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At least 1,135 records · Page 63Linked to original sources
PubMed1988

Sex change in cytochrome P-450 phenotype by growth hormone treatment of adult rat hepatocytes maintained in a culture system on matrigel.

Results of studies of hypophysectomized rats suggest that growth hormone serves as a final common mediator through which gonadal steroids and other modifiers of pituitary function alter the expression of gender-specific liver genes such as the sexually dimorphic pair of cytochrome P-450 isozymes, male-specific P-450h and female-specific P-450i. We tested the effects of growth hormone in a system for primary monolayer culture of adult rat hepatocytes on a laminin-rich extracellular matrix (matrigel), which permits sustained expression of both constitutive and inducible liver genes in a chemically defined medium. Cultures of freshly isolated hepatocytes prepared from untreated male rats and samples of the intact donor liver contained readily detectable quantities of immunoreactive P-450h protein (measured on immunoblots of cell microsomes) and P-450h mRNA (measured on Northern blots of cellular RNA). Neither P-450i immunoreactive protein nor P-450i mRNA were present. Addition of physiologic concentrations of human or bovine growth hormone, but not of prolactin, to culture medium lacking insulin or other hormones resulted in prompt induction of P-450i immunoreactive protein and P-450i mRNA. Induction of P-450i mRNA in male hepatocyte cultures was dependent on the concentration of growth hormone, required as little as 24 hr of exposure, and was markedly attenuated in cultures maintained on type I collagen rather than on matrigel. Growth hormone treatment also induced the level of mRNA for insulin-like growth factor I, whereas the amount of mRNA for the male-specific urinary protein alpha 2 mu-globulin was unaffected. Cultures of hepatocytes derived from untreated adult female rats retained high levels of P-450i mRNA but only if the culture medium contained growth hormone. None of the tested treatments with estrogens, androgens, glucocorticoids, or growth hormone induced P-450h mRNA or P-450h immunoreactive protein in cultures of female hepatocytes. We conclude that the somatogenic effects of growth hormone acting alone and directly on the hepatocyte in culture are sufficient to "feminize" the cytochrome P-450 phenotype. The present culture system offers a way to explore the molecular basis for hormonal control of liver gene expression.

Animals↗
PubMedDate not supplied

Expression and refolding of recombinant human fibroblast procathepsin D.

Procathepsin D is a precursor of the human lysosomal protease cathepsin D. Due to its short half-life, procathepsin D is difficult to obtain in quantities sufficient to allow structural and enzymatic studies. To obtain large quantities of this precursor, procathepsin D was expressed using the T7 promoter vector pET3a in bacteria that carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lac promoter. At high cell density in rich medium, basal levels of T7 RNA polymerase were sufficient to express recombinant procathepsin D without addition of an exogenous inducer of the lac promoter. The recombinant protein, constituting almost half of the total cell protein, accumulated in intracytoplasmic inclusion bodies and was isolated from the insoluble fraction of lysed cells. Antibodies prepared against the purified recombinant protein were shown to crossreact with native human placental and porcine spleen cathepsin D. Recombinant procathepsin D was solubilized in denaturants and was refolded. After extended preincubation of the denatured protein at acid pH to allow folding and activation of the zymogen, pepstatin inhibitable catalytic proteolysis was detected. These data demonstrated that the glycosylated aspartic protease, procathepsin D can be refolded and activated in an unglycosylated form and thus provides a system for the study of procathepsin D structure and function.

Amino Acid Sequence↗
PubMed1989

A novel method for the purification of the Xenopus transcription factor IIIA.

We have developed a quick and simple purification method that yields large quantities of the Xenopus zinc finger protein transcription factor IIIA (TFIIIA). The protein is purified in the form of the 7S storage particle (TFIIIA/5S RNA complex) found in the ovaries of immature Xenopus. Our method yields 0.5 to 1 mg of pure 7S particle per ovary. It involves a high speed centrifugation step, fractionation on a gel filtration column and a precipitation step using calcium chloride. TFIIIA purified using this protocol retains full DNA binding activity and has the expected zinc ion content.

Animals↗
PubMed1986

The peplomers of Berne virus.

