Regulation by glucocorticoids of rat-liver phenylalanine hydroxylase in vivo

Phenylalanine hydroxylase, a liver-associated enzyme, is induced markedly by glucocorticoids in two permanent rat-hepatoma cell lines. In order to gain evidence that this phenomenon also occurs in vivo, we examined the effect of adrenalectomy and/or hormone supplementation on the levels of phenylalanine hydroxylase in the livers of adult rats: glucocorticoid administration increases, and adrenal ablation reduces, the activity of the hepatic enzyme, and the diminution occurring in the latter instance is entirely prevented by concurrent hormone replacement. These results thus corroborate earlier findings from a single experiment and are consistent with the hypothesis that adrenal corticosteroid hormones participate in modulating phenylalanine-hydroxylase levels within the diploid hepatocyte.     

Integration is not necessary for expression of human immunodeficiency virus type 1 protein products.

A common feature in the life cycle of cytocidal retroviruses, including human immunodeficiency virus type 1 (HIV-1), is the accumulation of large amounts of unintegrated viral DNA. As yet, the role of unintegrated viral DNA in the cytopathogenesis of cytocidal retrovirus infections remains unresolved. HIV-1 mutants which were deleted in the integrase/endonuclease gene and which were unable to establish an integrated form of the virus were constructed. Despite an inability to integrate, these mutants were fully competent templates for HIV-1 core and envelope antigen production. HIV-1 antigen could be detected in the supernatants of lymphocyte cultures infected with HIV-1 integrase mutants. However, an inability to rescue infectious virus from these cultures indicated that HIV-1 integration was required for the production of infectious HIV-1. On the basis of the ability of unintegrated HIV-1 DNA to serve as a template for HIV-1 antigen production, it is plausible that unintegrated viral DNA can contribute to the HIV-1 antigen pool during HIV-1 replication.     

Cloning and characterization of human immunodeficiency virus type 1 variants diminished in the ability to induce syncytium-independent cytolysis.

The phenomenon of interference was exploited to isolate low-abundance noncytopathic human immunodeficiency virus type 1 (HIV-1) variants from a primary HIV-1 isolate from an asymptomatic HIV-1-seropositive hemophiliac. Successive rounds of virus infection of a cytolysis-susceptible CD4+ cell line and isolation of surviving cells resulted in selective amplification of an HIV-1 variant reduced in the ability to induce cytolysis. The presence of a PvuII polymorphism facilitated subsequent amplification and cloning of cytopathic and noncytopathic HIV-1 variants from the primary isolate. Cloned virus stocks from cytopathic and noncytopathic variants exhibited similar replication kinetics, infectivity, and syncytium induction in susceptible host cells. The noncytopathic HIV-1 variant was unable, however, to induce single-cell killing in susceptible host cells. Construction of viral hybrids in which regions of cytopathic and noncytopathic variants were exchanged indicated that determinants for the noncytopathic phenotype map to the envelope glycoprotein. Sequence analysis of the envelope coding regions indicated the absence of two highly conserved N-linked glycosylation sites in the noncytopathic HIV-1 variant, which accompanied differences in processing of precursor gp160 envelope glycoprotein. These results demonstrate that determinants for syncytium-independent single-cell killing are located within the envelope glycoprotein and suggest that single-cell killing is profoundly influenced by alterations in envelope sequence which affect posttranslational processing of HIV-1 envelope glycoprotein within the infected cell.     

JC virus-simian virus 40 genomes containing heterologous regulatory signals and chimeric early regions: identification of regions restricting transformation by JC virus.

