Expression of hepatitis B surface antigen with a recombinant adenovirus.

Early region 1 of the adenovirus type 5 genome was replaced with a DNA sequence containing the gene coding for the hepatitis B surface antigen (HBsAg) flanked by the major late promoter from adenovirus 2 and processing and polyadenylylation signals from simian virus 40. In one type of hybrid virus only the adenovirus 2 major late promoter, including just 33 base pairs of the adenovirus type 2 tripartite leader, preceded the coding region of the HBsAg gene. In another, this region was preceded by both the adenovirus major late promoter and almost the entire tripartite leader. The structure of the substituted sequence in each of the recombinant viral DNAs was identical to that in the plasmids used to construct the viruses. Approximately equivalent amounts of HBsAg-specific mRNA were produced late in infection with each recombinant virus. Although HBsAg production was detected late in infection of the hybrid virus not containing the full tripartite leader sequence, its level was 1/70th of that obtained with the hybrid virus containing this sequence. One likely interpretation is that the presence of the tripartite leader at the 5' end of this mRNA is critical for the synthesis of HBsAg polypeptide in the late stage of infection. HBsAg produced upon infection with the hybrid adenoviruses was glycosylated and secreted into the culture medium as particles that were essentially indistinguishable from the 22-nm particles found in human serum.     

Characterization of a preleukemic state induced by Moloney murine leukemia virus: evidence for two infection events during leukemogenesis.

A preleukemic state in mice inoculated with Moloney murine leukemia virus (Mo-MuLV) was characterized. Six to 10 weeks after neonatal inoculation, animals developed mild splenomegaly and generalized hematopoietic hyperplasia. The hyperplasia was evident from myeloid and erythroid progenitor assays. A nonleukemogenic variant, Mo+PyF101 Mo-MuLV, did not induce the hyperplasia; this suggests that the hyperplasia is a necessary event in Mo-MuLV leukemogenesis. Another variant, MF-MuLV, which contains the long terminal repeat of Friend MuLV and causes erythroid leukemia instead of T-cell lymphoma, also induced the preleukemic hyperplasia. A model for Mo-MuLV leukemogenesis is presented in which two infection events are necessary: the first leads to generalized hematopoietic hyperplasia, and the second results in site-specific insertion and long terminal repeat activation of cellular protooncogenes.     

Gamma-actin: unusual mRNA 3'-untranslated sequence conservation and amino acid substitutions that may be cancer related

beta-Actin mutations in chemically transformed human cell lines have been associated with tumorigenicity, an association consistent with other evidence suggesting that altered cytoskeletal proteins may have an important role in cancer initiation or progression. From a human promyelocytic leukemia cell line, we have isolated a gamma-actin cDNA clone with amino acid substitutions in a region highly conserved in the many actins analyzed. To our knowledge, this is the first example of a variant gamma-actin in a human neoplasm. A separate finding from the analysis of this clone is that the gamma-actin 3'-untranslated region is among the most highly conserved of all 3'-untranslated sequences so far reported, but is entirely different from the beta-actin 3'-untranslated region. The high degree of evolutionary conservation suggests that the 3'-untranslated regions of these two mRNAs have important and distinct functional roles that were already fully differentiated more than 100 million years ago. Mutations affecting four major cytoskeletal components have now been identified in human neoplastic cells. These findings suggest that mutated cytoskeletal genes may be members of a class of oncogenes, fundamentally different from both the nuclear-acting (e.g., myc and simian virus 40 large tumor antigen) and growth factor/receptor/protein kinase-related (e.g., sis, erbB, and ras) types of oncogenes.     

