Expression of Fc-gamma-RIII and fibronectin in peripheral polymorphonuclear neutrophils with increased response to Fc stimulation in patients with juvenile periodontitis. The nature of increased luminol-enhanced chemiluminescence (CL) in peripheral polymorphonuclear neutrophils (PMN) in juvenile periodontitis is of pathophysiological interest and may serve as a model for tissue damage caused by granulocytes. Activation of PMNs by opsonized Staphylococcus aureus was compared with that of Actinobacillus actinomycetemcomitans and Bacteroides gingivalis, regarded as being more specific for juvenile periodontitis. The CL was higher in the PMNs from the patients, independently of bacteria and mode of opsonization (autologous serum or gamma-globulin). Fc-gamma-RIII assessed on the washed fragments from peripheral PMNs was significantly (p < or = 0.005) lower in the patients with juvenile periodontitis than in their pair-matched healthy controls, while the content of fibronectin was higher (p < or = 0.032). However, when Fc-gamma-RIII and fibronectin were studied in fresh PMNs by flow cytometry no difference could be found between the two groups. The increased generation of CL of peripheral PMNs found in patients with juvenile periodontitis seems to be independent of humoral factors and of bacterial species and may be related to the properties of the PMN cell membranes.


[Use of an immunoenzyme histochemical method for the improvement of the etiologic diagnosis of Aujeszky's disease in swine]. With regard to the legal regulations for the diagnosis of Aujeszky's disease in pigs, a retrospective immunoenzymatic study was performed on brains of 20 pigs, in which Aujeszky's disease had been diagnosed based only on clinical, macroscopical and histological findings, although the etiological agent could not be demonstrated neither by immunofluorescent technique nor by virological cultivation. Applying the peroxidase-antiperoxidase-(PAP) method, in 6 of 20 animals (30%) viral antigen of porcine herpesvirus type 1 was demonstrated in situ in at least two of five different locations of the brain. Reasons for negative results in immunohistological and virological investigations are discussed and the significance of the performed immunoenzymatic technique is pointed out.


Molecular cloning and characterization of the promoter region of the calcineurin A alpha gene. The 5'-flanking region of the calcineurin A alpha gene was isolated from a rat genomic library. It lacked TATA and CAAT boxes but contained G+C-rich regions, and was demonstrated to function as a strong promoter in neuronal cell lines (NG108-15 mouse neuroblastoma x rat glioma hybrid cells or N1E115 mouse neuroblastoma cells), but not in nonneuronal cell lines (C6 rat glioma or L-M mouse fibroblastoid cells) in a transient chloramphenicol acetyltransferase expression assay. Deletion analysis of the 5'-flanking region revealed that the core promoter region, as well as the sequence critical for cell-type-specific-promoter function, reside within the fragment -107 to +157 with respect to the major transcription initiation site.


Protein tyrosine phosphorylation and regulation of the receptor for platelet-activating factor in rat Kupffer cells. Effect of sodium vanadate. Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) and sodium vanadate (a phosphotyrosine phosphatase inhibitor) induced a time- and concentration-dependent increase in phosphotyrosine in several proteins and stimulated prostaglandin (PG) E2 production in cultured rat Kupffer cells. In addition, vanadate induced a decrease in the surface expression of AGEPC receptors and, as a consequence, inhibited AGEPC-stimulated PGE2 production. The vanadate-induced decrease in the surface expression of AGEPC receptors was time- and concentration-dependent and was partially prevented by genistein, a putative tyrosine kinase inhibitor. Upon removal of vanadate from the culture medium and re-incubation of cells in vanadate-free medium, the surface AGEPC receptors were restored within 7 h after the removal of vanadate. Both AGEPC- and vanadate-stimulated PGE2 formation was attenuated by genistein. Thus the present investigation demonstrates that both AGEPC and sodium vanadate stimulate tyrosine phosphorylation of cellular proteins, and vanadate induces a decrease in the number of the surface AGEPC receptors. These results suggest that protein tyrosine phosphorylation may play a role, directly or indirectly, in the regulation of surface expression of AGEPC receptors as well as in PGE2 production in response to vanadate and AGEPC.


Analysis of [3H]inositol phosphate formation and metabolism in cerebral-cortical slices. Evidence for a dual metabolism of inositol 1,4-bisphosphate. Muscarinic-receptor-mediated phosphoinositide hydrolysis in rat cerebral cortex was investigated by analysis of the kinetics of [3H]inositol phosphate formation and degradation in myo-[2-3H]inositol-labelled tissue slices. Carbachol stimulated rapid (5 s) increases in the concentrations of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,4)P2. Stimulated accumulation of [3H]Ins(1,3,4)P3, [3H]Ins(1,3)P2 and [3H]Ins(3,4)P2 and [3H]Ins(1/3)P or of [3H]Ins(4)P occurred only subsequently and with a sequence indicating formation by successive dephosphorylation of [3H]Ins(1,3,4,5)P4 or of Ins(1,4)P2 respectively. A similar sequence was inferred from the order of rapidity with which the accumulations of [3H]inositol polyphosphates, resulting from sustained (5 min) carbachol stimulation in the presence of LiCl, were reversed when muscarinic receptors were subsequently blocked with atropine. During this latter period of receptor blockade, radiolabel lost from [3H]inositol polyphosphates was quantitively recovered as [3H]inositol monophosphates owing to effective inhibition of monophosphatase by Li+, and the rate of poly- into mono-phosphate conversion was similar to agonist-stimulated rates of monophosphate accumulation. This implies that, even during persistent stimulation, polyphosphoinositide, not PtdIns, is the substrate for phosphoinositidase C. Quantitative comparison of the degradation of [3H]inositol poly- to mono-phosphates after receptor blockade unexpectedly suggests the dual hydrolysis of [3H]Ins(1,4)P2 to [3H]Ins(1)P and [3H]Ins(4)P. This result advises cautious interpretation of the origin of [3H]Ins(1)P in stimulated tissue, but, with other data presented, allows calculation from the observed ratio of [3H]Ins(1/3)P:[3H]Ins(4)P that a minimum of approx. 50% of the [3H]Ins(1,4,5)P3 produced during persistent muscarinic-receptor stimulation is metabolized by Ins(1,4,5)P3 3-kinase.


On the mechanism by which a heat shock induces trehalose accumulation in Saccharomyces cerevisiae. When the temperature of exponential-phase cultures of Saccharomyces cerevisiae was abruptly raised from 28 to 40 degrees C, trehalose immediately accumulated, whereas the activities of trehalase and trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase complex increased after a lag period of about 10 min. Heat shock also induced a sudden rise in intracellular glucose, simultaneously with a decrease in the concentration of hexose phosphate and fructose 2,6-bisphosphate. The increase of trehalose-metabolizing enzymes, but not the accumulation of glucose and trehalose, was prevented by cycloheximide. Investigation of the kinetic properties of partially purified enzymes showed that both non-activated and cyclic AMP-dependent-protein-kinase-activated forms of trehalase are almost inactive in the absence of Ca2+ and that the concentration of free Ca2+ required for half-maximal activity increased with increasing temperature, being approx. 1 microM at 30 degrees C and 20 microM at 40 degrees C for the activated form of trehalase. In contrast, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase were three times more active at 40 degrees C. It is proposed that the rapid accumulation of trehalose induced by heat shock may be in part explained by changes in the kinetic properties of trehalase and trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase.


A comparison of the zinc contents and substrate specificities of the endothelial and testicular forms of porcine angiotensin converting enzyme and the preparation of isoenzyme-specific antisera. Angiotensin converting enzyme (ACE; EC 3.4.15.1) was purified from porcine kidney and lung (endothelial isoenzyme) and testis (testicular isoenzyme) by affinity chromatography on lisinopril-2.8 nm-Sepharose. Atomic-absorption spectroscopy revealed that ACE purified from kidney and lung contained 2.58 and 2.35 atoms of zinc per molecule of enzyme (M(r) 147,000) respectively. In contrast, ACE purified from testis contained only 1.58 atoms of zinc per molecule of enzyme (M(r) 80,000). Thus it would appear that both putative zinc-binding sites in endothelial ACE contain zinc and may therefore be catalytically active. No differences were observed in the pattern of products generated on hydrolysis of benzoyl (Bz)-Gly-His-Leu, substance P, luteinizing-hormone-releasing hormone (LH-RH) and its analogue, des-Gly10-LH-RH-ethylamide, by kidney and testicular ACE. There was also no difference in the initial rates of hydrolysis of Bz-Gly-His-Leu or substance P by the two isoenzymes, although LH-RH and its analogue were hydrolysed twice as rapidly by kidney ACE. It is therefore unlikely that the N-terminal catalytic site in porcine endothelial ACE is predominantly responsible for the atypical cleavage of LH-RH generating the N-terminal tripeptide. Two polyclonal antisera were raised to the affinity-purified forms of pig kidney and testicular ACE. Isoenzyme-specific antisera were then isolated from these by absorbing out those antibodies recognizing determinants on the other isoenzyme. Immunoelectrophoretic blot analyses and immunofluorescent staining of sections of pig kidney were used to demonstrate the specificity of the antisera. Immunofluorescent staining of sections of pig testis with the antiserum specific to testicular ACE localized testicular ACE solely to the lumen of the seminiferous tubules, whereas the antiserum specific to endothelial ACE revealed the presence of this isoenzyme only in blood vessels. The antiserum to endothelial ACE, which recognizes determinants in the unique N-terminal domain, was investigated as a possible specific inhibitor of the N-terminal catalytic site. Although this antiserum failed to inhibit testicular ACE, the effect on the activity of endothelial ACE appeared to be due to inhibition of both the N- and C-terminal catalytic sites.


Basal and phorbol-ester-stimulated turnover of phosphatidylcholine in HeLa cells involve different pathways. HeLa cells prelabelled with [3H]lysoplatelet-activating factor (lyso-PAF) accumulated phosphatidic acid (PtdOH) when incubated in the presence of either propranolol (an inhibitor of PtdOH phosphohydrolase) or phorbol ester. In the presence of ethanol, phorbol ester but not propranolol stimulated the accumulation of phosphatidylethanol, an index of phospholipase D activity. Incubation of cells with [3H]lyso-PAF led to a rapid accumulation of label in diacylglycerol (DG) followed by a delayed accumulation in PtdOH. It is concluded that propranolol-induced PtdOH accumulation is derived from DG by DG kinase and does not involve phospholipase D.


