Yeast artificial chromosomes for the molecular analysis of the familial polyposis APC gene region. Two yeast artificial chromosomes (YACs) spanning a total distance of 1.1 megabase pairs of DNA around the MCC (for mutated in colorectal carcinoma) and APC (for adenomatous polyposis coli) genes at 5q21 have been isolated and characterized. Starting from the MCC gene, a strategy was undertaken to identify constitutional submicroscopic deletions in familial adenomatous polyposis patients that might considerably narrow down the position of the APC gene. To this end, YACs identified by the MCC gene were screened across a chromosome 5-specific cosmid library to provide a source of DNA probes for genomic scanning. The cosmids isolated from these experiments were used to screen a panel of somatic cell hybrids containing chromosome 5 segregated from patients suspected to carry putative interstitial deletions. This screening approach led to the confirmation of a small heterozygous deletion in a polyposis patient that overlaps one of the two isolated YACs. This YAC has been shown to contain the entire APC gene, in addition to a significant portion of DNA flanking the 5' end of the gene, and should therefore prove a valuable resource for functional studies by transfer to colorectal tumor-derived cell lines.


Chemotactic sorting to cAMP in the multicellular stages of Dictyostelium development. Dictyostelium transformants that overproduce the extracellular form of cyclic nucleotide phosphodiesterase and so accumulate a reduced amount of cAMP are blocked in development after aggregation in the form of a tight mound, prior to formation of the apical tip. In such mounds, prespore cell differentiation is repressed, and the apical accumulation of prestalk cells is greatly retarded. When a source of cAMP is placed below the arrested mounds, prestalk cells that would normally migrate in an apical direction instead sort downwards to the substratum. Thus, by acting as the chemoattractant that draws prestalk cells to the apex, cAMP signaling directs the formation of a patterned structure.


Role for major histocompatibility complex class I in regulating natural killer cell-mediated killing of virus-infected cells. Target structures important for natural killer (NK) cell recognition of virally infected cells are not well defined. Since major histocompatibility complex (MHC) class I molecules bind viral peptides during acute infection, we evaluated whether an interaction between MHC and virus might influence the susceptibility of infected cells to NK cell-mediated lysis. To control for MHC class I expression on target cells, we used either HLA class I-deficient C1R cells or C1R sublines expressing transfected HLA class I gene products. Human NK cells were unable to preferentially lyse class I-deficient C1R cells after infection with herpes simplex virus (HSV). In contrast, HLA class I transfectants were significantly more susceptible to NK cell-mediated cytotoxicity after HSV infection. This occurred for HSV-infected C1R cells expressing any of the three HLA class I gene products tested (i.e., HLA-B27, HLA-A3, or HLA-Aw68), indicating that NK cell recognition in this system does not require "self" MHC and is not unique for a single haplotype. Productive HSV infection is required for the increased killing, since inoculation with UV-inactivated virus did not lead to increased lysis. In addition, since HSV infection of the transfectants did not significantly alter the level of class I expression, the change in susceptibility appears to be due to qualitative changes in the target structures on HSV-infected, HLA class I+ targets. These results demonstrate a role for MHC class I in regulating NK cell-mediated killing of virus-infected cells.


Endothelin regulation of neuropeptide release from nerve endings of the posterior pituitary. We have investigated the role of endothelin (ET) in the stimulus-secretion coupling mechanism in the posterior pituitary. We report that isolated nerve endings contain immunoreactive endothelin, the level of which is regulated by homeostatic mechanisms involved in control of water balance. ET-1 and ET-3 potentiate vasopressin release induced by depolarization through interaction with specific receptors of the ETA subtype and this response is antagonized by sarafotoxin S6b. The second messenger for this effect, however, remains unknown since the potentiation of depolarization-induced vasopressin release occurs in the absence of an increase in cellular calcium.


Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants. Transgenic plants were constructed that express two foreign calmodulins (VU-1 and VU-3 calmodulins) derived from a cloned synthetic calmodulin gene. VU-1 calmodulin, similar to endogenous plant calmodulin, possesses a lysine residue at position 115 and undergoes posttranslational methylation. VU-3 calmodulin is a site-directed mutant of VU-1 calmodulin that is identical in sequence except for the substitution of an arginine at position 115 and thus is incapable of methylation. Both calmodulin genes, under the control of the cauliflower mosaic virus 35S promoter, were expressed in transgenic tobacco. Foreign calmodulin protein accumulated in plant tissues to levels equivalent to that of the endogenous calmodulin. All transformed lines of VU-1 plants were indistinguishable from untransformed controls with respect to growth and development. However, all transformed lines of VU-3 plants were characterized by decreased stem internode growth, reduced seed production, and reduced seed and pollen viability. The data suggest that these phenotypes are the result of the expression of the calmodulin mutant rather than the position of transferred DNA insertion or the overall alteration of calmodulin levels. Analyses of the activity of the purified transgenic calmodulins suggest that calmodulin-dependent NAD kinase is among the potential targets that may have altered regulation in VU-3 transgenic plants.


Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition. To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule containing a single bound peptide, a complex of recombinant mouse H-2Kb, beta 2-microglobulin (beta 2m), and a fragment of the vesicular stomatitis virus (VSV) nuclear capsid protein, VSV-(N52-59) octapeptide (Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu), was prepared by exploiting a high-yield bacterial expression system and in vitro cocomplex formation. The structure of mouse H-2Kb revealed its similarity to three human class I HLA molecules, consistent with the high primary sequence homology and common function of these peptide-presenting molecules. Electron density was located in the peptide-binding groove, to which a single peptide in a unique conformation was unambiguously fit. The peptide extends the length of the groove, parallel to the alpha-helices, and assumes an extended, mostly beta-strand conformation. The peptide is constrained within the groove by hydrogen bonding of its main-chain atoms and by contacts of its side chains with the H-2Kb molecule. The amino-terminal nitrogen atom of the peptide forms a hydrogen bond with the hydroxyl group of Tyr-171 of H-2Kb at one end of the groove, while the carboxyl-terminal oxygen forms a hydrogen bond with the hydroxyl group of Tyr-84 at the other end. Since the amino acids at both ends are conserved among human and mouse MHC molecules, this anchoring of each end of the peptide appears to be a general feature of peptide-MHC class I molecule binding and imposes restrictions on its length. The side chains of residues Tyr-3, Tyr-5, and Leu-8 of the VSV octapeptide fit into the interior of the H-2Kb molecule with no appreciable surface exposure, a finding in support of previous biological studies that showed the importance of these residues for binding. Thus, the basis for binding of specific peptide sequences to the MHC class I molecule is the steric restriction imposed on the peptide side chains by the architecture of the floor and sides of the groove. The side chains of Arg-1, Val-4, and Gln-6 and the main-chain of Gly-7 of the octapeptide are exposed on the surface of the complex, thus confirming their availability for T-cell receptor contact, as previously demonstrated by T-cell recognition experiments.


Overproduction of four functionally active proteins, TnsA, B, C, and D, required for Tn7 transposition to its attachment site, attTn7. The bacterial transposon Tn7 encodes five trans-acting transposition genes, tnsA, B, C, D, and E. Tn7 requires four of these genes, tnsA, B, C, and D, for a novel transposition pathway: high-efficiency site-specific transposition to a chromosomal attachment site, attTn7. Plasmids that individually allow inducible overexpression of proteins from the first initiation codon of four of these genes were constructed. Escherichia coli strains carrying these plasmids were used to overexpress the TnsA, B, C, and D proteins. The abundance and the apparent relative molecular mass of these proteins were examined and the latter was compared to those predicted from wild-type Tn7. The functionality of these proteins, encoded by an overexpression construct, was demonstrated by the fact that they could efficiently trans-complement a defective mini-Tn7 carrying only the cis-essential Tn7 termini in an in vivo assay for transposition to attTn7.


The effects of cyclic imides on lipoprotein receptor binding and degradation of rat and human cells and effects on regulatory enzymes of lipid metabolism. A series of cyclic imides, which possess a bulkier N-ring structure than phthalimide and saccharin, were shown to suppress LDL receptor binding, internalization and degradation of isolated rat hepatocytes, foam cells, human fibroblasts and mouse macrophages. The HDL receptor binding and internalization was accelerated in hepatocytes but not in other tissue types. In general, the HDL receptor activity and degradation was reduced by the cyclic imides. The in vivo studies with selected cyclic imides supported this finding in that 125I-LDL was not cleared from serum as rapidly as the control after 14 days of treatment, whereas 125I-HDL was cleared more rapidly by treated rats. The tissue uptake of 125I-LDL amd 125I-HDL was generally reduced in the treated rat tissues after 14 days dosing. These agents did not suppress HMG-CoA reductase activity in any of the tissue cell lines. A correlation existed between lower LDL receptor activity and stimulated HMG-CoA reductase activity in cells. The cyclic imides suppressed the activities of acyl-CoA cholesterol-acyltransferase, sn-glycerol-3-phosphate acyl transferase, and heparin-induced tissue lipoprotein lipase. Neutral cholesterol ester hydrolase activity and protein synthesis were markedly stimulated by the cyclic imides in the aorta foam cells, but not the other cell types.


Epidermal growth factor stimulates the release of GDP induced by isoproterenol from isolated liver membrane. Epidermal growth factor (EGF) stimulated the release of GDP induced by isoproterenol from isolated liver membrane. This GDP release was not induced by EGF only. The stimulatory effect of EGF was dependent on the presence of Mg2+. These results suggests that EGF receptor indirectly couples with GS-protein.


