ATP and its metabolite adenosine act synergistically to mobilize intracellular calcium via the formation of inositol 1,4,5-trisphosphate in a smooth muscle cell line. Interactions between ATP and adenosine on the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and mobilization of intracellular calcium were investigated in the smooth muscle cell line DDT1 MF-2. Activation of adenosine A1 receptors with adenosine or cyclopentyladenosine (CPA) or of nucleotide receptors with ATP increased both Ins(1,4,5)P3 formation and intracellular calcium concentrations. The A1 receptor-induced Ins(1,4,5)P3 formation (EC50 10 nM) was antagonized by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and by pretreatment of the cells with pertussis toxin (PTX). ATP-stimulated Ins(1,4,5)P3 formation (EC50 21 microM) was attenuated, but still present, after PTX treatment. ATP and CPA had supraadditive effects on Ins(1,4,5)P3 accumulation and CPA increased ATP-induced Ins(1,4,5)P3 accumulation in a concentration-dependent manner with an EC50 of 3 nM, a concentration which per se had little or no effect on Ins(1,4,5)P3 accumulation. ATP (EC50 4 microM) and CPA (EC50 4 nM) both increased intracellular calcium levels. The effect of ATP was partially sensitive to PTX treatment, whereas the effect of CPA was blocked both by PTX and by DPCPX. Concentrations of ATP and CPA that by themselves were insufficient to raise intracellular calcium were able to do so when combined. The synergy between ATP and CPA on the mobilization of intracellular calcium was abolished after treatment of cells with PTX or when DPCPX was included in the experiment. Since ATP was metabolized by ecto-enzymes to ADP, AMP, and adenosine, we also examined whether adenosine formed from ATP could enhance the ATP effects on Ins(1,4,5)P3 accumulation. Indeed, the addition of the A1 receptor antagonist DPCPX or removal of endogenous adenosine by inclusion of adenosine deaminase in the experimental medium significantly attenuated the ATP response, and the two treatments did not have additive effects. The present study thus demonstrates that in a clonal cell line two types of receptors increase phospholipase C activity, but via different pathways; nucleotide receptors appeared to act via partially PTX-insensitive, and A1 receptors via PTX-sensitive G-proteins. ATP and CPA are not only able per se to induce formation of Ins(1,4,5)P3 and mobilize intracellular calcium, but they also act synergistically. Finally, it is demonstrated that endogenous adenosine, possibly formed from the rapid breakdown of ATP, can significantly enhance some ATP effects.


Specific phosphorylation of a COOH-terminal site on the full-length form of the alpha 1 subunit of the skeletal muscle calcium channel by cAMP-dependent protein kinase. The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium channels is present in full-length (212 kDa) and truncated (190 kDa) forms which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. In the present study, phosphorylation of the purified calcium channel by cA-PK followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed differential phosphorylation of the related 190- and 212-kDa forms. The 190-kDa form of the alpha 1 subunit was phosphorylated on three major and three minor tryptic phosphopeptides; the 212-kDa form was phosphorylated on all six of these phosphopeptides plus two that were unique. Time course experiments showed that a single site on the COOH-terminal portion of the full-length form of the alpha 1 subunit is most intensely and rapidly (within 10 s) phosphorylated. Phosphorylation occurs almost exclusively on this COOH-terminal site unless harsh conditions such as treatment with denaturing detergents are employed to expose phosphorylation sites within the 190-kDa segment of the molecule. Elution of phosphopeptides from the second dimension chromatograph followed by immunoprecipitation with an anti-peptide antibody (anti-CP1) directed against the COOH-terminal amino acid sequence enabled us to identify this major phosphorylation site as serine 1854. The nearby consensus sites for cA-PK phosphorylation at serines 1757 and 1772 were phosphorylated only after denaturation or proteolytic cleavage. Phosphorylation of serine 1854 may play a pivotal role in the regulation of calcium channel function by cA-PK.


Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C. Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.


Stimulation of mitogen-activated protein kinase by oncogenic Ras p21 in Xenopus oocytes. Requirement for Ras p21-GTPase-activating protein interaction. p21ras plays an important role in the control of cell proliferation. The molecular mechanisms implicated are unknown. We report that the injection of oncogenic Lys12 Ras into Xenopus laevis oocytes promoted the activation of mitogen-activated protein kinase (MAP kinase) after a lag of about 90 min. MAP kinase activity was 10-fold higher 4 h after injection of oncogenic Lys12 Ras than after injection of nononcogenic Gly12 Ras. The stimulated MAP kinase activity remained at a plateau for at least 18 h. Maximal stimulation was obtained with 5 ng of Lys12 Ras, which is similar to the amount that elicits germinal vesicle breakdown. DEAE-Sephacel chromatography of extracts from Lys12 Ras-injected oocytes showed one peak of MAP kinase. MAP kinase activation by Lys12 Ras was associated with tyrosine phosphorylation of MAP kinase (p42). As previously shown, the S6-kinase II (likely pp90rsk), which is activated in vitro by MAP kinase, was also activated by oncogenic Lys12 Ras. Lys12 Ras with an additional mutation (Glu38) in the effector region that binds GTPase-activating protein (GAP) did not promote MAP kinase or S6 kinase activations. Thus, GAP may be involved downstream to Ras in these activation processes. Our results indicate that the Ras-GAP complex promotes MAP kinase activation in oocytes. This supports the idea that Ras-GAP controls MAP kinase, a kinase implicated in the action of various stimuli.


Lipoxin recognition sites. Specific binding of labeled lipoxin A4 with human neutrophils. Lipoxin A4 stimulates rapid lipid remodeling and a pertussis toxin-sensitive release of arachidonic acid in polymorphonuclear leukocytes (PMN) (Nigam, S., Fiore, S., Luscinskas, F.W., and Serhan, C.N. (1990) J. Cell. Physiol. 143, 512-523) and has been shown to inhibit leukocyte responses in several systems. To examine the basis underlying these actions, we have prepared [11,12-3H]lipoxin A4 (LXA4) and characterized its interactions with human PMN. Time course studies (0-90 min) with intact PMN demonstrated cell association of 3H label which was specific and reversible. PMN bound [3H]LXA4 with a Kd of 0.5 +/- 0.3 nM, representing approximately 1,830 sites/PMN, and the Hill plot value of 1.9 suggests cooperative binding. [3H]LXA4 binding was stereoselective since neither leukotriene B4 (LTB4), lipoxin B4 (LXB4), (6S)-LXA4, 11-trans-LXA4, nor SKF 104353 competed for [3H]LXA4-specific binding while LTD4 and LTC4 partially competed. Subcellular fractionation revealed that specific binding with [3H]LXA4 was associated with membrane (42.1%)-, granule (34.5%)-, and nuclear (23.3%)-enriched fractions, a distribution distinct from that of [14,15-3H] LTB4 binding. [11,12-3H]LXA4-specific binding was modulated by guanosine analogs, suggesting the involvement of G proteins. A fluorescent LXA4 derivative (methyl-7-methoxycoumarin-LXA4) competed with [3H]LXA4 binding to intact PMN and showed specific and reversible binding as monitored by flow cytometric analysis. These results indicate that PMN possess specific recognition sites for LXA4 which may mediate its actions.


Subunit identity of the dimeric 17 beta-hydroxysteroid dehydrogenase from human placenta. Human placental 17 beta-hydroxysteroid dehydrogenase has been purified with a new rapid procedure based on fast protein liquid chromatography, yielding quantitatively a homogeneous preparation with high specific activity catalyzing the oxidation of 7.2 mumol of estradiol/min/mg of enzyme protein at 23 degrees C, pH 9.2. This preparation was shown to have a subunit mass of 34.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis while having a molecular mass of 68 kDa by both Superose-12 gel-filtration and native pore gradient gel electrophoresis. When 17 beta-hydroxysteroid dehydrogenase was expressed in HeLa cells or overproduced in insect cells using the baculovirus expression system, both from its cDNA encoding a protein of 34 kDa, the enzyme had the same migration in native and sodium dodecyl sulfate-gel electrophoresis as the purified one from human placenta and eluted from the Superose-12 column at the same elution volume. Moreover, all the above forms of this enzyme have similar specific activity. These results clearly demonstrate the identity of the three enzyme forms. The enzyme produced from the cDNA is expressed as a dimer, and its two subunits are identical. 17 beta-Hydroxysteroid dehydrogenase subunit identity is thus proved. The NH2-terminal analysis revealed a unique sequence of Ala-Arg-Thr-Val-Val-Leu-Ile for the purified enzyme from placenta, further confirming the above conclusion.


An inositol phosphate glycan derived from a Trypanosoma brucei glycosyl-phosphatidylinositol mimics some of the metabolic actions of insulin. Some of the acute actions of insulin may be mediated by an enzyme-modulating inositol phosphate glycan, produced by the insulin-sensitive hydrolysis of glycosyl-phosphatidylinositol (GPI) that is structurally similar to a membrane protein anchor. An inositol glycan fragment from the structurally characterized Trypanosoma brucei variant surface glycoprotein GPI anchor is evaluated for insulin-mimetic antilipolytic activity. The fragment specifically and dose-dependently inhibits isoproterenol-stimulated lipolysis. Like the effect of insulin, glycan-induced antilipolysis is blocked by the low Km cAMP phosphodiesterase inhibitor imazodan (CI-914) and the serine/threonine phosphatase inhibitor, okadaic acid, suggesting that the activation of both cAMP phosphodiesterase and serine/threonine protein phosphatases are necessary. Moreover, this fragment causes a specific and dose-dependent inhibition of both microsomal glucose-6-phosphatase (EC 3.1.3.9) and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activity. Additionally, direct addition of the glycan to hepatocytes caused marked inhibition of glucose production from pyruvate. These results suggest that the direct modification of the activities of these two gluconeogenic enzymes by an inositol glycan may play a role in the inhibition of glucose output by insulin and provide the first evidence for the insulin-mimetic properties of a chemically characterized inositol glycan.


Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II. The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation. The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps. Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship. Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 mM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0. The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 degrees C. The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions. A detailed kinetic analysis of the purified enzyme was performed using Triton X-100/phosphatidylinositol-mixed micelles. 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics. The interfacial Michaelis constant (Km) and the dissociation constant (Ks) for phosphatidylinositol in the Triton X-100 micelle surface were 1.3 mol % and 0.035 mM, respectively. The Km for MgATP was 0.36 mM. 55-kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions. The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP. The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase. This may suggest that the two forms of phosphatidylinositol 4-kinase from S. cerevisiae are regulated differentially in vivo.


Prolonged exposure to inositol 1,4,5-trisphosphate does not cause intrinsic desensitization of the intracellular Ca(2+)-mobilizing receptor. The rapid release of Ca2+ from intracellular stores stimulated with inositol 1,4,5-trisphosphate (InsP3) has required superfusion or stopped-flow techniques to resolve the kinetics of Ca2+ mobilization and made it difficult to determine whether the InsP3 receptor desensitizes during prolonged stimulation. Here we have overloaded the intracellular Ca2+ stores of permeabilized rat hepatocytes by incubating them with ATP and 45Ca2+ in the presence of pyrophosphate, which precipitates Ca2+ within the lumen of the stores. Subsequent ATP removal initiated slow 45Ca2+ efflux that followed zero-order kinetics, allowing us to examine the effects of InsP3 over a prolonged time course. InsP3 produced a concentration-dependent increase in the 45Ca2+ efflux rate that was sustained for several min. The rate rapidly returned to the unstimulated level after the addition of decavanadate, a competitive antagonist of InsP3 at its receptor. Prior incubation with a submaximal concentration of InsP3 (1 microM) did not affect the subsequent enhanced rate of 45Ca2+ efflux stimulated by a higher, but still submaximal, concentration of InsP3 (3 microM). We conclude that prolonged exposure to InsP3 does not desensitize the InsP3 receptor and that intrinsic receptor desensitization cannot provide an explanation for the quantal responses to InsP3 observed in several cell types.


Purification and characterization of the zeta isoform of protein kinase C from bovine kidney. The zeta isoform of protein kinase C (PKC zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC zeta. The enzymatic properties of PKC zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C.


Metal thiolate coordination in the E7 proteins of human papilloma virus 16 and cottontail rabbit papilloma virus as expressed in Escherichia coli. The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma virus (CRPV) have been purified from an expression system in Escherichia coli. The proteins as purified from E. coli contain one tightly bound Zn(II) ion per molecule. The metal site shows facile exchange with either Cd(II) or Cu(I). The HPV 16 E7 maximally bound one Cd(II) or two Cu(I) ions, while the CRPV E7 bound two Cd(II) or three Cu(I) ions. The Cd(II) and Cu(I) E7 molecules exhibited optical transitions in the ultraviolet suggestive of metal:thiolate coordination. E7 proteins from HPV 16 and CRPV contain 7 and 8 cysteines/molecule, respectively. Reaction of the E7 proteins with the sulfhydryl reagent, dithiodipyridine, revealed that all the cysteinyl sulfurs are present in the reduced thiol state. Cu(I)-E7 molecules are luminescent with maximal emission at 570 nm. The observed emission at room temperature is indicative of metal coordination within a compact protein environment shielded from solvent interactions. The emission maxima occurs at the same wavelength (570 nm) as Cu(I)-cysteinyl sulfur clusters in Cu(I)-metallothioneins. The single Zn(II) atom in each protein can be removed from E7 in the presence of EDTA. The resulting apoE7 molecules remain soluble and can be partially reconstituted with Cd(II) to regain the ultraviolet charge transfer transitions.


Methyltransferase and subunit association domains of vaccinia virus mRNA capping enzyme. RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7-)-methyltransferase activities are associated with the vaccinia virus mRNA capping enzyme, a heterodimeric protein containing polypeptides of M(r) 95,000 and 31,000. Although the RNA triphosphatase and RNA guanylyltransferase domains have been localized to a M(r) 59,000 fragment of the capping enzyme large subunit, the location of the methyltransferase domain within the protein and the catalytic role of individual subunits in methyl group transfer remain unclear. In the present work, through the study of methyltransferase activity of truncated forms of capping enzyme translated in vitro in a rabbit reticulocyte lysate, we have localized the methyltransferase domain to a complex consisting of the small subunit and the carboxyl-terminal portion of the large subunit. The M(r) 31,000 subunit translated alone was not sufficient for methyltransferase activity. This requirement for both subunits may explain the tight physical association of the two polypeptides in vivo. We have recreated the association of the large and small enzyme subunits in vitro through the translation of synthetic mRNAs encoding the two polypeptides. Study of the ability of deleted versions of the large subunit to bind the small subunit, as detected by co-immunoprecipitation, defined a 347-amino acid carboxyl-terminal region of the large subunit that was sufficient for heterodimerization. Colocalization within the large subunit of the methyltransferase and subunit association domains suggests that dimerization of the subunits may be required for methyltransferase activity.


The vaccinia virus mRNA (guanine-N7-)-methyltransferase requires both subunits of the mRNA capping enzyme for activity. Plasmid vectors capable of expressing the large and small subunits of the vaccinia virus mRNA capping enzyme were constructed and used to transform Escherichia coli. Conditions for the induction of the dimeric enzyme or the individual subunits in a soluble form were identified, and the capping enzyme was purified to near homogeneity. Proteolysis of the capping enzyme in bacteria yields a 60-kDa product shown previously to possess the mRNA triphosphatase and guanyltransferase activities (Shuman, S. (1990) J. Biol. Chem. 265, 11960-11966) was isolated and shown by amino acid sequence analysis to be derived from the NH2 terminus of D1R. The individual subunits lacked methyltransferase activity when assayed alone. However, mixing the D1R and D12L subunits permitted reconstitution of the methyltransferase activity, and this appearance in activity accompanied the association of the subunits. In contrast, mixing the D12L subunit with the D1R-60K proteolytic fragment failed to yield methyltransferase activity or result in a physical association of the two proteins. These results demonstrate that the methyltransferase active site requires the presence of the D12L subunit with the carboxyl-terminal portion of the D1R subunit. Furthermore, since the mRNA triphosphatase and guanyltransferase active sites reside in the NH2-terminal domain of the D1R subunit, and the methyltransferase activity is found in the carboxyl-terminal portion of this subunit and D12L, there must be at least two separate active sites in this enzyme.


Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2). Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. In such multiubiquitin chains, successive ubiquitins are linked by an isopeptide bond involving the side chain of Lys48 and the carboxyl group of Gly76. The crystal structure of a diubiquitin conjugate has been determined and refined at 2.3-A resolution. The molecule has internal approximate 2-fold symmetry with multiple hydrophobic and hydrophilic contacts along the 2-fold axis. The structure of the diubiquitin conjugate suggests determinants for recognition of multiubiquitin chains. A model for the interaction of diubiquitin and a ubiquitin conjugating enzyme (E2) is proposed.


Functional dissection of structural domains in the receptor for colony-stimulating factor-1. Receptor tyrosine kinases (RTKs) transduce external signals to the interior of the cell via a cytoplasmic kinase domain. We demonstrated previously that ligand-induced kinase activation of the colony-stimulating factor-1 receptor (CSF-1R) occurs via receptor oligomerization without propagation of conformational changes through the transmembrane (TM) domain (Lee, A. W., and Nienhuis, A. W. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7270-7274). We have now examined the role of the different subdomains in the metabolic and signaling properties of CSF-1R. Two types of chimeric receptors have been utilized, Glyfms A, with the extracellular and TM domains of glycophorin A (GpA) and the cytoplasmic domain of CSF-1R, and Glyfms B, where only the extracellular domain originates from GpA. Glyfms A was found to exhibit a higher basal level of in vitro kinase activity, an increased associated phosphatidylinositol (PtdIns) 3-kinase activity and to support enhanced cellular mitogenesis, compared with wild-type CSF-1R or to Glyfms B. The constitutive activation of Glyfms A is consistent with the hypothesis that the TM domain may play a role in receptor oligomerization. Cross-linking with anti-GpA antibodies activated the kinase function of Glyfms B leading to an increase in PtdIns 3-kinase association and to the transmission of a mitogenic signal. Our results indicate that an activated kinase domain contains the major determinant for coupling with PtdIns 3-kinase, independent of extracellular and TM sequences and of ligand binding. Both chimeric receptors underwent internalization in the presence of anti-GpA antibodies but were not degraded, including the tyrosine-phosphorylated and kinase-active population. These results suggest that structural determinants in the extracellular domain must be important for targeting internalized receptors for lysosomal degradation.


Organella-targeted expression of rat liver cytochrome P450c27 in yeast. Genetically engineered alteration of mitochondrial P450 into a microsomal form creates a novel functional electron transport chain. A modified rat cytochrome P450c27, whose mitochondrial targeting signal had been replaced by a possible microsomal targeting signal of bovine cytochrome P450c17, was expressed in yeast. The modified P450c27 hemoprotein was correctly localized on yeast microsomes and exhibited the P450c27-dependent monooxygenase activity by addition of bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR). Considering the previous observation that P450c27 with its own mitochondrial targeting signal was imported into yeast mitochondria (Akiyoshi-Shibata, M., Usui, E., Sakaki, T., Yabusaki, Y., Noshiro, M., Okuda, K., and Ohkawa, H. (1991) FEBS Lett. 280, 367-370), it is now suggested that the destination of P450c27 to either mitochondria or microsomes in yeast depends solely on the amino-terminal targeting signal. In addition, the modified P450c27 was simultaneously expressed in yeast with mature forms of bovine ADX and ADR. The recombinant yeast produced the P450 on the microsomes and mature forms of ADX and ADR in the cytoplasm, and showed the monooxygenase activity. Accordingly, a novel type of functional electron transport chain has been established between the cytoplasm and the microsomes in yeast.


