plasma osmolality (Posm)-plasma arginine vasopressin (PAVP) response relationships with particular characteristics of sensitivity, gain or response pattern (linear/non-linear) occur more frequently in hypertensive than normotensive subjects. Analysis of Posm-PAVP curves observed in individual normotensive and hypertensive subjects rather than whole groups was considered appropriate for the described objective. A sensitive and precise radioimmunoassay of PAVP in unextracted plasma was developed. Posm was raised in 11 normotensive and 19 hypertensive subjects by infusion of NaCl solution (0.86 mol/l, i.v.), and PAVP assayed at intervals. Best-fitting linear and non-linear equations for the Posm-PAVP relationship were established by computer. The mean rise in osmolality, blood pressure and blood volume was similar in the two groups. Hypertensive subjects showed a tendency towards higher rates of response (pmol AVP/l per mosm per kg) at Posm greater than 290 mosm/kg, leading to a non-linear response pattern compared with the normotensive subjects. The enhanced PAVP responses found in the hypertensive subjects at Posm greater than 290 mosm/kg may reinforce vascular and central nervous pathogenetic mechanisms.


Anoxia-induced changes in ventricular diastolic compliance in two models of hypertension in rats. The effects of a brief period of anoxia upon myocardial distensibility and coronary vascular resistance (CVR) were evaluated in two models of pressure overload in the rat. A similar degree of left ventricular hypertrophy was obtained by using deoxycorticosterone acetate (DOCA)-salt treatment and abdominal aortic stenosis (AS). The study was performed using an isolated, isovolumic cardiac preparation perfused at a constant coronary flow of approximately 20 ml/min per g left ventricular weight. In response to a 10-min period of anoxia, hypertrophied hearts manifested an exaggerated decrease in left ventricular diastolic chamber distensibility. Left ventricular diastolic blood pressure (DBP) rose in control, AS and DOCA-salt hearts, with no significant difference between the two hypertrophied groups. The anoxia-induced increase in left ventricular DBP correlated with the left ventricular weight: body weight ratio in all animals, but was higher in the two hypertrophied groups compared with controls when expressed per unit tissue mass. In response to anoxia, CVR increased in both groups of hypertrophied hearts and was correlated with enhanced left ventricular DBP. In addition, CVR increased in proportion to the degree of hypertrophy. Partial correlation analysis showed that this relationship depends upon the level of left ventricular DBP and disappears when this pressure is constant. The anoxia-induced increase in myocardial stiffness is exaggerated in two different models of cardiac hypertrophy. This exaggeration is primarily related to the degree of hypertrophy and results in a more pronounced limitation of the coronary flow in hypertrophied hearts.


Left ventricular diastolic function and responses to adrenergic stimuli in borderline arterial hypertension. To detect the existence of a possible relationship between arterial hypertension and adrenergic reactivity to pressure stimuli, and changes in left ventricular diastolic function (LVDF). Fifty-nine young subjects with borderline arterial hypertension and ten sex- and age-matched controls were investigated. After three medical examinations, the subjects were divided into hypertensive and borderline groups on the basis of the blood pressure reading at visit 3. A complete echocardiographic study was performed in 25 of the 59 subjects. Blood pressure was measured in baseline conditions and during pressure stimuli (mental stress, handgrip and cold pressor tests). LVDF was evaluated primarily by means of filling velocities during diastolic phases taken from the left ventricular volume curve (obtained from a complete echocardiographic study). No significant changes in blood pressure responses were observed for the borderline or hypertensive groups during the adrenergic test. The echocardiographic indices of diastolic function were statistically different for the two groups when compared with the control group. The LVDF parameters correlated significantly with systolic blood pressure and diastolic blood pressure measured at the time of the echocardiogram, but not with blood pressure measured occasionally. Blood pressure increases similarly during adrenergic stimuli in both the hypertensive and borderline groups. The correlation between systolic blood pressure, diastolic blood pressure and LVDF parameters may indicate a very early onset of reduced compliance of the left ventricle, even in a preclinical phase of hypertension.


