Nucleoside restoration of heat resistance and suppression of glucose-regulated protein synthesis by glucose-deprived L929 cells.

Depriving cultured cells of glucose increases glucose-regulated protein synthesis, suppresses heat shock protein synthesis, and increases sensitivity to killing by hyperthermia. The present study shows that supplementation of glucose-free culture medium with uridine or a number of other nucleosides reverses all these effects of glucose deprivation. Uridine is more effective in this regard than equimolar concentrations of glucose, and ribose is relatively ineffective. Uridine does not suppress glucose-regulated protein synthesis that has been induced by glycosylation inhibitors, calcium chelation, or anoxia. We infer from these data that the effects of glucose deprivation may result from inhibition of ribonucleoside synthesis and that ribonucleosides may be directly involved in regulating glucose-regulated protein and heat shock protein synthesis as well as in protecting cells against hyperthermic cytotoxicity.

Demonstration of pre-S polypeptides of hepatitis B virus in infected livers.

The large (pre-S1), middle (pre-S2) and major (P24) polypeptides of HBsAg have been defined in detail, but their role in hepatitis B virus infection is not known. Therefore, we studied the expression of pre-S1, pre-S2 and P24 in the liver of 15 patients with acute or chronic hepatitis B virus infection using monoclonal and polyclonal antibodies in a double staining immunofluorescence method. The pre-S and major HBsAg polypeptides were co-expressed in the hepatocyte cytoplasm of all patients except for one case of chronic active hepatitis and cirrhosis. HBcAg was present in hepatocyte nuclei of nine patients suggestive of active hepatitis B virus replication. These studies support the hypothesis that the pre-S polypeptides represent essential components of the envelope of hepatitis B virus.

Correlation of polyalbumin receptors with hepatitis B surface antigen polypeptides in human sera.

The molecular nature of the association between hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), polymerized human serum albumin receptors (pHSA-R), and HBsAg polypeptides is controversial. Therefore, we studied HBsAg, HBeAg, and pHSA-R by radioimmunoassay in sera of 27 patients with acute or chronic hepatitis B virus infection. In addition, the HBsAg polypeptides were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining and immunoblot analysis. As a group, HBeAg-positive sera contained significantly more HBsAg and higher pHSA-R activity than HBeAg-negative sera. At a given amount of HBsAg, pHSA-R activity was significantly higher in HBeAg-positive sera than in HBeAg-negative sera. However, there was no correlation between the presence or absence of a particular HBsAg polypeptide and pHSA-R activity, suggesting that posttranscriptional events such as glycosylation may modulate pHSA-R activity associated with native HBsAg.

Effects of glycosylation inhibitors on the expression of polyalbumin receptors by hepatitis B surface antigen produced in vitro.

In this study we examined the role of secondary modifications in the expression of polyalbumin receptors by hepatitis B surface antigen (HBsAg) produced in vitro. Several clones isolated from 3T3 mouse fibroblasts after transfection with the hepatitis B virus genome produced HBsAg with marked variation in the expression of polyalbumin receptors. Treatment of the cells with glycosylation inhibitors (tunicamycin, glucosamine) resulted in loss of the 27 000 mol. wt. HBsAg glycopolypeptide, concomitantly with a marked increase in polyalbumin receptors. These data suggest that glycosylation regulates the activity of polyalbumin receptors associated with native HBsAg.

Antagonistic effects of insulin and dexamethasone on glucose-regulated and heat shock protein synthesis.

The present study extends our previous observation (Kasambalides and Lanks, J. Cell. Physiol., 114:93-98, 1983), that dexamethasone inhibits the alterations in heat shock protein (HSP) and glucose-regulated protein (GRP) synthesis caused by glucose deprivation. We now show that insulin, even in the presence of high extracellular glucose concentrations, will induce 95K and 82K GRP synthesis while suppressing 85K and 69K HSP synthesis. Heat shock of insulin-treated cultures causes induction of the 82K GRP rather than the 85K and 69K HSP's. All of the insulin effects are antagonized by dexamethasone. These data suggest that the changes in GRP and HSP synthesis induced by glucose deprivation and heat shock, respectively, may reflect the operation of a normal physiological mechanism that regulates glucose metabolism.

