GB virus C (GBV-C/HGV) and E2 antibodies in children preliver and postliver transplant.

The association of GB virus type C (GBV-C) virus and clinical disease is uncertain. The role of GBV-C and (Envelope) E2 antibody in children with liver transplants has not been determined. This study's aim is to examine the prevalence of GBV-C in children with liver transplants, to assess the relationship of GBV-C to posttransplant hepatitis, and to determine the role of E2 antibodies. Sera from 34 children, preliver and postliver transplant, between 1989-1996 were tested for GBV-C (Ribonucleic acid) RNA by the automated Abbott LCx PCR assay. Anti-E2 antibodies were detected by an Abbott immunoassay. Recent posttransplant liver biopsies were examined for hepatitis. The results of the study determined that pretransplant, four children (12%) were GBV-C RNA positive. Posttransplant, 14 (42%) children were GBV-C RNA positive. The GBV-C RNA positive conversion rate was 33% (CI 17.2-55.7%). Patients received blood products from a mean of 68 +/- 34 donors, which correlated with GBV-C acquisition. There was no difference in the incidence (32%versus 36%; p = 0.726) or severity (grade 2.00 versus 0.68; p = 0.126) of posttransplant hepatitis in the liver biopsies of GBV-C RNA negative and/or positive children, respectively. Pretransplant, nine of 32 children were anti-E2 positive. Posttransplant, eight of 32 children were anti-E2 positive, including five children who were anti-E2 positive pretransplant. Of nine children who were anti-E2 positive and GBV-C RNA negative pretransplant, three became GBV-C RNA positive posttransplant. The results of this study conclude that the prevalence of GBV-C infection in children postliver transplantation is high and that blood product transfusions correlate with GBV-C acquisition. Also, no correlation was found between GBV-C RNA and the incidence or severity of posttransplant hepatitis. Finally, E2 antibody presence before transplantation failed to provide complete protection from GBV-C acquisition.

Detection of GB virus C by the RT-PCR LCx system.

The recent publication of representative genomic sequences of GBV-C has permitted the selection of PCR primers for detection of GBV-C in clinical samples by PCR techniques. Traditional amplification methodologies which couple reverse transcription polymerase chain reaction (RT-PCR) and Southern blot detection are slow, cumbersome, and can be technique dependent. This has hampered studies to determine the clinical significance of GBV-C. We report the selection of highly conserved PCR primers and a probe useful for semi-automated RT-PCR using the Abbott LCx system. This adaptation of the LCx system expands its capabilities to include the detection of RNA by RT-PCR, in addition to DNA detection by ligase chain reaction (LCR).

Multicenter trial on mother-to-infant transmission of GBV-C virus. The Lombardy Study Group on Vertical/Perinatal Hepatitis Viruses Transmission.

Evidence indicates that the GBV-C or hepatitis G virus can cause persistent infection in humans, but little is known on the importance of vertical transmission. To assess the risk of mother-to-infant transmission and the clinical outcome of infected babies, we investigated 175 anti-HCV positive mothers and followed-up their children for 3-33 months. GBV-C RNA was detected by RT-PCR and anti-E2 antibody was assayed by EIA. Thirty-four (19.4%) women were GBV-C RNA positive and transmission occurred to 21 (61.8%) babies; 20 (95.2%) acquired GBV-C alone, and one (4.8%) GBV-C and HCV. Maternal factors such as intravenous drug use, HIV coinfection, HCV-RNA positivity, and type of feeding were not correlated with GBV-C transmission. GBV-C RNA remained persistently positive in all infected babies but one baby who seroconverted to anti-E2. Seven (35%) babies with GBV-C alone developed marginally elevated ALT; the baby with HCV and GBV-C co-infection had the highest ALT peak value (664 IU/l). Seven of the 141 (5%) babies born to the GBV-C RNA negative mothers acquired HCV and six (85.7%) had abnormal ALT. The mean ALT peak value was significantly higher (P < 0.05) for babies with HCV than for those with GBV-C. None of the children with GBV-C or with HCV became icteric. GBV-C is frequently present in anti-HCV positive women. The infection is transmitted efficiently from mother to baby and rate of transmission is much higher than that for HCV. GBV-C can cause persistent infection in babies but usually without clear evidence of liver disease.

Detection of HCV RNA by the asymmetric gap ligase chain reaction.