Pressor dose responses and baroreflex sensitivity in quadriplegic spinal cord injury patients. To assess the relative contribution of impaired baroreceptor reflexes and enhanced cardiovascular reactivity to the exaggerated blood pressure rises which occur in quadriplegic spinal cord injury patients with automatic hyperreflexia. Pressor dose responsiveness was evaluated by determining the steady-state dose of phenylephrine, alpha-methylnoradrenaline and angiotensin II required to achieve a blood pressure rise of 20 mmHg and the steady-state dose of isoprenaline required to increase heart rate by 20 beats/min in eight quadriplegic spinal cord injury patients and eight control subjects. The dose of phenylephrine alpha-methylnoradrenaline and angiotensin II to achieve a rise in blood pressure of 20 mmHg was significantly reduced in the spinal cord injury group, whilst the dose of isoprenaline required to raise heart rate by 20 beats/min did not differ significantly from the control group. Baroreceptor sensitivity, assessed by straight line regression of change in heart period with change in blood pressure during steady-state infusions of phenylephrine, did not differ statistically between the two groups, but the results could not exclude some evidence of impairment in the spinal cord injury patients. Baroreceptor sensitivity was much less variable in spinal cord injury patients than in controls. These findings suggest that quadriplegic patients with spinal cord injury have exaggerated pressor responses with significantly less variability in baroreflex sensitivity. The former probably contributes to the autonomic hyperreflexia seen in these patients. The latter provides some support to the suggestion that centrally mediated psychogenic responses contribute to the variability in baroreceptor sensitivity seen in normal subjects.


Impaired microcirculation in mild-to-moderate essential arterial hypertension. The aim of this study was to correlate capillary morphology and erythrocyte velocity to blood pressure in mild-to-moderate essential arterial hypertension. Ambulatory blood pressure measurement may provide more precise information about a patient's mean blood pressure than office measurements. Fifteen patients with recently diagnosed, previously untreated mild-to-moderate essential hypertension underwent 24-h ambulatory blood pressure recording and a capillaroscopic examination of finger microcirculation. Erythrocyte velocity was determined by the flying spot technique. Both mean 24-h ambulatory systolic blood pressure (SBP) and mean 24-h ambulatory diastolic blood pressure (DBP) were significantly inversely correlated with capillary erythrocyte velocity. However, the correlation between erythrocyte velocity and office SBP and office DBP was less significant. Capillary length was related to 24-h ambulatory DBP but not to office DBP. Capillary number was not related to any blood pressure parameter. These results indicate that, in patients with mild-to-moderate essential hypertension, erythrocyte velocity is significantly lower than for matched controls. It is also inversely related to mean 24-h ambulatory SBP and 24-h ambulatory DBP.


Arterial smooth muscle contractions in spontaneously hypertensive rats on a high-calcium diet. To study the effects of a high-calcium diet upon blood pressure, vascular smooth muscle contractions and intracellular free calcium in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Eight-week old animals were placed on a normal-calcium diet (1.1% calcium; SHR and WKY rat groups) or a high-calcium diet (2.1% calcium; Ca-SHR and Ca-WKY rat groups) and observed for 12 weeks. Blood pressure was measured indirectly by the tail-cuff method and in vitro smooth muscle responses were studied using a standard organ bath chamber. Platelets were used as a cell model for analysis of intracellular free calcium concentration, measured by the fluorescent indicator Quin-2. The blood pressure of Ca-WKY and WKY rats did not differ, but increased systolic blood pressure was attenuated in Ca-SHR compared with SHR. The concentration-response curves of mesenteric arterial rings for potassium chloride and noradrenaline were not affected by the high-calcium diet in either SHR or WKY rats. The time required for total relaxation after washout of contractile agents (washout time) was shortest in WKY and Ca-WKY rats after both agonists, and shorter in Ca-SHR than in SHR after noradrenaline. Smooth muscle responses were also studied by contracting the preparations with noradrenaline and potassium chloride in a calcium-free solution, after which, calcium was added to the organ bath in increasing concentrations. Calcium contraction responses were similar in WKY and Ca-WKY rats; SHR displayed an attenuated response to calcium addition in mesenteric rings stimulated by both agonists. After potassium chloride as agonist, the responses of SHR and Ca-SHR did not deviate but, after noradrenaline, a significant shift in the calcium contraction curve towards the normotensive curve was observed in Ca-SHR. Intracellular free calcium was clearly lower in WKY rats than in SHR, and was significantly reduced by calcium supplementation in the hypertensive but not the normotensive animals. A reduction in intracellular free calcium concentration and an effect upon receptor-mediated vascular smooth muscle contraction and excitation-contraction coupling may participate in the blood pressure lowering effect of a high-calcium diet.