Low molecular weight GTP-binding proteins in cardiac muscle. Association with a 32-kDa component related to connexins. Low molecular weight GTP-binding proteins and their cellular interactions were examined in cardiac muscle. Heart homogenate was separated into various subcellular fractions by differential and sucrose density gradient centrifugation. Various fractions were separated by sodium dodecyl sulfate-gel electrophoresis, blotted to nitrocellulose, and GTP-binding proteins detected by incubating with [alpha-32]GTP. Three polypeptides of M(r) 23,000, 26,000, and 29,000 were specifically labeled with [alpha-32P]GTP in all the fractions examined and enriched in sarcolemmal membranes. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 26- and 29-kDa polypeptides. A polypeptide of M(r) 40,000 was weakly labeled with [alpha-32P]GTP in the sarcolemmal membrane and tentatively identified as Gi alpha by immunostaining with anti-Gi alpha antibodies. Cytosolic GTP-binding proteins were labeled with [alpha-32P]GTP and their potential sites of interaction investigated using the blot overlay approach. A polypeptide of 32 kDa present in sarcolemmal membranes, intercalated discs, and enriched in heart gap junctions was identified as a major site of interaction. The low molecular weight GTP-binding proteins associated with the 32-kDa polypeptide through a complex involving cytosolic components of M(r) 56,000, 36,000, 26,000, 23,000, and 12,000. A monoclonal antibody against connexin 32 from liver strongly recognized the 32-kDa polypeptide in heart gap junctions, whereas polyclonal antibodies only weakly reacted with this polypeptide. The low molecular weight GTP-binding proteins associated with a 32-kDa polypeptide in liver membranes that was also immunologically related to connexin 32. These results indicate the presence of a subset of low molecular weight GTP-binding proteins in a membrane-associated and a cytoplasmic pool in cardiac muscle. Their association with a 32-kDa component that is related to the connexins suggests that these polypeptides may be uniquely situated to modulate communication at the cell membrane.


The isolation of novel inhibitory polypeptides of protein phosphatase 1 from bovine thymus nuclei. Nuclei from bovine thymus contain a high level of partially latent protein phosphatase 1 (PP-1). More than 90% of this PP-1 is associated with the insoluble chromatin/matrix fraction and can be extracted with 0.3 M NaCl. The salt extract also contains three heat- and acid-stable inhibitory proteins of PP-1 that can be resolved on Mono Q. We have purified two of these nuclear inhibitors of PP-1 (NIPP-1a and NIPP-1b) until homogeneity. They are acidic proteins (pI = 4.4) with a molecular mass of 18 kDa (NIPP-1a) and 16 kDa (NIPP-1b) on SDS-PAGE. Judged from the larger molecular mass that was deduced from gel filtration (35 kDa), NIPP-1a and NIPP-1b appear to be asymmetric or dimeric proteins. The nuclear inhibitors totally inhibited the phosphorylase phosphatase activity of PP-1, but even at a 250-fold higher concentration they did not affect the activities of the other major serine/threonine protein phosphatases (PP-2A, PP-2B, and PP-2C). NIPP-1a and NIPP-1b inhibited the catalytic subunit of PP-1 with an extrapolated Ki of about 1 pM, which is some three orders of magnitude better than the cytoplasmic proteins inhibitor 1/DARPP-32 and modulator. The nuclear inhibitors were not inactivated by incubation with protein phosphatases that inactivate inhibitor 1 and DARPP-32. Unlike modulator, they were not able to convert the catalytic subunit of PP-1 into a MgATP-dependent form. Remarkably, the extent of inhibition of PP-1 by NIPP-1b depended on the nature of the substrate. The phosphorylase phosphatase and casein phosphatase activities of PP-1 were completely blocked by NIPP-1b, whereas the dephosphorylation of basic proteins was either not at all inhibited (histone IIA) or only partially (myelin basic protein). These data may indicate that the acidic NIPP-1b is inactivated through complexation by basic proteins. Indeed, nonphosphorylated histone IIA antagonized the inhibitory effect of NIPP-1b on the casein phosphatase activity of PP-1. Our data show that the nucleus contains specific and potent inhibitory proteins of PP-1 that differ from earlier described cytoplasmic inhibitors. We suggest that these novel proteins may control the activity of nuclear PP-1 on its natural substrate(s).


Molecular cloning of human macrophage capping protein cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages. Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. The human cDNA for MCP has been cloned and sequenced. The derived amino acid sequence predicts a polypeptide of 38.4 kDa. Human MCP expressed in Escherichia coli using a pET12a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+ sensitivity and ability to block monomer exchange at the barbed end of actin filaments. Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins. Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-1% of the total cytoplasmic protein. Northern blot analysis of U937 and HL60 cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and greater than 7 times initial levels, respectively. Human MCP displays a 93% amino acid sequence identity with two recently described mouse proteins, gCap39 and Mbh1. Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocyte development into macrophages indicate that MCP may play an important role in macrophage function.


Identification of a peptide antagonist for platelet-derived growth factor. A series of peptides derived from the primary sequence of the B-chain of platelet-derived growth factor (PDGF) was analyzed for their ability to inhibit the binding of 125I-PDGF-AA and 125I-PDGF-BB to PDGF alpha-receptors and PDGF beta-receptors, respectively. A 13-amino acid peptide (ANFLVWEIVRKKP), corresponding to amino acids 116-121 and 157-163 in PDGF B-chain, was found to compete with binding to both alpha- and beta-receptors. Modification of this peptide on the tryptophan residue increased its receptor competing activity. The peptide was found to be a receptor antagonist, since it inhibited dimerization and autophosphorylation of PDGF receptors. When analyzed on intact cells, the peptide was found to have, in addition to the specific inhibitory effect at the receptor level, a nonspecific inhibitory effect on [3H]thymidine incorporation. Our study has identified two regions in PDGF that are of importance for receptor interaction.


Molecular cloning and characterization of the inositol 1,4,5-trisphosphate receptor in Drosophila melanogaster. We isolated a cDNA encoding an inositol 1,4,5-trisphosphate receptor (InsP3R) of Drosophila melanogaster. The predicted Drosophila InsP3R (2,833 amino acids) has extensive sequence similarity to the mouse InsP3R. The polypeptide encoded by the cDNA was functionally expressed and showed characteristic InsP3-binding activity. The Drosophila InsP3R gene is located at the region 83A5-9 on the third chromosome and expresses throughout development but predominantly in the adult. Localization of the InsP3R mRNA in adult tissues suggests strong expression in the retina and antenna, indicating the involvement of the InsP3R in visual and olfactory transduction. In addition, the InsP3R mRNA is abundant in the legs and thorax, which are enriched with a muscular system. Such localization is apparently consistent with the quantitatively predominant sites for [3H]InsP3 binding in Drosophila and the fleshfly (Boettcherisca peregrina). The present study points to the likely functional importance of the InsP3/Ca2+ signaling system in Drosophila.


Receptor specificity of growth factor-stimulated synthesis of 3-phosphorylated inositol lipids in Swiss 3T3 cells. We have investigated synthesis of 3-phosphorylated inositol lipids in growth factor-stimulated Swiss 3T3 cells. Those growth factors tested which act via tyrosine kinase-containing receptors (platelet-derived growth factor (PDGF), insulin growth factor I (IGF-I), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF)) caused the rapid synthesis of [32P]PtdIns(3,4)P2 and [32P]PtdIns(3,4,5)P3 (PtdIns is phosphatidylinositol) in [32P]P(i)-prelabeled cells and the appearance of an inositol lipid 3-OH kinase in antiphosphotyrosine immunoprecipates. In contrast, those growth factors tested which act via G-protein-coupled receptors (bombesin, vasopressin, prostaglandin E1) were unable to stimulate either of the above responses. Furthermore, while PDGF was able to increase the formation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in streptolysin-permeabilized cells, guanosine 5'-3-(thio)triphosphate and guanyl-5'-yl imidodiphosphate were not. These results suggest that Swiss 3T3 cells possess the machinery for tyrosine kinase but not G-protein-mediated activation of PtdIns(4,5)P2 3-OH kinase; a situation which is the inverse to that recently described for human neutrophils. The tyrosine kinase-containing receptors differed markedly in their relative abilities to elevate the levels of [32P] PtdIns(3,4,5)P3 (ranked in the order PDGF greater than or equal to IGF-I greater than EGF greater than bFGF), [32P]Ptd-OH (PDGF greater than EGF greater than bFGF; undetectable for IGF-I), and [32P]PtdIns4P (EGF greater than bFGF greater than PDGF; undetectable for IGF-I) in [32P]P(i)-prelabeled cells. These differences are epitomized by IGF-I, which was the joint most powerful stimulus for [32P] PtdIns(3,4,5)P3 formation, but was unable to stimulate a measurable accumulation of [32P]Ptd-OH (and hence, by deduction, was unable to stimulate phospholipase C). These results indicate that there is a differential ability among the tyrosine kinase-containing receptors present in a single cell to recruit phospholipase C and PtdIns(4,5)P2 3-OH kinase into their signalling complexes and further emphasizes the notion that the rapid synthesis of PtdIns(3,4,5)P3 may be a signalling event.


Insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function. We have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (Tyr1158, Tyr1162, and Tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. A single-Phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. The double-Phe replacements showed a 60-70% reduction, and substitution of all 3 tyrosine residues with Phe or Ser reduced insulin-stimulated tyrosine autophosphorylation by greater than 80%. Phosphopeptide mapping each mutant revealed that all remaining tyrosine autophosphorylation sites were phosphorylated normally following insulin stimulation, and no new sites appeared. The single-Phe mutants showed insulin-stimulated kinase activity toward a synthetic peptide substrate of 50-75% when compared with wild-type receptor kinase activity. Insulin-stimulated kinase activity was further reduced in the double-Phe mutants and barely detectable in the triple-Phe mutants. In contrast to the wild-type receptor, all of the mutant receptor kinases showed a significant reduction in activation following in vitro insulin-stimulated autophosphorylation. When studied in intact Chinese hamster ovary cells, insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of the cellular substrate pp185 in the single-Phe and double-Phe mutants was progressively lower with increased tyrosine replacement and did not exceed the basal levels in the triple-Phe mutants. However, all the mutant receptors, including the triple-Phe mutant, retained the ability to undergo insulin-stimulated Ser and Thr phosphorylation. Thus, full activation of the insulin receptor tyrosine kinase is dependent on insulin-stimulated Tris phosphorylation of the kinase domain, and the level of autophosphorylation in the kinase domain provides a mechanism for modulating insulin receptor kinase activity following insulin stimulation. By contrast, insulin stimulation of receptor phosphorylation on Ser and Thr residues by cellular serine/threonine kinases can occur despite markedly reduced tyrosine autophosphorylation.


