Seroprevalence of GB virus C and persistence of RNA and antibody.

Exposure to GB virus C (GBV-C) was determined in several U.S. populations by both reverse-transcription-polymerase chain reaction (RT-PCR) and by an enzyme linked immunosorbent assay (ELISA) for antibodies to mammalian cell-expressed GBV-C envelope protein, E2 (GBV-C E2). Most individuals exposed to GBV-C were either RNA positive/ELISA negative or ELISA positive/RNA negative. Exposure, therefore, was measured as the sum of GBV-C RNA positive and GBV-C E2 antibody positive specimens, and was higher in commercial plasmapheresis donors (40.5%) than in volunteer blood donors (5.5%). In intravenous drug users (IVDUs), GBV-C exposure was 89.2%. Serial bleed specimens tested for GBV-C RNA indicate that some patients remain viremic for at least 3 years and fail to produce detectable antibodies to GBV-C E2. In other exposed individuals who tested negative for GBV-C RNA, antibodies to E2 appear to be similarly long-lived (greater than 3 years) with a fairly constant titer (ranging in reciprocal endpoint dilution from 336 to 21,504). Since the detection of GBV-C RNA and GBV-C E2 antibody are mutually exclusive in most exposed individuals, studies pertaining to incidence and prevalence of GBV-C infection require both antibody and nucleic acid detection.

GB virus C E2 glycoprotein: expression in CHO cells, purification and characterization.

A 315 amino acid recombinant segment of the GB virus C (GBV-C) E2 envelope glycoprotein (E2-315) was expressed and secreted from CHO cells. E2-315 was purified by affinity chromatography using a monoclonal antibody directed to a FLAG sequence genetically engineered onto the C terminus of the recombinant protein. The secreted protein had a molecular mass of 48-56 kDa and was shown to be N-glycosylated. Amino acid sequencing confirmed the expected N-terminal sequence. Purified E2-315 was used to develop an ELISA for detection of E2 antibodies in human sera. Antibodies to GBV-C E2 appeared to be directed toward conformational epitopes since human sera reactivity was detected in ELISA using native E2-315, but it was extremely weak or non-existent with denatured E2 protein. The use of an ELISA which can detect human GBV-C E2 antibodies will be important in further understanding of the clinical significance and epidemiology of GBV-C.

An ELISA for detection of antibodies to the E2 protein of GB virus C.

An ELISA was developed for detection of antibodies to GB virus C (GBV-C) using a recombinant E2 protein expressed in CHO cells. Seroconversion to anti-E2 positivity was noted among several persons infected with GBV-C RNA-positive blood through transfusion. Of 6 blood recipients infected by GBV-C RNA-positive donors, 4 (67%) became anti-E2 positive and cleared their viremia. Thus, anti-E2 seroconversion is associated with viral clearance. The prevalence of antibodies to E2 was relatively low (3.0%-8.1%) in volunteer blood donors but was higher in several other groups, including plasmapheresis donors (34.0%), intravenous drug users (85.2%), and West African subjects (13.3%), all of whom tested negative by GBV-C reverse-transcription polymerase chain reaction (RT-PCR). These data demonstrate that testing for anti-E2 should greatly extend the ability of RT-PCR to define the epidemiology and clinical significance of GBV-C.

Expression of the GB virus C E2 glycoprotein using the Semliki Forest virus vector system and its utility as a serologic marker.

A 336-amino-acid segment of the GB virus C second envelope protein (E2) has been produced in BHK-21 cells using the Semliki Forest virus vector system. Secretion of this protein was facilitated by deletion of a hydrophobic region at the C-terminus that may represent the membrane anchoring domain. The E2 protein recovered from the culture supernatant exhibited a molecular mass of approximately 52 kDa, with the increase in size relative to the polyprotein backbone being contributed by N-linked glycosylation. A radioimmunoprecipitation assay using GBV-C E2 was developed to test for the presence of antibodies against this protein in human sera. The prevalence of antibodies to E2 was high among injection drug users and other individuals at risk for acquiring parenterally transmitted agents. There was a much higher percentage of anti-E2 seropositivity in GBV-C RT-PCR negative compared to GBV-C RT-PCR positive samples from these populations. In addition, serial samples from patients transfused with blood containing GBV-C showed seroconversion to anti-E2 positivity and loss of GBV-C viremia as measured by RT-PCR within 11 months of transfusion in five of seven individuals. Thus, this system provided a rapid means to identify GBV-C E2 as a useful antigen for the study of GBV-C exposure.

Modulation of enzyme activity by antibody binding to an alkaline phosphatase-epitope hybrid protein.

An epitope from the HIV-1 gp120 protein V3 loop has been inserted onto the surface of bacterial alkaline phosphatase at different positions in the vicinity of the enzyme active site, creating hybrid proteins that can bind to an anti-gp120 monoclonal antibody. One of the hybrid proteins, API1, has a 13 amino acid V3 loop sequence inserted between residues 407 and 408 of alkaline phosphatase. The enzymatic activity of this protein is modulated upon antibody binding. API1 maintains the full activity of the wild type alkaline phosphatase but in the presence of the anti-gp120 antibody, the enzyme activity is inhibited by 40-50%. Thus, the hybrid enzyme can be used to detect the presence of antibody in solution. The concept of signalling proteins may have a wide application. Two models for the mechanism of modulation, steric hindrance and allosteric regulation, are discussed.