[Antiarrhythmic effects of magnesium on single ventricular myocytes]. Despite magnesium ions (Mg)'s widespread use for antiarrhythmic purposes, little is known concerning its antiarrhythmic mechanisms. We, therefore, examined Mg effects on delayed afterdepolarization (DAD), early afterdepolarization (EAD), triggered activity (TA) and aftercontraction (AC), using isolated ventricular cells and/or ventricular papillary muscle. In the experiments using the multicellular preparation, ACs were measured, with the use of a strain gauge. ACs were induced in isolated rat papillary muscle superfused with low K+ (0.5 mM) medium, after a train stimulation (2-5 Hz, 10-30 beats). In the experiments concerning cardiac myocytes, the effects of Mg on transient inward current (TI), which is responsible for DAD, were studied using a whole-cell voltage-clamp method applied on isolated guinea pig ventricular cells. And action potentials were also induced by current clamp (10 ms) and the effects of Mg on DAD, EAD and TA were examined. TI, DAD, EAD and TA were induced by use of pipette solution with high Ca2+ (0.7 mM) and low EGTA (0.1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)


The role of inhibitory amino acids in control of respiratory motor output in an arterially perfused rat. The respiratory effects of drugs affecting GABAergic and glycinergic transmission were examined in order to assess the role of synaptic inhibition in breathing rhythmogenesis. Experiments were performed in the arterially perfused in situ brainstem-spinal cord preparation from adult rats (Hayashi et al., 1991, J. Neurosci. Meth. 36:63-70). Administration to the perfusate of agonists of GABAA, GABAB, and glycine receptors reduced both the frequency and amplitude of the activity recorded from the phrenic and hypoglossal nerves. Similar effects were observed following the infusion of aminooxyacetic acid (a blocker of GABA-transaminase). Picrotoxin (0.1-2 microM), bicuculline (0.05-0.2 microM), strychnine (0.1-1 microM) and phaclofen (0.1-0.2 mM) usually increased the frequency and amplitude of inspiratory bursts. Perfusion with low Cl- (8 mM) solution elicited tonic discharge followed by reversible arrest of the respiratory activity. It is concluded that synaptic inhibition is involved in the respiratory rhythm generation process in the mature mammalian brain. As data from the literature indicate that interference with central inhibitory processes does not largely affect the rhythm generation process in newborn rats, a possibility is discussed that the brainstem respiratory generator undergoes a developmental change from a 'pacemaker' driven circuit at the neonatal stage to a network requiring post-synaptic inhibition in the mature brain.


Natural infection of dogs by influenza C virus. To date, only one seroepidemiological survey, carried out in Japan, gave a strong indication that dogs may be naturally infected by the influenza C virus, long considered to be exclusively human. In the present work, 134 serum samples were collected during the winter of 1988/89 from dogs aged 6 months to 16 years in northern France. Samples were tested for the presence of antibodies to influenza C virus by both haemagglutination inhibition (HI) and ELISA. Using antibody absorption by staphylococcal protein A, we demonstrated the specificity of the results. In 62% of cases, the results were identical using the two methods. Significant HI activity was found in 32% of the 134 tested sera and titres ranged from 20 to 320. Of the sera tested, 42% were positive by ELISA and titres ranged from 500 to 8,000. The discordant results are discussed. The population tested was divided into five age groups: less than 4 years, 4 to 6 years, 7 to 9 years, 10 to 11 years and greater than 12 years. The distribution of antibodies in the tested canine population, in contrast to that of humans, did not show a significant degree of association with age.


Staphylococcus albus-induced protein kinase C translocation in human neutrophils: the effect of opsonization, cytochalasin B, pertussis toxin, intra- and extracellular calcium, and R59022. Membrane-associated protein kinase C has been proposed to be essential in transmembrane signalling systems activating neutrophils. A main function of the neutrophil is phagocytosis and killing of microorganisms. Nevertheless, previously published reports mainly have described the effect of artificial or soluble stimulators upon neutrophil protein kinase C activity. Therefore, membrane-associated protein kinase C was studied in neutrophils stimulated by Staphylococcus albus. The bacteria were found to induce a striking increase in membrane-associated protein kinase C, an effect which depended upon a previous opsonization of the bacteria. Cytochalasin B, which inhibits phagocytosis, was shown to abrogate S. albus-induced protein kinase C translocation. Chelation of intracellular calcium totally abolished S. albus-induced protein kinase C translocation, a phenomenon that could not exclusively be ascribed to chelation of extracellular calcium. The diacylglycerol kinase inhibitor R59022, which has been reported to increase endogenous diacylglycerol accumulation, nearly doubled the effect of S. albus upon membrane-associated protein kinase C. Pertussis toxin in concentrations which completely inhibited fLMP-induced superoxide generation did not affect S. albus-induced protein kinase C translocation. It is concluded that phagocytosis of S. albus is accompanied by a translocation of protein kinase C to the cell membrane, a phenomenon that relies upon enhanced diacylglycerol production and calcium transients and occurs independently of pertussis toxin-inhibitable G-proteins.


Influence of zeolite on the availability of radiocaesium in soil to plants. Plant availability of radiocaesium is usually high immediately after incorporation in a soil. However, for some Swedish soils, e.g, peat soils, a high radiocaesium uptake in plants has also been observed during a second growth period after contamination. For these soils the reduction in the plant availability of the nuclide seems to be a slow process. In the last two years the mineral zeolite has become of interest and has been tested as a caesium binding agent in both animal and in soil-plant systems. The aim of this study was to evaluate the time dependency and the effect of zeolite on the plant availability of radiocaesium in a peat soil-plant system. The pot experiments designed for this purpose were carried out in a climate chamber, using winter wheat as the test crop. A significant reduction of the uptake of 134Cs was obtained in wheat when increasing amounts of zeolite were added. This reduction in plant uptake, up to a factor of 8, might depend on two factors. One is that zeolite has reduced the activity concentration of radiocaesium in the soil solution available to the plant roots. The other is that the potassium added with the natural zeolite increased the degree of dilution of caesium in the soil solution. Also, increasing equilibration time for caesium in soil before sowing brought about an effective reduction in the caesium uptake.


The cloning of a family of genes that encode the melanocortin receptors. Melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) regulate pigmentation and adrenal cortical function, respectively. These peptides also have a variety of biological activities in other areas, including the brain, the pituitary, and the immune system. A complete understanding of the biological activities of these hormones requires the isolation and characterization of their corresponding receptors. The murine and human MSH receptors (MSH-Rs) and a human ACTH receptor (ACTH-R) were cloned. These receptors define a subfamily of receptors coupled to guanine nucleotide-binding proteins that may include the cannabinoid receptor.


Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Extracellular signals that promote cell growth activate cascades of protein kinases. The kinases are dephosphorylated and deactivated by a single type-2A protein phosphatase. The catalytic subunit of type-2A protein phosphatase was phosphorylated by tyrosine-specific protein kinases. Phosphorylation was enhanced in the presence of the phosphatase inhibitor okadaic acid, consistent with an autodephosphorylation reaction. More than 90% of the activity of phosphatase 2A was lost when thioadenosine triphosphate was used to produce a thiophosphorylated protein resistant to autodephosphorylation. Phosphorylation in vitro occurred exclusively on Tyr307. Phosphorylation was catalyzed by p60v-src, p56lck, epidermal growth factor receptors, and insulin receptors. Transient deactivation of phosphatase 2A might enhance transmission of cellular signals through kinase cascades within cells.


Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors. The rate and extent of the agonist-dependent phosphorylation of beta 2-adrenergic receptors and rhodopsin by beta-adrenergic receptor kinase (beta ARK) are markedly enhanced on addition of G protein beta gamma subunits. With a model peptide substrate it was demonstrated that direct activation of the kinase could not account for this effect. G protein beta gamma subunits were shown to interact directly with the COOH-terminal region of beta ARK, and formation of this beta ARK-beta gamma complex resulted in receptor-facilitated membrane localization of the enzyme. The beta gamma subunits of transducin were less effective at both enhancing the rate of receptor phosphorylation and binding to the COOH-terminus of beta ARK, suggesting that the enzyme preferentially binds specific beta gamma complexes. The beta gamma-mediated membrane localization of beta ARK serves to intimately link receptor activation to beta ARK-mediated desensitization.


Influence of coexisting cirrhosis on long-term prognosis after surgery in patients with hepatocellular carcinoma. As one of the reasons for the poor prognosis of patients with hepatocellular carcinoma (HCC) and cirrhosis, the influence of cirrhosis itself has not been clarified. We compared the postoperative long-term courses of patients with HCC and cirrhosis with the courses of patients with HCC and without cirrhosis to determine how the coexisting cirrhosis affected the prognosis after surgery. The patients with HCC who underwent curative hepatic resection consisted of 142 with associated histologically confirmed cirrhosis and 48 without cirrhosis. The 5-, 7-, and 9-year survival rates were 44%, 32%, and 26%, respectively, in the patients with cirrhosis and 68%, 57%, and 57%, respectively, in the patients without cirrhosis. The prognosis of the group with cirrhosis was significantly worse than that of the group without cirrhosis. The main cause of death in both groups was cancer recurrence. The patients with cirrhosis had significantly lower recurrence-free survival rates at 3 years and later than had the patients without cirrhosis. A comparison of the background factors revealed no substantive disadvantages with regard to tumor-related and surgical factors in the patients with cirrhosis compared with the patients without cirrhosis. The recurrence-free survival rates after minor and major resection indicated fewer disadvantages of limited hepatectomy in the group with cirrhosis than in the group without cirrhosis. Moreover, the recurrence-free survival of the group with cirrhosis was shorter at less advanced stages than at more advanced stages when compared with that of the group without cirrhosis. The higher carcinogenic potential in cirrhosis could be presumed to be the most likely reason for the poorer prognosis after surgery in the patients with HCC and cirrhosis.


Mast cells and leukotrienes mediate neutrophil sequestration and lung edema after remote ischemia in rodents. Reperfusion of ischemic hindlimbs leads to leukotriene B4 (LTB4) and polymorphonuclear neutrophil (PMN)-dependent lung injury. Pulmonary mast cells are capable of synthesizing LTB4 and are potential mediators of this inflammatory response. This study tests their role in PMN sequestration and pulmonary edema after hindlimb ischemia. Anesthetized, mast cell-sufficient mice (n = 8) or their congeneic mast cell-deficient strain (n = 8) were subjected to 3 hours of hindlimb ischemia. After another 3 hours of reperfusion, plasma LTB4 levels rose to 651 pg/ml, higher than sham ischemic control (n = 8) values of 202 pg/ml (p less than 0.05). At this time there was sequestration of neutrophils in the pulmonary microcirculation (54 PMN/10 high-power fields [HPF]) and an increase in lung wet/dry weight ratio (W/D) of 4.4. Both these values were higher (p less than 0.05) than those in sham ischemic animals that showed sequestration of 18 PMN/10 HPF and a lung W/D of 3.1. In contrast, mast cell-deficient mice showed an attenuation of ischemia- and reperfusion-induced rise in plasma LTB4 (507 pg/ml), fewer sequestered neutrophils (34 PMNs/10 HPF), and a reduction in lung W/D to 3.9 (all p less than 0.05). To test the role of lung LTB4 in determining PMN sequestration, rats (n = 78) were subjected to 3 hours of hindlimb ischemia. After 3 hours of reperfusion, plasma and bronchoalveolar lavage (BAL) LTB4 concentrations rose to 956 and 211 pg/ml, respectively--higher than sham values of 460 and 121 pg/ml (both p less than 0.05). After 4 hours, plasma LTB4 levels had returned to baseline, whereas BAL LTB4 had increased further to 658 pg/ml, indicating lung origin. Treatment of other rats by localized lung lavage of the lipoxygenase inhibitor diethylcarbamazine (80 mg/kg in 0.1 ml twice) prevented the ischemia- and reperfusion-induced rise in BAL LTB4 (267 pg/ml) and limited local neutrophil sequestration (from 51 PMN/10 HPF after saline aspiration to 36 PMN/10 HPF) and lung W/D (from 4.5 to 4.1) (all p less than 0.05). The data indicate that after hindlimb ischemia pulmonary mast cells and localized LTB4 synthesis mediate, in part, the lung inflammatory response.