Lysine 356 is a critical residue for binding the C-6 phospho group of fructose 2,6-bisphosphate to the fructose-2,6-bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lysine 356 has been implicated by protein modification studies as a fructose-2,6-bisphosphate binding site residue in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Kitajima, S., Thomas, H., and Uyeda, K. (1985) J. Biol. Chem. 260, 13995-14002). However, Lys-356 is found in the fructose-2,6-bisphosphatase domain (Bazan, F., Fletterick, R., and Pilkis, S. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). In order to ascertain whether Lys-356 is involved in fructose-2,6-bisphosphatase catalysis and/or domain/domain interactions of the bifunctional enzyme, Lys-356 was mutated to Ala, expressed in Escherichia coli, and then purified to homogeneity. Circular dichroism experiments indicated that the secondary structure of the Lys-356-Ala mutant was not significantly different from that of the wild-type enzyme. The Km for fructose 2,6-bisphosphate and the Ki for the noncompetitive inhibitor, fructose 6-phosphate, for the fructose-2,6-bisphosphatase of the Lys-356-Ala mutant were 2700- and 2200-fold higher, respectively, than those of the wild-type enzyme. However, the maximal velocity and the Ki for the competitive product inhibitor, inorganic phosphate, were unchanged compared to the corresponding values of the wild-type enzyme. Furthermore, in contrast to the wild-type enzyme, which exhibits substrate inhibition, there was no inhibition by substrate of the Lys-356-Ala mutant. In the presence of saturating substrate, inorganic phosphate, which acts by relieving fructose-6-phosphate and substrate inhibition, is an activator of the bisphosphatase. The Ka for inorganic phosphate of the Lys-356-Ala mutant was 1300-fold higher than that of the wild-type enzyme. The kinetic properties of the 6-phosphofructo-2-kinase of the Lys-356-Ala mutant were essentially identical with that of the wild-type enzyme. The results demonstrate that: 1) Lys-356 is a critical residue in fructose-2,6-bisphosphatase for binding the 6-phospho group of fructose 6-phosphate/fructose 2,6-bisphosphate; 2) the fructose 6-phosphate binding site is responsible for substrate inhibition; 3) Inorganic phosphate activates fructose-2,6-bisphosphatase by competing with fructose 6-phosphate for the same site; and 4) Lys-356 is not involved in 6-phosphofructo-2-kinase substrate/product binding or catalysis.


A mitogen-activated protein (MAP) kinase activating factor in mammalian mitogen-stimulated cells is homologous to Xenopus M phase MAP kinase activator. The mitogen-activated protein (MAP) kinases, a family of 40-45-kDa kinases whose activation requires both tyrosine and threonine/serine phosphorylations, are suggested to play key roles in various phosphorylation cascades. A previous study of Krebs and co-workers (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) detected an activity in epidermal growth factor (EGF)-stimulated 3T3 cells that can stimulate inactive MAP kinases. We observed this activity in rat 3Y1 cells treated with various mitogenic factors and in PC12 cells treated with nerve growth factor (NGF). Its kinetics of activation and deactivation following EGF or NGF stimulation roughly paralleled that of MAP kinase. The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+ and Mg2+ and was inactivated by phosphatase 2A treatment in vitro. This activator has been isolated from EGF-stimulated 3Y1 cells by sequential chromatography and identified as a 45-kDa monomeric protein. It was able to activate mammalian and Xenopus MAP kinases in vitro and was very similar to Xenopus M phase MAP kinase activating factor, which was purified previously from mature oocytes (Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982), in terms of its functional, immunological, and physicochemical properties. Thus, the same or a similar upstream activating factor may function in mitogen-induced and M phase-promoting factor-induced MAP kinase activation pathways.


Expression and characterization of wild type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP beta. Human HPTP beta is unique among mammalian receptor-like protein tyrosine phosphatases in that it has only a single catalytic domain. The intracellular region of HPTP beta was expressed in bacteria, purified, and characterized. It exhibits high activity toward all substrates tested and is potently inhibited by zinc. Vanadate and polyanions also inhibited activity. The juxta-membrane segment of HPTP beta (residues 1622-1639) potentially functions as a negative regulatory sequence since its deletion can increase HPTP beta activity 5-fold. This segment contains up to two sites for protein kinase C phosphorylation, although in vitro phosphorylation by this kinase did not affect HPTP beta activity. The boundaries of the catalytic domain were delineated by truncation analyses. Successive deletion of N-terminal sequence prior to residue 1684 had little effect on substrate affinity and at most reduced activity about 6-fold. Further removal of residues 1684-1686 resulted in a marked 50-500-fold drop in activity, and loss of N-terminal sequence prior to residue 1690 abolished activity. Based on these analyses a highly conserved motif was identified in all mammalian tyrosine phosphatases (E/q) (F/y)XX(L/i), corresponding to positions 1684-1688 of HPTP beta. Mutation of residue 1684 or 1685 generally gave rise to proteins with marked temperature sensitivity. These mutant HPTP beta were active but had reduced activity compared to the wild type enzyme. In conjunction, these results suggest that this region represents the N-terminal border of the catalytic domain and is essential for correct phosphatase folding although not directly involved in catalysis. Parallel truncation studies have defined residues 1930-1939/40 as the C-terminal border of the catalytic domain.


Purification and characterization of smooth muscle myosin-associated phosphatase from chicken gizzards. Myosin light chain phosphatase associated with smooth muscle myosin (MAPP) was isolated from chicken gizzard. The MAPP was tightly associated with myosin and was not dissociated from myosin under the physiological ionic conditions. The phosphatase was dissociated from myosin in the presence of high MgCl2, i.e. 80 mM MgCl2. The binding site of the enzyme on the myosin molecule was the subfragment-2 region, since the enzyme did bind to the myosin rod and heavy meromyosin but not to the subfragment-1 affinity column. MAPP was purified with a heparin-Sepharose 6B column, and two activity peaks were obtained, i.e. MAPP I and MAPP II. The major activity peak, MAPP I, was further purified to homogeneity by thiophosphorylated myosin light chain-Sepharose 4B column chromatography. MAPP I was a tetramer composed of four 34-kDa subunits. The enzyme preferentially dephosphorylated the beta-subunit of phosphorylase kinase and was strongly inhibited by the heat- and acid-stable protein phosphatase inhibitor-1, whereas it was partially inhibited by the inhibitor-2. The IC50 (concentration of inhibitor giving 50% inhibition) value for the inhibition of the enzyme by okadaic acid was 70 nM which was about eight times higher than skeletal muscle type-1 and 390 times higher than type-2 protein phosphatase. These results demonstrate that the MAPP I is a type-1-like protein phosphatase, although the properties are not the same as type-I phosphatase. The properties of the myosin-associated phosphatase were distinct from the phosphatases reported previously, although some properties were similar to smooth muscle phosphatase-IV. Therefore, it is concluded that MAPP I is a novel smooth muscle protein phosphatase. Since it strongly associated with smooth muscle myosin, it is likely that MAPP I is responsible for the dephosphorylation of smooth muscle myosin in situ.


Elevation of large-T antigen production by sodium butyrate treatment of SV40-transformed WI-38 fibroblasts. The effects of sodium butyrate on simian virus 40 early gene expression were determined in SV40-transformed human embryonic lung fibroblasts (SVWI-38). Northern blot analysis and nuclear run-off transcription studies revealed that treatment of cells with millimolar concentrations of sodium butyrate (2.5 to 10 mM) resulted in increased levels of SV40 early gene transcripts, with a concomitant increase in their corresponding proteins (large-T and small-t antigens). Although sodium butyrate treatment enhanced the expression of the early genes, it was associated with a reduction in cell growth and total protein synthesis, as measured by cell number and incorporation of 3H-leucine into macromolecules, respectively. Immunoprecipitation of 35S-labelled cellular proteins with anti-p53 and anti-T antibodies revealed that the level of the cellular protein, p53, declined markedly in the presence of sodium butyrate. Furthermore, in control cells only 30% of the p53 was complexed with large-T antigen, whereas in butyrate-treated cells all the p53 was complexed with large-T antigen. The increased early gene expression was not due to altered methylation patterns, gene amplification, or rearrangement of the integrated SV40 genome. Sodium butyrate treatment did, however, result in the appearance of a new nuclear protein which bound specifically to a SV40 promoter fragment containing large-T antigen binding sites I and II.


Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects. The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)


Differential induction of heme oxygenase in the hepatocarcinoma cell line (Hep3B) by environmental agents. In situ hybridization and Northern analysis of heme oxygenase (HO) mRNA was used to determine the induction and expression of HO by various environmental agents. Exposure of Hep3B cells to hemin (10 microM) for as little as 5 min resulted in significant production of HO transcripts and mRNA expression as seen by in situ hybridization. We followed the pattern of HO transcript accumulation by heme and results indicate that the peak of induction of HO by heme was reached between 10 and 20 minutes. Other metalloporphyrins were all effective in inducing HO mRNA after 1 h exposure. On the other hand, CoCl2 caused accumulation of HO mRNA at a later time than seen with the metalloporphyrins. However, lipopolysaccharide (LPS) gave a more immediate effect on HO induction which was somewhat similar to heme in its time course. Direct measurements of HO activity revealed that enzyme activity could be detected after about 20 min exposure to hemin, and this activity was inhibited by tin protoporphyrin (SnPP). The different pattern of HO mRNA induction by LPS as contrasted with CoCl2 suggests that LPS may act through a different translational factor, or stimulate free radical formation and the subsequent release of heme and induction of HO. These results indicate that heme causes accumulation of HO mRNA by a different mechanism than that of CoCl2. Finally, LPS shares a concomitant effect on induction of HO as an acute phase reactant type protein.