A study of the interaction between the hypotensive actions of doxazosin and enalaprilat in anaesthetized rats. This study was designed to test whether previous work, which showed that the angiotensin converting enzyme (ACE) inhibitor enalaprilat potentiated the alpha 1-adrenoceptor antagonist activity of doxazosin in isolated rat tail arteries, could be extended to demonstrate a synergistic hypotensive effect of these two drugs. Groups of untreated or chronically deoxycorticosterone acetate (DOCA)-salt-treated female Sprague-Dawley rats were used. Rats were anaesthetized with Inactin (barbiturate); drugs were administered via a jugular venous catheter; blood pressure was monitored via a carotid arterial catheter. In previously untreated rats, pretreatment with enalaprilat shifted the dose-response curve for the hypotensive effect of doxazosin to the left, indicating synergism. In rats dosed with DOCA-salt (which suppresses renin and angiotensin II production): (1) there was no synergism between the hypotensive actions of enalaprilat and doxazosin; (2) doxazosin was more potent than in untreated rats; and (3) enalaprilat lowered blood pressure, suggesting a hypotensive mechanism separate from ACE inhibition. In the absence of angiotensin II (resulting from enalaprilat administration or from chronic DOCA-salt), doxazosin had a greater hypotensive action than in the presence of angiotensin II. This is consistent with the concept that angiotensin II modulates alpha 1-adrenoceptor activity.


Candoxatril, an orally active neutral endopeptidase inhibitor, raises plasma atrial natriuretic factor and is natriuretic in essential hypertension. Candoxatril (UK79,300) is an orally available inhibitor of the neutral endopeptidase (E.C.3.4.24.11) which degrades atrial natriuretic factor (ANF). This study was designed to establish initial safety and efficacy data in essential hypertension for this novel class of drug. A prospective, double-blind, placebo-controlled, single-dose comparison of candoxatril with placebo in a crossover manner. Three doses of candoxatril (10, 50 and 200 mg) were used, with four subjects at each dose level. The Medical Research Council Blood Pressure Unit, Western Infirmary, Glasgow, UK (a hospital clinical research unit). Twelve patients with untreated essential hypertension. Diastolic blood pressure was greater than 95 mmHg on three separate occasions before entry to the study. Candoxatril or matching placebo was administered orally in the fasting state. Serial measurements of urinary volume and electrolyte excretion were taken (on each hour, urine volume was replaced with an equivalent volume of water by mouth). Blood pressure and heart rate were recorded for 12 h after drug administration and serial blood samples were taken for measurement of plasma ANF and neurohormone concentrations. Urine volume and electrolyte concentration; blood pressure; heart rate; plasma atrial natriuretic factor. Plasma ANF concentrations rose significantly in all patients within 2 h of candoxatril administration compared with placebo although peak and integrated ANF levels were similar at all three doses. A significant natriuresis was only seen after 200 mg candoxatril, with a greater cumulative urine sodium excretion over 12 h compared with placebo; this was associated with a greater diuresis over 12 h compared with placebo. After a single oral dose of candoxatril, blood pressure and heart rate remained unchanged. Candoxatril in a single dose caused no adverse effects in essential hypertension. The drug caused a rise in basal ANF levels at all doses, but natriuresis was only seen with the highest dose used. No change in blood pressure was recorded after acute dosing, and the results of chronic studies with this compound are awaited. Oral inhibitors of ANF degradation may have therapeutic potential in cardiovascular disorders.