Using [3H]glucosamine and [3H]mannose labels, two virus-specific glycosylated polypeptide species with Mr values of about 200,000 (200K) and in the 75K to 100K range, respectively, were recognized in Berne virus-infected embryonic mule skin cells. In purified virions only the latter glycoprotein occurred. Concanavalin A was bound to the virion as evidenced by reduction in infectivity. Analyses using SDS-PAGE, blotting and glycoprotein identification with concanavalin A and horseradish peroxidase showed coincidence of the virion glycoprotein signals with the maximum infectivity and haemagglutinating activity in an isokinetic sucrose gradient. Polyclonal rabbit immune serum and a neutralizing and haemagglutination-inhibiting monoclonal antibody raised against Berne virus recognized both the 75K to 100K and the '200K' glycoproteins. Using tunicamycin, a concentration-dependent inhibition of infectivity was noted; however, non-infectious particles containing the two major polypeptides (20K and 22K) were released from the cells in small quantities. The glycoproteins were absent from cytoplasmic extracts and a novel polypeptide of about 150K was identified instead. Translation of poly(A)-selected intracellular RNA from infected cells in a rabbit reticulocyte cell-free system also resulted in the appearance of a new high Mr polypeptide (about 170K). Using pulse-chase labelling and radioimmunoprecipitation, suggestive evidence for a precursor-product relationship between the intracellular '200K' and the virion glycoproteins has been obtained. These experiments identify the N-glycosylated proteins in the 75K to 100K range as constituents of the peplomeric envelope projection of Berne virus; they probably arise by post-translational processing of a 150K to 170K precursor molecule involving glycosylation and subsequent cleavage.

Antibodies, Viral↗
PubMed1995

Identification of positive and negative splicing regulatory elements within the terminal tat-rev exon of human immunodeficiency virus type 1.

The requirement of human immunodeficiency virus type 1 to generate numerous proteins from a single primary transcript is met largely by the use of suboptimal splicing to generate over 30 mRNAs. To ensure that appropriate quantities of each protein are produced, there must be a signal(s) that controls the efficiency with which any particular splice site in the RNA is used. To identify this control element(s) and to understand how it operates to generate the splicing pattern observed, we have initially focused on the control of splicing of the tat-rev intron, which spans the majority of the env open reading frame. Previous analysis indicated that a suboptimal branchpoint and polypyridimine tract in this intron contribute to its suboptimal splicing (A. Staffa and A. Cochrane, J. Virol. 68:3071-3079, 1994). In this report, we identify two additional elements within the 3'-terminal exon, an exon-splicing enhancer (ESE) and an exon splicing silencer (ESS), that modulate the overall efficiency with which the 3' tat-rev splice site is utilized. Both elements are capable of functioning independently of one another. Furthermore, while both the ESE and ESS can function in a heterologous context, the function of the ESS is extremely sensitive to the sequence context into which it is placed. In conclusion, it would appear that the presence of a suboptimal branchpoint and a polypyrimidine tract as well as the ESE and ESS operate together to yield the balanced splicing of the tat-rev intron observed in vivo.

Animals↗
PubMed2000

Plasmid DNA for pharmaceutical applications.

With the advent and progress of recombinant DNA technology into a variety of fields such as medical therapy, preventive or curative vaccination or the induction of regeneration, the demand for large quantities of highly purified DNA is increasing. Traditional methods of purifying plasmids usually require sophisticated methodology if the DNA is to be separated from RNA and other contaminating organic components. In particular, methods for obtaining supercoiled covalently closed circular (CCC) plasmid DNA in pure form, cope with the requirement that other plasmid topologies also produced have to be separated from the final product. The innovative technology of capillary gel electrophoresis (CGE) contributes a sensitive tool to the short list of applicable quality control assays for clinical grade plasmid DNA.

Biotechnology↗
PubMedDate not supplied

Hypothesis: systemic lupus erythematosus as a disease secondary to polyclonal lymphocyte activation.

Systemic lupus erythematosus (SLE) is characterized by the concomitance of B cell polyclonal activation and the production of a wide spectrum of autoantibodies showing a marked preference for nuclear antigens. We propose the hypothesis that the production of the most pathogenic antinuclear antibodies with high affinity and IgG isotype is driven by antigens liberated by the death of rapidly proliferating lymphoid cells. The disease could evolve in three steps: (1) polyclonal B cell activation and proliferation caused by unknown etiologic factors (virus?, mitogen? ...); (2) primary activation of natural antibody-producing clones leading to the appearance in the serum of large amounts of natural autoantibodies with public idiotype; and (3) secondary immunization against molecules synthesized in large quantities during lymphocyte proliferation, released by dying cells and subsequently presented in an immunogenic form to T cells. These molecules include nucleic acids (DNA, RNA), nucleic acid-binding proteins (histones, Ro, La), and parts of the RNA splicing machinery (RNP). Other autoantibody activities could relate to cross-reactivities or the binding properties of DNA-anti-DNA immune complexes.