N-(3-oxohexanoyl)-L-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora. Erwinia carotovora A.T.C.C. 39048 produces the antibiotic 1-carbapen-2-em-3-carboxylic acid. A number of mutants with a carbapenem-non-producing phenotype were selected as part of an investigation into the molecular and genetic basis of carbapenem biosynthesis. Cross-feeding studies revealed that the mutants fell into two discrete groups. Group 1 mutants were found to secrete a diffusible low-molecular-mass compound which restored carbapenem production in group 2 mutants. This compound was isolated from the spent culture supernatant of a group 1 mutant using solvent extraction, hydrophobic-interaction chromatography and silica-gel chromatography, and finally purified by reverse-phase semipreparative h.p.l.c. M.s. and n.m.r. spectroscopy revealed that the compound was N-(3-oxohexanoyl)homoserine lactone. Both D- and L-isomers were synthesized, and subsequent analysis by c.d. established that the natural product has the L-configuration. Although carbapenem production was restored by both isomers, dose-response curves indicated that the L-isomer has greater activity, with an induction threshold of about 0.5 micrograms/ml. N-(3-Oxohexanoyl)-L-homoserine lactone is, therefore, an autoregulator of carbapenem biosynthesis rather than a biosynthetic intermediate. This compound is already known for its role in autoinduction of bioluminescence in the marine bacterium Vibrio fischeri. It is also structurally-related to the A- and I-factors which are known to regulate production of antibiotics in some Streptomyces species. Its association in this work with the regulation of carbapenem biosynthesis implies a broader role for autoregulator-controlled gene expression in prokaryotes.


The proto-oncogenes c-fos and c-jun modulate thyroid hormone inhibition of human thyrotropin beta subunit gene expression in opposite directions. The sequence from -1 to +6 bp in the hTSH beta gene contains overlapping putative thyroid hormone and AP-1 response elements. We demonstrate interaction between the AP-1 constituents c-fos and c-jun and thyroid hormone receptor in this region by transient transfection experiments using a -125 to +37 bp hTSH beta fragment. T3 inhibition was completely abolished by c-jun, but increased threefold by c-fos. A single transversion mutation at +2 bp restored T3 inhibition in the presence of c-jun and markedly reduced binding of purified c-jun by gel mobility shift assay. Thus, c-fos and c-jun influence T3 inhibition of hTSH beta expression in opposite directions acting through a response element shared with thyroid hormone receptor. Control of the relative cellular levels of these two proto-oncogenes may play a major role in modulating thyroid hormone inhibitory responses.


Differentiation-inducing activity of retinoic acid isomers and their oxidized analogs on human promyelocytic leukemia HL-60 cells. Retinoidal activity of retinoic acid isomers [all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9CRA) and 13-cis-retinoic acid (13CRA)] and their oxidized derivatives [19-hydroxy and 19-oxo derivatives of ATRA (19-hydroxy-ATRA and 19-oxo-ATRA), 19-oxo derivative of 9CRA (19-oxo-9CRA), and 19-hydroxy derivative of 13CRA (19-hydroxy-13CRA)] was evaluated by means of a human promyelocytic leukemia HL-60 cell differentiation induction assay. All the compounds examined showed this activity with ED50 values of 2-30 nM, which are in accordance with their binding activity to nuclear retinoic acid receptors (RARs).


Regulation of human calcitonin gene transcription by cyclic AMP. Transcription of the human calcitonin (CT) gene is markedly increased by cAMP in the TT line of medullary thyroid carcinoma. This response is conferred by 5' flanking DNA sequences located between -132 and -252 relative to the transcription initiation site. Within this region are an upstream cyclic AMP response element (CRE), a downstream CRE flanked by two octamer motifs, and two adjacent C-rich AP2-like sequences. In transfection experiments in TT cells, the downstream CRE, combined with CT promoter sequences, generated 70% of the maximal cAMP response. The upstream CRE and the C-rich elements conferred 10 and 30% of this response, respectively. In gel mobility shift assays, specific TT cell proteins bound to each of these sequences. Therefore, the cAMP response of the CT gene is complex, requiring multiple elements acting in concert.


Anti-viral activity of human recombinant heparin-binding proteins HBNF and MK. Herpes simplex viruses bind to cell surface heparan sulfate proteoglycans, as a first step of viral infection. We report here that two recombinant heparin-binding proteins HBNF and MK inhibit infectivity of human herpes simplex viruses types 1 and 2 and human cytomegalovirus. Carboxymethylated HBNF and MK, which retain affinity for heparin-Sepharose, do not exhibit anti-viral activities. Arguments are presented that anti-viral effects of HBNF and MK are due to the competition for the specific binding to the cell surface heparan sulfate proteoglycans.


Reactivity of [Fe] and [Ni-Fe-Se] hydrogenases with their oxido-reduction partner: the tetraheme cytochrome c3. In order to understand the electron transfer mechanisms for the [Fe] and [Ni-Fe] hydrogenases, a kinetic study of cytochrome c3 reduction has been undertaken. Cyclic voltammetry and controlled-potential amperometry techniques have been used to investigate the intermolecular electron-transfer reaction between cytochrome c3 and [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough. Electron-transfer cross-reactions between [Fe] or [Ni-Fe-Se] hydrogenase and cytochrome c3 from Desulfovibrio vulgaris Hildenborough or Desulfovibrio desulfuricans Norway have been studied. Some structural implications are considered from these experimental data.


A Na pump inhibitor from bovine posterior pituitary: purification, structure determination and its cardiovascular effect in rat. We examined the hypothesis that hypothalamo-hypophysial tissue contains an endogenous Na pump inhibitor. From bovine posterior pituitary, we purified a substance which inhibits Rb uptake by human erythrocytes. This inhibitory activity was found in the eluate of 10% acetonitrile from a C18 flash column and purified by subsequent three steps of reversed-phase high-performance liquid chromatography (HPLC). Sequence analysis revealed that this substance was identical to joining peptide, one of the major products of proopiomelanocortin (POMC). This peptide had hypertensive and tachycardiac effects in spontaneously hypertensive rats (SHR) after central administration, with weak Na,K-ATPase inhibitory activity (IC50 = 0.5 mM).


Two types of inositol trisphosphate binding in cardiac microsomes. Two distinct types of [3H]IP3 binding were found in canine cardiac microsomes with high (Kd = 21 nM, Bmax = 0.66 pmol/mg) and low affinity (Kd = 230 nM, Bmax = 2.9 pmol/mg). Also found were low affinity [3H]IP4 binding (Kd = 190 nM, Bmax = 4.5 pmol/mg) and high affinity [3H]IP6 binding (Kd = 10 nM, Bmax = 4.9 pmol/mg). The rank order of potency to displace these radioligands indicates that binding of IP3 and IP6 is ligand-specific. Sucrose gradient centrifugation of the detergent-solubilized cardiac microsomes indicates that the molecular size of the cardiac high affinity IP3 receptor is similar to that of the aortic smooth muscle IP3 receptor and smaller than that of the ryanodine receptor which migrates more rapidly. The IP4 and IP6 binding migrates more slowly than the IP3 receptor.


Enhancement by staurosporine of platelet-activating factor formation in N-formyl peptide-challenged human neutrophils is mediated by intracellular platelet-activating factor binding sites. Staurosporine potentiates the formation of platelet-activating factor (PAF) and causes a sustained elevation of intracellular Ca2+ ([Ca2+]i). WEB 2086, a specific PAF-receptor antagonist, inhibits both potentiation of PAF formation and elevation of [Ca2+]i by 78% and 65%, respectively. Moreover, the PAF produced by FMLP and/or Staurosporine was completely retained in the cell. This suggests that the effect of staurosporine in FMLP-stimulated neutrophils may be mediated by the action of endogenously produced PAF, which in turn leads to an increase in [Ca2+]i and PAF formation. We conclude that PAF is the major product of human neutrophils which reacts via specific intracellular PAF binding sites to stimulate the phospholipase A2, and its synthesis is under control of a staurosporine-sensitive protein kinase.


Protein kinase C activity is rate limiting for shedding of the interleukin-6 receptor. An analysis of the mechanism of generation of the soluble interleukin-6 receptor (IL-6R) has been performed. The membrane-bound receptor is proteolytically cleaved to release a soluble receptor form which retained its ligand binding capacity. Furthermore, the soluble IL-6R is unique in its ability to induce a biological signal in complex with the ligand interleukin-6 (IL-6) on cells which by themselves do not bind IL-6. Shedding of the IL-6R is strongly activated by PMA and can be inhibited by the protein kinase inhibitor staurosporine. The generation of the IL-6R is not dependent on protein synthesis. The inactive PMA analogue 4-alpha-phorbol-12,13-didecanoate fails to induce shedding of the IL-6R. Transfection of a protein kinase C expression plasmid into IL-6R expressing cells leads to enhanced shedding of the receptor. These experiments clearly show that protein kinase C regulates shedding of the IL-6R.


Discordant estimates of heterologous promoter activity as determined by reporter gene mRNA levels and enzyme activity. In this study, the human cytomegalovirus (CMV) promoter fused to the lacZ (beta-gal) reporter gene was transfected into neuroblastoma SK-N-BE(2)-C cells, and phorbol ester-stimulated promoter activity assessed by both PCR quantitation of reporter gene mRNA levels and enzyme activity. Surprisingly, significant differences were observed in the induction profile of CMV promoter activity as judged by these two independent methods of analysis. For example, at 24 hrs post-transfection beta-gal activity was elevated 7.3-fold in phorbol ester-treated cells, whereas 2.4-fold increases were observed in the cognate mRNA levels. These findings demonstrate the efficacy of quantitative PCR methodology to evaluate promoter activity in DNA-mediated cell transfection analyses, and raise a cautionary note on the reliance of reporter gene enzyme activity to estimate the transcriptional activity of heterologous promoters.


Expression and regulation of cytosolic phosphoenolpyruvate carboxykinase in 3T3-L1 adipocytes. Animal studies have shown that the gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK) is controlled by unique mechanisms in fat. For example, a unique cis-acting DNA sequence located 1-2 kilobase pairs upstream of the promoter is required for PEPCK gene expression in adipocytes but not in other cell types. Moreover, glucocorticoids repress PEPCK gene transcription in fat whereas these steroids induce the same gene in liver and kidney. An in vitro system of cultured adipocytes would greatly facilitate studies of PEPCK gene regulation in fat cells. In this study, we report that cultured 3T3-L1 cells activate the PEPCK gene upon differentiation from fibroblasts to adipocytes. In addition, we report that cAMP induces and insulin and dexamethasone repress PEPCK mRNA in 3T3-L1 adipocytes. Thus these cells may provide a useful model system for future studies.