Blood pressure response to acute changes in dietary sodium in young Zimbabwean men. The primary objective of this study was to determine the effect of acute alterations in sodium intake upon the blood pressure and hormone levels of young Zimbabwean men. Blood pressure, 24-h urinary electrolyte excretion and plasma concentrations of angiotensin II, aldosterone, and atrial natriuretic peptide were measured in normotensive black medical students. Three sets of measurements were taken: (1) during free access to sodium (baseline); (2) after 4 days on a low-sodium diet (10 mmol/day); and (3) after 4 days on a high-sodium diet (800 mmol/day). Blood pressure was measured by random zero sphygmomanometry, hormone levels by radioimmunoassay, and urinary electrolytes by flame photometry. The low-sodium diet caused the range of pulse pressure to narrow, with a decrease in systolic blood pressure (SBP) and an increase in diastolic blood pressure (DBP). With the introduction of the high-sodium diet, SBP increased and DBP decreased. Mean arterial pressure did not change. At the same time, angiotensin II and aldosterone decreased. Plasma atrial natriuretic peptide did not change. A subgroup of the men on the high-sodium diet also received 100 mmol potassium/day. The increase in SBP associated with high sodium was significantly attenuated by the presence of added potassium. SBP of young black Zimbabwean men is lowered by dietary sodium restriction and rises with a large increase in dietary sodium for a short duration, but mean arterial pressure does not change due to the opposing decreases in DBP.


A factorial study of salt restriction and a low-fat/high-fibre diet in hypertensive subjects. To compare the independent and additive effects of sodium restriction and a low-fat, high polyunsaturated: saturated fatty acids (P:S) ratio, high-fibre diet upon blood pressure. A randomized, parallet, double-blind, placebo-controlled (for sodium) 2 x 2 factorial trial. Clinical. Ninety-five hypertensive subjects (mean blood pressure, 137/83 mmHg), mean age 53.5 years, consuming less than 30 ml ethanol/day were selected from community volunteers. Seventy-nine treated and twelve untreated hypertensives completed the trial. Subjects followed either a low-sodium, low-fat/high-fibre diet (less than 60 mmol sodium/day; 30% fat energy; P:S ratio = 1; 30-50 g fibre/day) or a low-sodium, normal-fat/normal-fibre diet (less than 60 mmol sodium/day; 40% fat energy; P:S ratio = 0.3; 15 g fibre/day) for 8 weeks. Half of each group received 100 mmol/day NaCl and the remainder received placebo. Blood pressure and blood lipids. Sodium restriction significantly reduced standing and supine systolic blood pressure, with no effect upon diastolic blood pressure. The low-fat/high-fibre diet had no effect upon blood pressure, but significantly reduced total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol. Sodium restriction reduced blood pressure and did not raise low-density lipoprotein cholesterol. A low-fat/high-fibre diet did not reduce blood pressure but lowered cholesterol levels. A combination of the two regimes has the greater potential for reducing cardiovascular risk in hypertensives.


Granulocyte-macrophage colony-stimulating factor (GM-CSF) production by glioblastoma cells. Despite the presence of inducing signals GM-CSF is not expressed in vivo. One of the morphologic hallmarks of human gliomas are inflammatory infiltrates with accumulation of macrophages in the tumor site. The signals leading to the macrophage response are only at the beginning of being understood. Novel chemotactic factors that have recently been characterized as secretory products of glioblastoma cells may attract mononuclear cells from the blood. Within the tumor tissue blood-derived monocytes and macrophages of the brain tissue, the microglial cells, may increase in cell numbers due to tumor-derived growth factors. Both astrocytoma cell lines and cultured astrocytes have been shown recently to produce granulocyte-macrophage (GM)-CSF. We show that in vitro not only astrocytoma but also glioblastoma cell lines secrete GM-CSF when stimulated with TNF-alpha or IL-1. However, there is no evidence for GM-CSF production by glioblastoma cells in vivo: fresh tumor samples lack the mRNA for GM-CSF and the protein is not detectable in the tumor cyst fluids or the cerebrospinal fluids of glioblastoma patients. This contrasts IL-1 and IL-6 that are detectable in the tumor cyst fluids and IL-6 also in the cerebrospinal fluids of the patients. Unlike GM-CSF, transforming growth factor-beta 2 mRNA is expressed in ex vivo tested glioblastoma tissues. Absence of GM-CSF in vivo may be explained by the presence of tumor-derived inhibitory factors, such as transforming growth factor-beta 2 and PGE which suppress GM-CSF production by glioblastoma cells in vitro. The accumulation of macrophages at the tumor site may be due to local elaboration of chemoattractants and/or not yet defined growth factors rather than due to GM-CSF production.


