A multigene family encodes the human cytomegalovirus glycoprotein complex gcII (gp47-52 complex).

The HXLF (HindIII-X left reading frame) gene family is a group of five genes that share one or two regions of homology and are arranged in tandem within the short unique component of the human cytomegalovirus genome (K. Weston and B.G. Barrell, J. Mol. Biol. 192:177-208, 1986). These genes were cloned into an SP6 expression vector in both the sense and antisense orientations. An abundant 1.62-kilobase (kb) bicistronic mRNA, predicted to originate from HXLF1 and HXLF2, was detected in the cytoplasm of infected human fibroblast cells by Northern (RNA) blot analysis. Less abundant RNAs of 1.0 and 0.8 kb, predicted to originate from the HXLF5 and HXLF2 genes, respectively, were also detected. Monocistronic, bicistronic, and polycistronic RNAs synthesized in vitro by using SP6 polymerase were translated in rabbit reticulocyte lysates with or without canine pancreatic microsomal membranes. The HXLF1 or the HXLF1 and HXLF2 translation products were detected when the above mRNAs were used. The HXLF3, HXLF4, and HXLF5 gene products were not detected by in vitro translation of the SP6-derived polycistronic mRNA. Nonglycosylated or glycosylated HXLF1 and HXLF2 gene products were immunoprecipitated by monoclonal antibody 9E10, which is specific for a virion envelope glycoprotein complex designated gcII (gp47-52 complex). In addition, the monoclonal antibody 9E10 immunoprecipitated a diffuse glycoprotein band, designated gp47-52, from HCMV-infected cell lysates. The amino acid composition of gp47-52 purified from viron envelopes has the highest similarity to the predicted amino acid composition of the HXLF1 plus HXLF2 open reading frames, but it is more similar to HXLF2 than to HXLF1. The Northern blot results imply that gp47-52 is synthesized predominantly from the abundant 1.62-kb bicistronic mRNA encoded by the HXLF1 and HXLF2 genes. However, the glycoprotein could also be synthesized by the monocistronic 0.8-kb mRNA encoded by the HXLF2 gene as well as by the mRNAs predicted from the other HXLF genes.     

Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus

Several disulfide-linked glycoprotein complexes were identified in the envelope of human cytomegalovirus (HCMV). These glycoprotein complexes were fractionated by rate-zonal centrifugation in sucrose density gradients in the presence of detergents. Fractionated glycoproteins and complexes were immunoprecipitated with three different monoclonal antibodies specific for HCMV glycoproteins and a rabbit polyclonal antiserum prepared against detergent-extracted virion and dense-body envelope glycoproteins. Three distinct families of disulfide-linked glycoprotein complexes were observed and designated glycoprotein complex gcI, gcII, and gcIII. The gcI family, recognized by monoclonal antibody 41C2 under nonreducing conditions, consisted of three complexes with approximate molecular masses of 250 to 300, 190, and 160 kilodaltons (kDa). These complexes consistently sediment more rapidly than other HCMV glycoproteins or complexes in sucrose density gradients. Upon reduction of the gcI family, two size classes of glycoproteins with average molecular masses of 93 to 130 and 55 kDa were observed. The gcII family was recognized by monoclonal antibody 9E10. Under nonreducing conditions, as many as six electrophoretic forms were observed for gcII. When reduced, the major component of the gcII family was a heterogeneous glycoprotein designated gp47-52. The gcIII family was recognized by monoclonal antibody 1G6. It consisted of a complex of approximately 240 kDa without reduction of disulfide bonds. When reduced, two glycoprotein size classes with average molecular masses of 145 and 86 kDa were observed. Polyclonal antiserum R-7 reacted strongly with the gcI and gcIII families, but weakly with the gcII family.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Delimitation of the maritime boundary in the gulf of Maine area

The compulsory dispute settlement regime included in the 1982 Law of the Sea Convention is recognized as one of the most comprehensive in a modern international convention. Yet, in the recent application of this regime, the question has arisen as to whether the procedural prerequisites associated with the LOS Convention's compulsory dispute settlement mechanism are so arduous as to avoid binding and compulsory jurisdiction in most instances. This article addresses that question by examining, in particular, the reasoning of the Southern Bluefin Tuna arbitration tribunal, which found Article 281 of Section 1 of the LOS Convention to bar jurisdiction to the compulsory dispute settlement mechanism prescribed by the Convention, and offers suggestions as to how states might distinguish or overcome the barriers imposed by the Southern Bluefin Tuna tribunal in future cases.     