The ligase chain reaction (LCR) and the gap ligase chain reaction (gLCR) are exponential amplification techniques for the detection of DNA sequences in a sample. Both techniques depend on the enzyme, DNA ligase, to join adjacent probes annealed to a DNA molecule. However, DNA ligase joins DNA inefficiency on an RNA target. Consequently, LCR and gLCR cannot amplify RNA efficiency. RNA detection methods using LCR or gLCR require a cDNA synthesis step. The carryover of four dNTPs from the cDNA reaction inhibits gLCR. Although LCR can use cDNA reaction products directly, background generated by blunt-end ligation does not allow the high sensitivity typically needed for HIV or HCV detection. The asymmetric gap ligase chain reaction (AGLCR) is a modification of gLCR that allows for the detection of RNA by using < or = 3 of the 4 nucleotides in the cDNA step and the gLCR step. Fewer than 50 copies of synthetic RNA transcript can be reproducibly detected. HCV, an RNA virus with no DNA intermediate, was chosen as the initial RNA model system. HCV antibody-positive and normal samples were analyzed, and the results were found to correlate with the results obtained using nested RNA-PCR. AGLCR provides a new nucleic acid amplification technique that can aid in the diagnosis of disease when the detection of RNA is critical.

The ligase chain reaction in DNA-based diagnosis.

Clinical specimens containing a suspected pathogen often have too little of the pathogen's DNA to be detected directly. It is generally necessary to first amplify the DNA and then to detect the amplification products. An amplification technique called the ligase chain reaction (LCR) is described, which in conjunction with an automated, nonradioactive readout format allows less than 10 molecules of target DNA to be detected. A prototype HIV assay and two prototype Chlamydia assays have sensitivities and specificities equivalent to PCR.

Isolation and sequence determination of a cDNA clone for rat peroxisomal urate oxidase: liver-specific expression in the rat.

Urate oxidase (UOxase; urate:oxygen oxidoreductase, EC 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, is present in most mammals but is absent in humans and certain primates. A cDNA clone for UOxase containing an insert of 1.3 kilobases (kb) was isolated from a lambda gt11 cDNA library prepared from rat liver mRNA. This recombinant clone with a 1283-nucleotide insert has sequence for 97% of the coding region together with 401 nucleotides of the 3'-untranslated region of the mRNA. The identity of UOxase cDNA clone was verified by analyzing the fusion protein, immunocytochemical localization with epitope-selected antibody, and hybrid-select translation analysis and by comparing sequences of four CNBr-cleaved peptides of the protein. Blot analysis revealed that the probe hybridizes to a single 1.5-kb mRNA species in the rat liver and a transplantable hepatocellular carcinoma. No UOxase mRNA was detected in 11 nonhepatic tissues of rat, suggesting tissue specificity of expression of this UOxase gene. Blot analysis of RNA from livers of rats treated with a peroxisome proliferator showed 2- to 3-fold increase in UOxase mRNA content, whereas the fatty acyl-CoA oxidase mRNA increased over 30-fold. Southern blot analysis of restriction enzyme digests of rat DNA suggests that there is a single copy of UOxase gene. Analysis of human genomic DNA revealed restriction fragments that are homologous to rat UOxase cDNA, although no UOxase mRNA was detected in human liver.

Studies of cystic fibrosis in Hutterite families by using linked DNA probes.

Several multigenerational S-leut Hutterite families with cystic fibrosis (CF) were ascertained. Linkage studies with DNA marker loci MET and pJ3.11 (D7S8) were performed to determine whether (1) the CF gene in this inbred population is linked to DNA markers on chromosome 7, as it is in outbred populations of European origins, and (2) ancestral origin(s) of the CF gene could be determined. Our results indicate that the CF gene in Hutterite families segregates with chromosome 7 markers, identified by probes metH and pJ3.11. Thus, the CF mutation in Hutterites is likely to be either at the same locus as or at one closely linked to that reported in outbred populations. Heterozygous carriers could be distinguished from normal homozygous sibs of affected individuals. In the families studied, three different chromosome 7 haplotypes carried the CF mutation, raising the possibility that the CF gene may have been introduced into this population by as many as three different ancestors.

Cell-cycle-regulated translation of histone mRNA in Physarum plasmodia.

In Physarum polycephalum, histone mRNA begins to accumulate 3 h before the S phase. We show that histone synthesis was limited to the S phase and that the mRNA that accumulated in the G2-phase cytoplasm could be translated in vitro. Thus, recruitment of stored mature histone mRNA did not occur until the S phase began.

Distinct replication-independent and -dependent phases of histone gene expression during the Physarum cell cycle.