Inhibition of tumor necrosis factor (TNF)-mediated NF-kappa B activation by selective blockade of the human 55-kDa TNF receptor. The numerous biologic activities of TNF appear mediated by two types of specific cell surface receptors of 55 to 60 kDa (TR55) and 75 to 80 kDa (TR75) molecular mass, respectively. The role of TR55 in the activation of the nuclear transcription factor kappa B (NF-kappa B) was investigated using an antagonistic, mAb, H398, specific for the human TR55. The human leukemic T cell line, Jurkat, which expresses both types of receptors at comparable levels, was used to test for NF-kappa B activation by electrophoretic mobility shift assays using as a probe an oligonucleotide encompassing the two tandemly arranged kappa B sites of the HIV-1 LTR enhancer. mAb H398 is shown to efficiently block not only TNF- but also lymphotoxin-mediated activation of NF-kappa B. Furthermore mAb H398 also impeded TNF- or lymphotoxin-mediated activation of chloramphenicol acetyl transferase gene expression from the HIV-1-LTR as determined by transient transfection assays. These findings indicate that both, induction of NF-kappa B binding to DNA, and transcriptional activity can be efficiently inhibited by selective blockade of TR55. Finally it is shown, that human TR55 confers NF-kappa B inducibility when expressed in the mouse pre-B cell line 70Z/3, which does not respond to TNF in its parental state. Together, the results of this study indicate that TR55 is both necessary and sufficient for mediating TNF activation of NF-kappa B.


IL-8 stimulates phosphatidylinositol-4-phosphate kinase in human polymorphonuclear leukocytes. IL-8 is a neutrophil-specific chemoattractant and cellular activator which exists in at least three forms, 69, 72, and 77 amino acids. The predominant monocyte product has 72 amino acids, whereas endothelial cells secrete the 77-amino acid form. The 72-amino acid form has been shown to increase intracellular calcium in neutrophils, but the exact biochemical pathways involved in stimulation of these cells is unknown. N-formyl peptide chemoattractants in neutrophils stimulate the formation of phosphatidylinositol-4,5-bisphosphate (PIP2), a reservoir for second messenger molecules and regulator of actin assembly through its association with the actin-binding proteins, profilin, and gelsolin. The present study examined whether IL-8 altered the enzyme which synthesizes PIP2, phosphatidylinositol-4-phosphate (PIP) kinase. Incubation of intact neutrophils with 10 nM IL-8 caused approximately a twofold increase in the activity of the enzyme. All forms of IL-8 stimulated PIP kinase activity in concentrations ranging from 1 to 50 nM, and the dose-response curves exactly correlated with the order of potency of these cytokines for interacting with the IL-8R on the surface of neutrophils. Lineweaver-Burk analysis of the kinetics of PIP kinase assayed in the presence of 0.03 to 0.7 mM ATP showed that 10 nM IL-8 increased the Vmax of the enzyme 38 to 70.5%, with no significant change in the apparent Km for ATP or for PIP. The stimulation of PIP kinase activity could not be explained by decreased degradation of PIP2 by phospholipase C or phosphomonoesterase activity in the membranes isolated from cells treated with IL-8 or by a decrease in the degradation of ATP. The microfilament disrupter, cytochalasin b, inhibited IL-8 induced stimulation of PIP kinase. These findings demonstrate that all forms of IL-8 stimulate PIP kinase in human neutrophils. This event may provide molecular signals to these cells that are necessary to maintain or change the state of microfilament assembly during cellular activation.