Animals↗
PubMed1986

In vitro translation of rat liver and Novikoff hepatoma cytokeratin mRNAs.

Cytoplasmic poly(A)+ RNA was isolated from normal rat liver and Novikoff ascites hepatoma cells, translated in vitro using rabbit reticulocyte lysate system and the translational products were assayed by immunoprecipitation with antibodies specific for Novikoff hepatoma principal cytokeratins p39, p49 (a group of hepatic cytokeratins C, D, and E) and p56. The identity of the precipitated antigens was further confirmed by two-dimensional polyacrylamide gel electrophoresis. Only the Novikoff hepatoma poly(A)+ RNA contained translatable mRNA coding for the p39 cytokeratin while the p49 and p56 cytokeratins were translated from both the normal rat liver and Novikoff hepatoma poly(A)+ RNAs. Immunoprecipitations employing monoclonal antibody specific for p39 also recovered significant quantities of p56 and 49K cytokeratins, presumably due to oligomeric associations of these proteins with p39 immediately after in vitro synthesis. Similar results were observed after experiments with anti-p56 monoclonal antibody in which p39, not reactive with this antibody, was recovered in immunoprecipitates. Overall, the two-dimensional gel fluorograms of cytokeratins synthesized in vitro from NAH or liver poly(A)+ RNA are quite similar to isolated antigenic and cytokeratin profiles reported previously. These results suggest that overt posttranslational processing is not likely responsible for the diversity of cytokeratins observed in the liver.

Animals↗
PubMed1987

Phosphorylation of RNA polymerase I-associated polypeptides of Tetrahymena pyriformis.

In the ciliate Tetrahymena pyriformis phosphorylation of RNA polymerase I [EC 2.7.7.6] and of polymerase-associated polypeptides was investigated in growing and growth-arrested cultures which differ widely in their rates of rRNA synthesis. Several putative subunits of RNA polymerase I (of 180, 21.5, and 19.5 kDa) and a polymerase-associated polypeptide of 27 kDa were found to be phosphorylated, independent of the growth conditions. However, an additional enzyme-associated polypeptide of 26 kDa was intensively labeled with 32P only after arrestment of growth by starvation. The molar quantities of both phosphorylated, enzyme-associated polypeptides thereby did not differ in growing and growth-arrested cultures, and the specific 32P-labeling of cellular ATP remained nearly unchanged under the different culture conditions. These findings indicate a selective, reversible phosphorylation of the RNA polymerase I-associated 26 kDa polypeptide correlated with conditions of repressed rRNA synthesis induced by the starvation procedure. In vitro phosphorylation in macronuclei isolated from growing and growth-arrested cultures using [gamma-32P]ATP revealed essentially the same pattern of labeling of the enzyme-associated polypeptides of 27 and 26 kDa as it was found in vivo.

Adenosine Triphosphate↗
PubMed1989

Heat shock response in Escherichia coli promotes assembly of plasmid encoded RNA polymerase beta-subunit into RNA polymerase.

Escherichia coli cells, carrying a rifampicin sensitive RNA polymerase beta-subunit gene in the chromosome and a rifampicin resistant beta-subunit gene placed under the control of a strong promoter in a multicopy plasmid, are unable to grow in the presence of rifampicin, despite the accumulation of large quantities of the resistant subunit. A major portion of the overproduced subunit is found in an insoluble form. Conditions known to induce the heat shock proteins (hsps), e.g. elevated temperature or the presence of ethanol in the growth medium, increase the amount of the plasmid-borne beta-subunit which apparently assembles into active RNA polymerase and makes the plasmid bearing cells rifampicin resistant. Alternatively, plasmid-borne subunits assemble into RNA polymerase with low efficiency in rpoH mutant cells known to have reduced level of hsps. We suggest that the plasmid-borne subunit is poorly assembled into RNA polymerase and that hsps promote the assembly by interfering with beta-subunit aggregation.

DNA-Directed RNA Polymerases↗
PubMed1998

Motion of RNA polymerase along DNA: a stochastic model.