Regulation of cyclic AMP metabolism in bovine adrenal medullary cells. The capacity of cultured bovine adrenal medullary cells to metabolize and export cyclic AMP has been studied. Basal cellular cyclic AMP levels were increased 50% by 100 microM 3-isobutyl-1-methylxanthine (IBMX) and rolipram, a class IV (cyclic AMP-specific) phosphodiesterase (PDE) inhibitor. They were not affected by inhibition of class I (Ca2+/calmodulin-dependent), class III (cyclic GMP-inhibited) or class V PDE (cyclic GMP-specific) with vinpocetine or 3-isobutyl-8-methoxymethyl-1-methylxanthine (8-methoxymethyl-IBMX), SK&F 94120, or MB 22,948, respectively, all at 100 microM. Furthermore, only IBMX and rolipram enhanced the cyclic AMP response to 0.3 microM forskolin. Rolipram had an EC50 of < or = 1 microM and was equally effective at 100 microM and 1 mM. IBMX enhanced cyclic AMP levels significantly more at 1 mM than at 100 microM. Neither vinpocetine nor 8-methoxymethyl-IBMX (100 microM) enhanced the Ca(2+)-dependent cyclic AMP response to K+ depolarization. Elevation of cyclic GMP levels with sodium nitroprusside (10 or 100 microM), to activate any cyclic GMP-stimulated class II PDE and to inhibit any cyclic GMP-inhibited class III PDE, also had no effect on basal or forskolin-stimulated cyclic AMP levels. In the presence of IBMX (1 mM), forskolin (5 microM) caused a rapid and large increase in cellular cyclic AMP levels which was maximal after about 5 min and declined slightly over 3 hr. Over this period, extracellular cyclic AMP levels rose almost linearly reaching levels 2-3 times those in the cells. The results indicate bovine adrenal medullary cells have a high capacity for sustained cyclic AMP export. Furthermore, two PDE isozymes appear to degrade cyclic AMP in these cells, a rolipram-sensitive, cyclic AMP-specific, class IV isozyme and a rolipram-insensitive isoform.


A carboline derivative as a novel mammalian DNA topoisomerase II targeting agent. The DNA intercalating, ellipticine analog drug, 5,11-dimethyl-5H-indol[2,3-b]quinoline, is able to stabilize in vitro the topoisomerase II-DNA cleavable complex and to induce DNA breaks in BPV I episome in rat fibroblasts. Cytotoxicity studies with DC3F cells resistant to ellipticine strongly suggest that topoisomerase II is a cellular target involved in the mechanism of cytotoxic action of this carboline derivative.


8-(4-Chlorophenyl)thio-cyclic AMP is a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA). 8-(4-Chlorophenyl)thio-cyclic AMP (8-CPT-cAMP), extensively used as selective activator of cyclic AMP-dependent protein kinase, has been found to be a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA). Indeed, 8-CPT-cAMP (IC50 = 0.9 microM) inhibited PDE VA with a potency identical to that of zaprinast. 8-CPT-cAMP was also metabolized by PDE VA at a rate half that of cyclic GMP. The cyclic GMP-inhibited phosphodiesterase (PDE III) (IC50 = 24 microM) and the cyclic AMP-specific phosphodiesterase (PDE IV) (IC50 = 25 microM) were also inhibited by 8-CPT-cAMP. In contrast, most of the other cAMP-derivative studies showed little inhibition of any phosphodiesterase isoenzyme. These observations provide further reasons why the mechanism of the physiological effects of 8-CPT-cAMP should be interpreted with caution.


Prostaglandin E2-induced arachidonic acid release and catecholamine secretion from cultured bovine adrenal chromaffin cells. We recently reported that prostaglandin E2 (PGE2) and arachidonic acid (AA) each induced a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells in the presence of ouabain by stimulation of phosphoinositide metabolism. In the present study, we examined the relationship between phospholipase A2 and C activation and catecholamine secretion by PGE2 in chromaffin cells. The phospholipase A2 inhibitors p-bromophenacyl bromide and mepacrine did not affect the basal and ouabain-induced release, but dose-dependently blocked PGE2-evoked phosphoinositide metabolism and the consequent catecholamine release at an IC50 value of 3 microM. PGE2 induced rapid hydrolysis of [3H]AA from prelabeled phospholipid pools: the release of [3H]AA could be detected at as early as 15 sec and reached a plateau after 1 min. While the phospholipase C inhibitor neomycin did not inhibit PGE2-induced AA release, phospholipase A2 inhibitors dose-dependently inhibited it at IC50 values comparable to those for catecholamine release. Pretreatment of intact cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, but not with pertussis toxin, prevented AA release by PGE2. These results demonstrate that PGE2 activates phospholipase A2 as well as phospholipase C in a pertussis toxin-insensitive manner and suggest that the released arachidonic acid may be involved in PGE2-induced catecholamine release from chromaffin cells.


Effect of gallium on the tyrosyl radical of the iron-dependent M2 subunit of ribonucleotide reductase. Gallium, a pharmacologically important metal which resembles iron, was shown in previous studies to inhibit ribonucleotide reductase. To better understand its mechanism of action, we have examined the interaction of gallium with the iron-dependent M2 subunit of ribonucleotide reductase. In its active form, M2 contains an iron center and a tyrosyl free radical which is detectable by ESR spectroscopy. In the present study, cytoplasmic extracts prepared from murine leukemic L1210 cells after an 18-hr incubation with 960 microM gallium nitrate displayed a > 60% inhibition in their M2 tyrosyl radical ESR signal. However, this signal was restored within 15 min to levels greater than that of controls by the addition of increasing concentrations of ferrous ammonium sulfate. Gallium citrate added directly to cytoplasmic extracts from control cells also decreased the tyrosyl radical signal, an effect which could be reversed by iron. Immunoblot analysis revealed that incubation with gallium did not diminish the amount of M2 protein in cells, thus indicating that the decrease in the tyrosyl radical signal was not due to a decrease in cellular M2 content. In immunoprecipitation studies of 59Fe-labeled M2, gallium displaced 55-60% of the 59Fe incorporated into M2. Our studies suggest that gallium displaces iron from the M2 subunit of ribonucleotide reductase, resulting in a loss of the tyrosyl radical and an accumulation of inactive M2 within the cell.


[Intracomplex alkylation of octanucleotide pd(TGTTTGGC) by a 5'-(4-N-methyl-N-(2-chloroethyl)-amino)benzylphosphamide derivative of heptanucleotide pd(CCAAACA). Preparation of a covalent adduct and study of its spatial structure in an aqueous solution by two-dimensional (1)H-NMR spectroscopy]. Covalent adduct--the product of intracomplex alkylation at N-3-position of dC-8-nucleoside residue of target octanucleotide pd[TGTTTGGC] was completely synthesized by means of 4-[N-methyl-N-(2-chloroethyl)amino]benzyl-5'-phosphamido derivative of heptanucleotide pd[CCAAACA]. Its melting temperature was shown to be 70 degrees C. Tm did not depend on covalent adduct concentration and was by 40 degrees C higher than that for unmodified duplex pd[TGTTTGGC].pd[CCAAACA] at concentration of 0.5 x 10(-4) M. The spatial structure of the covalent adduct in aqueous solution was investigated by two-dimensional 3H-NMR spectroscopy. The assignment of oligonucleotide protons as well as protons of a modifying group was carried out using COSY, COSY-DQF and NOESY experiments. Conformational analysis of proton-proton coupling constants for H1', H2'a, H2'b and H3' protons showed the sugar residues to be in 2'-endo conformation. Analysis of NOE connectivities observed between the protons of the alkylating group and oligonucleotide protons yielded conclusion, regarding the 4-[N-methyl-N-(2-chloroethyl)amino]benzylamido 5'-residue being localized in the region of the lacked nucleoside residue of the heptanucleotide chain about 5 A apart from the dC-1 residue and from cytosine base of the alkylated dC-8 residue.


[Approach to studying natural oligopeptides in the area of structure relationship to function]. Structure and structure-function relations of naturally occurring oligopeptides and peptide receptors are discussed. An approach to inferring function of low-molecular peptides in the direction from their structure is postulated. Diverse biological activities of oligopeptides supposedly arise from a limited number of preferable spatial structures which may exist under physiological conditions. Each particular function of an oligopeptide is connected with a definite spatial structure, belonging to the set of the low-energy conformations. A method is suggested for constructing a synthetic analogue with a predetermined physiologically active conformation, prior to all chemical and biological tests.


General pharmacological properties of the main metabolite of flosequinan. The general pharmacological profile of 7-fluoro-1-methyl-3-(methylsulfonyl)- 4(1H)-quinolone BTS 53 554, CAS 76568-68-8), the main metabolite of a new vasodilator, flosequinan (BTS 49 465), was investigated. 1. The central nervous system: BTS 53 554 at the dose of 30 mg/kg i.v. caused an increase in respiratory rate and a sedation in general behavior in rats. The drug also inhibited acetic acid-induced writhing and slightly decreased normal body temperature in mice. However, the drug at the doses up to 30 mg/kg i.v. had little effect on the spontaneous movement, hexobarbital-induced hypnosis, reserpine-induced hypothermia and motor coordination in mice. The drug showed neither anticonvulsant nor analgesic actions in mice. Furthermore, it had no effect on the spontaneous EEG, sleep-wakefulness cycle and EEG arousal response in rabbits at doses up to 10 mg/kg intravenously. 2. The somatic nervous system: BTS 53 554 induced no muscle relaxation in mice and exerted no local anesthetic action in guinea pigs by corneal reflex method. In addition, it had little effect on the neuromuscular transmission in cats. 3. The autonomic nervous system and smooth muscle: BTS 53 554 showed no effect on the sympathetic ganglionic transmission in cats. In isolated smooth muscles, at doses up to 10(-3) mol/l it showed little effect on the acetylcholine- or barium chloride-induced contraction of guinea-pig ileum, norepinephrine-induced contraction of rat vas deferens or oxytocin-induced contraction of nonpregnant rat uterus. However, it inhibited non-competitively norepinephrine-induced contraction of isolated rat aorta at 10(-4) mol/l or more and serotonin-induced contraction of isolated rat fundus at 3 x 10(-4) mol/l or more. In the isolated guinea-pig ileum, the drug slightly inhibited the histamine-induced maximal contraction at 10(-3) mol/l. These results suggest BTS 53 554 had no specific effect on norepinephrine, serotonin, acetylcholine or histamine. The drug relaxed isolated guinea-pig trachea at 3 x 10(-5) mol/l or more and inhibited the spontaneous movement of isolated pregnant rat uterus at 10(-4) mol/l or more, although these actions were extremely weaker than those of isoproterenol (isoprenaline). BTS 53 554 also showed a slight inhibition of uterus movement in anesthetized rats at 30 mg/kg intravenously. 4. The digestive system: BTS 53 554 tended to inhibit the gastrointestinal propulsion in mice and showed a slight inhibition of gastric and intestinal motilities in rats at 10 mg/kg intravenously.(ABSTRACT TRUNCATED AT 400 WORDS)


Bovine viral diarrhea virus-specific neutralizing antibodies induced by anti-idiotypic antibodies. Two murine neutralizing monoclonal antibodies (MAbs), 4D8 and 6D11, recognizing epitopes on gp53, a surface glycoprotein of bovine viral diarrhea virus (BVDV), were used to generate anti-idiotypic antibodies (anti-ids) in a calf. The polyclonal anti-ids were isolated from serum by affinity chromatography on their respective Ab-1-Sepharose columns, followed by repeated adsorption on isotype-matched antibody-Sepharose columns. The anti-ids reacted specifically with their respective Ab-1, but not with isotype-matched controls. They also inhibited the binding of their Ab-1 to BVDV in a concentration-dependent manner. Mice immunized with the two anti-id preparations developed antibodies to BVDV, which neutralized the virus in vitro.