On employing Leishmania promastigotes for systemic lupus erythematosus diagnosis. Based on having definite double stranded DNA determinants in the kinetoplast, it was attempted to use Leishmania promastigotes as an IFAT substrate for the detection of serum anti ds-DNA autoantibodies which are the prominent diagnostic marker of SLE. Serological inspection of different Leishmania preparations revealed that Leishmania is not analogous to Crithidia lucilia as an appropriate ds-DNA substrate. It seems likely that the species-specific molecular characters of the kinetoplast-DNA are influencing the reaction. The issue is an evidence of the distinct differentiation of the K-DNA antigenic determinants in haemoflagellates.


A method for the assay of "difficult" interferons exemplified with recombinant equine interferon-beta 1. We wished to assay recombinant equine interferon-beta 1 (rEqIFN-beta 1) but could not obtain satisfactory results with previously described methods. Therefore, we developed a yield-reduction assay, using primary horse peripheral blood mononuclear cells (PBMC) with vesicular stomatitis virus (VSV) for challenge, which proved consistently satisfactory and highly sensitive. It is suggested that this method of assay may be useful for IFNs from other animals where problems are encountered.


Interferon-gamma potentiates the antiviral activity and the expression of interferon-stimulated genes induced by interferon-alpha in U937 cells. Binding of type I interferon (IFN-alpha/beta) to specific receptors results in the rapid transcriptional activation, independent of protein synthesis, of IFN-alpha-stimulated genes (ISGs) in human fibroblasts and HeLa and Daudi cell lines. The binding of ISGF3 (IFN-stimulated gene factor 3) to the conserved IFN-stimulated response element (ISRE) results in transcriptional activation. This factor is composed of a DNA-binding protein (ISGF3 gamma), which normally is present in the cytoplasm, and other IFN-alpha-activated proteins which preexist as latent cytoplasmic precursors (ISGF3 alpha). We have found that ISG expression in the monocytic U937 cell line differs from most cell lines previously examined. U937 cells express both type I and type II IFN receptors, but only IFN-alpha is capable of inducing antiviral protection in these cells. Pretreatment with IFN-gamma potentiates the IFN-alpha-induced protection, but IFN-gamma alone does not have any antiviral activity. ISG15 mRNA accumulation in U937 cells is not detectable before 6 h of IFN-alpha treatment, peaks at 24 h, and requires protein synthesis. Although IFN-gamma alone does not induce ISG expression, IFN-gamma pretreatment markedly increases and hastens ISG expression and transcriptional induction. Nuclear extracts assayed for the presence of ISRE binding factors by electrophoretic mobility shift assays show that ISGF3 is induced by IFN-alpha within 6 h from undetectable basal levels in untreated U937 cells. Activation of ISGF3 alpha, the latent component of ISGF3, occurs rapidly. However, the increase in ISGF3 activity ultimately correlates with the accumulation of ISGF3 gamma induced by IFN-alpha or IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)


A high frequency of human papillomavirus DNA sequences in cervical carcinomas of Indian women as revealed by Southern blot hybridization and polymerase chain reaction. Ninety-six colposcopically directed biopsies from squamous epithelial carcinoma of the uterine cervix and 22 age-matched normal control biopsy specimens were examined by both Southern blot hybridization and polymerase chain reaction (PCR) for the presence of different human papillomavirus (HPV) DNA types. Cancer of the uterine cervix, which is the most common malignant disease in Indian women, showed a high frequency (98%) of HPV as compared to those reported from other parts of the world. HPV type 16 was found to be the dominant (64%) type while the frequency of HPV type 18 was very low (3%). On individual typing of HPV, no biopsy was found to contain any other known HPV types under stringent conditions of hybridization except a single case of HPV type 11. Only one case of double infection with HPV types 16 and 18 was recorded. Under low stringency conditions of hybridization with a mixed probe of HPV types 16 and 18, 29 additional biopsies were found to be positive. Southern blot hybridization alone detected HPV DNA in 92% of the cases but none in the controls. By PCR, six (6.25%) more cases and four (18.18%) healthy women were found to be positive for HPVs. Analysis of the physical state of HPV 16 indicated integration in about 70% of carcinoma cases while 30% of them were in episomal form. The findings suggest that infection with HPV is an important etiologic factor for the development of cervical cancer, that a number of such tumours may arise without HPV infection, and that integration of the viral DNA into host genome is not always essential for malignant progression.(ABSTRACT TRUNCATED AT 250 WORDS)