Percutaneous interstitial laser therapy of a patient with recurrent hepatoma in a transplanted liver. We report in situ treatment, by focal hyperthermia, of a recurrent hepatoma in the transplanted liver of a 53-year-old man. Hyperthermia was generated by neodymium-yttrium-aluminum-garnet laser, which was delivered through a fiber placed inside a 19-gauge needle and inserted percutaneously into the liver under ultrasound guidance. The effect was monitored in real time by ultrasound and subsequently confirmed by computerized tomography and needle core biopsy. Objectively the tumor growth was halted for 3 months, indicating partial response to this minimally invasive treatment.


Autoantibodies to thrombomodulin: development of an enzyme immunoassay and a survey of their frequency in patients with the lupus anticoagulant. In order to investigate the possibility that autoantibodies to thrombomodulin (TM) may exist in patients with the lupus anticoagulant (LA) and perhaps be implicated in the pathogenesis of recurrent thrombosis seen in such patients, we developed an enzyme-immunoassay to screen serum samples for anti-human TM activity. The major technical problem encountered in developing this assay was to reduce the non-specific binding of serum components from both the LA positive and the negative population. Considerable reduction of non-specific binding was achieved by use of a phosphate/citrate buffer at pH 8.0 and the use of an optimal sample dilution of 1/40. In addition, samples were always tested in parallel in blank wells and results are expressed as an OD ratio. Samples from 113 patients with the LA were assayed and compared to 78 patients referred for LA testing but found to be negative. The mean OD values for the LA positive patients (+/- SD) was 1.36 (0.44) with a range of 0.78-2.57. This was virtually identical to the values for the LA negative population (1.38 +/- 0.40, range 0.76-2.77). The results of this study indicate that there is no evidence for the presence of a significant autoantibody activity to TM in patients with the LA when compared to LA negative patients. If such autoantibodies do exist their frequency must be quite low.


Insertions/deletions in the antithrombin gene: 3 mutations associated with non-expression. We have investigated the molecular basis of antithrombin deficiency in 3 individuals, 2 of whom had a proven family history of thromboembolic disease. An approximate 50% reduction in functional and immunologic levels of antithrombin was detected in plasma from the propositi indicating an allelic deficiency of antithrombin. In each case direct sequencing of amplified DNA revealed a novel mutation involving single bases: two being insertions, of a T in codon 48 and an A in codon 208, and the third being the deletion of an A in codon 370. The three mutations, which were confirmed by cloning and sequencing the normal and variant alleles, all caused frameshifts leading to premature termination of protein translation. In no case could a truncated antithrombin be detected in plasma from the propositus suggesting either that it fails to be secreted, or is rapidly degraded.


Expression and characterization of recombinant human protein S in heterologous cells--studies of the interaction of amino acid residues leu-608 to glu-612 with human C4b-binding protein. Mouse C127 epithelioid cells were genetically engineered to produce biologically active gamma-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of gamma-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully gamma-carboxylated and fully beta-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gln, respectively and this variant was designated C variant.(ABSTRACT TRUNCATED AT 250 WORDS)


Heparin-associated thrombocytopenia: the antibody is not heparin specific. In this study the hypothesis was assessed whether heparin-associated thrombocytopenia (HAT) may be caused by an antibody dependent on polysulfated oligosaccharide epitopes, present not only on heparin but also on different polysulfated substances such as dextran sulfate and pentosan polysulfate. We found that the major factor for eliciting platelet activation with sera of HAT type II patients is neither the structure nor the AT III binding capacity of an oligosaccharide, but rather its grade of sulfation. This was shown by in vitro crossreactivity studies with 40 sera of HAT type II patients using unfractionated heparins, LMW heparins (Fragmin, Fraxiparin), enoxaparin, LMW heparinoid (Org 10172 and its subfractions), de-N-sulfated heparin, dermatan sulfate, dextran sulfate, pentosan polysulfate and dextran. Platelet activation was measured by the heparin induced platelet activation (HIPA) assay and the serotonin release assay (SRA). The platelet activating factor was isolated with the IgG fraction, but did not bind to heparin and dextran sulfate fixed to a solid phase. By isoimmune fixation electrophoresis a monoclonal gammopathy was ruled out in the three sera assessed. The in vivo effect of different LMW heparins and the heparinoid Org 10172 was observed in 10 patients with HAT type II. In a prospective study, a compatible heparin-like anticoagulant was selected for 10 HAT patients for whom further parenteral anticoagulation was required. The only substance that showed no crossreactivity in vitro was the LMW heparinoid Org 10172, which differs from heparin and LMW heparins by its low-grade sulfation. Upon treatment with the heparinoid, all 10 patients had a good clinical outcome, even if they had previously developed thromboembolic complications under LMW heparin administration.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacodynamic properties of long lasting butyryl heparin derivatives in the rabbit. This paper reports on the pharmacodynamic properties of butyryl derivatives of unfractionated heparin (C4-UH) and of low molecular weight heparin (C4-CY 216) after bolus intravenous injection, constant infusion and subcutaneous administration to rabbits. The pharmacodynamic properties of the two butyryl derivatives were compared to those of the parent compounds, unfractionated heparin (UH) and low molecular weight heparin (CY 216). After bolus intravenous injection of increasing doses, the disposition of the butyryl derivatives were comparable to that of their parent compounds up to 3 mg kg-1. Over this dose, their clearances became 2 to 3 times lower. These long lasting properties were confirmed by constant intravenous infusion experiments. After subcutaneous administration, the bioavailability of C4-UH remained low (10%) at any dose while that of C4-CY 216 ranged from 42 to 120%. If these findings are confirmed in man, these new derivatives open the possibility of treating established deep vein thrombosis with only one daily injection of a butyryl derivative of low molecular weight heparin.


On the relationship between molecular mass and anticoagulant activity in a low molecular weight heparin (enoxaparin). A low molecular weight heparin (enoxaparin, mean molecular weight approximately 4,400) was separated by gel chromatography into eight different fractions with a narrow distribution around the following mean molecular weights: 1,800, 2,400, 2,900, 4,200, 6,200, 8,600, 9,800 and 11,000. We compared the influence of enoxaparin on the generation of thrombin in plasma to that of the eight fractions. We determined: a) the % of material with high affinity to antithrombin III (HAM) and the % of HAM above the critical chain length necessary to allow for thrombin inhibition (ACLM), b) the specific catalytic activity on the decay of endogenous thrombin, and c) the inhibition of over-all thrombin formation in the extrinsic and the intrinsic pathway. From b and c we calculated the inhibition of prothrombin conversion in these pathways. We found that a) there is a gradual decrease of the HAM fraction with decreasing molecular weight; b) the specific catalytic activity for the inactivation of thrombin does not vary significantly between the fractions when expressed in terms of ACLM; c) the potency to inhibit prothrombin conversion does not vary significantly between the fractions when expressed in terms of HAM.


Differential involvement of membrane (Na-K)ATPase in thrombin- and trypsin-mediated platelet activation. The possibility that platelet activation may also involve membrane (Na-K)ATPase was investigated by testing the effects of both proteinases on platelet shape change and aggregation in the absence and presence of the specific (Na-K)ATPase inhibitor ouabain. Ouabain (8 to 80 microM) completely antagonized trypsin-induced platelet shape change and aggregation when it was preincubated with platelet suspension before the addition of trypsin. Unlike trypsin, thrombin-induced platelet activation was significantly enhanced by ouabain. It was also observed that on partially purified beef heart (Na-K)ATPase preparation, thrombin significantly enhanced the enzyme inhibition caused by submaximal inhibitory concentrations of ouabain. Soybean trypsin inhibitor (4 micrograms/ml) employed as the agent capable to counteract proteinase effects on the (Na-K)ATPase, was shown both to prevent and antagonize the platelet activation induced by trypsin (0.3 to 1.5 micrograms/ml), but it failed to modify the responses evoked by thrombin. It is concluded that membrane (Na-K)ATPase is involved differently in platelet activation by trypsin and thrombin probably because receptor sites to which either proteinase on the platelet surface binds, are distinct. Direct enzyme involvement is indeed apparent only in trypsin-induced platelet activation.


Formation of the semiquinone anion radical from tert-butylquinone and from tert-butylhydroquinone in rat liver microsomes. The tert-butylsemiquinone anion radical is formed from tert-butylhydroquinone and from tert-butylquinone in rat liver microsomes. In the presence of oxygen, the quinone and, less extensively, the hydroquinone induce excess production of superoxide in microsomes. It is concluded that autoxidation of the semiquinone formed from the quinone by microsomal enzyme activity is responsible for superoxide formation and that the hydroquinone enters the redox cycle via autoxidation. tert-Butylquinone induces injury of the hepatocyte plasma membrane. tert-Butylhydroquinone and tert-butylquinone are metabolites of the antioxidant butylated hydroxyanisole. The semiquinone-dependent superoxide formation may contribute to the toxic actions of butylated hydroxyanisole.


Effects of Buthus martensii (Karsch) scorpion venom on sodium currents in rat anterior pituitary (GH3/B6) cells. The effects of two fractions (II, containing anti-insect toxins, and III, containing eight anti-mammal toxins) isolated from the venom of the Old World scorpion Buthus martensii (Karsch) on Na+ currents of rat anterior pituitary cells (GH3/B6 cells) were investigated using the whole-cell configuration of the patch clamp technique. Fraction II induced a temporary, and fraction III a permanent increase of the Na+ current amplitude. Application of each of the venom fractions resulted in a flattening of the curve relating steady state Na+ inactivation to membrane potential. In addition, the two fractions had specific effects. Fraction II shifted the voltage dependence of Na+ current activation by -42 mV, and the voltage dependence of Na+ inactivation by -25 mV in the absence of a conditioning depolarizing pre-pulse. Slowing of Na+ inactivation was most prominent at negative membrane potentials, resulting in a steady Na+ inward current at the holding potential of -80 mV. Fraction III induced a pronounced slowing of Na+ inactivation leading to an increase of peak Na+ currents and to incomplete steady state Na+ inactivation even at positive membrane potentials.