The papovavirus JC virus (JCV) is highly oncogenic in experimental animals but, unlike simian virus 40 (SV40), is severely restricted in its ability to transform cells in culture. We exploited the close genetic relatedness of these two viruses to delimit region(s) of the T protein which can restrict transforming activity. Novel chimeric genomes were produced by exchanging various segments of the JCV and SV40 T-protein-coding regions. These DNA constructs specified early proteins with in-frame substitutions of analogous amino acid sequences. A second set of genomes was prepared which, in addition to chimeric early proteins, contained substituted regulatory regions. The transformation efficiencies of these chimeric genomes were intermediate between those of SV40 and JCV, with the source of T protein exerting a greater effect than that of the regulatory region. The ability of certain constructs to induce efficient transformation required the presence of an SV40 regulatory region or specific sequences within the SV40 early coding region. Cloned cell lines prepared from representative transformants were characterized; the ability to form colonies in soft agarose was investigated, and the presence of viral T and cellular p53 proteins was determined. The various T proteins differed in amount, stability, and the ability to form stable complexes with p53.     

Analysis of ordered arrays of adsorbed lysozyme by scanning tunneling microscopy.

Scanning tunneling microscopy (STM) has been used to observe lysozyme at a graphite surface directly in order to gain mechanistic information about the molecular events involved in protein adsorption. The experiments were performed using an insulated tip in an aqueous protein solution, allowing the time course of the adsorption process to be followed, including the evolution of ordered arrays. Ordered arrays of protein molecules were observed, with lattice spacings that varied with bulk protein concentration and salt strength. Fourier analysis was used to determine the average cell dimensions of an array. From the observed lattice spacings, it was possible to estimate the surface coverage of the protein, and thus, by varying the conditions, adsorption isotherms could be obtained. These isotherms compare well with adsorption isotherms measured using total internal reflectance fluorescence (TIRF) spectroscopy on a hydrophobic surface. Since the protein is charged and the electrolyte has an effect on the isotherms, electrostatics are a likely controlling factor. Molecular electrostatics computations were thus used to investigate the possible origins of the lattice structure, and they suggest that favorable intermolecular interactions among adsorbed molecules are consistent with hydrophobically dominated protein-surface interactions.     

Kinetics of the onset of catabolite repression in Escherichia coli as determined by lac messenger ribonucleic acid initiations and intracellular cyclic adenosine 3'',5''-monophosphate levels.

The rates of synthesis of beta-galactosidase (EC 3.2.1.23) and the intracellular levels of cyclic 3',5'-adenosine monophosphate (cAMP) soon after the addition of glucose or glycerol to exponentially growing cultures of Escherichia coli have been determined. Within 10 s of its addition, glucose, but not glycerol, lowered the apparent initiation frequency of lac messenger ribonucleic acid. The glucose-generated reduction in initiations is identified as catabolite repression by its reversibility with cAMP. The intracellular cAMP levels respond virtually identically to glucose and glycerol additions. Thus, no correlation was observed between the rate of messenger ribonucleic acid initiation and the level of cAMP.     

Targeting of p300 to the interleukin-2 promoter via CREB-Rel cross-talk during mitogen and oncogenic molecular signaling in activated T-cells

In this report, we explore the mechanisms of targeting of p300 to the interleukin-2 (IL-2) promoter in response to mitogenic and oncogenic molecular signals. Recruitment of p300 by cAMP-responsive element-binding protein-Rel cross-talk at the composite CD28 response element (CD28RE)-TRE element of the IL-2 promoter is essential for promoter inducibility during T-cell activation, and CD28RE-TRE is the exclusive target of the human T-cell lymphotropic virus type I oncoprotein Tax. The intrinsic histone acetyltransferase activity of p300 is dispensable for activation of the IL-2 promoter, and the N-terminal 743 residues contain the minimal structural requirements for synergistic transactivation of the CD28RE-TRE, the IL-2 promoter, and endogenous IL-2 gene expression. Mutational analysis of p300 reveals differential structural requirements for the N-terminal p300 module by individual cis-elements within the IL-2 promoter. These findings provide evidence that p300 assembles at the IL-2 promoter to form an enhanceosome-like signal transduction target that is centrally integrated at the CD28RE-TRE element of the IL-2 promoter through specific protein module-targeted associations in activated T-cells.