Further characterization of a broad-spectrum antiviral substance in human serum. A broadly active antiviral glycoprotein (UTI beta) occurs naturally in human sera at an average antiviral titer of 50 U/ml. This inhibitor is active against all virus families tested to date, including representative poxviruses, herpesviruses, enteroviruses, paramyxoviruses, alpha-viruses, flaviviruses, bunyaviruses, and rhabdoviruses. It is a glycoprotein of approximately 60,000 +/- 10,000 Da, which is stable at pH 2 to 10 and at 80 degrees C for up to 10 min. Mild oxidation with NaIO4 and treatment with glycosidases inactivates the material. Proteolytic degradation of the inhibitor molecule releases small active components of < 1000 Da, which retain antiviral activity. This activity of the small components has increased heat stability (120 degrees C for 15 min) and is inactivated by glycosidases. The antiviral activity thus appears to reside mainly in the oligosaccharide moiety of the glycoprotein. The inhibitor does not neutralize virions, but prevents attachment of most viruses to cells. These properties occur also in highly purified preparations. These findings indicate that human serum contains significant concentrations of a broadly active antiviral glycoprotein, which is distinct from interferon and other antiviral substances naturally found in human body fluids and tissues.


Neuroethological evaluation of audiogenic seizures and audiogenic-like seizures induced by microinjection of bicuculline into the inferior colliculus. II. Effects of nigral clobazam microinjections. Male Wistar rats were classified as susceptible (S) and resistant (R) to audiogenic seizures (AS) by evaluation of their response to high-intensity sound stimulation (110.3 dB). R rats injected with bicuculline into the inferior colliculus (IC) preferentially displayed audiogenic-like seizures with gyri, jumping and atonic falling, without important tonic-clonic components but with postictal contralateral asymmetry and hyperreactivity. These audiogenic-like seizures were blocked by clobazam microinjection into the substantia nigra (SN) and partially modified by SN vehicle injection. Injection of vehicle or clobazam into the SN of susceptible rats (S) did not modify the occurrence of AS. This may suggest the participation of GABAergic regulation in the development of audiogenic-like seizures in R rats and a defect in GABAergic neurotransmission in S rats.


Neuroethological evaluation of audiogenic seizures and audiogenic-like seizures induced by microinjection of bicuculline into the inferior colliculus. I. Effects of midcollicular knife cuts. Audiogenic seizures (AS) are a model of generalized tonic-clonic seizures. The inferior colliculus (IC) and the GABAergic neurotransmission seems to be the most critical site and neurotransmitter system, respectively, of the auditory midbrain involved in AS origin and development. Thus, audiogenic-like seizures are evoked by GABAA antagonists such as bicuculline (BIC). Wistar audiogenic AS resistant (R) rats were sham-transected through the midcollicular line and microinjected with IC bicuculline (BIC; 80 ng/0.2 microliters) (n = 8); transected through the midcollicular line and microinjected with IC saline 0.9% (n = 8); transected through the cortex above the midcollicular line and microinjected with IC BIC (n = 3); transected through the midcollicular line up to 6.0 mm depth and microinjected with IC BIC (80 ng/0.2 microliters or 120 ng/0.3 microliters (n = 8). Wistar AS susceptible (S) rats were submitted to cortical transections (n = 8) and midcollicular transections (n = 7). Animals were studied by means of an ethological method before and after microinjections and/or transections in order to evaluate possible pathways in the AS-like evoked seizures. Bicuculline-evoked seizures were very similar to those evoked by acoustic stimulation, but lacked the tonic-clonic component. No modification in animal behavior was observed in the presence of sound, once the AS-like behavior was initiated. A small percentage of the animals, however, presented procursive behavior which was increased by sound. The IC BIC-evoked patterns were almost totally blocked by midcollicular but not cortical transections. Furthermore, midcollicular but not cortical transections blocked the tonic-clonic component of AS in genetically S animals without modifying the wild running component. These data suggest that the inferior colliculus-superior colliculus connection may be involved in the sensorimotor transduction necessary for AS-like behaviors.


Differential involvement of gustatory insular cortex and amygdala in the acquisition and retrieval of conditioned taste aversion in rats. Lesion studies of the role of the gustatory insular cortex (GC) and amygdala (Am) in conditioned taste aversion (CTA) are confounded by the irreversibility of the intervention. Functional ablation methods allow more specific influencing of different phases of CTA acquisition and retrieval. Bilateral tetrodotoxin (TTX) blockade of GC (10 ng) or Am (3 ng) before or after saccharin drinking in rats with chronically implanted intracerebral cannulae showed that GC is indispensable for the initial processing of the taste stimulus but not for the association of the gustatory trace with the symptoms of LiCl poisoning. Gustatory signals can by-pass the blocked Am, the inactivation of which, however, impairs the gustatory trace-poisoning association. TTX injection into both GC and Am impairs CTA retrieval more than isolated blockade of either of these structures. It is argued that GC and Am implement processing of gustatory and visceral signals, respectively, but that formation and consolidation of the CTA engram proceeds outside forebrain, probably at the level of the brainstem.


Inhibition of ingestive behavior following fourth ventricle bombesin injection in chronic decerebrate rats. To test the hypothesis that the effects of 4th ventricle bombesin (BN) injection on feeding require interaction with forebrain neural systems, intraoral sucrose (0.1 M) was measured in tube-fed control and tube-fed supracollicular decerebrate rats after 4th ventricle injections of 1, 5, 10, 20, and 50 ng BN. Fourth ventricle injections of all doses of BN reliably suppressed sucrose intake in both control and chronic decerebrate rats. These results indicate that caudal brain stem afferent signals produced by 4th ventricle BN injections are integrated by the local neural circuitry of the caudal brain stem, independent of the forebrain systems, to modulate ingestive behavior.


Paraventricular hypothalamic clonidine increases rather than decreases susceptibility to activity-based anorexia in the rat. Anorexia has been related to reduced activity of the paraventricular hypothalamic (PVN) noradrenergic-feeding system. In this study we determined whether clonidine (an alpha 2-adrenergic agonist) infused into the PVN reduced susceptibility to activity-based anorexia (ABA) in the rat. In Experiment 1, clonidine (6 doses) was chronically infused into the PVN of male Sprague-Dawley rats. All animals were exposed to ABA (1.5 hr/day food access; 22.5 hr/day running wheel access) until a 25% body weight loss was reached. Dose-related increases in susceptibility to ABA and decreases in food intake were observed. In Experiment 2, for which heavier animals and 3 doses of clonidine were used, we found no difference in food intake and wheel activity but increased susceptibility to ABA. Chronic clonidine infused into the PVN does not produce hyperphagia and exacerbates rather than attenuates susceptibility to ABA.


Reversible lesions of the cerebellar interpositus nucleus during acquisition and retention of a classically conditioned behavior. Previous lesion, recording, and stimulation studies have implicated the cerebellum and its associated circuitry as essentially involved in classical conditioning of discrete, somatic muscle responses. In 2 experiments, the interpositus nucleus of the cerebellum was assessed for the plasticity associated with learning and memory of the nictitating membrane (NM) response by using local cooling as a reversible lesion technique. In well-trained animals (Experiment 1), NM conditioned responses (CRs) were abolished during cooling of the interpositus but reappeared when the interpositus returned to body temperature. This cooling-warming protocol could be repeated many times. Cooling could be prolonged (one session, approximately 1 hr) with recovery of NM CRs as tested on the next day. Multiple-unit recordings related to learning were also absent in the interpositus and red nucleus during cooling. In naive animals (Experiment 2), both behavioral and unit CRs did not develop while training with cooling. There was no evidence of savings when training continued without cooling: Behavioral and unit CRs developed as if the animals were still naive. These results support the idea that the interpositus nucleus of the cerebellum is the critical locus for learning and memory of this classical CR.


An auditory conditioned stimulus modulates unconditioned stimulus-elicited neuronal activity in the cerebellar anterior interpositus and dentate nuclei during nictitating membrane response conditioning in rabbits. The effects of the presentation of an auditory conditioned stimulus (CS) on unconditioned stimulus (US)-elicited neuronal activity in the anterior interpositus (AIPN) and dentate (DN) nuclei of the cerebellum were investigated during the initial stages of classical conditioning of the nictitating membrane (NM) response in rabbits. In Experiment 1, a 500-ms CS (but not a 30-ms CS) facilitated US-elicited single-unit activity in the AIPN and depressed US-elicited activity in the DN during training. In Experiment 2, lesions of the AIPN but not of the DN prevented acquisition of conditioned NM responses. The results are interpreted within the framework of a model of classical conditioning that proposes that conditioned neuronal activity that underlies behavioral plasticity develops from the modulation of US-elicited neuronal activity by the CS.


Lateralized contributions of the cerebral cortex, parabrachial nucleus, and amygdala to acquisition and retrieval of passive avoidance reaction in rats: a functional ablation study. Reversible blockade of the neocortex by spreading depression and of the parabrachial nucleus (PBN) and amygdala (AMG) by tetrodotoxin (TTX) injection was used to study lateralization of passive avoidance reaction (PAR) engrams in rats. In Experiment 1, PAR acquisition was not disrupted by posttrial TTX applied into the PBN or AMG or into both of these structures in the same hemisphere but was impaired when the AMG and PBN were blocked in opposite hemispheres. In Experiment 2, retrieval of PAR acquired with intact brain was not impaired by unilateral TTX blockade of the AMG. In Experiment 3, PAR was acquired with posttrial TTX blockade of the AMG and PBN in one hemisphere. Full or partial block of PAR retrieval was induced by functional decortication of the contra- or ipsilateral hemisphere, respectively, whereas blockade of either AMG was ineffective. It is concluded that PAR acquisition requires ipsilateral interaction of the AMG and PBN and that the two hemicortices contribute differentially to the retrieval of engrams formed under such conditions.


Biophysical and behavioral correlates of memory storage, degradation, and reactivation. Neural correlates of associative memory and "forgetting" were observed 1, 6, and 14 days after acquisition of a conditioned response (CR) in the marine snail Hermissenda. Behavioral expression of a light-rotation association, as indexed by contraction of the animal's foot in response to light, dissipated throughout the 14-day interval such that a CR was observed 1 and 6 days after conditioning but was absent 14 days later. In relation to naive or pseudoconditioned animals, membrane resistance (inversely related to neuronal membrane conductance and directly related to excitability) of the isolated Type B photoreceptor (B cell) was elevated in conditioned animals on Days 1 and 6, whereas no elevation was detectable on Day 14. However, both the behavioral response and the elevated membrane resistance in conditioned animals were hypersensitive to light-rotation pairings (i.e., exhibited "savings") on Day 14, which is indicative of a latent memory trace. In a second experiment, a current-induced depolarization of the B cell after 14 retention days resulted in an increase in input resistance of the B cell membrane in previously conditioned animals but a weaker, transient rise in resistance in B cells from animals exposed to the nonassociative control procedure 14 days earlier. This effect was Ca(2+)-dependent because no rise in resistance was observed if Ca2+ was removed from the extracellular bath. These results indicate that modification of membrane conductance (i.e., elevated resistance), although apparently critical for the behavioral expression of the memory, is not essential for the maintenance of the latent memory trace, whereas Ca2+ hypersensitivity may be a principal contributor to the storage of a latent memory trace and memory reactivation.