RNA polymerase is a key transcription enzyme that moves along a DNA double helix to polymerize an RNA transcript. Recent progress in micromechanical experiments permits quantitative studies of forces and motion generated by the enzyme. We present in this paper a chemical kinetics description of RNA polymerase motion. The model is based on a classical chemical kinetics description of polymerization reactions driven by a free energy gain that depends on forces applied externally at the catalytic site. The RNA polymerase controlled activation barrier of the reaction is assumed to be strongly dependent on inhibitory internal strains of the RNA polymerase molecule. The sequence sensitivity of RNA polymerase is described by a linear coupling between the height of the activation barrier and the local DNA sequence. Our model can simulate optical trap experiments and allows us to study the dynamics of chemically halted complexes that are important for footprinting studies. We find that the effective stall force is a sequence-dependent, statistical quantity, whose distribution depends on the observation time. The results are consistent with the experimental observations to date.

Base Sequence↗
PubMed1996

Phenotypic characterization of mutants in vaccinia virus gene G2R, a putative transcription elongation factor.

The phenotypic defects of two mutants of vaccinia virus, the lesions of which map to gene G2R, were characterized in vivo, and the results suggest a role for the G2R protein in viral transcription elongation. Both a temperature-sensitive mutant, Cts56, and an isatin-beta-thiosemicarbazone-dependent deletion mutant, G2A, in gene G2R have a characteristic and unique defect in late viral gene expression. The G2R mutants synthesize early viral RNA, early viral proteins, and viral DNA normally under nonpermissive conditions. In G2R mutants, late viral protein synthesis begins at the normal time, low-molecular-weight viral proteins are synthesized in normal quantities, but synthesis of high-molecular-weight viral proteins is reduced in amount. Intermediate and late promoter utilization is normal in G2R mutants, but intermediate and late RNAs are reduced in size. The reduction in length of the intermediate and late mRNAs represents a truncation of mRNA 3' ends. Thus, intermediate and late RNAs are too short to encode large proteins but long enough to encode small proteins, therefore accounting for the protein synthesis phenotype. These results suggest that the G2R protein acts to regulate the elongation potential of the viral RNA polymerase late during a vaccinia virus infection.

Animals↗
PubMed1996

Prognosis in HIV-1 infection predicted by the quantity of virus in plasma.

The relation between viremia and clinical outcome in individuals infected with human immunodeficiency virus-type 1 (HIV-1) has important implications for therapeutic research and clinical care. HIV-1 RNA in plasma was quantified with a branched-DNA signal amplification assay as a measure of viral load in a cohort of 180 seropositive men studied for more than 10 years. The risk of acquired immunodeficiency syndrome (AIDS) and death in study subjects, including those with normal numbers of CD4+ T cells, was directly related to plasma viral load at study entry. Plasma viral load was a better predictor of progression to AIDS and death than was the number of CD4+ T cells.

Acquired Immunodeficiency Syndrome↗
PubMed1988

[Morphometric research on the nuclei and argentophilic structures of the nucleoli of granulocytopoietic cells in rats after long-term cyclophosphamide exposure].

The Ag-staining intensity of interphase bone marrow granulopoietic cells decreases during their maturation as evidenced by TV computerized morphometry. These data are in accordance with the phenomenon of RNA transcription reduction in mature granulocytes. The Ag-positive material amount increases during S and G2 phases of proliferative pool granulocytes. 8 days after every day injection of 20 mg/kg cyclophosphamide an integral area of silver grains in proliferative pool granulocytes decreased 1.5 times. In mature granulocytes it decreased and was two times higher than in the control. The nuclear area of all granulocytes increased. There was a positive correlation (p less than 0.01) between the quantity of the silver grains and nuclear areas. It seems that mature granulocytes inherit the increased Ag-positive material amount from G2 blocked progenitors (promyelocytes and myelocytes) and this increase is not due to the RNA transcriptional activity.

Animals↗
PubMed1994

Identification of drug-related genotypic changes in HIV-1 from serum using the selective polymerase chain reaction.