1,25-Dihydroxycholecalciferol modulates 3H-thymidine incorporation in FRTL5 cells. 1,25-dihydroxycholecalciferol (1,25(OH)2D3) possesses proliferation and differentiation modulating effects in many cell types in vitro. We studied the effect of 1,25(OH)2D3 on 3H-thymidine incorporation in FRTL5 cells, a cultured rat thyroid follicular cell line. 1,25(OH)2D3 alone at 10(-11) and 10(-9) M exerted no effect on 3H-thymidine incorporation. However, at 10(-7) M, 1,25(OH)2D3 slightly enhanced 3H-thymidine incorporation. In the presence of 5% calf serum, 1,25(OH)2D3 increased 3H-thymidine incorporation induced by calf serum in a dose-dependent manner. 1,25(OH)2D3 also enhanced 3H-thymidine incorporation induced by PMA, an extrinsic stimulator of protein kinase C, without directly affecting PMA-induced protein kinase C translocation. In contrast to the stimulatory effects of 1,25(OH)2D3 on the calf serum and PMA-induced 3H-thymidine incorporation, 1,25(OH)2D3 inhibited the increase in 3H-thymidine incorporation induced by TSH in a dose-dependent manner. This effect of 1,25(OH)2D3 on TSH-induced 3H-thymidine incorporation may be, in part, due to post-cAMP pathways since 1,25(OH)2D3 also inhibited the increase in 3H-thymidine incorporation induced by Bu2cAMP without affecting the TSH-induced increase in cAMP. The stimulatory effect of insulin on 3H-thymidine incorporation, a cAMP-independent process, was also inhibited by 1,25(OH)2D3. We conclude that 1,25(OH)2D3 affects 3H-thymidine incorporation in FRTL5 cells raising the possibility of a physiologic role for 1,25(OH)2D3 in the growth and function of thyroid follicular cells.


Principles of neuromuscular transmission. Activation of skeletal muscles, small and large alike, occurs when spinal cord neurons send action potentials to groups of fibers. The central role of acetylcholine and its receptors in this activation is well established. The mechanisms involved and the implications for understanding neuromuscular disorders and for rationalizing and improving therapy are discussed.


High-performance affinity chromatography of oligonucleotides on nucleic acid analogue immobilized silica gel columns. The nucleic acid analogues poly(9-vinyladenine) (PVAd), poly(9-adenylethyl methacrylate) and poly(thymylethyl methacrylate) (PTM) were chemically bonded to porous silica gel, which had been pretreated with 3-trimethoxysilylpropyl methacrylate, by free radical copolymerization to produce novel packing materials for affinity chromatographic columns. The columns separated nucleosides and nucleotide dimers on the basis of hydrophobic interaction using an aqueous buffer and complementary hydrogen bonding interaction in methanol as an eluent. The PVAd- and PTM-silica gel columns gave a nucleobase-selective separation of oligonucleotides differing in length from mixtures of oligoadenylic and oligouridylic acids. On the PVAd-silica gel column terminal phosphate isomers of oligouridylic acid up to seven mer were resolved and the elution order of the isomers was different from that on an ODS column.


Separation of oligogalacturonic acids by high-performance gel filtration chromatography on silica gel with diol radical. Oligogalacturonic acids (OLGAs) ranging from two to nineteen residues in length were separated using high-performance gel filtration chromatography on a silica gel with diol radical. The optimum conditions (eluent, column temperature) for separation of OLGAs by high-performance gel filtration chromatography were investigated. The column used in this experiment allowed a high pressure of 4900 p.s.i. and a flow-rate of 2 ml/min. The stationary phase of silica gel stabilized the separation of OLGAs. The peaks of OLGAs separated using this column were assigned by comparing retention times with standards, and the molecular weights of the corresponding OLGAs were determined by fast atom bombardment mass spectrometry.


Solventless determination of caffeine in beverages using solid-phase microextraction with fused-silica fibers. Caffeine concentrations in beverages were determined using a simple and rapid method based on microextraction of caffeine onto the surface of a fused-silica fiber. The uncoated fiber was dipped into the beverage sample for 5 min after the addition of isotopically labeled (trimethyl 13C)caffeine. The adsorbed caffeine was then thermally desorbed in a conventional split/splitless injection port, and the concentration of caffeine was determined using gas chromatography with mass spectrometric detection. Quantitative reproducibilities were ca. 5% (relative standard deviation) and the entire scheme including sample preparation and gas chromatographic analysis was completed in ca. 15 min per sample. The potential of the microextraction technique for the analysis of flavor and fragrance compounds in non-caffeinated beverages is also demonstrated. Since no solvents or class-fractionation steps are required, the method has good potential for automation.


Detection of CMV DNA in clinical samples of AIDS patients by chemiluminescence hybridization. A sensitive chemiluminescence dot-blot hybridization assay for the detection of CMV DNA in clinical samples of AIDS patients is described. In the chemiluminescence hybridization assay, digoxigenin-labelled CMV DNA probes were used and when hybridized they were detected by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. Adamantyl 1,2-dioxetane phenyl phosphate was used as the chemiluminescent substrate. The results were recorded by instant photographic films. The results obtained with the chemiluminescence hybridization assay were compared with the results obtained by hybridization with colourimetric detection. The chemiluminescent assay proved specific, sensitive and reliable and thus can be used as a valuable routine diagnostic test for the detection of CMV DNA in clinical samples.


Microheterogeneity of S-glycoprotein of mouse hepatitis virus temperature-sensitive mutants. Mouse hepatitis virus (MHV) strain JHM (MHV-JHM) is a neurotropic coronavirus that causes acute fatal encephalomyelitis in 75-99% of infected mice. The surviving animals may subsequently develop demyelinating disease. We compared the S peplomer protein of the wild type (wt) and five temperature-sensitive (ts) mutants of MHV-JHM. In contrast with the wt, none of these five cause fatal disease (mortality less than 10%). Three of these ts mutants did not induce any demyelinating disease, a fourth caused demyelinating disease in 5% of the animals and a fifth, designated ts8, exhibited strong demyelinating properties and caused demyelination in 99% of the animals. SDS-PAGE analysis revealed no differences in the molecular weight of S peplomer protein of wt or ts MHV-JHM mutants. However, isoelectric focusing of the S protein of these five ts mutants and the wt MHV-JHM, followed by transfer to nitrocellulose sheets and immunoblotting with anti-S specific antibody revealed significant differences in the microheterogeneity of the S protein.


Distribution of type A and type B EBV in normal individuals and patients with head and neck carcinomas in Taiwan. The subtypes of Epstein-Barr virus (EBV) according to the EBNA 2 gene were investigated in Taiwan by the polymerase chain reaction (PCR) and by Southern blot hybridization. The materials included 53 nasopharyngeal carcinoma (NPC) biopsies, 49 other head and neck cancers and 32 throat washings of normal individuals. EBV DNA was found in all NPC biopsies, 27 of 49 other head and neck carcinomas and 81% of normal individuals. Type A EBV was the predominant type of EBV in both normal individuals and patients with head and neck carcinomas in Taiwan. Type B EBV or coexistence of the A and B types comprised a small number of samples in this study.


Differential polymerase chain reaction for detection of wild-type and a vaccine strain of Aujeszky's disease (pseudorabies) virus. A polymerase chain reaction (PCR) strategy for differentiating between a vaccine mutant strain and wild-type (WT) strains of Aujesky's disease (pseudorabies) virus (ADV) was evaluated. With this approach, a single virus or a concurrent WT and vaccine virus infection could be distinguished by targeting the genomic alteration within the vaccine strain. PCR primers were designed for a recombinant vaccine virus that has almost all of the WT gX gene replaced by the lacZ gene. One primer, corresponding to a conserved sequence upstream of the altered region, was selected for common use. The differentiating primers were chosen from the unique WT gX and vaccine lacZ gene sequences. The sensitivity of the differential PCR was analyzed using extracted viral DNA and in vitro infected cell lysates. Approximately 10 and between 10 to 100 molecules of WT and vaccine viral DNAs, respectively, could be detected, regardless of the presence or absence of uninfected cell lysates. Detection of viral DNA from in vitro infected cell cultures approximated this level of sensitivity. The specificity of the amplifications was verified by restriction endonuclease analysis and Southern hybridization. Although the vaccine primer pair target was amplified to a lesser degree as compared to the WT primer pair product, utility of the differential PCR was demonstrated using trigeminal nerve ganglia from swine infected with vaccine virus and WT virus. Both viral targets were detected only by their specific primer pair, in either the single or dual infection.


Increased cytomegalovirus infection of human fibroblast and endothelial cells by electroporation. Electroporation of fibroblasts and endothelial cells in the presence of HCMV caused an increase in the infection of these cells by the virus. This method could be applied to cells that are difficult to infect by ordinary laboratory methods and also in cases when synchronized infection of the cells by the virus is needed.


Restriction analysis of the prototype strain of enteric adenovirus type 41 using exonuclease III. Enteric adenoviruses 40 and 41 (Ad40 and Ad41) are a prominent cause of gastroenteritis in young children. Diagnosis of these enteric types by conventional means is complicated by their fastidious growth characteristics. Enteric adenovirus growth was enhanced by cocultivation. Typing of enteric isolates currently entails analysis of the extracted viral DNA with restriction enzymes. Restriction endonuclease fragments of the Ad41 strain Tak genome were ordered by (i) double digestion, (ii) release of restriction fragments from plasmids containing 84% of the Ad41 genome in EcoRI fragments A, B and C, (iii) hybridization of Southern blotted Ad41 fragments with EcoRI fragment containing plasmids and various segments of the Ad2 genome, (iv) sequential reduction of the genome beginning with terminal restriction fragments with exonuclease III and S1 nuclease. The termini of adenovirus genomes are difficult to clone and the use of exonuclease III is a useful alternative to conventional restriction mapping. DNA restriction patterns, fragment sizes and restriction maps of the Ad4 1 strain Tak with enzymes BamHI, BglII, ClaI, EcoRI, HindIII, PstI, SalI, SmaI and XhoI are presented. Prototype strain restriction maps should enable better understanding of adenovirus type 41 and its epidemiology.