Induction of endothelial NO synthase by hydrogen peroxide via a Ca(2+)/calmodulin-dependent protein kinase II/janus kinase 2-dependent pathway

We have recently demonstrated that hydrogen peroxide (H(2)O(2)) is an extremely potent stimulus of endothelial NO synthase (eNOS) gene expression. The present study was designed to identify the signaling mechanisms mediating this response. Induction of eNOS expression by H(2)O(2) was found to be Ca(2+) dependent, inasmuch as it was blocked by BAPTA-AM. Further studies have indicated that Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) plays a critical role in mediating this response. Immunocytochemical staining with an anti-CaM kinase II antibody confirmed the expression of CaM kinase II in cultured bovine aortic endothelial cells. H(2)O(2) induced autophosphorylation of CaM kinase II and increased the activity of the enzyme, as assessed by an in-gel kinase assay. A specific inhibitor for CaM kinase II, KN93, and a calmodulin antagonist, W-7, attenuated eNOS induction by H(2)O(2). Further studies have indicated that janus kinase 2 is important in mediating increased eNOS expression in response to H(2)O(2) and likely is downstream from CaM kinase II. In conclusion, these data provide the first evidence that CaM kinase II plays a critical role in endothelial redox signaling. Regulation of eNOS via this pathway may represent an important vascular adaptation to oxidant stress.     

Ethanol metabolism in the generation of new antigenic determinants on liver cells.

Antibodies directed against ethanol altered liver cell components have been detected in the serum of nearly 50% of patients with alcoholic liver disease although the pathogenetic mechanisms are unclear. The importance of ethanol metabolism in the generation of new antigenic determinants on liver cells was investigated by in vivo inhibition of alcohol or acetaldehyde dehydrogenase and an induced cytotoxicity assay. There was a significant reduction in cytotoxicity to hepatocytes isolated from rabbits treated with ethanol 1 g/kg when the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was inhibited. In contrast when the oxidation of acetaldehyde was inhibited by disulfiram cytotoxicity was significantly enhanced. These results show that ethanol metabolism is integral to the expression of the ethanol related determinant and suggest that an impaired ability to metabolism acetaldehyde could lead to the development of immunological reactions to alcohol altered liver membrane antigens.     

Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion.

The synthetic peptides DP-107 and DP-178 (T-20), derived from separate domains within the human immunodeficiency virus type 1 (HIV-1) transmembrane (TM) protein, gp4l, are stable and potent inhibitors of HIV-1 infection and fusion. Using a computer searching strategy (computerized antiviral searching technology, C.A.S.T.) based on the predicted secondary structure of DP-107 and DP-178 (T-20), we have identified conserved heptad repeat domains analogous to the DP-107 and DP-178 regions of HIV-1 gp41 within the glycoproteins of other fusogenic viruses. Here we report on antiviral peptides derived from three representative paramyxoviruses, respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV-3), and measles virus (MV). We screened crude preparations of synthetic 35-residue peptides, scanning the DP-178-like domains, in antiviral assays. Peptide preparations demonstrating antiviral activity were purified and tested for their ability to block syncytium formation. Representative DP-178-like peptides from each paramyxovirus blocked homologous virus-mediated syncytium formation and exhibited EC50 values in the range 0.015-0.250 microM. Moreover, these peptides were highly selective for the virus of origin. Identification of biologically active peptides derived from domains within paramyxovirus F1 proteins analogous to the DP-178 domain of HIV-1 gp4l is compelling evidence for equivalent structural and functional features between retroviral and paramyxoviral fusion proteins. These antiviral peptides provide a novel approach to the development of targeted therapies for paramyxovirus infections.     

Angiotensin II- and glucose-stimulated extracellular matrix production: mediation by the insulin-like growth factor (IGF) axis in a murine mesangial cell line