The specificity of antibodies induced by infection with rhinovirus type 2. Mouse monoclonal antibodies (MAbs) against three distinct antigenic sites on rhinovirus type 2 have been obtained and the sites identified. We describe how these MAbs were used in a blocking test to detect antibodies in human sera directed against the same three defined sites. Sera from twelve volunteers were studied. All had been exposed to rhinovirus type 2 by intranasal inoculation, four had been uninfected, eight were infected of whom four developed a cold while four did not. Blocking antibodies were high and did not increase in the resistant volunteers, and were lower and increased in the infected volunteers. The antibodies were almost as sensitive as other antibody assays for detecting infection. The responses to all three sites were similar. Correlations between the results of all tests were calculated and the results are summarised. Tests were also devised to measure the Ig subclass of antibodies against the whole virus particle. The A1, G1, and G4 classes showed most frequent rises in response to infection. Correlations between these results and other antibody assays were found and are presented.


Association of hepatitis C virus carrier state with the occurrence of hepatitis C virus core antibodies. An enzyme immunoassay (EIA) was developed for the determination of antibodies against the "putative" core protein of hepatitis C virus (HCV). Antigens used were recombinant fragments (amino acids 6-77 or 6-143) of the HCV core protein, produced in Escherichia coli with truncated hepatitis B core (HBc) as fusion protein. Evaluation of 385 sera positive for HCV antibodies by first generation EIA, revealed 98 (25.4%) with HCV core antibodies. HCV-RNA, determined by the polymerase chain reaction (PCR), was exclusively found in the sera positive for HCV core antibodies (89 PCR positives). In random screening of 3,708 sera, 3 sera with HCV core antibodies were found PCR positive. Only 2 of these sera were positive in the first generation EIA. It is concluded that HCV core antibody determination is a reliable test for identifying HCV carriers among blood donors.


High rate of multiple genital HPV infections detected by DNA hybridization. Cervical smears collected from 450 patients involved in a clinical follow-up of cervical human papillomaviruses (HPV) infections were screened for the presence of HPV 6b, 11, 16, and 18 DNA by both dot blot and southern blot hybridization methods. Using very high stringency hybridization assays, the four HPV types could be easily distinguished by dot blotting. After a preliminary clinical sorting, 42.9% of the samples were found to be HPV-positive. Among the samples infected by a single HPV, type 16 was the most frequent (25.4% of the positive samples) followed by 6b (19.7%), 11 (8.3%), and 18 (7.2%). Double or even multiple infections by the different HPV types were detected at a very high rate (39.4% of the positive samples).


Prevalence of high risk genital papillomaviruses in the Belgian female population determined by fast multiplex polymerase chain reaction. Because in situ/filter hybridisation is not sensitive enough and because classical polymerase chain reaction (PCR) protocols are generally not sufficiently reproducible and specific, there is little accurate information on the prevalence of human papillomaviruses (HPV) 16, 18, and 33 infections in women without dyskaryotic changes of the cervix. In our hands, our Fast Multiplex PCR protocol has always been the most sensitive, specific, and reproducible DNA detection assay in all the microbiological and haematological applications we attempted (Vandenvelde C, Verstraete M, Van Beers D [1990]: Journal of Virological Methods 30:215-228; Vandenvelde C, Scheen R, Corazza F, Van Beers D [1991a]: Journal of Experimental and Clinical Hematology 33:293-297; Vandenvelde C, Scheen R, Van Beers D, Fondu P [1991b]: Journal of Experimental and Clinical Hematology 30:25-29). Using this new technique, cervical scrapes from 336 Belgian women attending the cervical cancer screening clinic were examined for the presence of these three high-risk genital papillomaviruses. Positive results were confirmed using another set of HPV-specific primers. Exactly one sixth of our population was found positive for one or more of these HPVs. Types 33 and 16 were significantly more prevalent than type 18. The nonparametric statistical analysis of the data suggests that some risk factors such as particular sexual habits, that are inversely related to age, must exist.