Nucleotide sequencing of 5' and 3' termini of bovine viral diarrhea virus by RNA ligation and PCR. Genomic RNA was extracted from cytopathic (CP) bovine viral diarrhea virus (BVDV) strain NADL, CP strain 72, and noncytopathic (NCP) strain SD-1 purified by ultracentrifugation. Assuming the presence of a cap structure, de-blocking of the 5' capped end of the genomic RNA was done by treatment with tobacco acid pyrophosphatase (TAP). Following decapping, the RNA molecules were ligated using T4 RNA ligase and the ligated tandem RNA templates were then amplified by primer-directed amplification (PCR). cDNA synthesis was done using reverse transcriptase with random primers and cDNA amplification was done using a negative sense primer 231-248 and a positive sense primer 12434-12451. The nucleotide sequence of the amplified product was determined by double-stranded sequencing using the Sanger di-deoxy chain termination method and an additional 'CCCCC' nucleotide sequence was identified at the ligation site. Following dATP tailing of cDNA and amplification across the 5' terminus and nucleotide sequencing, no additional nucleotides were identified on the 5' terminus. The 5' terminus as published by Collett et al., 1988b was confirmed as previously reported. Therefore, the 3' terminus includes an additional 'CCCCC' nucleotide sequence to that previously reported. Identical results were obtained when the BVDV genomic RNA was not decapped prior to RNA ligation and amplification.


Rapid sequencing of the Sendai virus 6.8 kb large (L) gene through primer walking with an automated DNA sequencer. The determination of the complete DNA sequence of the large (L) polymerase gene of Sendai virus strain Fushimi was used to explore the potential and feasibility of primer walking with fluorescent dye-labelled dideoxynucleotide terminators on an automated ABI DNA sequencer. The rapid identification of the complete sequence demonstrated that this approach is a time- and cost-saving alternative to classical sequencing techniques. Analysis of the data revealed that the L gene of Sendai virus strain Fushimi consists of exactly 6800 nucleotides and that the deduced amino acid sequence identifies a single open reading frame encoding a protein of 252.876 kDa. In contrast to Sendai virus strain Enders, the L mRNA of strain Fushimi is monocistronic. The comparison of the deduced amino acid sequences of the L genes of three different Sendai virus strains confirmed the existence of conserved as well as variable regions in the L protein and revealed a high grade of conservation in the carboxyterminal third. Furthermore, functional amino acid sequence motifs, like elements of RNA-dependent RNA polymerases and ATP-binding sites as postulated previously, were identified.


Comparison of several PCR procedures for detection of serum HCV-RNA using different regions of the HCV genome. Different methods for the isolation of hepatitis C virus RNA and several sets of primers from the 5' untranslated, core, NS3, NS3/NS4 and NS5 regions of the hepatitis C virus genome were used for the detection of the viral genome by the polymerase chain reaction. Serum samples from 10 patients with chronic hepatitis C and 10 healthy controls were studied. The best method for the RNA extraction was with cold guanidinium isothiocyanate followed by a denaturation step prior to the polymerase chain reaction. Using this method, the percentage of positivity to hepatitis C virus sequences in serum depending on the region amplified were: 5' untranslated, 90%; Core, 20%; NS3, 80%; NS3/NS4, 60% and NS5, 60%.


Characterization of nonradioactive hybridization probes for detecting infectious bursal disease virus. Reverse transcription followed by the polymerase chain reaction was used to amplify a fragment of infectious bursal disease virus (IBDV) strain P3009 genome. The amplified DNA fragment was annealed into the plasmid pUC18 and used to transform Escherichia coli strain JM109. A clone that contained IBDV-specific nucleotide sequences was selected and designated pC23. The DNA fragment within pC23 was 320 base pairs in length and designated C23. Radiolabeled probes prepared from C23 hybridized to genome segment A of strain P3009 by a northern-blot hybridization assay. Biotin-labeled probes prepared from C23 and pC23 either by using nick translation (designated C23/NT and pC23/NT, respectively) or by direct introduction of biotin molecules into C23 and pC32 (designated C23/BH and pC23/BH, respectively) were used in the dot blot hybridization assay for detecting IBDV strains. All four biotinylated probes detected three serotype 1 viruses and one serotype 2 IBDV. However, they did not cross-react with nucleic acids extracted from mock-infected cells or from seven unrelated avian viruses. Probe pC23/BH detected as little as 0.04 ng of IBDV RNA, while the other three probes were less sensitive and detected approximately 1 ng of IBDV RNA. In addition, the probe pC23/BH detected IBDV RNA in bursa tissues from commercial broiler raising farms following the dot blot hybridization.


Psoralen preparation of antigenically intact noninfectious rotavirus particles. The use of the synthetic psoralen 4'-aminomethyl-4,5',8-trimethylpsoralen hydrochloride (AMT) is described for the inactivation of infectious rotavirus, a member of the viral family Reoviradae with a double-stranded RNA genome. This method not only provides complete inactivation of the virus but leaves antigenically intact particles. The lack of viral replication following inactivation was determined with an immunohistochemical focus assay. The antigenic authenticity of the particles was determined by monoclonal antibody ELISA and a viral hemagglutination assay.


Effects of atrial natriuretic peptide and vasopressin on chloride transport in long- and short-looped medullary thick ascending limbs. Recent studies have suggested a selective effect of atrial natriuretic peptide (ANP) in regulating NaCl reabsorption in juxtamedullary nephrons. We examined (a) functional differences between medullary thick ascending limbs from long and short loops of Henle (lMAL and sMAL, respectively) and (b) the interaction of ANP and arginine vasopressin (AVP) on Cl- transport (JCl) in these two segments. AVP-, glucagon-, and calcitonin-stimulated cAMP accumulation was higher in lMAL than in sMAL. 10(-10) M AVP increased JCl in lMAL but not in sMAL. ANP-stimulated cGMP production was higher in lMAL than in sMAL. 10(-10) and 10(-8) M ANP inhibited AVP-stimulated JCl in lMAL by 26-30% (from 70.3 +/- 11.4 to 51.7 +/- 13.6 pmol/mm per min and from 88.1 +/- 10.1 to 61.8 +/- 11.7 pmol/mm per min, respectively), and this effect was mimicked by 10(-5) to 10(-4) M cGMP. This effect of ANP in lMAL could account for a large part of the ANP-induced natriuresis and diuresis in vivo, in that the rate of NaCl reabsorption in MAL is the largest among distal nephron segments, providing the chemical potential energy for the renal countercurrent multiplication system.


Blockade of cardiac sodium channels. Competition between the permeant ion and antiarrhythmic drugs. A number of basic and clinical studies suggest that elevation of external sodium concentrations, [Na]o, may reverse the cardiotoxic effect of local anesthetic-class drugs. The mechanisms of reversal are uncertain. The blocking action of lidocaine and disopyramide were studied over a range of [Na]o. Both whole-cell voltage clamp and single-channel recordings were performed on isolated rabbit myocytes at 17 and 22 degrees C, respectively. In the presence of lidocaine, an inactivated channel blocker, the level of steady-state block in response to pulse train stimulation was not affected by variations in [Na]o from 20 to 150 mM. Estimates of the rate of dissociation of drug from the channel also were unaffected. In contrast, steady-state block by disopyramide, a drug that blocks open channels, was decreased as [Na]o was increased. Single-channel measurements suggest that the influence of [Na]o on channel current amplitude was small, 12% for a 25 mM increase in [Na]o. This increase in single-channel current amplitude would affect drug-free channels only, in that our studies suggest that drug-associated channels do not conduct. The association rate constant of disopyramide with open single sodium channels was decreased from 10 x 10(6) to 5 x 10(6)/M per s by an increase in [Na]o from 120 to 180 mM. Elevation of [Na]o may reverse the blocking action of local anesthetic-class drugs by an increase in single-channel current amplitude or by a decrease in drug association rate with the sodium channel. The occurrence of the latter action depends on the mode of block of the specific agent.


Suppression of metalloproteinase biosynthesis in human alveolar macrophages by interleukin-4. To study the interaction of lymphocytes and macrophages in the control of extracellular matrix turnover, we determined the effects of several soluble T cell products on mononuclear phagocyte production of metalloproteinases. Cytokines including IL-2, IL-4, IL-6, tumor necrosis factor alpha (TNF alpha), GM-CSF, and IFN-gamma were each tested for capacity to modulate macrophage metalloproteinase and tissue inhibitor of metalloproteinases (TIMP) expression. The addition of IL-4 to cells cultured under basal conditions caused a dose-dependent suppression in the release of 92-kD type IV collagenase without affecting TIMP production. 92-kD enzyme secretion was inhibited by 50% with 1-2 ng/ml of IL-4 and by 90% with 10 ng/ml of IL-4. When cells were first exposed to killed Staphylococcus aureus to induce metalloproteinase production, IL-4 potently blocked the stimulated release of both interstitial collagenase and 92-kD type IV collagenase, again without effect upon TIMP. Metabolic labeling experiments and Northern hybridizations demonstrated that IL-4 exerted its action at a pretranslational level. Furthermore, IL-4 possessed the capacity to inhibit metalloproteinase expression even in the relatively immature peripheral blood monocyte. As reported previously (Shapiro, S. D., E. J. Campbell, D. K. Kobayashi, and H. G. Welgus. 1990. J. Clin. Invest. 86:1204), IFN-gamma suppressed constitutive macrophage production of 92-kD type IV collagenase. Despite the frequent antagonism observed between IL-4 and IFN-gamma in other systems, the combination of these two agents lowered metalloproteinase biosynthesis dramatically, whereas IL-4 opposed the IFN-gamma-stimulated production of cytokines (IL-1 and TNF alpha). IL-6 had only minimal effect upon metalloproteinase production, but appeared to specifically augment TIMP release. In summary, cytokines released by activated T cells may profoundly reduce the capacity of the macrophage to mediate extracellular matrix degradation.


Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells. Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ uptake averaged 272 +/- 35 nmol min-1 mg protein-1 and were inhibited 40% by chlorothiazide (CTZ, 10(-4) M). Control rates of 36Cl- uptake averaged 340 +/- 50 nmol min-1 mg protein-1 and were inhibited 50% by CTZ. CTZ stimulated 45Ca2+ uptake by 45% from resting levels of 2.86 +/- 0.26 nmol min-1 mg protein-1. Bumetanide (10(-4) M) had no effect on 22Na+, 36Cl-, or 45Ca2+ uptake. Control levels of intracellular calcium activity ([Ca2+]i) averaged 91 +/- 12 nM. CTZ elicited concentration-dependent increases of [Ca2+]i to a maximum of 654 +/- 31 nM at 10(-4) M. CTZ reduced intracellular chloride activity ([Cl-]i), as determined with the chloride-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10(-5) M) abolished the effect of CTZ on [Cl-]i. NPPB also blocked CTZ-induced increases of 45Ca2+. Resting membrane voltage, measured in cells loaded with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], averaged -72 +/- 2 mV. CTZ hyperpolarized cells in a concentration-dependent and reversible manner. At 10(-4) M, CTZ hyperpolarized MDCT cells by 20.4 +/- 7.2 mV. Reduction of extracellular Cl- or addition of NPPB abolished CTZ-induced hyperpolarization. Direct membrane hyperpolarization increased 45Ca2+ uptake whereas depolarization inhibited 45Ca2+ uptake. CTZ-stimulated 45Ca2+ uptake was inhibited by the Ca2+ channel blocker nifedipine (10(-5) M). We conclude that thiazide diuretics block cellular chloride entry mediated by apical membrane NaCl cotransport. Intracellular chloride, which under control conditions is above its equilibrium value, exits the cell through NPPB-sensitive chloride channels. This decrease of intracellular chloride hyperpolarizes MDCT cells and stimulates Ca2+ entry by apical membrane, dihydropyridine-sensitive Ca2+ channels.


Receptor-specific induction of insulin-like growth factor I in human monocytes by advanced glycosylation end product-modified proteins. Normal tissue homeostasis requires a finely balanced interaction between phagocytic scavenger cells (such as monocytes and macrophages) that degrade senescent material and mesenchymal cells (such as fibroblasts and smooth muscle cells), which proliferate and lay down new extracellular matrix. Macrophages and monocytes express specific surface receptors for advanced glycosylation end products (AGEs), which are covalently attached adducts resulting from a series of spontaneous nonenzymatic reactions of glucose with tissue proteins. Receptor-mediated uptake of AGE-modified proteins induces human monocytes to synthesize and release cytokines (TNF and IL-1), which are thought to contribute to normal tissue remodeling by mechanisms not entirely understood. We now report that AGEs also induce human monocytes to generate the potent progression growth factor insulin-like growth factor I (IGF-I), known to stimulate proliferation of mesenchymal cells. After in vitro stimulation with AGE-modified proteins, normal human blood monocytes express IGF-IA mRNA leading to the secretion of IGF-IA prohormone. The signal for IGF-IA mRNA induction seems to be initiated via the monocyte AGE-receptor, and to be propagated in an autocrine fashion via either IL-1 beta or PDGF. These data introduce a novel regulatory system for IGF-I, with broad in vivo relevance, and provide an essential link to the chain of events leading from the spontaneously formed tissue AGEs, hypothesized to act as markers of protein senescence, to their replacement and to tissue remodeling by the locally controlled induction of growth factors.


Herpes simplex virus-2 (HSV-2) type-specific antibody correlates of protection in infants exposed to HSV-2 at birth. Western blot analysis was used to compare the herpes simplex virus (HSV)-2 antibody profiles of 40 infants less than 2 wk of age who had been exposed to maternal genital HSV-2 at birth. 4 mothers were HSV seronegative at delivery and seroconverted to HSV-2 ("primary infection"), 9 had HSV-1 antibodies and seroconverted to HSV-2 ("nonprimary first episode infection"), and 27 were HSV-2 seropositive ("recurrent infection"). Neonatal herpes infections developed in 1 of 4 infants of women with primary infection, in 3 of 9 infants of women with nonprimary first episode infection, and in none of the 27 infants of women with recurrent HSV-2. Antibodies to HSV-2 proteins gG-2, VP5, and ICP35 were detected in 83, 89, and 72% of the 36 uninfected infants, respectively. None of the four infected infants had detectable antibodies to gG-2 and only one (25%) had antibodies to VP5 or ICP35. The more limited profiles of the 13 infants born to mothers with first episodes of HSV-2 were then analyzed separately; these profiles were similar among infected and uninfected infants except for gG-2, which elicits antibodies that are type specific for HSV-2. None of the infected infants versus seven of nine (78%) uninfected infants were gG-2 seropositive. These comparisons suggest that maternal type-specific antibodies may play a role in preventing neonatal infection after exposure to HSV-2.


Release of soluble receptors for tumor necrosis factor (TNF) in relation to circulating TNF during experimental endotoxinemia. Serial plasma samples from human volunteers obtained after intravenous administration of Escherichia coli endotoxin were analyzed for the presence of circulating soluble tumor necrosis factor receptors (sTNFR). A four- to fivefold increase of type A (p75) and type B (p55) sTNFR was observed 3 h after endotoxin challenge. Pretreatment of the volunteers with ibuprofen before the injection of endotoxin resulted in a slight increase (3.87 +/- 0.2 vs. 3.27 +/- 0.3 ng/ml) and temporal shift of sTNFR-A release concurrent to a marked augmentation of TNF levels (603 +/- 118 vs. 338 +/- 56 pg/ml) as compared to the group without ibuprofen pretreatment. There was a significant correlation between peak sTNFR-A levels and peak TNF levels in the individual probands (r = 0.52, P = 0.04). On the contrary, release kinetics and plasma concentrations of sTNFR-B were identical in both groups (7.38 +/- 0.69 vs. 7.44 +/- 0.33 ng/ml) and no correlation with individual TNF levels was observed. The amount of sTNFR liberated upon endotoxin challenge was not sufficient to block TNF-mediated cytotoxic effects. Our data indicate that the release in vivo of type A and type B sTNFR upon a short exposure to endotoxin is regulated differently.


Differing roles for platelet-activating factor during inflammation of the lung and subarachnoid space. The special case of Streptococcus pneumoniae. Although well-characterized in the lung, the role of platelet-activating factor (PAF) in inflammation in the central nervous system is undefined. Using rabbit models of meningitis and pneumonia, PAF was found to induce significant blood-brain barrier permeability and brain edema at doses five times lower than those required to generate leukocyte recruitment to the subarachnoid space. Both leukocytosis and increased vascular permeability occurred in response to PAF in the lung. Antibody to the CD-18 family of leukocyte adhesion molecules inhibited leukocyte recruitment in response to PAF in the brain (greater than 80%); a similar level of inhibition in the lung required treatment with a combination of a PAF receptor antagonist (L-659,989) and anti-CD18 antibody. Treatment with L-659,989 decreased abnormal cerebrospinal fluid cytochemical values induced by intracisternal challenge with pneumococci but not Haemophilus influenzae, indicating a special role for PAF in pneumococcal disease. Antibodies directed at phosphorylcholine, a unique, shared determinant of bioactivity of PAF and pneumococcal cell wall, obviated the inflammatory potential of both agents. However, no evidence for a direct PAF-like activity of pneumococcal cell wall components was detected in vitro by bioassay using platelets or neutrophils. It is concluded that PAF can induce inflammation in the subarachnoid space. In brain, PAF effects appear to be mediated through CD-18-dependent events, while in lung, PAF effects independent of CD-18 are also evident. At both sites, PAF is of particular clinical importance during inflammation induced by pneumococci apparently due to a unique proinflammatory relationship between the pneumococcal cell wall teichoic acid and PAF.


Association of virulent and avirulent strains of bluetongue virus serotype 11 with premature births of late-term bovine fetuses. Two strains of bluetongue virus serotype 11 (BTV 11), UC-2 avirulent and UC-8 neurovirulent in newborn mice, were inoculated into late-term bovine fetuses to investigate whether infection with these two BTV strains in late gestation would produce congenital infection and pathological changes. Fetuses were inoculated by intramuscular injection through the uterine wall at 243 days gestation and recovered after spontaneous delivery. In calves inoculated with UC-8, births occurred 15 to 27 days before expected parturition, resulting in small, weak calves. These calves had a mild encephalitis and were unthrifty at birth. Calves inoculated with UC-2 appeared healthy when born 7 to 11 days prior to expected parturition. No lesions were found in these calves at necropsy. All calves seroconverted by the time of birth. Viraemia was present in the calves inoculated with UC-8 and in one calf inoculated with UC-2. Plasma cortisol concentrations were prematurely elevated, particularly in the calves inoculated with UC-8, indicating that they were stressed by the infection. The elevated cortisol, associated with an active congenital infection caused by bluetongue virus serotype 11 strain UC-8, is capable of causing premature delivery of low birth-weight, weak calves.


The effect of mixed live vaccines of Newcastle disease and infectious bronchitis on the chicken respiratory tract. The effect of mixed live vaccines of Newcastle disease (ND) and infectious bronchitis (IB) on specific pathogen-free chickens aged 7 days was investigated. The chickens were inoculated intranasally with mixed live vaccines with and without Escherichia coli, or with E. coli alone. "Vaccine 1" consisted of ND virus strain B1 and IB virus strain ON; "vaccine 2" consisted of ND virus strain B1 and IB virus strain H120. The tracheas of chickens inoculated with the vaccines and E. coli and with the vaccines alone showed multiplication of E. coli and histological lesions (loss of cilia, degeneration and hyperplasia in epithelial cells and cellular infiltration of subepithelial tissues). In the tracheas of chickens inoculated with vaccines and E. coli, multiplication of E. coli was greater than in chickens given vaccine alone. There were no histological lesions, and only mild, transient multiplication of E. coli in chickens inoculated with E. coli alone. The results suggest that these mixed live vaccines, especially vaccine 2, play a role in inducing or enhancing colibacillosis in the chicken.