The role of bovine lipoproteins in the regulation of steroidogenesis and HMG-CoA reductase in bovine adrenocortical cells. The sources of cholesterol for steroid hormone production were examined using bovine adrenocortical (BAC) cells in primary culture. The experiments were designed to determine the effects of lipoproteins on cortisol production and the level of BAC cell 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Most studies on BAC cell lipoprotein requirements have been conducted using human low-density lipoprotein (hHDL); none have used the homologous bovine lipoproteins. BAC cells treated with corticotropin (ACTH) in a medium devoid of lipoproteins increased and maintained cortisol production 7- to 20-fold above basal levels. Under such conditions ACTH also increased the rate of HMG-CoA reductase activity. Inhibition of HMG-CoA reductase with mevinolin inhibited cortisol production by 85%, indicating that the cells were using cholesterol synthesized de novo for steroid production. Cortisol production was increased almost 40-fold above basal levels if hLDL (100 micrograms/ml) was included in the incubation medium. Human LDL also suppressed the levels of HMG-CoA reductase in a concentration-dependent fashion. Human HDL was without effect on either BAC cell steroidogenesis of HMG-CoA reductase. Addition of bovine LDL (bLDL) to the incubation medium also caused an increase in cortisol production and inhibited cholesterol synthesis. By contrast to hHDL, bHDL (100 micrograms/ml) increased the ability of BAC cells to produce cortisol production. Bovine HDL (bHDL) also was able to decrease HMG-CoA reductase, but not to the extent caused by hLDL or bLDL. These data demonstrate that bovine adrenal cells can use bHDL as a source of cholesterol for steroid hormone production. These findings may be of particular importance when one considers that in vivo, the bHDL content of bovine serum greatly surpasses the level of bLDL.


Edema, cation content, and ATPase activity after middle cerebral artery occlusion in rats. Reduction of cerebral blood flow results in several acute metabolic disturbances, including a reduction in Na,K-ATPase activity. The relation between this reduction and the onset of edema is unknown, as is the effect of restoration of blood flow. Therefore, we investigated the role of decreased Na,K-ATPase activity in the pathogenesis and time course of ischemic brain edema and reperfusion. The middle cerebral arteries of rats were occluded by cannulation with a nylon suture for 30, 60, 120, or 240 minutes. The animals were then decapitated (permanent occlusion) or the suture was withdrawn to allow 24 hours of reperfusion before decapitation (temporary occlusion). Na,K-ATPase activity and Na+, K+ and water contents were measured at various intervals. In the ischemic hemisphere, Na,K-ATPase activity was significantly decreased at 30, 60, 120, and 240 minutes of permanent occlusion (p less than 0.05). There was also a significant decrease in rats subjected to 60 or 120 minutes of temporary occlusion followed by 24 hours of reperfusion. Water content increased after 60, 120, or 240 minutes of permanent occlusion (p less than 0.01); after 24 hours of reperfusion, water content remained elevated (p less than 0.01). The Na+ content increased after both permanent and temporary occlusion, and the K+ content decreased only after permanent occlusion. Increases in water content correlated with decreases in Na,K-ATPase activity after temporary occlusion and with the Na+:K+ ratio after permanent occlusion. Reduction in Na,K-ATPase activity resulting in disruption of cellular ionic homeostasis may account for early development of cytotoxic brain edema after permanent occlusion of the middle cerebral artery. Such edema is also present 24 hours after 60 and 120 but not 30 minutes of temporary occlusion.


Actions of 6-epitetrodotoxin and 11-deoxytetrodotoxin on the frog skeletal muscle fiber. 6-Epitetrodotoxin (6-epiTTX) and 11-deoxytetrodotoxin (11-deoxyTTX), isolated from an Okinawan newt, Cynops ensicauda, were tested for sodium-channel blocking effects on the voltage-clamped frog skeletal muscle fiber. In 6-epiTTX, the C-6 -OH is in an epimeric position; in 11-deoxyTTX, C-11 has a methyl in place of a hydroxymethyl group. At pH 7.25, the ED50s for reducing INa are: 4.1 nM (TTX), 96 nM (6-epiTTX), and 445 nM (11-deoxyTTX). In each analogue, the lowered potency can be attributed energetically to the loss of a hydrogen bond. By complementarity, in the sodium-channel receptor for TTX, there must be a hydrogen-acceptor group for the C-6 -OH, and another for the C-11 -OH. Therefore, the TTX molecule is bound to the receptor through an ion-pair (for the guanidinium), and five hydrogen bonds, one each for the -OH on C-9, C-10, C-4, and, as now identified, for C-6 and C-11. Considering the three-dimensional structure of the toxin molecules, these binding sites must be located in a fold or a crevice of the channel protein. If glutamate 387 of rat brain sodium channel II is the ion-pairing site for the guanidinium group, then the carbonyl oxygen of asparagine 388 is the hydrogen acceptor for the C-9 and C-10 -OHs.


Expedient liquid chromatographic analysis of azathioprine in plasma by use of silica solid phase extraction. We report an expedient analysis of azathioprine (AZN) in plasma by use of a radial compression liquid chromatography and silica solid phase extraction. The separation of the drug and internal standard (antipyrine) was achieved on a C18 cartridge using a mixture of 0.01 M aqueous solution of sodium phosphate (pH = 4.5) and methanol (87:13; vol/vol) as the mobile phase. The assay is highly specific with no interference from any endogenous substance or concomitantly used drug. The peak height ratio of drug to internal standard was linearly (r greater than 0.09976) related to concentration in the range 0.005-2 micrograms/ml, and the within-run and between-day coefficient of variation did not exceed 6.9% at any concentration. This study measured the steady-state concentration of azathioprine in plasma of renal transplants patients. Also, the applicability of this method to study the pharmacokinetics of AZN following a single intravenous administration was examined, using the rabbit as an in vivo model.


Actions of decarbamoyloxysaxitoxin and decarbamoylneosaxitoxin on the frog skeletal muscle fiber. Two new analogues of the decarbamoyl series of paralytic shellfish toxins have been isolated through improved HPLC methods. In decarbamoyloxysaxitoxin (doSTX), the -OH function at C-13 of decarbamoylsaxitoxin (dcSTX) is changed to -CH3. In decarbamoylneosaxitoxin (dcneoSTX), the carbomyl side-chain of neosaxitoxin (neoSTX) has been removed. The new analogues were assayed on voltage-clamped frog skeletal muscle fiber for their potency in reducing the sodium current. Compared with neoSTX, the relative potencies of dcneoSTX are: 0.003 (at pH 6.50), 0.004 (pH 7.25), and 0.005 (pH 8.25). The influence of pH on the potency is the same in neoSTX and dcneoSTX. The fractional loss of potency caused by decarbamoylation is much greater in neoSTX than in STX, possibly because of an intramolecular interaction between the N-1 -OH in neoSTX and the -OH on C-13. Compared with STX, the ED50 for reducing INa by doSTX is 618 nM, making its relative potency 0.008 that of STX. Energetically, the decreased potency can be accounted for by the loss of two hydrogen bonds, one at the C-13 -OH of dcSTX, and the other at the amino group in the carbamoyl function of STX. These two groups resemble the C-6 and C-11 -OHs in tetrodotoxin, and probably bind to the same site-points. Thus, the near-identical actions of STX and TTX can be attributed to the common sharing of one ion-pair site and four hydrogen-bonding sites. If glutamate 387 of rat brain sodium channel II were the anionic site which ion-pairs with the 7, 8, 9 guanidinium of STX, then the carbonyl oxygen of asparagin 388 is the hydrogen-acceptor for the C-12 gem-diols.


Induction of thymus-reestablishing lymphoid tumors by a murine leukemia virus carrying the avian v-myc oncogene. Primary lymphoid tumor cells induced by wild type or recombinant Moloney murine leukemia viruses were inoculated intravenously into sublethally irradiated, syngeneic mice and compared for their ability to reestablish in the recipient thymus and spleen. Lymphoid tumors induced by one recombinant, M-MuLV(myc), containing the v-myc oncogene from the avian MC29 retrovirus consistently reestablished in the thymus and spleen of recipient mice. Since tumors in the recipient thymus and spleen could have arisen by infection of host cells, or could reflect an expansion of a minor subpopulation from the donor tumor, we verified the donor and clonal origin of these tumors by Southern blot analysis using a virus-specific probe. As few as 500 of these tumor cells could migrate to the thymus via the blood and flourish in that microenvironment. In contrast, the majority of tumors induced by wild type M-MuLV or two other M-MuLV recombinants not containing v-myc sequences, inefficiently reestablished in the thymus of recipient mice following i.v. inoculation, although splenic tumors developed. These tumors could, however, multiply within the recipient thymus when inoculated intrathymically. Thus, the inability of tumors induced by wild type M-MuLV or M-MuLV recombinants lacking v-myc to reestablish in the thymus following i.v. inoculation appears to reflect inefficient 'thymic homing' rather than the ability to grow in the thymic microenvironment. Collectively, our data suggest that the thymic homing ability of M-MuLV(myc)-induced tumor cells is related to the presence of v-myc sequences or to the cell type transformed by M-MuLV(myc). Thus, we suggest that these cells may provide a useful model for studying how blood-borne metastatic cells, and possibly normal lymphocytes, enter the thymus by way of the microvasculature.


Failure to detect human cytomegalovirus DNA in peripheral blood leukocytes of healthy blood donors by the polymerase chain reaction. The transmission of cytomegalovirus (CMV) by blood transfusion may have a major effect on certain immunocompromised patients. To protect susceptible blood recipients from infection, it is advisable to use blood components from CMV-seronegative donors. However, serologic tests are not capable of indicating which blood component actually harbors infectious virus and can transfer it to the recipient. Therefore, a sensitive method is needed for the detection of the virus itself. There have been three reports on the detection of CMV in healthy volunteer blood donors by the polymerase chain reaction (PCR). CMV DNA was found in all seropositive and most seronegative blood donors. However, many other authors have failed to confirm these data. A highly sensitive and specific PCR assay was developed for the detection of CMV DNA in peripheral blood leukocytes. With this protocol, blood samples from 116 volunteer blood donors were investigated. None of these samples proved to be positive for CMV DNA. In contrast, CMV DNA was detected in 10 of 10 renal transplant patients early in the course of active CMV infection. It can be concluded that the CMV genome copy number in the peripheral blood leukocytes of healthy individuals is beyond the detection limit of current PCR technology.