We attempted to detect drug-related HIV-1 pol gene mutations by selective polymerase chain reaction (PCR) using both proviral DNA and viral RNA isolated from patients (pts) with AIDS or ARC receiving antiretroviral therapy. Peripheral blood mononuclear cell (PBM)-associated proviral DNA and serum-derived viral RNA were obtained from eight patients before and after receiving an alternating regimen of AZT and ddC for 15-41 months or ddI monotherapy for 12-26 months. These specimens were examined for the presence of mutations at positions 70, 74, 215 and 219. We noted that selective PCR results can be ambiguous depending on the quantity of DNA template employed. We, therefore, used the minimal quantity of DNA templates that yielded evaluable PCR products in this study. For all the eight pairs of pre- and post-therapy proviral DNA samples, selective PCR results agreed with independently determined nucleotide sequences. Results of reverse transcription of serum-derived viral RNA followed by selective PCR differed in some cases from those using the proviral DNA. In particular, the use of serum viral RNA appeared to allow earlier detection of changes in drug-related mutations than the use of PBM-associated proviral DNA. We conclude that (i) selective PCR using the minimum and sufficient number of PBM-associated proviral DNA and serum viral RNA copies successfully detects the presence of known pol gene mutations; (ii) drug-related mutations may be distinguished earlier in virions in serum (or plasma) than in proviral DNA in PBM; and (iii) quantification of HIV-1 prior to selective PCR may be an important component in monitoring the therapy of HIV-1 infection.

Antiviral Agents↗
PubMed2006

Analysis of RNA recovery and gene expression in the epidermis using non-invasive tape stripping.

BACKGROUND: The recovery of RNA from the upper epidermis by tape stripping yields variable RNA mass but has not been evaluated for its dependence on anatomical location. Gene expression at different body locations and the origin of RNA recovered by tape stripping have not been investigated. OBJECTIVES: To characterize the recovery of RNA from different anatomical locations by tape stripping; to correlate the recovery of RNA and removal of barrier by tape stripping, as assayed by transepidermal water loss; and to investigate gene expression in the upper epidermis at different body locations. METHODS: Twelve subjects were tape stripped at 15 body locations. RNA mass was evaluated and gene expression assayed. Subjects were tape stripped 4, 8 and 12 times on the upper back and transepidermal water loss and RNA recovery assayed. RESULTS: Ranked by median RNA recovery, the following order was observed: mastoid>forehead>chest>upper back>mid back>cheek>lower back>deltoid>forearm>abdomen>ventral thigh>inner arm>shin>dorsal thigh>lower leg. Expression of the housekeeping gene mRNAs is found to be uniform and reproducible while IL-8 and TNFalpha mRNAs are expressed in different quantities both at different body sites within an individual and between individuals at a specific anatomical site. Data show a significant and high correlation between the number of tapes used to strip a site and transepidermal water loss but no strong correlation between transepidermal water loss and RNA recovery or number of tapes used to strip a site and RNA recovery. CONCLUSIONS: Subjects and anatomical location are shown to be significantly different for the ability to recover RNA by tape stripping. We hypothesize that RNA recovered by tape strip is not derived from corneocytes but from cells associated with the stratum corneum.

Abdomen↗
PubMed2003

Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli.

The 5'-AMP-activated protein kinase (AMPK) plays a critical role in the regulation of cellular energy homeostasis. AMPK is a heterotrimer composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). To date, purified AMPK has only been obtained in small, microgram quantities from tissues. Here, we describe an expression and purification system for production of functional AMPK in Escherichia coli. A plasmid carrying all three subunits of AMPK (alpha1, beta1, and gamma1) for T7 RNA polymerase-driven transcription of a single tricistronic messenger was constructed, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. AMPK was purified from the bacterial lysates by single-step nickel-ion chromatography, utilizing a poly-histidine tag fused to the N-terminus of the alpha-subunit. The recombinant AMPK complex was monodisperse, as shown by gel filtration chromatography with elution of a single peak at a Stokes radius of 52A. Bacterially expressed AMPK was entirely inactive, yet it could be activated by upstream kinase in the presence of AMP. Sufficient quantities of purified functional AMPK should prove to be an invaluable tool to solve many of the pertinent questions about its molecular structure and function, in particular facilitating protein crystallization for X-ray structure analysis.

Adenosine Monophosphate↗
PubMed1999

Human S100A11 exhibits differential steady-state RNA levels in various tissues and a distinct subcellular localization.

In order to analyze the steady-state RNA levels of S100A11 in different tissues, a cDNA fragment of human S100A11 was isolated from a cDNA library. The obtained fragment was labeled and hybridized to RNA isolated from various tissues. The Northern blot analysis revealed that S100A11 RNA levels varied from high in placenta, through intermediate in heart, lung, kidney, and most muscle samples, to barely detectable in brain. An efficient purification method for recombinant S100A11 yielding high quantities was developed. Furthermore, to examine the subcellular localization of this protein, the human polypeptide S100A11 antibodies were raised in rabbit. S100A11 was found to have a localization distinct from other S100 proteins examined, and is mostly localized in the nucleus, with slight variations among different glioblastoma cell types.

Adult↗