Elevated levels of interleukin-8 in serum are associated with hepatitis C virus infection and resistance to interferon therapy

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). We have recently found that the HCV NS5A protein induces expression of the proinflammatory chemokine IL-8 to partially inhibit the antiviral actions of IFN in vitro. To extend these observations, in the present study we examined the relationship between levels of IL-8 in serum, HCV infection, and biochemical response to IFN therapy. Levels of IL-8 were significantly elevated in 132 HCV-infected patients compared to levels in 32 normal healthy subjects and were also significantly higher in patients who did not respond to IFN therapy than in patients who did respond to therapy. This study suggests that HCV-induced changes in levels of chemokine and cytokine expression may be involved in HCV antiviral resistance, persistence, and pathogenesis.     

In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease

Liver failure from chronic hepatitis C is the leading indication for liver transplantation in the United States. However, the pathogenesis of liver injury resulting from chronic hepatitis C virus (HCV) infection is not well understood. To examine the relationship between HCV replication in liver tissue and hepatocellular injury, a strand-specific in situ hybridization procedure was developed. The sensitivity and specificity of digoxigenin-labeled riboprobes were optimized by analyzing Northern blots and cell lines expressing HCV RNAs. For the current study, both genomic (sense) and replicative-intermediate (antisense) HCV RNAs were detected and quantified in 8 of 8 liver tissue specimens from infected patients versus 0 of 11 liver tissue specimens from noninfected controls. The distribution pattern for HCV replicative-intermediate RNA in liver was different from that for HCV genomic RNA. HCV genomic RNA was variably distributed throughout infected livers and was located primarily in the cytoplasm of hepatocytes, with some signal in fibroblasts and/or macrophages in the surrounding fibroconnective tissue. However, HCV replicative-intermediate RNA showed a more focal pattern of distribution and was exclusively localized in the cytoplasm of hepatocytes. There was no significant relationship between the distribution pattern for HCV genomic RNA and any indices of hepatocellular injury. However, a highly significant correlation was observed between the percentage of cells staining positive for replicative-intermediate RNA and the degree of hepatic inflammatory activity (P, < 0.0001). Furthermore, the ratio of cells staining positive for HCV replicative-intermediate versus genomic RNA correlated with the histological severity of liver injury (P, 0. 0065), supporting the hypothesis that active replication of HCV in liver tissue may be a significant determinant of hepatocellular injury.     

Prospective characterization of full-length hepatitis C virus NS5A quasispecies during induction and combination antiviral therapy

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR(237-276)) sequence associated with IFN resistance was not found, although the presence of Ala(245) within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P<0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P<0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P<0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.     

A spatial analytical approach for selecting reintroduction sites for burbot in English rivers

1. Availability of suitable habitat is a prerequisite for species reintroduction success, and to ensure population persistence, investigations of a species’ habitat utilisation throughout its life history should be conducted as part of a feasibility study. 2. Habitat utilisation models for burbot, Lota lota, developed using data from field studies conducted in France and Germany and information from the literature were used to assess the feasibility of reintroducing burbot into rivers of its former native range in eastern England. 3. Per cent tree roots, aquatic vegetation and flow types were important predictors of adult burbot abundance. Furthermore, the habitat utilisation models were supplemented with information from the literature, which suggested that off‐channel habitat such as wetlands and backwaters is important for spawning and nursery stages. 4. An assessment of the habitat availability in the rivers of the burbot’s former native range using variables related to spawning and nursery and adult life stages showed that although adult habitat was widely distributed, the availability of spawning and nursery habitat was less abundant, potentially limiting successful reestablishment. 5. Potential suitable habitat was concentrated in the central and southern areas of the species’ former English distribution. Overall, rivers of the burbot’s former range potentially afford suitable habitat to sustain a reintroduced population. However, sites should be preferentially selected on the basis of having appropriate spawning and nursery areas.