Geographic risk factors for viral hepatitis and cytomegalovirus infection among United States Armed Forces blood donors. In an effort to determine whether residence in a foreign country increases the risk of hepatitis B and C and cytomegalovirus (CMV) infection in United States (US) Armed Forces blood donors, 5719 volunteer donors at four US Navy blood banks were evaluated. Most participants were repeat donors (68%) and were young (mean age, 25 years), male (88%), and white (80%), black (10%), or Hispanic (7%). Birth outside of the United States was reported by 6 percent of subjects, and 34 percent had lived in a foreign country for more than 3 months. Twenty (0.3%) subjects had hepatitis B surface antigen (HBsAg), and 100 (1.7%) had antibody to hepatitis B core antigen (anti-HBc). Thirty-four (0.6%) were repeatably reactive in enzyme-linked immunosorbent assay for antibody to hepatitis C virus (anti-HCV); 11 (0.2%) had anti-HCV in immumoblot assay. Of the 3484 donors tested for anti-CMV, 1117 (32.1%) were positive. When demographic characteristics were controlled for both anti-HBc and anti-CMV seropositivies were independently associated in male blood donors with residence in the Philippines. Geographic factors were not associated with HBsAg and anti-HCV positivity. These findings indicate that the prevalence of serologic markers for viral hepatitis is low in military blood donors, but that residence in the Western Pacific is a risk factor for hepatitis B and CMV infection.


Blood donation and transfusion practices: the 1990 American Association of Blood Banks Institutional Membership Questionnaire. Responses to the 1990 American Association of Blood Banks (AABB) Institutional Membership Questionnaire were submitted by 2126 regional blood centers, hospital-based blood banks, and transfusion facilities. Data from 2117 of these facilities were considered to be valid. The questionnaire included information on blood donor demographics, number of units collected, and collection procedures; services performed; usage of blood components; and transfusion-transmitted diseases reported during 1989. Institutional members collected 7.4 million whole blood units, of which 90.8 percent were donated for allogeneic use, 6.0 percent were donated for autologous use, and 3.2 percent were donated for directed use. Approximately 630,546 allogeneic and directed-use blood donors were deferred, most often for low hemoglobin or hematocrit values. Approximately 225,205 full allogeneic and directed-donor units were discarded, primarily for elevated alanine aminotransferase levels or the presence of hepatitis B core antibody. The 14.3 million transfused components included 56.7 percent red cell-containing components, 27.4 percent platelets, 11 percent fresh-frozen plasma, and 4.8 percent cryoprecipitate. Institutional members reported 1397 cases of transfusion-associated hepatitis. In this group, 921 patients were tested for hepatitis B surface antigen after the transfusion; 339 (36.8%) were found to be hepatitis B surface antigen positive. The AABB Institutional Questionnaire results provide recent data on blood donor and transfusion-related activities that are vital to the evaluation of current transfusion medicine practices.


Analysis of the amino terminal presequence of the feline immunodeficiency virus glycoprotein: effect of deletions on the intracellular transport of gp95. The envelope glycoprotein of feline immunodeficiency virus (FIV) consists of two noncovalently associated subunits, the surface glycoprotein (SU; gp95) and the transmembrane glycoprotein (TM; gp40). An unusual feature of the open reading frame (ORF) encoding the FIV glycoprotein is the presence of an unusually long amino terminal sequence (149 amino acids, "L" region or n-region of the signal sequence) preceding the predicted hydrophobic signal sequence. To examine the role of this n-region in the biosynthesis of gp95, the gene-encoding signal sequence and the surface glycoprotein (gp95) were expressed using recombinant vaccinia viruses. Glycoprotein mutants were constructed with 25, 42, 73, 102, and 147 amino acids removed from the n-region. Expression studies revealed that deletion of 25-102 amino acids did not appreciably effect the biosynthesis, intracellular transport, and release of gp95 from the cell surface. In contrast, removal of 147 of 149 amino acids resulted in the gp95 that was blocked in release from the cell. These results indicate that between 3 and 47 amino acids of the n-region are required for the proper biosynthesis, processing, and release of the FIV gp95 from infected cells.


Human papillomavirus type 13 and pygmy chimpanzee papillomavirus type 1: comparison of the genome organizations. Human papillomavirus type 13(HPV-13) is associated with oral focal epithelial hyperplasia (FEH) in humans. A recent epidemic of a FEH-like disease in a pygmy chimpanzee (Pan paniscus) colony allowed us to clone a novel papillomavirus genome. To assess the homology between HPV-13 and the pygmy chimpanzee papillomavirus type 1 (PCPV-1), the complete nucleotide sequences of both FEH-related viruses were determined. In both viruses, all eight major open reading frames were located on one strand and the genomic organization was similar to that of other mucosal papillomaviruses. The genomes of PCPV-1 and HPV-13 showed extensive overall sequence homology (85%). They could be classified, using phylogenetic analysis, together with HPV types 6, 11, 43, and 44 in a group associated with benign orogenital lesions. These data indicate that two phylogenetically related papillomaviruses can elicit similar pathology in different primate host species, reflecting viral genomic similarities.


Genetic relationships between bovine herpesvirus 4 and the gammaherpesviruses Epstein-Barr virus and herpesvirus saimiri. The overall arrangement of genes in the unique central part of the bovine herpesvirus type 4 (BHV-4) genome has been deduced by analysis of short DNA sequences. Twenty-three genes conserved in at least one of the completely sequenced herpesviruses have been identified and localized. All of these genes encoded amino acid sequences with higher similarity to proteins of the gammaherpesviruses Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simplex virus type 1 or the betaherpesvirus human cytomegalovirus. The genome organization of BHV-4 had also an overall colinearity with that of the gammaherpesviruses EBV and HVS. Furthermore, the BHV-4 genes content and arrangement were more similar to those of HVS than to those of EBV, suggesting that BHV-4 and HVS are evolutionarily more closely related to each other than either are to EBV. BHV-4 DNA sequences were generally deficient in CpG dinucleotide. This CpG deficiency is characteristic of gammaherpesvirus genomes and suggests that the BHV-4 latent genome is extensively methylated. Despite several biological features similar to those of betaherpesviruses, BHV-4 displays the molecular characteristics of the representative members of the gammaherpesvirinae subfamily.


Bovine herpesvirus 1 as a live virus vector for expression of foreign genes. DNA sequence analysis and 5'-end mapping of mRNA were used to identify and clone DNA fragments which contain the presumptive promoter (Pgl) and poly(A) site (An) of the bovine herpesvirus 1 (BHV1) gl glycoprotein. To confirm the presence of these regulatory regions in the above fragments, they were cloned together with a chloramphenicol acetyltransferase (CAT) reporter gene into pUC19. The recombinant plasmid formed, pEC3, was capable of inducing CAT activity when transfected into bovine cells demonstrating that the Pgl-CAT-An sequence constituted a functional CAT expression cassette. The cassette was excised from pEC3, transferred to a plasmid insertion vector (pIV3A) and subsequently inserted into the thymidine kinase (tk) gene of BHV1. Insertion in either orientation, relative to the tk gene, gave rise to BHV1 recombinants which expressed CAT activity in infected cells. Analysis of RNA from infected cells indicated that CAT transcripts were present in multiple species of RNA. This unexpected result was found to reflect temporal shifts in promoter and poly(A) site usage during infection. Although the poly(A) site which forms part of the expression cassette was used extensively early in infection, most CAT transcripts synthesized at late times read through this promoter-proximal site and terminated at the distal site normally used for tk mRNA synthesis.


Noninfectious L-particles supply functions which can facilitate infection by HSV-1. The tegument of herpes simplex virus contains proteins important for the efficient initiation of infection. L-particles are noninfectious virion-related particles that lack the nucleocapsid but do contain tegument and envelope. Their ability to deliver functional proteins into cells was compared to that of virions by testing the biological activities of two tegument proteins which are present in both types of particle. Results indicated that L-particles are as efficient as virions in supplying these in a fully functional state. Thus, L-particles are biologically competent and have the potential to participate in the early stages of virus infection.


Sequencing and 5'- and 3'-end transcript mapping of the gene encoding the small subunit of ribonucleotide reductase from bovine herpesvirus type-1. The complete nucleotide sequence of the gene encoding the small subunit of ribonucleotide reductase (RNR) from bovine herpesvirus type-1 (BHV-1) was determined. The genomic DNA fragment sequenced also represented regions corresponding to the carboxy termini of RNR large subunit and of a virion protein causing host shut-off. The small subunit polypeptide was constituted of 314 amino acid residues totalling 35.25 kDa. The major transcription initiation and termination sites of the small subunit mRNA were located 95 bases upstream and 88 nucleotides downstream from the coding region, respectively. These findings indicate that the mRNA was 1128 bases long which correlated well with the size of the polyadenylated transcript detected in Northern blot analysis (1.3 kb). Within the RNR large subunit coding region, a TATA box and two CAAT box motifs were found 26, 104, and 190 nucleotides, respectively, upstream from the transcription initiation site of the small subunit mRNA. In contrast to previous studies (Slabaugh et al., J. Virol. 1988, 62, 519-527; Boursnell et al., Virology 1991, 184, 411-416), our comparative analysis of five herpesviruses, one iridovirus, and one poxvirus small subunit protein sequences suggested that the seven viruses arose from a common lineage.


Genetic analysis of the herpes simplex virus type 1 UL9 gene: isolation of a LacZ insertion mutant and expression in eukaryotic cells. HSV-1 host range mutants in complementation group 1-36 (hr27 and hr156) whose mutations map in the UL9 gene, encoding the origin binding protein, are unable to form plaques or synthesize viral DNA or late viral proteins when grown in nonpermissive Vero cells (Carmichael, E. P., Kosovsky, M. J., Weller, S. K., 1988, J. Virol. 62, 91-99). These defects are complemented efficiently by growth in the permissive cell line, S22, which contains the wild type version of several HSV genes including UL9. In this report the precise nature and location of the lesions in host range mutants hr27 and hr156 were determined by DNA sequencing; both mutants were found to contain identical single-base-pair substitutions at codons 309 and 311 in the UL9 open reading frame. This region lies within the putative helicase domain of the UL9 protein. The UL9 gene was disrupted by the insertion of an insertional mutagen ICP6::lacZ in which the Escherichia coli lacZ gene is expressed under control of the viral ICP6 promoter. Hr94, a viral mutant containing this insertion, does not form plaques or synthesize viral DNA when grown in Vero cells, although both defects are complemented efficiently on permissive cell lines. These results confirm that the UL9 gene product is essential for viral growth and DNA replication. Furthermore, since no detectable UL9 protein is synthesized in hr94-infected cells, this virus provides a useful genetic background for further structure-function analysis since no potentially interfering nonfunctional UL9 protein will be expressed. We have expressed the UL9 open reading frame under the control of the strong and inducible HSV-1 ICP6 promoter and have derived Vero cell lines containing variable copy numbers of the ICP6::UL9 construct. Cells whose copy number of this construct exceeded approximately 120 are unable to support efficient plaque formation by wild-type virus. Cell lines with low copy numbers of this construct are able to complement hr27, hr156, and hr94.