Morphine- and naltrexone-induced modification of palatability: analysis by the taste reactivity test. We used the taste reactivity (TR) test, a direct measure of the hedonic properties of a tastant, to assess in Sprague-Dawley rats the ability of morphine (an opiate agonist) and naltrexone (an opiate antagonist) to modify the palatability of a bitter quinine solution and a sweet sucrose solution. Morphine reduced the aversive hedonic properties of both novel and familiar quinine solution (0.05% and 0.1%) but did not modify the palatability of 20% sucrose solution. Naltrexone reduced the positive hedonic properties of sucrose solution (2% and 20%) but did not modify the palatability of 0.05% quinine solution. The pattern of results suggests that the modification of feeding produced by opiate agonists and antagonists may be mediated by an hedonic shift in the palatability of the tastant.


Frequent oropharyngeal shedding of Epstein-Barr virus in homosexual men during early HIV infection. To determine the frequency of Epstein-Barr virus (EBV) oropharyngeal shedding during HIV infection in homosexual men in the Multicenter AIDS Cohort Study. The cohort consisted of 210 men who were HIV-seronegative at their baseline study visit, 39 of whom seroconverted to HIV at a later date, and 73 asymptomatic and mildly symptomatic men with HIV infection of indeterminate duration. EBV in throat washings was detected by transformation of newborn cord blood lymphocytes. EBV was isolated from 49% (35 out of 71) of the HIV-seropositive and 16% (33 out of 204) of the HIV-seronegative homosexual men tested at their baseline visit. Elevated EBV shedding frequency was noted 6 months before, as well as during the first HIV-seropositive clinic visit, in the men who seroconverted to HIV. Seronegative men who shed EBV at their baseline visit seroconverted to HIV within a shorter period than did non-shedders during 5 years of follow-up. Shedding of EBV was not significantly associated with either abnormal T-cell numbers, clinical symptoms or risk for development of AIDS. There is an increased rate of EBV shedding in HIV-seropositive homosexual men that occurs very early in the course of HIV infection.


HIV infection and primary resistance to antituberculosis drugs in Abidjan, Côte d'Ivoire. To determine the prevalence of Mycobacterium tuberculosis resistance to antituberculosis drugs, and to relate this resistance to HIV serologic status. Cross-sectional prevalence study. The two major outpatient tuberculosis clinics in Abidjan, Côte d'Ivoire, West Africa. Sixty individuals with newly diagnosed pulmonary tuberculosis and sputum smears positive for acid-fast bacilli. HIV serologic status and in vitro testing for susceptibility of M. tuberculosis isolates to antituberculosis drugs. M. tuberculosis was isolated from 82% (49 out of 60) of sputum specimens. Thirty-five per cent (17 out of 49) were obtained from HIV-seropositive and 65% (32 out of 49) from HIV-seronegative patients. There was no statistically significant difference in the proportion of resistant isolates from HIV-seropositive versus HIV-seronegative patients, although the relatively small sample size limited power. Of the total number of isolates, 17% were resistant to isoniazid; resistance was less to streptomycin (7%), rifampin (2%), pyrazinamide (0%), and ethambutol (0%). Eighteen and 21% of mycobacterial isolates from HIV-seropositive and HIV-seronegative individuals, respectively, were resistant to one or more of these drugs. Surveys of this type are useful in planning and evaluating tuberculosis preventive therapy in individuals with dual infection.


Macrophage association of polyomavirus in progressive multifocal leukoencephalopathy: an immunohistochemical and ultrastructural study. Case report. Progressive multifocal leukoencephalopathy (PML) in a patient following autologous bone marrow transplantation for a non-Hodgkin's centroblastic lymphoma was studied by immunohistochemistry and transmission electron microscopy. Our observations indicate that a large amount of polyomavirus, most probably JC virus, is taken up and segregated within vacuoles of macrophages by phagocytosis. A relevant role of macrophages in the pathogenesis of PML is emphasized.


The cell biology of blastocyst development. Preimplantation development encompasses the "free"-living period of mammalian embryogenesis, which culminates in the formation of a fluid-filled structure, the blastocyst. Cavitation (blastocyst formation) is accompanied by the expression of a novel set of gene products that contribute directly to the attainment of cell polarity with the trophectoderm, which is both the first epithelium of development and the outer cell layer encircling the inner cell mass of the blastocyst. Several of these gene products have been identified and include the tight junction (ZO-1), Na/K-ATPase (alpha and beta subunits), uvomorulin, gap junction (connexin43), and growth factors such as transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF). This review will examine the role(s) of each of these gene products during the onset and progression of blastocyst formation. The trophectodermal tight junctional permeability seal regulates the leakage of blastocoel fluid and also assists in the maintenance of a polarized Na/K-ATPase distribution to the basolateral plasma membrane domain of the mural trophectoderm. The polarized distribution of the Na/K-ATPase plays an integral role in the establishment of a trans-trophectoderm Na+ gradient, which drives the osmotic accumulation of water across the epithelium into the nascent blastocoelic cavity. The cell adhesion provided by uvomorulin is necessary for the establishment of the tight junctional seal, as well as the maintenance of the polarized Na/K-ATPase distribution. Growth factors such as TGF-alpha and EGF stimulate an increase in the rate of blastocoel expansion, which could, in part, be mediated by secondary messengers that result in an increase in Na/K-ATPase activity. Insight into the mechanism of cavitation has, therefore, directly linked blastocyst formation to trophectoderm cell differentiation, which arises through fundamental cell biological processes that are directly involved in the attainment of epithelial cell polarity.


Quantitative structure-activity relationship studies on benzodiazepine receptor binding: recognition of active sites in receptor and modelling of interaction. A quantitative structure-activity relationship study was carried out for the binding of a series of 'classical' benzodiazepines (BZs) and some beta-carbolines with BZ receptors to investigate the active sites in the latter and the nature of the binding of compounds with them. Using the Hansch approach, an attempt was made to correlate binding affinities of compounds with various physico-chemical and electronic properties of substituents. The correlations obtained showed the main roles were played by the hydrophobic constant pi and the Hammett constant sigma (an electronic parameter) of various substituents. This led to the suggestion that BZ receptors have many additional hydrophobic, hydrogen bonding and polar sites other than those suggested by Hollinshead et al. (1990). From the present study, the Hollinshead model of interaction was found to be inadequate to account fully for the binding of all types of compounds.


Isolation and identification of vitamin D3, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3 in Solanum malacoxylon incubated with ruminal fluid. It has been shown that Solanum malacoxylon contains 1 alpha,25-dihydroxyvitamin D3-glycoside. The presence of vitamin D3 and 25-hydroxyvitamin D3 has also been suggested. In the present study vitamin D3 and three of its metabolites, including 1 alpha,25-dihydroxyvitamin D3, were detected in plant leaf extracts preincubated with ruminal fluid (SMRF). Extraction of SMRF with non-polar organic solvents and purification of the lipid extract by TLC followed by HPLC yielded nine ultraviolet-absorbing (264 nm) peaks. Four of them comigrated on a Zorbax-Sil HPLC column with synthetic standards of vitamin D3, 25-hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3, respectively. These compounds were unequivocally identified by means of mass spectrometry. The results confirm that Solanum malacoxylon contains, in addition to 1 alpha,25-dihydroxyvitamin D3, vitamin D3, 25-hydroxyvitamin D3 and possibly other as yet unidentified derivatives. As 1,24,25-trihydroxyvitamin D3 is absent in plant extracts not incubated with ruminal fluid, the data also indicate that rumen microbes may convert 1 alpha,25-dihydroxyvitamin D3 into 1,24,25-trihydroxyvitamin D3.


Hormonal and stressor-associated changes in porcine adrenocortical cholesterol ester hydrolase activity. Cholesterol ester hydrolase (CEH) activity was characterized in the porcine adrenal gland and experiments conducted to determine the nature of its hormonal regulation. CEH activity was studied in the 14,000 gmax pellet (F4) and in the 192,000 gmax supernatant (F6). Characteristics associated with pH optima, product formation with time, linearity with increasing protein concentration, and equilibration of exogenous cholesterol esters added in acetone with endogenous cholesterol esters were determined. Scatchard analyses of saturation data demonstrated two-site models, which indicated the presence of lower velocity lower Km enzymes (catalytic sites) (L-VKm) and higher velocity higher Km enzymes (catalytic sites) (H-VKm) in both subcellular fractions. Neither ACTH (0.4 micrograms/kg body weight) nor 30-min restraint affected CEH activities at 0.5, 2, and 5 h after injection or initiation of restraint. However, 1 h after a longer restraint period (45 min), F4 H-VKm CEH activity increased concomitantly with decreased F6 L-VKm (P = 0.003). More modest increases in F4 H-VKm (P = 0.03) were still apparent 1 h after the last of nine daily 45-min restraints. Bromocriptine (CB154, a dopamine agonist) administration for 6 days (9.6 mg/daily) reduced plasma prolactin (PRL) by 53% (P < 0.05), but had no effect on CEH activities. ACTH treatment to CB154-induced hypoprolactinemic barrows dramatically reduced F4 (63%) and F6 (49%) L-VKm CEH activity (P = 0.03). These data are the first concerned with regulation of adrenal CEH activity in swine, and are the first to evaluate in vivo treatments on in vitro CEH activity in any species evolutionarily higher than rodents. In vivo regulation of porcine adrenal CEH activity appears complex. Stressor-associated hormonal perturbations apparently must surpass a certain threshold of duration and/or magnitude before they alter CEH activity. Differing Km and Vmax of CEH within and between the two subcellular fractions studied and the differential responses to restraint stressor suggest that as many as four different enzymes with CEH activity are involved. Additionally, the combined effect of ACTH and CB154-induced hypoprolactinemia argues for an interrelated modulatory function of ACTH and PRL (or dopamine) on specific porcine adrenal CEH activities.


Alcohol stimulation of lipid peroxidation and esophageal tumor growth in mice immunocompromised by retrovirus infection. Tumor appearance can be accelerated in the immunodeficient and immunosuppressed animal. The role of lipid peroxidation and immune dysfunction induced by retrovirus and ethanol treatments on cancer promotion were investigated. Following the initiation of esophageal cancer by methylbenzylnitrosamine, ethanol consumption and retrovirus infection individually and concomitantly increased growth of esophageal tumors. Dietary supplementation with vitamin E reduced the size and frequency of the developed tumors. Tumor growth modifications in the vitamin E supplemented animals may be due to changes in T-cell numbers and functions stimulated by vitamin E. In addition, increased production of free radicals following ethanol treatment and retrovirus infection, and the suppression of these formations lipid peroxide by vitamin E is accompanied by lower incidence and size of tumors. Thus, the mechanisms of tumor enhancement observed in immunocompromised animals may include a combination of immunomodulation and modification of oxidant production by ethanol consumption and retrovirus infection.