During the S phase of the cell cycle, histone gene expression and DNA replication are tightly coupled. In mitotically synchronous plasmodia of the myxomycete Physarum polycephalum, which has no G1 phase, histone mRNA synthesis begins in mid-G2 phase. Although histone gene transcription is activated in the absence of significant DNA synthesis, our data demonstrate that histone gene expression became tightly coupled to DNA replication once the S phase began. There was a transition from the replication-independent phase to the replication-dependent phase of histone gene expression. During the first phase, histone mRNA synthesis appears to be under direct cell cycle control; it was not coupled to DNA replication. This allowed a pool of histone mRNA to accumulate in late G2 phase, in anticipation of future demand. The second phase began at the end of mitosis, when the S phase began, and expression became homeostatically coupled to DNA replication. This homeostatic control required continuing protein synthesis, since cycloheximide uncoupled transcription from DNA synthesis. Nuclear run-on assays suggest that in P. polycephalum this coupling occurs at the level of transcription. While histone gene transcription appears to be directly switched on in mid-G2 phase and off at the end of the S phase by cell cycle regulators, only during the S phase was the level of transcription balanced with the rate of DNA synthesis.

Negative control of periodic tubulin gene expression in the mitotic cycle of Physarum polycephalum.

Naturally synchronous syncytial plasmodia of Physarum polycephalum provide an ideal model for studying the eukaryotic cell cycle. The tubulin genes of Physarum provide a striking example of cell cycle-regulated gene expression: synthesis of both tubulin mRNA and the tubulin proteins is limited to the last three hours of G2 phase. Fusion of late G2-phase plasmodia, in which the tubulin genes are actively expressed, and mid-S phase plasmodia, in which the tubulin genes are not expressed, is used to demonstrate the presence of a diffusible negative regulator of tubulin gene expression in S-phase plasmodia. It is inferred that this factor is responsible for the turning-off of tubulin gene transcription that follows mitosis.

Evidence for a protonmotive force related regulatory system in Escherichia coli and its effects on lactose transport.

Strains of Escherichia coli with mutations in the eup (energy-uncoupled phenotype) locus do not grow on nonfermentable carbon sources, have reduced growth yields on limiting glucose, are insensitive to colicins A and K, exhibit resistance to aminoglycoside antibiotics, and are defective in protonmotive force coupled active transport. eup mutations do not result in lowered protonmotive force. Here we show that deenergization of a eup+ strain results in the appearance of a new low KT, low Vmax form of the lactose carrier; in a strain deleted of the eup locus, deenergization does not evoke the low KT, low Vmax form of the lactose carrier. Cells bearing a eup point mutation and exhibiting the Eup- phenotype possess the low KT, low Vmax form of the lactose carrier even when energized. In addition to affecting the kinetic parameters of the lactose carrier, the eup point mutation also reduces the KT and Vmax of the proline carrier. On the basis of these findings, we suggest that the normal eup gene product mediates a novel regulation of lactose carrier function following deenergization. The defect in proline and lactose transport caused by eup point mutations may stem from an altered eup product aberrantly mediating the regulation under energized conditions. Finally, the pleiotropy associated with eup point mutations may be indicative of those protonmotive force driven functions that are subject to eup regulation.

Transcription of alpha-tubulin and histone H4 genes begins at the same point in the Physarum cell cycle.

In naturally synchronous plasmodia of Physarum polycephalum, both tubulin and histone gene transcription define periodic cell cycle-regulated events. Using a slot-blot hybridization assay and Northern blot analysis, we have demonstrated that a major peak of accumulation of both alpha-tubulin and histone H4 transcripts occurs in late G2 phase. Nuclear transcription assays indicate that both genes are transcriptionally activated at the same point in the cell cycle: mid G2 phase. While the rate of tubulin gene transcription drops sharply at the M/S-phase boundary, the rate of histone gene transcription remains high through most of S phase. We conclude that the cell cycle regulation of tubulin expression occurs primarily at the level of transcription, while histone regulation involves both transcriptional and posttranscriptional controls. It is possible that the periodic expression of both histone and tubulin genes is triggered by a common cell cycle regulatory mechanism.

Transcription regulation of peroxisomal fatty acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in rat liver by peroxisome proliferators.

The structurally diverse peroxisome proliferators ciprofibrate, clofibrate, and bis(2-ethylhexyl) phthalate [(EtHx)2 greater than Pht] increase the activities of hepatic catalase and peroxisomal fatty acid beta-oxidation enzymes in conjunction with profound proliferation of peroxisomes in hepatocytes. In order to delineate the level at which these enzymes are induced in the liver, the transcriptional activity of specific genes for fatty acyl-CoA oxidase (FAOxase) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE), the first two enzymes of the peroxisomal beta-oxidation system, and for catalase were measured in isolated hepatocyte nuclei obtained from male rats following a single intragastric dose of ciprofibrate, clofibrate, or (EtHx)2 greater than Pht. All three peroxisome proliferators rapidly increased the rate of FAOxase and PBE gene transcription in liver, with near maximal rates (9-15 times control) reached by 1 hr and persisting until at least 16 hr after administration of the compound. FAOxase and PBE mRNA levels, measured by blot-hybridization analysis and FAOxase and PBE protein content, analyzed by immunoblotting, increased concurrently up to at least 16 hr following a single dose of peroxisome proliferator. The catalase mRNA level increased about 1.4-fold, but the transcription rate of the catalase gene was not significantly affected. The results show that the peroxisome proliferators clofibrate, ciprofibrate, and (EtHx)2 greater than Pht selectively increase the rate of transcription of peroxisomal fatty acid beta-oxidation enzyme genes. Whether the transcriptional effects are mediated by peroxisome proliferator-receptor complexes remains to be elucidated.