Secretion of polyalbumin receptors in vitro.

The nature of hepatitis B surface antigen (HBsAg)-associated receptors for polymerized human serum albumin (pHSA-R) and their relationship to hepatitis B e antigen (HBeAg) and human serum proteins have not been defined. We studied by radioimmunoassay and by electron microscopy HBsAg-associated pHSA-R secreted in vitro by a human hepatocellular carcinoma cell line (PLC/PRF/5) and by mouse 3T3 fibroblasts after transfection with cloned hepatitis B virus (HBV) DNA (4.10 cells). PLC/PRF/5 cells expressed only HBsAg, whereas 4.10 cells secreted also HBeAg. There was no significant difference in the production of HBsAg, HBeAg, and pHSA-R when the cells were cultured in the presence or absence of fetal calf serum. Secretion of pHSA-R by the two cell lines for a given amount of HBsAg was equal irrespective of the presence or absence of HBeAg. Supernatants from both cell lines grown in serum-free medium did not contain any Clq or albumin when tested by immunodiffusion. The ability of a transfected mouse cell line to produce HBsAg with pHSA-R activity strongly suggests that pHSA-R is coded by the HBV genome and does not depend on the presence of human serum proteins. In addition, our findings fail to demonstrate any correlation between HBeAg production and pHSA-R.

Dexamethasone induces gelsolin synthesis and altered morphology in L929 cells.

When L929 cells are exposed to 5 mug/ml dexamethasone, synthesis of a 90,000 M(r) polypeptide is induced within 12 h. Flattening of the cells begins at about this time and progresses to become quite prominent after 48 h of exposure. Two-dimensional PAGE and partial proteolytic fingerprints identify the 90,000 M(r) polypeptide as gelsolin, a Ca(++)-dependent inhibitor of actin polymerization. Thus, this system provides evidence that gelsolin may have a role in regulating cell shape in response to physiological agents such as glucocorticoids.

A major cytoplasmic glucose-regulated protein is associated with the Rous sarcoma virus pp60src protein.

Previous studies have established that glucose deprivation of murine cells suppresses the synthesis of an Mr = 85,000 polypeptide. A protein of approximately the same molecular weight has been found to be associated with the transforming protein of Rous sarcoma virus, pp60src. The present study compares the Mr = 85,000 glucose-regulated protein with the pp60src-associated protein. By the criteria of mobility in sodium dodecyl sulfate-polyacrylamide gels and one-dimensional partial proteolytic peptide mapping the two proteins appear to be identical. It has previously been shown that the pp60src-associated protein is also identical with one of several proteins whose synthesis is induced after growth of cells at elevated temperatures or in the presence of arsenite and canavanine. Considering that it is involved in a number of complex response patterns, the name syndromin is proposed for this protein. These findings open the possibility of a fundamental interrelationship among the heat shock effect, regulation of protein synthesis by glucose deprivation, and oncogenic transformation by Rous sarcoma virus.

Purification and characterization of a major component from the cytoplasmic matrix of cultured murine L cells.

This study describes the purification and preliminary characterization of a 94 000 molecular weight protein (S-94) previously known only as a major band in SDS-polyacrylamide gels of total proteins from cultured murine cells. Native S-94 is a soluble constituent of the cytoplasm and sediments at 5.0 S in sucrose gradients, behavior compatible with that of a 94 000 molecular weight monomer. S-94 is purified more than 20-fold from the 100 000 X g supernatant of murine L or L1210 lymphoma cells by DEAE-Sephadex (A-50) chromatography in the presence of 2-mercaptoethanol followed by Sephadex G-200 chromatography in 8 M urea. The protein prepared by this procedure is extensively aggregated, but is essentially homogeneous by SDS-polyacrylamide gel electrophoresis. S-94 preparations from the two types of murine cells behave identically during the purification procedure and are presumed to be the same protein. It appears that these cells contain only one major protein of 94 000 molecular weight. Purified S-94 yields a distinctive pattern of fragments following CNBr degradation, including one peptide of 36 100 molecular weight. The amino acid composition is distinguished by being relatively rich in threonine, glycine and lysine, but poor in valine, leucine and tyrosine.