In diabetic nephropathy, glomerular mesangial cells exhibit aberrant anabolic activity that includes excessive production of extracellular matrix (ECM) proteins, leading to crowding of filtration surface areas and possible renal failure. In the present study, a murine mesangial cell line (MES-13 cells) was studied to determine the roles of the renin-angiotensin system (RAS) and the insulin-like growth factor (IGF) axis in the anabolic response to elevated glucose levels. Culture of MES-13 cells in medium containing supra-physiological glucose concentrations (>5.5 mmol/l) resulted in increased production of ECM proteins including laminin, fibronectin, and heparan sulfate proteoglycan with concurrent increases in IGF-binding protein (IGFBP)-2 production. These responses were blocked by the angiotensin receptor antagonists saralasin and losartan, while exogenous angiotensin II (Ang II) treatment directly stimulated increases in ECM and IGFBP-2. In all experiments, IGFBP-2 levels were correlated with anabolic activity implicating IGFBP-2 as a possible mediator in cellular responses to high glucose and Ang II. Such mediation appears to involve IGFBP-2 modulation of IGF-I signaling, since all responses to high glucose or Ang II were blocked by immuno-neutralization of IGF-I. These data suggest alterations in the IGF axis as key mechanisms underlying nephropathic responses of mesangial cells to Ang II and high glucose.     

Carboxyl-terminal deletion and point mutations decrease the transforming potential of the activated rat neu oncogene product.

The rat neu oncogene encodes a constitutively activated growth factor receptor/transmembrane tyrosine kinase, p185Tneu, that is structurally similar to yet distinct from the epidermal growth factor receptor. To explore the role of the carboxyl-terminal region and of putative autophosphorylation sites in regulating the activity of the rat p185Tneu (T, transforming) protein, we used site-directed mutagenesis to generate a p185Tneu mutant in which a putative tyrosine autophosphorylation site (residue 1253) at the extreme carboxyl terminus was replaced by a phenylalanine residue and a mutant in which the carboxyl-terminal 122 amino acids were deleted. These proteins were expressed in NIH 3T3 cells at comparable levels and exhibited similar autophosphorylation activity, exogenous substrate phosphorylation ability, oligomerization levels, and responsiveness to a partially purified neu-activating factor. However, the mutant p185Tneu proteins displayed a decreased transforming capacity both in vitro and in vivo. This analysis demonstrated that the carboxyl-terminal domain and at least one putative tyrosine autophosphorylation site of p185Tneu play a role in positively regulating the cell growth-regulating properties of the neu protein.     

Effect of drug and diet treatment of mild hypertension on diastolic blood pressure. The TAIM Research Group.

The Trial of Antihypertensive Interventions and Management is a multicenter randomized trial designed to examine the diastolic blood pressure response of various combinations of pharmacological and dietary interventions in the treatment of mild hypertension (diastolic blood pressure 90-100 mm Hg). Eight hundred and seventy-eight participants at 110-160% of ideal weight were randomly allocated to nine drug/diet treatment groups receiving either a placebo, chlorthalidone (25 mg), or atenolol (50 mg), combined with a usual, a weight loss, or a low sodium/high potassium diet. The primary outcome was diastolic blood pressure change from baseline to 6 months. Seven hundred and eighty-seven participants had follow-up data. The mean baseline diastolic blood pressure was 93.8 mm Hg; 55.9% of the participants were male, and the weight loss diet group lost an average of 4.7 kg. Multiple comparisons were accounted for in the analysis. A significantly greater lowering of diastolic blood pressure (12.4 mm Hg) was achieved in the atenolol group compared with either the low sodium/high potassium diet group (7.9 mm Hg, p = 0.001) or weight loss group (8.8 mm Hg, p = 0.006). Adding weight loss to chlorthalidone significantly enhanced blood pressure lowering (15.1 mm Hg) when compared with the diuretic alone (10.8 mm Hg, p = 0.002), but adding a low sodium/high potassium diet (12.2 mm Hg, p = 0.029) did not. In the short-term treatment of mild hypertension where diastolic blood pressure is the sole consideration, drugs outperform diet, and weight loss is beneficial, especially with diuretics.     

Dissection of Mycobacterium tuberculosis antigens using recombinant DNA.

A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies. M. tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large lambda gt11 recombinant DNA expression library. Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome. Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes. This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M. tuberculosis. The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines.