RESUMO
Rat osteo-testicular protein tyrosine phosphatase (OST-PTP), expressed in osteoblasts and testis, is a receptor-like transmembrane protein with two tandemly repeated phosphatase domains in the cytoplasmic region. In this report, we show that the first domain (CD1) is enzymatically active and appears to be influenced by the catalytically inactive second domain (CD2). The activity of CD1 is specific to phosphorylated tyrosine. Full-length OST-PTP protein expressed in COS cells has a molecular mass of approximately 185 kDa, and immunoprecipitates of this protein using OST-PTP-specific antisera show strong tyrosine phosphatase activity. Expression of OST-PTP mRNA in primary rat calvarial osteoblasts is temporally regulated, and peak expression is found at approximately day 15, which correlated well with the appearance of OST-PTP protein and its associated tyrosine phosphatase activity. Treatment of osteoblasts in culture with antisense oligonucleotides directed against the 5' untranslated region of OST-PTP results in abrogation of differentiation, confirming the functional importance of OST-PTP expression in osteoblast development.
Assuntos
Osteoblastos/enzimologia , Proteínas Tirosina Fosfatases/química , Testículo/enzimologia , Animais , Células COS , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Chlorocebus aethiops , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Feto , Inibidores do Crescimento/genética , Inibidores do Crescimento/farmacologia , Masculino , Mutagênese Sítio-Dirigida , Oligonucleotídeos Antissenso/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The herpes simplex virus type 1 (HSV) single-stranded DNA-binding protein (SSB, ICP8) stimulates the viral DNA polymerase (Pol) on an oligonucleotide-primed single-stranded DNA template. This stimulation is non-specific since other SSBs also increase Pol activity. However, only ICP8 was stimulatory when Pol activity was dependent upon priming by the viral helicase-primase complex. ICP8 also specifically stimulated the primer synthesis and ATPase activities of the helicase-primase. The mechanism of stimulation was different from that of Pol; helicase-primase stimulation required much lower amounts of ICP8 than the amount that saturates the DNA and optimally stimulates Pol. Furthermore, ICP8 did not act by removing secondary structure as stimulation also occurred on homopolymer templates. While the UL8 component of the helicase-primase is not required for enzymatic activities by a subassembly of the UL5 and UL52 proteins, only the holoenzyme (UL5/8/52) was stimulated by ICP8. These results identify a unique, functional interaction between the ICP8 SSB and the helicase-primase complex, mediated by the UL8 subunit.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Virais/metabolismo , DNA Primase , Ativação Enzimática , Humanos , Cinética , Dados de Sequência MolecularRESUMO
A panel of monoclonal antibodies (MAbs) directed against the herpes simplex virus type 1 (HSV-1) DNA polymerase (Pol) accessory protein, UL42, was developed and characterized. Thirteen different MAbs were isolated which exhibited varied affinities for the protein. All MAbs reacted with UL42 in ELISA, Western blot and immunoprecipitation analyses. Competitive ELISA was used to show that 6 different epitopes within UL42 were recognized by the MAbs. Immunoprecipitation of amino- and carboxy-terminal truncations of UL42 mapped the epitopes to regions containing amino acids 1-10, 10-108, 338-402, 402-460, and 460-477. All but one of these epitopes were outside the minimal active portion of the protein previously mapped to amino acids 20-315. None of these MAbs, alone or in combination, specifically neutralized the ability of UL42 to stimulate Pol activity in vitro. These results are consistent with structure-function studies that showed that N- and C-terminal regions of the UL42 protein, those recognized by the MAbs, are not involved in UL42 function in vitro.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , DNA Polimerase Dirigida por DNA , Epitopos/imunologia , Exodesoxirribonucleases , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Relação Estrutura-AtividadeRESUMO
The herpes simplex virus type 1 protease and its substrate, ICP35, are involved in the assembly of viral capsids. Both proteins are encoded by a single open reading frame from overlapping mRNAs. The protease is autoproteolytically processed at two sites. The protease cleaves itself at the C-terminal site (maturation site) and also cleaves ICP35 at an identical site, releasing a 25-amino-acid (aa) peptide from each protein. To determine whether these 25 aa play a role in capsid assembly, we constructed a mutant virus expressing only Prb, the protease without the C-terminal 25 aa. Phenotypic analysis of the Prb virus in the presence and absence of ICP35 shows the following: (i) Prb retains the functional activity of the wild-type protease which supports virus growth in the presence of ICP35; (ii) in contrast to the ICP35 null mutant delta ICP35 virus, the Prb virus fails to grow in the absence of ICP35; and (iii) trans-complementation experiments indicated that full-length ICP35 (ICP35 c,d), but not the cleaved form (ICP35 e,f), complements the growth of the Prb virus. The most striking phenotype of the Prb virus is that only unsealed aberrant capsid structures are observed by electron microscopy in mutant-infected Vero cells. Our results demonstrate that the growth of herpes simplex virus type 1 requires the C-terminal 25 aa of either the protease or its substrate, ICP35, and that the C-terminal 25 aa are involved in the formation of sealed capsids.
Assuntos
Capsídeo/química , Serina Endopeptidases/fisiologia , Proteínas Virais/fisiologia , Animais , Sequência de Bases , Chlorocebus aethiops , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/fisiologia , Células VeroRESUMO
The herpes simplex virus helicase-primase complex, a heterotrimer of the UL5, UL8, and UL52 proteins, displays a single predominant site of primer synthesis on phi X174 virion DNA (Tenney, D. J., Hurlburt, W. W., Micheletti, P. M., Bifano, M., and Hamatake, R. K. (1994) J. Biol. Chem. 269, 5030-5035). This site was mapped and found to be deoxycytosine-rich, directing the synthesis of a primer initiating with several guanine residues. The size and sequence requirements for primer synthesis were determined using oligonucleotides containing variations of the predominant template. Although the efficiency of primer synthesis on oligonucleotides was influenced by template size, it was absolutely dependent on nucleotide sequence. Conversely, the ATPase activity on oligonucleotide templates was dependent on template size rather than nucleotide sequence. Furthermore, only oligonucleotides containing primase templates were inhibitory in a coupled primase-polymerase assay using phi X174 DNA as template, suggesting that primer synthesis or primase turnover is rate-limiting. Additionally, stimulation of helicase-primase by the UL8 component and that by the ICP8 protein were shown to differ mechanistically using different templates: the UL8 component stimulated the rate of primer synthesis on phi X174 virion DNA and oligonucleotide templates, while ICP8 stimulation occurred only on phi X174 virion DNA.
Assuntos
DNA Helicases/metabolismo , Primers do DNA/biossíntese , RNA Nucleotidiltransferases/metabolismo , Simplexvirus/enzimologia , Adenosina Trifosfatases/metabolismo , Sequência de Bases , DNA Helicases/fisiologia , DNA Primase , Proteínas de Ligação a DNA , Dados de Sequência Molecular , Proteínas Virais/fisiologiaRESUMO
The herpes simplex virus type 1 ICP35 assembly protein is involved in the formation of viral capsids. ICP35 is encoded by the UL26.5 gene and is specifically processed by the herpes simplex virus type 1 protease encoded by the UL26 gene. To better understand the functions of ICP35 in infected cells, we have isolated and characterized an ICP35 mutant virus, delta ICP35. The mutant virus was propagated in complementing 35J cells, which express wild-type ICP35. Phenotypic analysis of delta ICP35 shows that (i) mutant virus growth in Vero cells was severely restricted, although small amounts of progeny virus was produced; (ii) full-length ICP35 protein was not produced, although autoproteolysis of the protease still occurred in mutant-infected nonpermissive cells; (iii) viral DNA replication of the mutant proceeded at wild-type levels, but only a very small portion of the replicated DNA was processed to unit length and encapsidated; (iv) capsid structures were observed in delta ICP35-infected Vero cells by electron microscopy and by sucrose sedimentation analysis; (v) assembly of VP5 into hexons of the capsids was conformationally altered; and (vi) ICP35 has a novel function which is involved in the nuclear transport of VP5.
Assuntos
Herpesvirus Humano 1/ultraestrutura , Proteínas Virais/fisiologia , Animais , Capsídeo/metabolismo , DNA Viral/genética , Genes Virais , Teste de Complementação Genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crescimento & desenvolvimento , Técnicas In Vitro , Mapeamento por Restrição , Deleção de Sequência , Células Vero , Proteínas Estruturais Virais/genética , Replicação ViralRESUMO
The herpes simplex virus type 1 protease and related proteins are involved in the assembly of viral capsids. The protease encoded by the UL26 gene can process itself and its substrate ICP35, encoded by the UL26.5 gene. To better understand the functions of the protease in infected cells, we have isolated a complementing cell line (BMS-MG22) and constructed and characterized a null UL26 mutant virus, m100. The mutant virus failed to grow on Vero cells and required a complementing cell line for its propagation, confirming that the UL26 gene product is essential for viral growth. Phenotypic analysis of m100 shows that (i) normal amounts of the c and d forms of ICP35 were produced, but they failed to be processed to the cleaved forms, e and f; (ii) viral DNA replication of the mutant proceeded at near wild-type levels, but DNA was not processed to unit length or encapsidated; (iii) capsid structures were observed in thin sections of m100-infected Vero cells by electron microscopy, but assembly of VP5 into hexons of the capsid structure was conformationally altered; and (iv) nuclear localizations of the protease and ICP35 are independent of each other, and the function(s) of Na, at least in part, is to direct the catalytic domain N(o) to the nucleus.