The detection of Alcelaphine herpesvirus-1 DNA by in situ hybridization of tissues from rabbits affected with malignant catarrhal fever. Tissue sections and cultured lymphocytes from rabbits clinically affected following experimental infection with Alcelaphine herpesvirus-1 (AHV-1) were assessed for the presence of viral DNA by in situ hybridization with the cloned major HindII repeat sequence of this virus. Small numbers of virus-infected cells were consistently detected only in submandibular lymph nodes, while other tissues showed no evidence of viral DNA. Virus titration in culture suggested that there were higher titres of virus in the lymph nodes, spleen and lung of infected animals than in the kidney or peripheral blood lymphocytes and confirmed the low level of virus in these animals. Substantially more viral DNA was detected by in situ hybridization in lymphocytes following at least 24 h of culture, suggesting that viral replication is normally repressed by the host.


Neuromyoblastoma in the rat.

Neuromyoblastoma in the rat. We report three tumours arising in the brain stem or adjacent cranial nerves of the Alderly Park rat. Light microscopy with special stains, immunocytochemistry and electron microscopy supported the conclusion that both neuronal and myoid differentiation occurred in all neoplasms. This was shown by the presence in neural cells of axon-like cell processes and in myoblastic cells of striated myofibrils. Although medulloblastomas occur in man and rats the tumours reported here were not related to the cerebellum. We have termed this tumour neuromyoblastoma.


Itraconazole in the treatment of cryptococcal meningitis. The results of an open study of the efficacy of oral itraconazole therapy in 8 patients with cryptococcal meningitis are reported. Therapy is monitored by clinical response with culture, indian ink stain and cryptococcal antigen testing of CSF. Four (50%) of eight patients were cured, two (25%) had partial response, and two (25%) failed. One patient had a relapse but responded to retreatment. No toxicity was observed. Itraconazole is, therefore, suggested as a new treatment for cryptococcal meningitis.


Treatment of dermatophytosis with new systemic antifungal agent, itraconazole. Thirty patients with the clinical and laboratory proven diagnosis of either tinea corporis or tinea cruris were treated with a new systemic antifungal agent, itraconazole. Itraconazole, a triazole derivative, interacts specifically with fungal cytochrome P-450 and has high affinity for skin and mucous membrane. In this open non-comparative clinical trial, itraconazole was given orally in the dosage of 100 mg per day for 14 consecutive days. Twenty-nine cases (96.6%) had good clinical response with 83.3 per cent mycological cure rate. Complete healing of cutaneous lesions was 56.6 per cent at the end of therapy (14 days) and increased to 83.3 per cent two weeks later. No significant side effects were noted. Itraconazole is highly effective in treatment of dermatophytosis with a shorter treatment period than other conventional antifungal agents.


Universal precautions in the family physician's office. The risk of occupational exposure to the human immunodeficiency virus (HIV) may be one of the important issues facing family physicians in the 1990s. The use of universal precautions has been shown to reduce the incidence of exposures to bloodborne pathogens. Studies indicate, however, that these guidelines are not being followed consistently by physicians or their staffs. A survey of 3568 randomly sampled members of the American Academy of Family Physicians was performed using a questionnaire that was designed by the authors. Three mailings were conducted. The total response rate was 39%. Approximately 80% of the respondents reported that they used gloves appropriately and disposed of sharp instruments in a puncture-resistant container. Only 39% "always" or "almost always" used eye protection when indicated, and only 35% "almost never" or "never" recapped used needles. There was a significant number of physicians who reported that they or their office staff had had an occupational exposure to bloodborne pathogens within the last year. Family physicians and their staffs do not uniformly follow universal precaution guidelines and, as a result, many have been exposed to blood products. If the physician or the office staff would not recap used needles and would place used sharp instruments in a puncture-resistant container, the greatest risks of occupational exposure would be reduced.


Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization. Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.


Exogenous siderophore-mediated iron uptake in Pseudomonas aeruginosa: possible involvement of porin OprF in iron translocation. In addition to the two siderophores pyoverdine and pyochelin synthesized by Pseudomonas aeruginosa ATCC 15692 (strain PAO1), several siderophores produced by other bacteria or fungi, namely cepabactin, salicylic acid, desferriferrichrysin, desferriferricrocin, desferriferrioxamine B, desferriferrioxamine E and coprogen, were able to promote iron uptake with variable efficiencies into this bacterium. For most of these siderophores, these results were consistent with the growth stimulation produced by the same compounds in a plate bioassay. Desferriferrichrome A, enterobactin and desferriferrirubin, however, did not promote iron uptake, although enterobactin and desferriferrirubin stimulated bacterial growth. These paradoxical data are discussed in view of siderophore-inducible iron uptake systems, as demonstrated recently for enterobactin. Among the strains tested, including the wild-type PAO1, the pyoverdine-less mutant PAO6606 and the two porin-mutants P. aeruginosa H636 (oprF::omega) and P. aeruginosa H673 (oprD::Tn501), only for the porin-OprF mutant were fewer siderophores able to promote iron uptake compared to the other strains. Such results suggest that beside specific routes for iron uptake P. aeruginosa is also able to take up siderophore-liganded iron through OprF.


Induction and inhibition of bovine leukaemia virus expression in naturally infected cells. Bovine leukaemia virus (BLV) resides in infected lymphocytes in a latent, repressed state but becomes expressed a few hours after the cells are cultured in vitro. We have identified several conditions and factors affecting the expression of BLV in short-term cultures of naturally infected lymphoid cells. The presence of foetal calf serum in the culture medium greatly stimulates virus expression. This stimulation is not due to cellular proliferation. Transcription of BLV RNA and synthesis of p25 in the cultures of peripheral blood lymphocytes are preceded by a lag period of several hours. Synthesis of BLV p25 in these cultures takes place almost immediately after viral RNA synthesis. Extending previous results, we demonstrate that the plasma and lymphatic fluid of cattle contain factors that suppress and stimulate BLV expression. As a result of systematic examination of several parameters, we have developed reproducible assays for the detection of these factors. It is very likely that their relative concentration in the host is an important determinant of susceptibility and resistance to the development of lymphosarcoma and persistent lymphocytosis in BLV-infected cattle.


Scheme for the generation of a truncated endogenous murine leukaemia virus, the Fv-4 resistance gene. The Fv-4 resistance (Fv-4r) gene is a truncated endogenous murine leukaemia virus (MuLV) containing a 3' portion of pol, the entire env gene and the 3' long terminal repeat. Env expression renders mice resistant to infection by ecotropic MuLVs, probably via receptor interference. Previous studies have suggested that the flanking cellular sequences are also important for Fv-4 env gene expression. To establish how the truncated retrovirus was generated and the nature of the cellular sequences involved, the Fv-4 susceptible (Fv-4s) allele DNA was cloned, and its restriction map and nucleotide sequence were compared with those of the Fv-4r allele. A likely mechanism for generation of the truncated endogenous MuLV is suggested by the results; integration of a prototype MuLV provirus at a site within the Fv-4s allele about 6 to 8 kb downstream of a non-retroviral promoter region, followed by deletion of the 5' half of the provirus, with an accompanying loss of only 7 or 10 bp of cellular flanking sequences. The deletion may have led to the expression of the Fv-4r env gene under control of the non-retroviral promoter.


A VP4 sequence highly conserved in human rotavirus strain AU-1 and feline rotavirus strain FRV-1. The primary amino acid sequence of the VP4 proteins of human rotavirus strain AU-1 and feline rotavirus strain FRV-1 was deduced from nucleotide sequence analysis of full-length genome segment 4 cDNAs produced by a combined reverse transcription-polymerase chain reaction. The VP4 genes were 2359 nucleotides in length and contained one long open reading frame capable of encoding a protein of 775 amino acids. Strain AU-1 and FRV-1 VP4s were 98.8% similar at both the nucleotide sequence and amino acid level. Given that most of the genome segments of strains AU-1 and FRV-1 formed hybrids under stringent hybridization conditions, the relationship between their VP4 gene sequences is best explained by feline rotavirus being transmitted to human hosts as whole virions relatively recently. Of added interest is that AU-1 and FRV-1 VP4 both exhibit high degrees of similarity (96.0% nucleotide identity and 97.2 to 97.5% amino acid identity) with serotype G1 human rotavirus strain K8 VP4, which is distinct from any other sequenced VP4 allele. This suggests that strain K8 VP4 was derived by natural gene reassortment from a feline rotavirus or a strain AU-1-like human rotavirus.


Flow cytometric analysis of in vitro bluetongue virus infection of bovine blood mononuclear cells. Cultures of adherent and non-adherent bovine peripheral blood mononuclear (PBM) cells were inoculated with bluetongue virus (BTV) serotype 10. Some cultures of non-adherent cells were stimulated with interleukin 2 (IL-2) and concanavalin A for 24 h prior to virus inoculation. Cells were harvested at various intervals up to 72 h after inoculation. A panel of leukocyte differentiation antigen-specific monoclonal antibodies (MAbs), specific for bovine CD2, CD4 or CD8, monocytes and granulocytes, B cells, gamma delta T cells or the IL-2 receptor (IL-2r), was directly conjugated to fluorescein isothiocyanate, and a MAb specific for the BTV major core protein VP7 was directly conjugated to phycoerythrin. Cells were labelled with conjugated MAbs in single- and double-label immunofluorescence studies to identify specifically the BTV-infected cells in inoculated cultures. The viability of cells was determined by propidium iodide exclusion, and all analyses were done using flow cytometry. Productive infection of cultures of PBM cells was confirmed by virus titration. The data revealed a clear difference between subsets of bovine PBM cells in susceptibility to infection with BTV in vitro. Monocytes were readily infected with BTV, as were stimulated CD4+ cells, and infection was cytopathic to monocytes and stimulated lymphocytes. The proportion of infected cells decreased after 24 h and virus titres dropped markedly by 72 h in all cultures. CD4+ cells in cultures of unstimulated non-adherent cells inoculated with BTV showed increased expression of IL-2r. The possible relevance of these findings to the pathogenesis of BTV infection of cattle is discussed.