Coordinate expression of Wilms' tumor genes correlates with Wilms' tumor phenotypes. The cloning and molecular characterization of two putative tumor genes, WT1 and WIT1, from the chromosome 11p13 region has provided a means of evaluating their role in the generation of Wilms' tumor heterogeneity. A series of 29 tumors were analyzed for WT1 and WIT1 expression by Northern blot or RNase protection analyses, and results were compared with tumor histopathology. Tumors were scored for the percentage of mesenchymal and epithelial derived tissue components. Homotypic tumors comprised blastema, tubular epithelium, and a fibroblast-like mesenchyme. In addition to these tissue components, the group of tumors designated as heterotypic also contained ectopic cell phenotypes such as muscle and squamous epithelium. The analyses suggest that heterotypic differentiation patterns occur when WT1 and WIT1 expression is low relative to normal fetal kidney. In situ hybridization using antisense RNA probes showed that WT1 and WIT1 were concordantly expressed in normal fetal kidney and in the blastema of tumors. The ratio of WT1:WIT1 expression remained relatively constant in homotypic tumors but deviated significantly in heterotypic tumors. These results suggest that expression patterns of the WT1 and WIT1 genes can be closely correlated to Wilms' tumor histopathology.


Nonuniform expression of a mouse mammary tumor virus-driven int-2/Fgf-3 transgene in pregnancy-responsive breast tumors. We have developed transgenic mice in which expression of the mouse int-2/Fgf-3 gene is regulated by a single long terminal repeat from mouse mammary tumor virus. Such mice contain and transmit a replica of the activated int-2/Fgf-3 allele present in a spontaneous mammary tumor from a BR6 mouse. Although free of infectious mouse mammary tumor virus and with a different genetic background, the transgenic mice develop pregnancy-responsive mammary epithelial proliferations that are similar to the early stages of tumorigenesis in the BR6 strain. Histological examination revealed that most of these tumors showed pronounced tubular and acinar structures, features usually associated with morphological differentiation. In some cases, the tumors were locally invasive, causing disruption of the dermis which manifested itself as local hair loss. In situ hybridization showed that patterns of transgene expression in the abnormal glands were markedly nonuniform. In contrast, mouse mammary tumor virus-induced neoplasms showed more uniform expression of int-2/Fgf-3, as did the urogenital epithelial proliferations that occur among males of this transgenic line. These data suggest that mammary tumors in virally infected animals may depend primarily on autocrine stimulation by the int-2/Fgf-3 gene product, whereas both autocrine and paracrine mechanisms may contribute to tumors and hyperplasias found in transgenic animals.


Accumulation of multiple T cell clonotypes in the synovial lesions of patients with rheumatoid arthritis revealed by a novel clonality analysis. T cell activation in the characteristic synovial lesions of rheumatoid arthritis may play a major role in the pathogenesis of this autoimmune disease. Analysis of T cell clonal diversity in these sites remains equivocal. Using the PCR and subsequent single-strand conformation polymorphism analysis it is possible to assess the degree of junctional diversity in the TCR with minimal selection bias. Concentrating on the beta-chain of the TCR, a paucity of clonotypic T cell expansion is demonstrated in the peripheral blood of healthy individuals. After polyclonal stimulation in vitro (with concanavalin A or phytohemagglutinin) this pattern does not change. In contrast, some T cell clonotypes appear following in vitro stimulation with purified protein derivative. Analysis of the peripheral blood, synovial fluid, and synovial tissue of patients with rheumatoid arthritis indicated many dominant T cell clonotypes. These data argue for a clonally diverse T cell response in the affected tissues of rheumatoid arthritic subjects.


Structure-activity relationships of mu-conotoxin GIIIA: structure determination of active and inactive sodium channel blocker peptides by NMR and simulated annealing calculations. A synthetic replacement study of the amino acid residues of mu-conotoxin GIIIA, a peptide blocker for muscle sodium channels, has recently shown that the conformation formed by three disulfide bridges and the molecular basicity, especially the one around the Arg13 residue, are important for blocking activity. In the present study, we determined the three-dimensional structure of an inactive analog, [Ala13]mu-conotoxin GIIIA, and refined that of the native toxin by NMR spectroscopy combined with simulated annealing calculations. The atomic root-mean-square difference of the mutant from the native conotoxin was 0.62 A for the backbone atoms (N, C alpha, C') of all residues except for the two terminal residues. The observation that the replacement of Arg13 by Ala13 does not significantly change the molecular conformation suggests that the loss of activity is not due to the conformational change but to the direct interaction of the essential Arg13 residue with the sodium channel molecules. In the determined structure, important residues for the activity, Arg13, Lys16, Hyp(hydroxyproline)17, and Arg19, are clustered on one side of the molecule, an observation which suggests that this face of the molecule associates with the receptor site of sodium channels. The hydroxyl group of Hyp17 is suggested to interact with the channel site with which the essential hydroxyl groups of tetrodotoxin and saxitoxin interact.


Non-transferrin-bound iron species in the serum of hypotransferrinaemic mice. Serum from homozygous hypotransferrinaemic mice (a mixed group of males and females, aged 6-8 wk) was found to contain low levels of iron (mean 0.9 +/- 0.5 microM (SEM, n = 4), as assayed by conventional serum iron assays. Similarly, low levels of non-transferrin-bound iron were determined with a nitrilotriacetate chelation assay (1.3 +/- 0.4 microM, n = 4) (Singh, S., Hider, R.C. and Porter, J.B. (1990) Analytical Biochemistry 186, 320-323). Mononuclear Fe (citrate) was undectable by electron paramagnetic resonance spectroscopy (EPR). Significantly larger quantities of iron (16 +/- 5 microM, n = 8) were detected by the bleomycin assay (Gutteridge, J.M.C., Rowley, D.A. and Halliwell, B. (1981) Biochemical Journal 199, 263-265), while non-haem iron assay or atomic absorption spectrophotometry revealed up to 96 microM iron. Haemoglobin iron was detectable at approximately 10 microM by spectrophotometry. Ferri-haem was undetectable by EPR spectroscopy. Serum ferritin levels of 641 +/- 128 micrograms/l (n = 14) in hypotransferrinaemic mice (wild-types 44 +/- 6 micrograms/l, n = 14) were observed and these cannot account for the non-transferrin-bound iron. Hypotransferrinaemic mouse serum therefore contains large quantities of non-transferrin-bound iron which is unreactive in some assays used to detect such iron in human iron overload. Fractionation by Sephadex G200 chromatography revealed three distinct species with apparent molecular weights of > or = 150 kDa, 40-80 kDa and 1-5 kDa. The iron may be distinguished from known extracellular iron proteins and haem-proteins by its availability to hot acid extractions.


Decay kinetics of O2.- studied by direct spectrophotometry. Interaction with catalytic and non-catalytic substances. The kinetic behaviour of O2.- during spontaneous dismutation and in the presence of Cu,Zn-superoxide dismutase and other compounds, was studied by monitoring the decrease in absorbance (A250nm-A360nm) on a time-scale of > or = 1 min, at pH 9.5. O2.- was generated from KO2, and calculations were performed between 25 and 4 microM of O2.-. An algorithm for the simultaneous calculation of the 1st and 2nd-order rate constants from the decay curve, was evolved. The respective fractions of O2.- which interacted with catalysts or disappeared spontaneously, in various experimental situations, could be estimated. Substances could be classified as inert, catalysts or scavengers. The high assay pH excluded examination of the effect of alkali sensitive substances, e.g., Mn-superoxide dismutase. However, the high pH minimized the interfering effect of trace amounts of Cu(II). Therefore a metal chelator was superfluous and even the effect of metals and metal complexes could be tested. The extremely high sensitivity of the method allowed minute concentrations of reagents to be used, including proteins absorbing in the UV-region. The rate constants found by this simple method, agreed with those obtained by more sophisticated and inaccessible techniques like pulse radiolysis and stopped-flow spectrophotometry.


Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry. A detailed procedure for the assay of free pyrroloquinoline quinone (PQQ) in human and rat samples by gas chromatography/mass spectrometry (GC/MS) has been established with stable-isotopic PQQ as internal standard. PQQ was extracted from the samples, after addition of the internal standard, with butanol under acid conditions and with Sep-Pak C18 cartridges. After derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ by selected ion monitoring, respectively. Trace amounts of free PQQ were detected in eight organs, plasma and urine of the human, and in three organs of the rat. The PQQ level was highest in the human spleen (5.9 +/- 3.4 ng/g tissue, followed by the pancreas and lung, and it was below detection limits for human brain and heart. Trace levels of PQQ were also found in rat small intestine, liver and testis. Our data are far below those measured by the redox cycling method of Gallop's group for human plasma, adrenal and urine.


[Interaction of proflavin with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT): thermodynamic analysis from (1)H NMR data]. Temperature relationships of chemical shifts of protons of proflavin mixed with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in water solution were investigated on impulse NMR spectrometer (500 MHz). Procedure is suggested for calculating values of mole fractions of various associates in solution as a function against temperature. Free energies of Gibbs, entalpy and entropy were determined in the reactions of complex formation 1:1, 1:2, 2:1 of proflavin with tetranucleotide. The results point to a significant role of hydrophobic interactions during the formation of dye--tetramere duplex complexes.


[Nonenzymatic reduction of Met-Hb under the action of NAD radicals generated in the presence of HAD.H and Fe(II) salts. Antioxidant properties of Met-Hb]. It has been found that superoxide radicals formed at autooxidation of Fe(II) ions or of their complexes with EDTA in the presence of NAD.H cause reduction of met-Hb into deoxy or oxy-form. Under the effect of hydroxyl radicals generated in the ultrasonic field in the presence of NADH reduction of met-Hb into the ferro-form (carboxy-Hb) was observed in Co atmosphere and not in O2 atmosphere. It was induced by a high oxidation rate of oxy-Hb by hydroxyl radicals into met-Hb as compared to carboxy-Hb. Reduction of met-Hb was shown to be accompanied with the formation of NAD+. The role of NAD. radicals in the reduction of met-Hb which acts as an antioxidant in respect to organic free radicals was discussed.


[Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein]. A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre).


Action potential refractory period in axonal demyelination: a computer simulation. Axonal demyelination leads to an increase in the refractory period for propagation of the action potential. Computer simulations were used to investigate the mechanism by which changes in the passive properties of the internodal membrane increase the refractory period. The properties of the voltage dependent ion channels can be altered to restore conduction in demyelinated nerve fibers. The ability of these alterations to decrease the refractory period of demyelinated model nerve fibers was compared. The model nerve fiber contained six nodes. The action potential was stimulated at node one and propagated to node six. The internode between nodes three and four was demyelinated in a graded manner. The absolute refractory period for propagation of the action potential through the demyelinated internode increased as the number of myelin wraps was reduced to less than 25% of the normal value. The increase in refractory period was found to be due to a reduction in the rate or repolarization of the action potential at node three. The delay in repolarization reduced the rate of recovery of inactivated Na channels and slowed the closing of K channels. The rate of repolarization of node three was reduced by the conduction delay for the depolarization of node four caused by demyelination of the preceding internode. In these simulations the increase in refractory period due to demyelination was eliminated by slowing the onset of Na channel inactivation. A small reduction of the K conductance also decreased the refractory period. However, larger reductions eliminated this effect.