Low water potentials affect expression of genes encoding vegetative storage proteins and plasma membrane proton ATPase in soybean.

We have examined growth, water status and gene expression in dark-grown soybean (Glycine max L. Merr.) seedlings in response to water deficit (low water potentials) during the first days following germination. The genes encoded the plasma membrane proton ATPase and two proteins of 28 kDa and 31 kDa putatively involved in vegetative storage. Water potentials of stems and roots decreased when 2-day-old seedlings were transferred to water-saturated air. Stem growth was inhibited immediately. Root growth continued at control rates for one day and then was totally inhibited when the normal root-stem water potential gradient was reversed. Expression of mRNA for the 28 kDa and 31 kDa proteins, measured independently using specific 3'-end probes, occurred about equally in stems. However, only the mRNA for the 31 kDa protein was detected in roots and at a lower abundance than in stems. Low water potentials increased the mRNA only for the 28 kDa protein in stems and the 31 kDa protein in roots. This differential expression followed the inhibition of stem growth but preceded the inhibition of root growth. The expression of the message for the ATPase, measured using a probe synthesized from a partial oat ATPase clone, was low in stems and roots but there was a 6-fold increase at low water potentials in roots. The increase followed the inhibition of root growth. This appears to be the first instance of regulation of ATPase gene expression in plants and the first demonstration of differential expression of the 28 kDa, 31 kDa, and ATPase messages. The correlation with the differential growth responses of the stems and roots raises the possibility that the differential gene expression could be involved in the growth response to low water potentials.

Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana.

In plants, the transport of solutes across the plasma membrane is driven by a proton pump (H+-ATPase) that produces an electric potential and pH gradient. We have isolated and sequenced a full-length cDNA clone that encodes this enzyme in Arabidopsis thaliana. The protein predicted from its nucleotide sequence encodes 959 amino acids and has a molecular mass of 104,207 Da. The plant protein shows structural features common to a family of cation-translocating ATPases found in the plasma membrane of prokaryotic and eukaryotic cells, with the greatest overall identity in amino acid sequence (36%) to the H+-ATPase observed in the plasma membrane of fungi. The structure predicted from a hydropathy plot contains at least eight transmembrane segments, with most of the protein (73%) extending into the cytoplasm and only 5% of the residues exposed on the external surface. Unique features of the plant enzyme include diverged sequences at the amino and carboxyl termini as well as greater hydrophilic character in three extracellular loops.

Antigenic sites of human growth hormone and related molecules detected by monoclonal antibodies to human growth hormone.

Four major antigenic sites for human growth hormone (hGH) were identified by 27 mouse monoclonal antibodies to hGH. Sites 1 and 2 are spatially close whereas sites 3 and 4 are located in other parts of the molecule. There also appears to be a subdivision of antigenic sites. A panel of 10 monoclonal antibodies, which included representatives from each antigenic site group, were used to determine cross-reactivities between hGH and human placental lactogen (hPL), human prolactin (hPRL), the 20,000 mol. wt variant of hGH (hGH20K) and a disulfide-linked dimer of hGH (diS-dimer). The data suggest a high conformational dependence of antigenic sites in hGH. DiS-dimer retains all four antigenic sites of hGH, although all have been altered. hGH20K retains sites 2-4 but site 1 has been dramatically altered. hPL retains site 3, whereas sites 1 and 4 have been dramatically altered and site 2 may be lacking. The extremely low cross-reactivity observed for hPRL is consistent with the dissimilarity between hGH and hPRL. Antigenic site 3 is the most conserved of all sites. The lack of structural similarity compared with hGH of site 1 in hGH20K and of a portion of site 3 in diS-dimer suggests that it may be possible to develop specific radioimmunoassays for these structural variants of hGH.

Size heterogeneity of prolactin secreted from and stored in the rat anterior pituitary gland in vitro.

The size heterogeneity of rat pituitary prolactin was investigated using anterior pituitary glands from female rats incubated in vitro and gel filtration on Sephadex G-100. Monomeric prolactin was preferentially secreted compared with dimeric and 'trimeric' material. When glands were incubated with dopamine, prolactin secretion was inhibited and the relative proportion of dimer in the gland (but not the medium) was decreased. Morphine sulphate reversed the effect of dopamine on prolactin secretion and on the proportion of prolactin in the gland that was in the dimeric form. The results suggest that monomeric prolactin is more readily secreted than dimer, and that dopamine decreases the production or stability of the dimer.

Binding specificity of monoclonal antibodies towards the size variants of human growth hormone.

The expression of antigenic determinants on size variants of human growth hormone (hGH) has been investigated using monoclonal antibodies of distinct combining-site specificity. Monomeric, dimeric, trimeric and polymeric (very high-molecular-weight) forms of hGH were separated by gel filtration on Sephadex G-100, and their antigenic potency was determined quantitatively by competition with 125I-labelled hGH for binding to each of four different monoclonal antibodies. With three of these antibodies the potencies of monomeric, dimeric and trimeric hGH were not significantly different, and the polymeric material was 11-13% as potent as the monomer. However, using one antibody (NA 71) the antigenic potencies of dimeric and trimeric hGH were lower (30-50%) than that of the monomer, and the polymeric material was only about 5% as potent as the monomer. These results suggest that the determinant with which antibody NA 71 interacts is close to the site of interaction between hGH monomers and apparently partially 'masked' in dimers, trimers and polymeric hGH.