Interaction of the human polyomavirus, JCV, with human B-lymphocytes. The human polyomavirus, JCV, is the causative agent of the central nervous system demyelinating disease progressive multifocal leukoencephalopathy (PML). The principal target of JCV infection in the central nervous system (CNS) is the myelinating oligodendrocyte. However, the site of JCV multiplication outside of the CNS and the mechanism by which virus gains access to the brain are not known. Recently, JCV infected B-lymphocytes have been demonstrated in PML patients in several lymphoid organs, in circulating peripheral lymphocytes, and in brain, suggesting a possible role of B-lymphocytes in the dissemination of virus to the brain. The experiments reported here were undertaken to understand more about the interactions of JCV with human B-lymphocytes. The data show that JCV is able to multiply in either Epstein-Barr virus transformed (EBV) or EBV negative human B cell lines resulting in production of infectious, progeny virions. In addition, nuclear proteins extracted from these B cells bind to similar nucleotides within the JCV regulatory region that are bound by nuclear proteins extracted from human fetal glial cells, the most susceptible host and principal target cell for JCV infection in vitro. It is not known, however, whether these DNA binding proteins from susceptible B cells and glial cells are similar.


Independent association of antibodies against human papillomavirus type 16 E1/E4 and E7 proteins with cervical cancer. The E4 open reading frame (ORF) of human papillomaviruses (HPVs) is transcribed in abundant mRNAs encoding an E1/E4 fusion gene during the productive infection, and the HPV 16 E7 ORF encodes an oncoprotein detectable in the cell lines derived from cervical carcinoma. We examined 421 human sera, which included 108 samples from the patients with cervical carcinoma, for the presence of IgG antibodies against the HPV 16 E4 and E7 proteins by enzyme-linked immunosorbent assay. Bacterially expressed fusion protein lac-E1/E4 and nonfusion protein E7 were purified and used as antigens. All of the 22 serum samples positive for anti-E7 antibody and the 11 out of 15 samples positive for anti-E1/E4 antibody were from the patients with cervical carcinoma, but only one sample was found to contain both anti-E1/E4 and anti-E7 antibodies. These findings show specific and independent association of these antibodies with cervical carcinoma.


Proteolytic processing of the cardioviral P2 region: primary 2A/2B cleavage in clone-derived precursors. The primary 2A/2B cleavage within cardiovirus polyprotein was examined by construction of cDNA plasmids which linked fragments from the P2 region of encephalomyocarditis virus (EMCV) and Mengovirus genomes to the EMCV 5' nontranslated region. When RNA transcripts from these clones were tested in reticulocyte extracts, the synthesized proteins were cotranslationally processed at the 2A/2B site. No viral segments outside of the P2 region were required for this activity. Engineered deletions which removed the amino-terminal two-thirds of protein 2A or the carboxyl half of protein 2B had no effect on this scission, nor did insertions into a Ser-Ala-Phe sequence (SAF) within 2B, which is conserved in most cardio- and aphthoviruses. In contrast, mutations which disrupted a conserved Asn-Pro-Gly-Pro (NPGP) sequence abolished primary scission. Precursors thus inactivated were unable to serve as substrate when simultaneously expressed with active (wild-type) 2AB sequences. Microsequencing placed the EMCV primary cleavage site between the Gly/Pro pair within the NPGP sequence. It was also determined that endogenous viral protease 3C is the previously unidentified agent responsible for cardiovirus 1D/2A scission, a cleavage that is part of the primary processing reaction in poliovirus.


Cell surface ligands for rotavirus: mouse intestinal glycolipids and synthetic carbohydrate analogs. Rotaviral binding to receptors on epithelial cells in the small intestine is thought to be a key event in the infection process and may be carbohydrate-mediated. Strain SA11 of rotavirus bound in vitro both to glycolipids isolated from mouse small intestine and to authentic glycolipids using thin layer chromatography overlay and microtiter well adsorption assays. Neutral mouse intestinal glycolipids which bound rotavirus were GA1 (Gal beta 1----3GalNAc beta 1---4Glc beta 1----4Glc beta 1----1-ceramide) and pentaosylceramides with terminal N-acetylgalactosamine, while acidic lipids which bound rotavirus included cholesterol 3-sulfate and two compounds termed bands 80 and 81. Digestion with ceramide glycanase suggested that bands 80 and 81 have lactosyl ceramide cores and an unidentified acidic moiety(s). No sialic-acid-containing glycolipids tested were active in viral binding. Band 81, which may have a ganglio core, bound rotavirus with greatest avidity, followed by GA1. Of authentic glycolipids assayed, only GA1 and GA2 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1-ceramide) displayed rotaviral binding. A phosphatidylethanolamide dipalmitoyl-containing neoglycolipid analog of GA2 bound rotavirus with avidity similar to native GA2. Substitution of beta 1----4-linked GlcNAc or beta 1----3-linked GalNAc for terminal GalNAc of GA2 neoglycolipid supported rotaviral binding, while other substitutions abrogated it. These findings suggest that a carbohydrate epitope similar to that of GA2 is sufficient for in vitro rotaviral binding, although binding may be enhanced by galactose and/or an acidic moiety in a secondary epitope.


Role of exonuclease in the specificity of bacteriophage T7 DNA packaging. During morphogenesis in vivo, bacteriophage T7 packages and cuts to mature size an end-to-end concatemer of its nonpermuted, terminally repetitious, double-stranded, mature DNA. Efficient production (90-100%) and packaging (20-35%) of concatemers has also been demonstrated in extracts of T7-infected cells (in vitro) (Son, M., Hayes, S. J., and Serwer, P. [1988] Virology 162, 38-46). By use of both this procedure of in vitro DNA packaging and in-gel hybridization to packaged DNA fractionated by agarose gel electrophoresis, the specificity of packaging in vitro is found to depend on the presence of T7 gene 6 exonuclease (p6). In the absence of p6 in vitro, no concatemerization is detected and packaging of DNA nonhomologous to T7 DNA (bacteriophage P22 DNA) is as efficient (0.05-1.1%) as the packaging of monomeric T7 DNA. Addition of p6 in vitro both stimulates the concatemerization-packaging of T7 DNA and suppresses the packaging of P22 DNA. The packaging efficiency for concatemeric T7 DNA is 29-611 x higher than that for monomeric T7 DNA. Inhibition of the packaging of P22 DNA by p6 is correlated with the formation of single-stranded P22 DNA ends. These data are explained by the hypothesis that a DNA molecule with a single-stranded end is packaged less efficiently than the same DNA without the single-stranded end. Testing this hypothesis in vivo reveals that both p6 and gene 3 endonuclease contribute to suppressing the packaging of host DNA.


In vitro accurate transcription from the cap site of bovine leukemia virus (BLV) dependent on the BLV-infected cell nuclear lysate. The cell-free transcriptional system initiating from the cap site in bovine leukemia virus (BLV) LTR by RNA polymerase II was constructed. The transcription was completely dependent on the template DNA and the nuclear lysate isolated from BLV-infected bat lung cells (TB1Lu). The relative transcriptional rates estimated using several deletion mutants around the promoter sequence in BLV LTR as templates closely corresponded to that obtained by transient expression assay in cultured cells using these plasmids and tax-producing plasmid. The partial purification of the factor(s) involving to the transcriptional activation from the nuclear lysate suggested that the factor(s) was different from tax and rex, the regulatory factors encoded on viral genome. The transcription from the caps site of adenovirus E3 was also stimulated in the presence of the nuclear lysate from BLV-infected cells.


Identification of heat shock protein 70 in the rabies virion. We investigated a 73-kDa polypeptide (p73), a minor component of the rabies virion (HEP-Flury and ERA strains), accounting for as much as 1% of total virion proteins. Two-dimensional gel electrophoresis and immunoblotting with the antiserum against the heat shock protein 70 (hsp70) demonstrated that p73 was identical to a constitutive type of cellular hsp70. The antiserum also detected p73/hsp70 in the purified virions of other negative-stranded RNA viruses, such as vesicular stomatitis virus (New Jersey serotype), Newcastle disease virus (Miyadera strain), and influenza A virus (PR8 strain), among which, however, the contents were variable.


Synergism between bovine papillomavirus type 4 and the flavonoid quercetin in cell transformation in vitro. Bovine papillomavirus type 4 (BPV-4) morphologically transforms primary bovine cells in vitro only in the presence of an activated ras gene. The transformed cells are capable of anchorage-independent growth, but are not immortal and are incapable of inducing tumors in nude mice, suggesting that other events are needed to convert the cells to the fully transformed phenotype. We show here that treatment of the cells with a single dose of the flavonoid quercetin leads to full oncogenic transformation of cells transfected with BPV-4 and ras. Quercetin is one of the most potent mutagens found in bracken fern, the environmental cofactor in BPV-4-associated carcinogenesis of the upper alimentary canal of cattle. Our results point to quercetin as the probable in vivo cocarcinogen synergizing with BPV-4 in malignant progression.


5' avian leukosis virus sequences and osteopetrotic potential. Recombinants of Rous-associated virus-0 and Br21 have been used to localize 5' viral sequences that affect the osteopetrotic potential of avian leukosis viruses. Rous-associated virus-0 is a benign subgroup E virus of endogenous origin that does not cause osteopetrosis. Br21 is a constructed subgroup E virus with high osteopetrotic potential. 5' sequences that affected osteopetrotic potential resided in an 834-bp region near the 5' LTR. Sequence analysis of this region revealed differences between Br21 and RAV-0 in the mRNA leader and codons for MA.


Changing the spacing between metal-binding motifs decreases stability and transforming activity of the human papillomavirus type 18 E7 oncoprotein. Spacing composed of 29 amino acids (AAs) between a pair of metal-binding motifs, Cys-X-X-Cys, is common to human papillomavirus (HPV) zinc-binding oncoproteins E6 and E7 from various HPV types. From the HPV 18 E7 gene encoding a 105-AA protein with the motifs at AAs 65-68 and 98-101, we constructed expression plasmids for two mutants, 18del73 and 18ins84, with varied spacing between the motifs. Mutant proteins 18del73 and 18ins84 had a deletion from AAs 74 to 88 and an insertion of three AAs substituting for AA 85, respectively. The mutations lowered the efficiency of E7-mediated focal transformation of rat 3Y1 cells, approximately parallel to the reduced level of steady-state E7 expressed in COS-1 cells. It is likely that, besides the presence of the motifs, the conserved spacing between the motifs is important for a stable structure of E7, but is not essential for the E7-transforming activity.


Genetic drift of hepatitis C virus during an 8.2-year infection in a chimpanzee: variability and stability. Extensive variability in genomic sequence, especially at "hypervariable regions" within the NS1/E2 region of the long open reading frame, has been reported for RNA cloned from hepatitis C virus (HCV)-infected humans and chimpanzees. However, genetic changes of HCV occurring during the course of chronic infections in humans and animals have been evaluated only for partial sequences of the HCV genome. We compared two full-length cDNA sequences of HCV obtained from a chimpanzee that was experimentally infected with the HC-J4 strain of HCV: one during the early acute phase and another during a chronic phase 8.2 years afterward. Both isolates had 9412 nucleotides plus the 3' poly(U) tail with varying length organized as follows: 5'UTR (1-341); C (342-914); E (915-1490); NS1/E2 (1491-2528); NS2 (2529-3359); NS3 (3360-5186); NS4 (5187-6380); NS5 (6381-9371); and 3'UTR (9372-9412). We found that 111 (1.18%) of the 9412 nucleotides differed between the two isolates and estimated the mutation rate as approximately 1.44 x 10(-3) base substitutions per site per year. Changes in amino acid coding were associated with 42 mutations, 8 of which were clustered at 5' end of NS1/E2 coding region, so-called "HVR-1." We analyzed the HVR-1 and HVR-2 sequences during the course of infection and found that homologous populations were present at the beginning of infection, and sequence heterogeneity within the region had developed 3.5 years later. Two regions of the HCV genome were characterized by a high degree of conservation of nucleotide sequence: 5'UTR and the 3' half of the NS4 region. The possible secondary structure of the 5'UTR suggests a region for internal ribosomal entry. The 3' half of the NS4 region may also have some specific function which depends upon a strict conservation of nucleotide sequence.


Seropositivity to hepatitis C virus in Saudi haemodialysis patients. Seropositivity to hepatitis B, C and D viruses was studied in 66 Saudi haemodialysis patients from Al Baha region, south of Saudi Arabia, and was compared to that in 380 healthy Saudis from the same region. The results showed that HCV is endemic in this region (3.6%) and the prevalence of anti-HCV in the haemodialysis patients was 45.5%, which is the highest reported so far in this group of patients. Anti-HCV was related to the amount of blood transfused (greater than 5 units) and the duration of dialysis (greater than 3 years). The region is also hyperendemic for hepatitis B virus (67.4% overall exposure rate in controls compared to 75.7% in haemodialysis patients) and hepatitis D virus (HDV; 11.4% in controls compared to 12.5% in haemodialysis patients). The similarity in profile of HBV markers and HDV between haemodialysis patients and controls indicates that the current strategy regarding HBV is quite effective, in preventing the transmission of HBV and hence HDV but not HCV in haemodialysis patients. A strategy for preventing HCV is recommended.


A study of hepatitis C virus antibodies and serum alanine amino transferase in blood donors in Hong Kong Chinese. The reference range of serum alanine amino transferase (ALT) for the local population was established by testing 5,000 random voluntary Chinese blood donors of various age groups of both sexes. In addition, 1,769 serum samples with elevated ALT levels were also collected for anti-HCV assays using both the Abbott and Ortho anti-hepatitis C virus (HCV) assay kits. The relationship between serum ALT and anti-HCV tests was studied and the performances of both kits used were compared. It was found that while the prevalence of serum anti-HCV was 0.4% among hepatitis B surface antigen-negative donors with normal ALT, subjects with ALT between 2 and 3 standard deviations (SD) and greater than 3 SD above the mean level had respective prevalence of anti-HCV 3 and 9.5 times that of the normal ALT subjects. Both anti-HCV kits were found to identify in majority the same positive population among the different groups of subjects studied. In addition, it was observed that for subjects who were anti-HCV-positive, the higher the serum ALT level, the higher the mean anti-HCV ELISA ratio and this observation was similar for both anti-HCV kits used. We conclude that: (1) there is a direct relationship between serum ALT level and anti-HCV positivity by EIA; (2) there is a direct correlation between serum ALT level and anti-HCV ELISA ratio, and (3) both Abbott and Ortho anti-HCV kits perform similarly in the identification of positive serum samples.


[Cytopathology of the breast. 2. Puncture cytology]. The fine needle aspiration cytology of the breast is the only way for the morphological examination of suspicious palpable lesions without surgical procedure. The cytological examination is able to produce clinical relevant results on the basis of typical microscopical pictures especially in breast carcinoma, fibroadenoma, and cysts. However it must be taken into account that not all breast tumors can be detected by fine needle aspiration cytology. Therefore negative and doubtful results have to be interpreted in connection with clinical and mammographic findings. An essential condition for good results is an effective punction technique to obtain representative probes of cell material. In case punction and cytological examination are not performed by the same investigator there is the risk that an inadequate punction technique is not noticed and leads to unsatisfactory cytological results. In connection with clinical and mammographic examinations the fine needle aspiration cytology can be used as alternative diagnostic way and replace the conventional breast tumor diagnostic procedure by surgical biopsy and frozen section, if the necessary requirements are fulfilled. This includes a high standard of clinical, mammographic and cytological examinations and a critical summarizing interpretation of the examination results.


[Condylomata acuminata and associated infections--possibilities for therapy with interferon]. Recombinant alpha-Interferon can be used in primary therapy of condylomata acuminata. In our patients we found a response rate of 53%. Besides use in primary therapy the significant low rate of recurrences is important, thus offering the possibility of adjuvant interferon therapy after primary surgical treatment, where laser-vaporisation is best applied. Possible applications for primary and secondary treatment are shown. The rate of additional infections is of great importance; besides common agents for vaginal infections we found an elevated rate of HPV 16/18 with 47%.


Immunohistological diagnosis of rabbit haemorrhagic disease (RHD). In the present study the diagnostic use of a biotinylated serum from an immune rabbit was investigated by means of an Avidin-Biotin-Complex (ABC)-Peroxidase method on paraffin sections. 15 cases of RHD which had been verified histologically and/or by haemagglutination test (HA), 4 suspected cases and 3 cases without history of RHD were included (cases 1 to 22). From 5 prospective cases a wider tissue range was examined (cases 23 to 25 and 29 to 30). Furthermore lungs, liver and placenta of 3 fetuses from a RHD affected dam were investigated (cases 26 to 28). The 20 typical cases had intense intranuclear and diffuse intracytoplasmic immunostaining of hepatocytes, predominantly in the periportal areas. In some cases there was also positive staining of macrophages in the lungs (4 cases), spleen (4 cases) and in lymph nodes (1 case). Positive granular staining in the renal mesangial cells of the glomeruli was observed in 1 case. No positive staining was observed in the 3 negative cases. In contrast to other reports (4), crossreactivity of these antigens to Porcine Parvovirus (PPV) could not be confirmed. Furthermore the RHD virus (RHDV) seems not to crossreact with Feline and Bovine Parvoviruses.


Sero-epidemiological survey of porcine respiratory coronavirus (PRCV) infection in breeding herds in southeastern Spain. In 1987 we conducted a sero-epidemiological survey in the Murcia Region (South-East Spain) to discover the prevalence and spread of PRCV-infection among breeding pigs and farms and determine the association between herd size and geographical zone with PRCV-infection. The Murcia Region was divided into four geographical zones and the farms classified by size in four categories. The random sample was statistically representative of both the breeding stock and farms in each geographical zone. We analysed 6,000 breeding pigs from 480 farms. The immunological techniques employed were indirect ELISA and blocking ELISA. The prevalence (P +/- IC) of PRCV-seropositive breeding pigs and infected breeding farms was 14.53 +/- 0.89% and 21.87 +/- 7.83% respectively. On 55% of the infected farms, the prevalence of seropositive breeding pigs was 60-100%. PRCV-infection appears spread throughout the four geographical zones of the Murcia Region. However, a significant association (p less than 0.01) was observed between geographical zone and the prevalence of PRCV-infection. A herd size of greater than 50 breeding pigs had a greater risk (p less than 0.01) of PRCV-infection.


Serotyping and antigenic comparison of some animal rotaviruses isolated in China. Eight strains of rotaviruses isolated from diarrheal animals (4 from calves and 4 from piglets) in China were compared by serotyping with reference animal rotavirus strains (bovine NCDV, porcine OSU and simian SA-11 and human rotavirus Wa strain). Two-way cross neutralization test showed no antigenic difference between all 4 local strains of bovine rotavirus (BRV007, BRV014, HN-7 and BRV6555) and reference NCDV, so they belonged to rotavirus serotype 6 (bovine rotavirus serotype 1 or NCDV-serotype). Meanwhile, the four strains of Chinese porcine rotavirus could be determined into 2 different serotypes. One (Li99) was neutralised to a high titer with the antiserum against reference OSU virus and probably related to OSU (serotype 5 or porcine serotype 1). The other three strains (Lin71, Nan86 and Jiang150) were antigenically obviously different from Li99 and did not react with the antiserum against OSU. They were tentatively considered as porcine rotavirus serotype 2. All the strains of bovine and porcine rotavirus did not cross-neutralise with simian SA-11 and human Wa strain. There was also no antigenic relationship between bovine rotaviruses and porcine rotaviruses.


Detection of bovine adenovirus nucleic acid sequences in nasal specimens by biotinylated DNA probes. Bovine adenovirus infection has been detected in experimentally infected lambs by applying the rapid and simple DNA detection method of direct filter hybridization (DFH). Three fragments of BAV-2 DNA were obtained by molecular cloning and labelled with biotin. Clone 207, representing an EcoRI fragment between coordinates 35 to 55 of the viral genome, showed the highest sensitivity to detect adenovirus DNA, thus, this probe was applied on nasal cells from the lambs. Comparative tests revealed a concordance over 97% between the results of conventional virus isolation and of the DFH method. Virus isolation required several weeks to be completed, whereas the DFH method gave results within 10 hours. Considering its simplicity, speed and economy, the DFH method is recommended as a practical diagnostic tool for the direct detection of adenoviruses in clinical specimens.