Transcription of the genes encoding the small heat shock protein ubiquitin is unchanged in patients with systemic lupus erythematosus. The heat shock proteins hsp90 and hsp70 have been shown to be over-expressed in peripheral blood mononuclear cells from SLE patients. We show, however, that transcription of the three genes encoding the small hsp ubiquitin is not enhanced in these patients. The over expression of hsp90 and hsp70 in SLE is likely therefore to reflect cellular events resulting in the specific induction of these hsps rather than a generalized induction of all hsp synthesis due to the stress of the disease or the presence of fever.


[Titration of SH-groups of histone H3 in a DNP preparation of normal and neoplastic animal cells with a mercury-containing spin marker and with a DTNB preparation]. DNP samples isolated from the cells of calf thymus and Ehrlich ascite carcinoma of mice were examined. SH-groups of histone H3 of chromatin from these cells were titrated with mercury-containing spin label and with DTNB under joint action of different salt and sarcosyl concentrations on DNP. The results revealed differences in accessibility and titration of histone H3 SH-groups in DNP of normal and tumor cells with DTNB, as well as in molecular dynamics of the mercury-containing spin label introduced to these SH-groups.


Anti-Epstein-Barr virus-nuclear antigen-1, -2A and -2B antibodies in rheumatoid arthritis patients and their relatives. We have examined serum antibodies to Epstein-Barr virus Nuclear Antigen (EBNA)-1, -2A and -2B, in addition to antibodies to viral capsid antigen and early antigen in 100 rheumatoid arthritis patients and 50 of their relatives. Using indirect immunofluorescence on transfected cells and Western-blot technique, we have found increased frequency and titres of antibodies to EBNA-2B in patients and, to a lesser degree, in their family members, whereas other anti-Epstein-Barr virus antibodies appeared to be similar to controls. Cross-inhibition experiments were carried out and show that antibodies to EBNA-2A are distinct from those to -2B, and vice versa.


[Evaluation of distances in yeast phosphoglycerate kinase molecules using saturation effect ESR spectra at 77 K]. Distances between a spin label attached to the single SH-group of yeast phosphoglycerate kinase and paramagnetic ions bound to the active centre in the complex with ATP were estimated by means of saturation of ESR spectra of the spin label at 77K. The calculations were performed for the ions with g-factor approximately equal to the g-factor of the spin label on IBM PC/AT. Distances between the SH-group of phosphoglycerate kinase and the middle of the active centre of the ATP binding site were estimated by using the stable complex of CrATP.


Suckling reverses mammotrope responsiveness to TRH. It is well established that suckling can alter the responsiveness of mammotropes to the prolactin (PRL) releasing actions of thyrotropin-releasing hormone (TRH). The purpose of the present study was to more critically characterize this phenomenon and to determine whether this effect was manifested at the point of TRH receptor binding and effector coupling. Cultured anterior pituitary (AP) cells from suckled and nonsuckled rats were subjected to reverse hemolytic plaque assays for PRL in the absence or presence of TRH. Treatment with TRH (100 nM for 2 h) significantly stimulated PRL secretion by pituitary cells from transiently suckled females (to 147.0 +/- 0.7% of control value; P < 0.05). Surprisingly, this same dose of the secretagogue caused a 30% inhibition (P < 0.05) of PRL release by AP cells obtained from nonsuckled lactators. In order to gain some insight into the possible mechanisms that govern these drastic changes in mammotrope responsiveness to TRH, we evaluated the ability of this secretagogue to activate the phosphoinositidase pathway. To this end, we chose to measure the intracellular accumulation of inositol monophosphate (IP1), a degradative metabolite of the inositol 1,4,5-triphosphate (Ins 1,4,5-P3) second messenger molecule, in the presence of LiCl (10 mM) which prevents further metabolism of IP1. We found that a 2 h exposure to 100 nM TRH resulted in a 3-fold increase in IP1 accumulation by AP cultures derived from both suckled and nonsuckled dams. Our results indicate that TRH effectively activates its receptor and couples to the phosphoinositidase pathway in AP cells from both nonsuckled and suckled females. Thus, we conclude that the suckling-induced increase in mammotrope responsiveness to TRH is regulated subsequent to TRH receptor activation.


Effect of FSH, LH, LH-RH and arginine-vasotocin on the production of steroids, nonapeptide hormones and cGMP by rabbits granulosa cells isolated at different stages of reproductive cycle. Granulosa cells isolated from ovaries of non-cycling, cycling and pregnant rabbits of the same age were cultured in vitro either without or with pFSH (1 micrograms/ml), bLH (1 IU/ml), LH-RH (25 ng/ml) or arginine-8-vasotocin (100 ng/ml). The production of immunoreactive progesterone, estradiol-17 beta, oxytocin, arginine-8-vasopressin and cGMP was analyzed. The gonadotropins did not show any significant effects on the cells isolated from non-cycling and cycling rabbits, but not from these of pregnant ones. LH-RH inhibited and vasotocin stimulated progesterone production. All hormones used stimulated estradiol release from cells of non-cycling rabbits, while in a case of cycling animals no change was found. In the cell from pregnant females the release of estradiol was enhanced after LH treatment only. The treatment with FSH and LH (but not with LH-RH or vasotocin) resulted in a remarkable rise of granulosa vasopressin surge irrespectively to the reproductive stage. Oxytocin production by granulosa cells incubated either without or with LH, LH-RH or vasotocin was undetectable. However, FSH strongly stimulated oxytocin release. FSH and in lesser extent, LH or LH-RH (but not vasotocin) activated granulosa cGMP production in the cells from cycling and pregnant (but not from non-cycling) animals. It was also found that, in contrast to other reproductive stages, basal progesterone release from the cells of pregnant rabbits was increased, while in a case of non-cycling animals the basal estradiol release was decreased and that of cGMP was increased.(ABSTRACT TRUNCATED AT 250 WORDS)


[Osmotic regulation of the sodium pump in rat brain synaptosomes]. Effect of medium osmolarity on 3H-ouabain binding and the rate of ouabain-sensitive 86Rb+ transport in the rat brain synaptosomes was studied. A decrease in tonicity to 230 mOsm increases both parameters indicating the activation of the sodium pump upon synaptosome swelling. The effect is retained in the absence of inside-oriented Na+ gradient, i. e. a rise in Na(in) is not responsible for hypoosmotic activation. Colchicine (5mM) decreased and cytochalasin B (40 microM) increased the ouabain binding. In the presence of cytochalasin B the inhibition of binding observed under hypotonic conditions was shifted to higher osmolarity values. It is suggested that volume regulation of the sodium pump is controlled by the cytoskeleton elements.


The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. The complete nucleotide sequence of the inverted repeat component (IR; 12,776 bp each) of the genome of equine herpesvirus type 1 (EHV-1) has been determined. Transcription analyses have revealed that the EHV-1 IR sequence encodes at least 6 genes. In this report, we present the DNA sequence and transcriptional characterization of a gene (IR3) that maps entirely within the IR sequences. The IR3 open reading frame (ORF) is located between nucleotides (nt) 6123-6411 of the IR sequence and possesses an ORF of 95 amino acids. Interestingly, this ORF does not show homology to any known herpesvirus gene, suggesting that the IR3 gene is unique to EHV-1. Moreover, the location of the IR3 gene between the immediate-early (IR1) gene and the origin of replication is unique in comparison to the IR gene arrangement of other alphaherpesviruses such as herpes simplex virus type 1 and varicella zoster virus. Putative cis-acting elements flanking the IR3 ORF include a TATA box (nt 5648-5652), a GC box (nt 5600-5605), and three polyadenylation signals (nt 6533-6538, 6648-6653, and 6663-6668). Northern blot analyses identified a 1.0 kb mRNA that exhibits characteristics of a late gene of the gamma-1 class. Northern blot, S1 nuclease, and primer extension analyses revealed that transcription of IR3 initiates within the intron of the immediate-early gene (IR1) on the opposite stand of the genome. Thus, the 5' end of IR3 transcript is antisense to the 5' end of the IR1 mRNA and promoter, and IR3 transcription may regulate the expression of IR1 during late times of infection.


[The effect of electromagnetic radiation with extremely high frequency and low intensity on cytotoxic activity of human natural killer cells]. EHF electromagnetic radiation under short-time action suppresses the cytotoxical activity of the natural killer cells from granulocyte fraction and peripheral blood of healthy volunteers; the observed effect is non-linear. Under the long-time irradiation of the natural killer cells from the mononuclear fraction of blood, the suppressing effect gets a practically linear character after the 20-30 minutes action. Under the long-time irradiation of peripheral blood the insignificant stimulation of natural killers was observed. It is assumed that the radiation applied can suppress the cytotoxic activity of the natural killers, breaking the normal metabolic pathway of phosphatidylinositphosphate.


The World Reference Laboratory for Foot and Mouth Disease: a review of thirty-three years of activity (1958-1991). The range of activities and contributions of the World Reference Laboratory for Foot and Mouth Disease in Pirbright, Surrey, United Kingdom, from 1958 to 1991 is reviewed. The countries for which a service has been provided, the number of samples submitted for investigation and the serotypes identified are recorded. Factors which have influenced the number of samples received are outlined. The developments and improvements made in the laboratory diagnosis of vesicular virus diseases over the thirty-three-year period are described.


A review of foot and mouth disease in Nepal. Foot and mouth disease (FMD) causes substantial economic losses to the predominantly agricultural community of the Kingdom of Nepal. FMD is endemic in the country and four of the seven serotypes of FMD virus have been isolated (O, A, C and Asia 1). The epidemiology of FMD and the factors which play a role in its prevalence and spread are outlined. The National Epidemiological Laboratory for FMD has been established in Kathmandu and its diagnostic capabilities and activities are described. The important points to be considered in the formulation of any future regional or national control programme for FMD in Nepal are discussed.


Bluetongue virus infection in India: a review. The history and epizootiology of bluetongue (BT) in India are reviewed. BT has become endemic in India. The first outbreak of BT in sheep and goats in the country was recorded in 1964 in Maharashtra State. Since then, several outbreaks of BT have been reported in sheep. Exotic sheep are more susceptible than indigenous and cross-bred sheep. A serological survey has indicated the presence of bluetongue virus (BTV) antibodies in cattle and buffalo in several states in India. However, clinical BT has not been observed in cattle or buffalo to date. Of the 24 known serotypes of BTV, 18 have been reported in India. Although BTV has been isolated from Culicoides midges, the particular species responsible for transmission has not yet been identified.