The effect of heat shock on the cell cycle regulation of tubulin expression in Physarum polycephalum.

In the myxomycete Physarum polycephalum, tubulin synthesis is subject to mitotic cycle control. Virtually all tubulin synthesis is limited to a 2-h period immediately preceding mitosis, and the peak of tubulin protein synthesis is accompanied by a parallel increase in the level of tubulin mRNA. The mechanism by which the accumulation of tubulin mRNA is turned on and off is not clear. To probe the relationship between tubulin regulation and cell cycle controls, we have used heat shocks to delay mitosis and have followed the pattern of tubulin synthesis during these delays. Two peaks of tubulin synthesis are observed after a heat shock. One occurs at a time when synthesis would have occurred without a heat shock, and a second peak immediately precedes the eventual delayed mitosis. These results are clearly due to altered cell cycle regulation. No mitotic activity is detected in delayed plasmodia at the time of the control mitosis, and tubulin behavior is shown to be clearly distinct from that of heat shock proteins. We believe that the tubulin family of proteins is subject to regulation by a thermolabile mitotic control mechanism but that once the cell has been committed to a round of tubulin synthesis the "tubulin clock" runs independently of the heat sensitive system. In delayed plasmodia, the second peak of synthesis may be turned on by a repeat of the commitment event.

The periodic synthesis of tubulin in the Physarum cell cycle. Characterization of Physarum tubulins by affinity for monoclonal antibodies and by peptide mapping.

Polypeptides preferentially labeled in the G2 phase of the synchronous nuclear replication cycle of Physarum macroplasmodia were compared in electrophoretic mobility and peptide map with the tubulins enriched from Physarum myxamoebae. One major and one minor fluorographic species match the myxamoebal alpha and beta chains, respectively. Thus, tubulins are among the proteins of Physarum selectively synthesized before nuclear division. A third species P, prominently labeled in premitotic plasmodia, is distinct from the two myxamoebal tubulins even though it co-polymerizes with microtubules. The nature of P remains unknown. Two rat monoclonal antibodies directed against yeast tubulin were found to bind selectively to the alpha tubulin of porcine brain. These served to confirm the assignment of the 50,000-dalton Physarum myxamoebal tubulin as an alpha-like polypeptide.

Periodic synthesis of microtubular proteins in the cell cycle of Physarum.

Periodic polypeptide labeling over the naturally synchronous nuclear replication cycle of Physarum polycephalum was analyzed by fluorography of two-dimensional electropherograms. Two sets of polypeptides, denoted as P and Q, showed strong periodicity; they were maximally labeled just prior to mitosis. This periodicity was shown to reflect synthesis rather than turnover or recovery. Both P and Q copolymerized with porcine microtubular proteins and displayed electrophoretic properties similar to those of porcine tubulins. The significance of the periodic synthesis of these microtubular proteins is discussed as a possible component within the chain of events that establishes the high mitotic synchrony of Physarum syncytia.

Temperature-sensitive mutants of Physarum polycephalum: viability, growth, and nuclear replication.

Using a selfing strain of Physarum polycephalum that forms haploid plasmodia, we have isolated temperature-sensitive growth mutants in two ways. The negative selectant, netropsin, was used to enrich for temperature-sensitive mutants among a population of mutagenized amoebae, and, separately, a nonselective screening method was used to isolate plasmodial temperature-sensitive mutants among clonal plasmodia derived from mutagenized amoebae. Complementation in heterokaryons was used to sort the mutants into nine functional groups. When transferred to the restrictive temperature, two mutants immediately lysed, whereas the remainder slowed or stopped growing. Of the two lytic mutants, one affected both amoebae and plasmodia, and the other affected plasmodia alone. The growth-defective mutants were examined for protein and deoxyribonucleic acid synthesis and for aberrations in mitotic behavior. One mutant may be defective in both protein and deoxyribonucleic acid synthesis, and another only in deoxyribonucleic acid synthesis. The latter shows a striking reduction in the frequency of postmitotic reconstruction nuclei at the restrictive temperature. We believe that this mutant, MA67, is affected in a step in the nuclear replication cycle occurring late in G2. Execution of this step is necessary for both mitosis and chromosome replication.