Some physicochemical properties of the virus of rabbit haemorrhagic disease. Purified and concentrated preparations of virus from liver extracts of infected rabbits contain virus specific components with sedimentation coefficients of about 175, 110 and sometimes 133S and more slow units. Full and empty virus particles with a diameter of about 34 nm were shown electron microscopically in the corresponding 175 and 110S fractions of the sucrose density gradient. The average of buoyant density of the 175, 133, 110S and more slow units are 1.36, 1.32 and 1.31 g/ml respectively. The extinction coefficient E260 nm is 4.3 +/- 0.7 cm2/mg. The RNA content is 17 +/- 4%. SDS-PAGE shows a "65" kD protein as a single or major component. Beside smaller polypeptides with lower intensities, the 67 kD polypeptide reacts positively in the Western blot with polyclonal antibodies of rabbits. The molecular weight of the virus is 15 +/- 4 x 10(6)D. The pH stability of the 175S unit was also tested.


Monitoring of effects induced by recombinant equine interferon-beta 1 in whole blood and separated fractions of peripheral blood of horses. Interferon is known to induce antiviral mechanisms and to exert immunoregulatory capacities on various cell types. The antiviral capacity of recombinant equine interferon-beta 1 (rEqIFN-beta 1) is most sensitively monitored by indirect quantitation of multiplication of vesicular stomatitis virus (VSV) in blood cells of horses. As few as 0.5 pg rEqIFN-beta 1/ml can be assessed by means of 90% reduction of VSV-replication in whole blood (w.b.) as well as in isolated mononuclear blood cells (MNC) in spite of individual variations. The immunoregulatory influence of 20-50 pg rEqIFN-beta 1/ml is sufficient to cause at least a 50% reduction of mitogen-induced lymphocyte proliferation in MNC, while higher concentrations are needed in w.b. Of the mitogens tested the best stimulation of proliferation on the equine lymphoid cells was obtained with staphylococcal enterotoxin B (SEB). Release of reactive oxygen species (ROS) from phagocytic cells in w.b. or from isolated polymorphonuclear cells (PMN) as monitored by chemiluminescence (CL) does not seem suitable for evaluation of rEqIFN-beta 1-induced immunoregulation as only very high rEqIFN-beta 1-concentrations (10(3)-10(4) pg/ml) result in a minute increase (up to 20%) of CL. Comparative studies on w.b. and isolated leukocyte fractions from identical specimens of individual horses suggest that monitoring of antiviral and distinct immunoregulatory capacities of rEqIFN-beta 1 can be performed on w.b. without loss of information and sensitivity as compared to isolated MNC.


Field trials of an inactivated virus vaccine against porcine parvovirus. Serological response and reproductive performance were estimated in field trials of an inactivated virus vaccine against porcine parvovirus. Experiments were carried out in 10 selected pig breeding herds. A total of 277 seronegative gilts were used. Two hundred and twenty animals were vaccinated twice before mating, fourteen days apart and revaccinated after farrowing. Blood samples were obtained from both vaccinated and non-vaccinated (57 animal) control gilts, one week after the 2nd dose of vaccination, at farrowing time and one week after revaccination. Although there were considerable variations among the herds, the number of returns to oestrus in all herds was higher in vaccinated gilts (11.81%) than in the controls (10.52%). This difference, however, was not statistically significant. The reproductive performance results revealed the absence of an increase in the total born, as pooled values, in vaccinated gilts compared to controls. However, when these results are interpreted in relation to serological data, many control gilts were already seropositive before mating, or remained seronegative at farrowing. According to our results, the duration of immunity with this vaccine is apparently short, as there is a clear decrease in the titres between the 1st and the 2nd sampling times (2.35 +/- 0.14 and 1.97 +/- 0.08, respectively).


Oral fowlpox vaccination in chickens.

Oral fowlpox vaccination in chickens. Chickens were given various fowlpox vaccines on food pellets--a commercial vaccine (strain M), and the same strain after a single passage on chorio-allantoic membrane or in chicken embryo fibroblasts. All three oral vaccines induced antibodies at levels similar to those induced by commercial strain M administered to the wingweb. The oral vaccine derived from chorio-allantoic membrane gave protection similar to that obtained with vaccine administered by the wingweb, but this required a thousandfold more virus.


Inactivation of the thyrotropin releasing hormone. Thyrotropin Releasing Hormone (TRH; pyroGlu-His-Pro-NH2) is rapidly hydrolyzed at the pyroGlu-His bond by a heterogeneously distributed ectoenzyme and a TRH-degrading serum enzyme. For these enzymes, the high degree of substrate specificity, the tissue specific hormonal regulation, the unusual tissue and the heterogenous cellular distribution strongly suggests that they serve very specialized functions for the transmission of TRH signals at specific target sites.


Effects of uridine in the treatment of diabetic neuropathy: an electrophysiological study. The authors performed a controlled double-blind neurophysiological study (uridine vs placebo) in 40 diabetic patients with peripheral neuropathy. Twenty subjects were treated with uridine and 20 with placebo. The neurophysiological evaluation consisted of a study of the MCV of the median nerve, the common Peroneal, the posterior Tibial, the SCV of the radial nerve, the median and the sural as well as the amplitudes of the motor and sensory responses. The nerves examined were on the dominant side. The evaluations were performed at baseline and after 60, 120, 180 days of therapy with a follow up control after 90 days from the completion of therapy. No statistically significant modifications were observed in the placebo group. In the drug group, the neurophysiological parameters improved significantly from the 120th day post therapy compared with baseline and were maintained through to follow up. The authors discuss the results which demonstrated that treatment with uridine can bring about a neurophysiological improvement in peripheral nerves.


Protection against ischemic hippocampal CA1 damage in the rat with a new non-NMDA antagonist, NBQX. Two glutamate antagonists were tested in a rat model of complete, transient cerebral ischemia. Six days after 10 min ischemia the mean loss of hippocampal CA1 pyramidal neurones was 73%. Administration of the AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) antagonist NBQX (2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) reduced the pyramidal neurone loss to 1%, 11% and 15%, when given before, immediately after or 1 h after ischemia, respectively. MK-801 (dizocilpine), a competitive NMDA antagonist gave no protection in this model. We suggest that the AMPA receptor transduction mechanisms are sensitized by ischemia and that the postischemic blockade of the main glutamatergic input to the CA1 cells with NBQX impairs the deleterious effect of "normal" postischemic excitatory transmission.


Search for a retrovirus in multiple sclerosis by enzymatic amplification of DNA from brain capillaries, brain tissue and mononuclear cells from peripheral blood. Attempts were made to trace a possible retrovirus in multiple sclerosis (MS) by means of polymerase chain reaction. The primers derived from conserved sequences in the genome of human T cell leukemia/lymphoma virus type I (HTLV-I) and the amplifications were carried out at varying stringency conditions (i.a. annealing at low stringency to enhance the cross-reactivity of the primers). Included in the study were samples of DNA from brain capillaries (five MS patients), brain tissue (three MS patients) and peripheral blood mononuclear cells (three MS patients). Using an HTLV-I derived probe at relaxed conditions of stringency, hybridization signals were not recorded with amplified material from any of these samples. Aspects of infections with neurotropic retroviruses that could be relevant to the search for a retrovirus in MS are reviewed.


Seroreactivity to human T cell leukemia/lymphoma virus type 1 and related retroviruses in multiple sclerosis patients from Denmark and the Faroes. A total of 40 multiple sclerosis (MS) patients from Denmark and 10 from the Faroes were examined for antibodies with affinity to human T cell leukemia/lymphoma virus type 1 (HTLV-I) and human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2). Using ELISA, MS patients and a group of healthy controls did not differ significantly in their reactivities to HTLV-I. However, elevated reactivities were recorded with 5 MS sera, whereas only 2 of the sera from the controls produced highly values. Ten patients with other neurological diseases all seemed to exhibit low reactivity in HTLV-I ELISA. The reactivities of 2 MS sera decreased considerably by absorption with an HTLV-I lysate. In immunofluorescence assay, two other MS sera reacted with HTLV-I transformed cell lines as well as with non-infected cells. Examined by Western blotting (WB), a single MS serum produced a distinct HTLV-I p19 band. With ELISA for detection of HIV-1 and HIV-2 antibodies, 2 MS sera exhibited borderline reactions. Further examination of these two sera by WB revealed weak reactivities against p24 and p53 of HIV-1. One the whole, the present observations do not suggest that a putative MS retrovirus would be closely related with HTLV-I, HIV-1 or HIV-2.


Fluoride interactions with stimulus-secretion coupling of normal and pathological parathyroid cells. Effects of the GTP binding protein (G-protein) activator NaF on parathyroid hormone (PTH) release, cytoplasmic Ca2+ concentration ([Ca2+]i) and cAMP content of bovine as well as normal and pathological human parathyroid cells were studied using precautions to avoid CaF2 precipitation. In 0.5 mM external Ca2+, NaF inhibited PTH release and lowered the cAMP content by 50-70% of the effects attained with 3.0 mM Ca2+. The NaF-induced increase of [Ca2+]i was considerably smaller than that obtained with rise of external Ca2+. It seems likely that NaF activates the inhibitory G1-protein involved in the regulation of cAMP generation. However, it is unclear whether the sluggish rise of [Ca2+]i induced by NaF is due to a direct effect of a G-protein on Ca2+ entry, or somehow related to the G-protein mediated formation of inositol 1,4,5-trisphosphate, which is part of the signal transduction pathway normally initiated by Ca2+ binding to its receptor on the parathyroid cell surface. Inhibition of PTH release by NaF probably results from the combined effects on [Ca2+]i and cAMP content. In hyperparathyroidism (HPT) the actions of NaF were not markedly affected despite severe impairments of Ca(2+)-inhibited PTH release and Ca2+ triggered increase of [Ca2+]i. Consistent with observations of down regulation of the parathyroid Ca2+ receptor in HPT, the present results indicate that the disease perturbs signal transduction at a level proximal to the site of action for NaF.


Hepatocellular carcinoma: a multivariate analysis of prognostic features in patients treated with hepatic arterial embolization. The prognosis after hepatic arterial chemoembolization was retrospectively analyzed in relation to therapeutic modalities, stage of tumor, and degree of liver cirrhosis in 150 patients with solitary tumors of hepatocellular carcinoma. The analyses of life-table methods revealed that adjunct hepatectomy, tumor size, bilirubin, albumin, globulin, and the 15-min retention rate of indocyanine green are statistically significant prognostic factors for hepatic arterial chemoembolization. Results of Cox's proportional hazard analyses disclosed that adjunct hepatectomy (p = 0.0001), serum albumin level (p = 0.0032), and stage of tumor (p = 0.0194) are statistically significant and independent prognostic factors. These findings suggest that the prognosis after hepatic arterial chemoembolization depends on the hepatic functional reserve and stage of tumor in patients with hepatocellular carcinoma, and adjunct hepatectomy improves the prognosis in these patients.