A comparison of type O foot and mouth disease virus field isolates from northern Thailand. A survey of type O foot and mouth disease (FMD) virus isolates from northern Thailand was undertaken to determine the relationship between field viruses and the vaccine in use, and to gauge the range of antigenic variation among field viruses. Isolates were collected from the two most recent epizootics, 1986-1987 and 1989-1990, and assessed using a two-dimensional neutralisation test to determine their relationship to FMD type O1 Bangkok 1960 (O BKK/60) reference (vaccine challenge) virus. The critical r value for the survey was 0.259 and all isolates tested were found to have an r value considerably greater than this (range 0.66 to 0.80). The results showed close antigenic relationships between the isolates and the reference virus, and indicated a relatively small range of antigenic variation between the isolates.


Identification of the diacylglycerol kinase structural gene of Rhizobium meliloti 1021. The cyclic beta-1,2-glucans of Rhizobium may function during legume nodulation. These molecules may become highly substituted with phosphoglycerol moieties from the head group of phosphatidylglycerol; diglyceride is a by-product of this reaction (K. J. Miller, R. S. Gore, and A. J. Benesi, J. Bacteriol. 170:4569-4575, 1988). We recently reported that R. meliloti 1021 produces a diacylglycerol kinase (EC 2.7.1.107) activity that shares several properties with the diacylglycerol kinase enzyme of Escherichia coli (W. P. Hunt, R. S. Gore, K. J. Miller, Appl. Environ. Microbiol. 57:3645-3647, 1991). A primary function of this rhizobial enzyme is to recycle diglyceride generated during cyclic beta-1,2-glucan biosynthesis. In the present study, we report the cloning and initial characterization of a single-copy gene from R. meliloti 1021 that encodes a diacylglycerol kinase homolog; this homolog can complement a diacylglycerol kinase deficient strain of E. coli. The sequence of the rhizobial diacylglycerol kinase gene was predicted to encode a protein of 137 amino acids; this protein shares 32% identity with the E. coli enzyme. Analysis of hydropathy and the potential to form specific secondary structures indicated a common overall structure for the two enzymes. Because diglyceride metabolism and cyclic beta-1,2-glucan biosynthesis are metabolically linked, future studies with diacylglycerol kinase mutants of R. meliloti 1021 should further elucidate the roles of the cyclic beta-1,2-glucans in the Rhizobium-legume symbiosis.


The administration of foot and mouth disease vaccine with oil adjuvant and its influence on the diagnosis of bovine tuberculosis. The possible influence of vaccination with oil adjuvanted foot and mouth disease vaccines on the tuberculin response was investigated in 32 normal guinea pigs and 190 non-tuberculous bovines. Circulating anti-Mycobacterium bovis IgG antibodies were analysed by an enzyme-linked immunosorbent assay (ELISA), in order to determine the effect of the vaccination on the humoral response against mycobacteria in cattle. Control animals were either nonvaccinated or injected with aluminium hydroxide adjuvanted vaccine. Administration of foot and mouth disease vaccine had no apparent influence on the tuberculin responses of either guinea pigs or cattle, nor did it influence the level of anti-M. bovis antibodies in cattle.


Pseudorabies (Aujeszky's disease) in Argentina. Various methods have been employed for the diagnosis of pseudorabies in Argentina. A large serological survey was carried out by means of enzyme-linked immunosorbent assay (blocking ELISA) and virus neutralisation (VN). An outbreak was studied by virological and immunohistochemical methods and in situ nucleic acid hybridisation.


A novel subunit ISCOM vaccine against bovine virus diarrhoea virus. The preparation and preliminary testing of a subunit ISCOM (immunostimulating complex) vaccine against bovine virus diarrhoea virus (BVDV) is described. Vaccination of calves with this vaccine yields high neutralising titres against a panel of Danish BVDV field isolates. The serological difference between virus isolates and vaccine strain selection is discussed.


Pestivirus infection of ruminants in Australia. Pestivirus infections are commonly diagnosed in cattle but are relatively uncommon in other ruminant species in Australia. Virus isolation is a very reliable technique for detecting pestivirus in specimens, especially when group reactive monoclonal antibodies are used with immunoperoxidase staining to detect non-cytopathogenic virus. Care must be taken to prevent adventitious pestivirus contamination of serum or cells used for cell culture. A recently developed antigen capture enzyme-linked immunosorbent assay has been extensively evaluated and found to be extremely accurate. This test is also much quicker and less expensive than virus isolation. Procedures are outlined to reliably certify animals to be free of pestivirus infection for export or as donors of semen or embryos.


Thermal and pH stability of pestiviruses. Three strains/isolates of hog cholera virus (HCV) and two strains/isolates each of cytopathogenic (cp) and non-cytopathogenic (ncp) biotype of bovine virus diarrhoea virus (BVDV) were each exposed to pH 3, 3.5 and 4 at 4 degrees C, 21 degrees C and 37 degrees C in a number of combinations. Infectivity titration and half-life determinations following correlation and regression analysis showed a significant temperature-dependent shortening of half-lives within the pH range investigated. At pH 3, mean half-lives were more than tenfold lower when HCV was kept at an ambient temperature of 21 degrees C rather than at 4 degrees C. Additionally, in some of the strains/isolates tested, half-lives of HCV kept at 4 degrees C were four to ten times lower when the pH was raised from 3 to 4. BVDV appeared more sensitive at 4 degrees C and pH 3 than HCV, but equally sensitive at 21 degrees C. Differences in temperature or pH stability between cp and ncp biotypes of BVDV could not be statistically verified although, in general, the cp biotypes seemed to be more stable than the ncp strains/isolates.


Immunokinetics of equine herpesvirus 1 in donkey mares: suppression of secondary cell-mediated response. To study the immunokinetics of equine herpesvirus 1 (EHV1), donkey mares were immunised with a laboratory strain of EHV1, or with recommended doses of Pneumabort-K vaccine (EHV1 Army 183 strain, formalin-inactivated, with an oil adjuvant) and a booster was given after three months. Humoral immune responses were studied by employing a virus neutralisation (VN) test. A leucocyte migration inhibition test (LMIT) was employed for the assay of cellular immune responses. The VN antibody titre reached 1:64 or 1:128 after primary immunisation and showed a marginal increase (1:256) after secondary immunisation with either of the immunogens. After the primary dose of immunogen, there was a gradual increase in host cellular response which persisted for up to three months. However, on secondary immunisation, cell-mediated immune response was short-lived and weak compared to the primary response with both immunogens. This could be one possible explanation for breakdown of anti-EHV1 immunity leading to abortion in immunised mares.


Establishing a diagnosis of adrenal metastasis from hepatocellular carcinoma by 99mTc-PMT hepatobiliary scintigraphy. A 69-year-old woman was diagnosed as having hepatocellular carcinoma (HCC) with liver cirrhosis in October, 1984 and treated by transcatheter arterial embolization (TAE). In June, 1990 she was found to have a huge mass in the left hypochondrium which ultrasonography and computed tomography (CT) scan revealed to be a lett adrenal mass. A 99mTc pyridoxyl-5-methyl tryptophan (99mTC-PMT) hepatobiliary scintigraphy was positive and confirmed metastatic HCC. Although the adrenal mass was large, the HCC itself was controlled well with TAE. The adrenal mass was removed surgically in July, 1990 and the histological findings were compatible with HCC metastasized to the adrenal gland.


Protein phosphorylation regulates the mouse sperm acrosome reaction induced by the zona pellucida. Recently, the ligand-receptor signal transduction mechanism has been implicated in mediating the zona pellucida (ZP)-induced acrosome reaction. Little is known about the role of protein phosphorylation in this specific event. We examine whether modification of protein phosphorylation and dephosphorylation affects the kinetics of the acid-solubilized ZP-induced acrosome reaction of mouse sperm. Mouse epididymal sperm were incubated in modified Krebs-Ringer bicarbonate medium for a period of 90 to 120 min and then treated with 2 acid-solubilized ZP/microliters for an additional 60 min. The chlortetracycline fluorescence assay was used to monitor the acrosome reaction. Capacitated sperm were inhibited from undergoing acid-solubilized ZP-induced acrosome reaction in the presence of an inhibitor of cyclic nucleotide-dependent protein kinase, H8; activators of the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C); an inhibitor of phosphatases 1 and 2A, okadaic acid; or an inhibitor of protein tyrosine kinases, genistein. The addition of inhibitors of protein kinase C, such as staurosporine, H7, and protein kinase C [19-36] pseudosubstrate, inhibited the phorbol ester-dependent inhibition of the acid-solubilized ZP-induced acrosome reaction. The present study suggests that protein phosphorylation and dephosphorylation play a regulatory role in the process of the ZP-induced acrosome reaction.


Effects of modulators of protein kinases and phosphatases on mouse sperm capacitation. We examined effects of modulators of protein kinases and phosphatases on the kinetics of mouse sperm capacitation. The chlortetracycline fluorescence assay was used to monitor the process of capacitation (in terms of the appearance of the B pattern). The treatment of sperm with dibutyryl cyclic AMP (cAMP) or dibutyryl cGMP resulted in a higher percentage B pattern at various times during capacitation compared with the control. The addition of 100 microM H8 inhibited the cyclic nucleotide-dependent stimulation of capacitation. Tumor promotors, 12-O-tetradecanoyl phorbol 13-acetate (TPA; a stimulator of protein kinase C) and okadaic acid (an inhibitor of protein phosphatases 1 and 2A), induced a rapid appearance of the B pattern (15 min after addition) and maintained a percentage B pattern similar to that of the control in the later period of capacitation. An inhibitor of protein kinase C, staurosporine, inhibited the TPA-dependent acceleration of capacitation. Furthermore, the addition of genistein, an inhibitor of protein tyrosine kinases, resulted in a strong inhibition of capacitation. All agents tested did not affect sperm motility. These data suggest that protein phosphorylation and dephosphorylation may play regulatory roles in mediating mouse sperm capacitation.


Analysis of the promoter of the cytochrome P-450 2B2 gene in the rat. About 3 kb of the promoter region of the gene encoding cytochrome P-450 2B2 (CYP2B2) in the rat were sequenced and searched for potential cis-acting elements. Apart from putative binding sites for (liver-specific) protein factors, a region showing homology with the LINE 1 retrotransposon element was also found. Three proximal promoter fragments, encompassing nucleotides -579 to -372, -372 to -211, and -211 to +1, respectively, were shown to contain binding sites for multiple protein factors by bandshift analyses. The strongest protein-binding element, designated BRE (basic regulatory element), occurs between -103 to -66. Its structure is very similar to a negative control element in the murine cmyc promoter and displays a composite feature having a tandemly repeated sequence homology with the BTE (basic transcription element; Yanagida et al., 1990) separated by a CCAAA-box. The use of a deletion series of this template in in vitro transcription assays, provided evidence that the BRE serves as a major cis-acting element in the (regulated) transcription activation of the CYP2B2 gene.