Assuntos
Capsídeo/biossíntese , Endopeptidases/fisiologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Proteínas Virais , Animais , Sequência de Bases , Capsídeo/genética , Capsídeo/metabolismo , Proteínas do Capsídeo , Linhagem Celular , Células Clonais , Cricetinae , Primers do DNA/genética , DNA Viral/genética , Endopeptidases/genética , Genes Virais , Teste de Complementação Genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Microscopia Eletrônica , Dados de Sequência Molecular , Mutagênese , Fenótipo , Processamento de Proteína Pós-Traducional , Temperatura , Células VeroRESUMO
The herpes simplex virus type 1 (HSV) UL5, UL8, and UL52 proteins form a helicase-primase complex in infected cells. Several laboratories have demonstrated that helicase and nucleoside triphosphatase activities of the heterotrimer (UL5/8/52) are indistinguishable from that of a subassembly of UL5 and UL52 (UL5/52). Although the UL5/52 subassembly functions in coupled primase-polymerase assays on homopolymeric templates, its activity on natural DNA templates has been reported to require UL8. To determine the role of UL8 in primase assays, the activity of the UL5/52 subassembly was compared to that of the heterotrimer reconstituted by adding UL8 to UL5/52. We detected significant activity of the UL5/52 subassembly in coupled primase-polymerase and oligoribonucleotide primer synthesis assays on phi X174 and M13 virion DNAs. However the addition of UL8 to UL5/52 stimulated this activity in a dose-dependent manner. We demonstrate that stimulation occurred at the level of primer synthesis. UL8 did not affect the amount or size of primers annealed to template, their utilization by DNA polymerase, or the use of specific initiation sites within the template. In kinetic studies, the rate of primer synthesis was increased by UL8 but the Km for phi X174 DNA template was unchanged. These results suggest that a function of the UL8 component of the HSV helicase-primase complex is to increase the efficiency of primer synthesis by UL5/52.
Assuntos
DNA Helicases/metabolismo , Herpesvirus Humano 1/enzimologia , RNA Nucleotidiltransferases/metabolismo , Animais , Baculoviridae , Células , Cromatografia de Afinidade , DNA Helicases/biossíntese , DNA Helicases/isolamento & purificação , DNA Primase , Cinética , Substâncias Macromoleculares , Mamíferos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas ViraisRESUMO
Herpes simplex virus type 1 encodes its own DNA polymerase (Pol), the product of the UL30 gene, and a polymerase accessory subunit, the product of the UL42 gene, both of which are required for viral DNA replication. Pol and the UL42 protein associate to form a heterodimeric complex (Pol/UL42) which is more active and has a higher processivity than the Pol catalytic subunit alone. The Pol/UL42 complex has been reconstituted by mixing together highly purified Pol and UL42 subunits obtained from recombinant baculovirus-infected cells. We have used polymerase activity on poly(dA):oligo(dT20), a template that the Pol subunit utilizes with low efficiency, to measure the formation of the Pol/UL42 complex. Our data indicate that the association constant for the Pol/UL42 complex is 1 x 10(8) M-1. Proteolytic digestions of UL42 were performed to determine whether structural domains of UL42 could be disclosed by differential amino acid accessibilities. The ability of these protease-resistant domains to form a functional complex with Pol was determined by measuring their ability to stimulate Pol activity on poly(dA):oligo(dT20). We have found that trypsin digestion of UL42 in the presence of DNA generates protease-resistant fragments of 28K and 8K which co-elute from a MonoQ column and are able to stimulate Pol activity on poly(dA):oligo(dT20). Complex formation of the 28K and 8K tryptic fragments with Pol was also shown by their co-immunoprecipitation with antibody to Pol. It was determined that the 28K fragment of UL42 comprised amino acids 1 to 245 or 1 to 254 of UL42, whereas the 8K fragment started at amino acid 255. Thus, controlled proteolysis of UL42 revealed two closely contiguous structural domains that retained the ability to complex with Pol and stimulate Pol activity.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases , Herpesvirus Humano 1/química , Proteínas Virais/química , Herpesvirus Humano 1/enzimologia , Humanos , Tripsina/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The herpes simplex virus type 1 UL42 protein was synthesized in reticulocyte lysates and assayed for activity in vitro. Three functional assays were used to examine the properties of in vitro-synthesized UL42: (i) coimmunoprecipitation to detect stable complex formation with purified herpes simplex virus type 1 DNA polymerase (Pol), (ii) a simple gel-based assay for DNA binding, and (iii) a sensitive assay for the stimulation of Pol activity. UL42 synthesized in reticulocyte lysates formed a stable coimmunoprecipitable complex with Pol, bound to double-stranded DNA, and stimulated the activity of Pol in vitro. Carboxy-terminal truncations of the UL42 protein were synthesized from restriction enzyme-digested UL42 gene templates and gene templates made by polymerase chain reaction and assayed for in vitro activity. Truncations of the 488-amino-acid (aa) UL42 protein to aa 315 did not abolish its ability to bind to Pol and DNA or to stimulate Pol activity. Proteins terminating at aas 314 and 313 showed reduced levels of binding to Pol, but these and shorter proteins were unable to bind to DNA or to stimulate Pol activity. These results suggest that all three of the biochemical functions of UL42 colocalize entirely within the N-terminal 315 aas of the UL42 protein. Amino acid sequence alignment of alpha herpesvirus UL42 homologs revealed that the N-terminal functional domain corresponds to the most highly conserved region of the protein, while the dispensable C terminus is not conserved. Conservative aa changes at the C terminus of the 315-aa truncated protein were used to show that conserved residues were important for activity. These results suggest that 173 aa of UL42 can be deleted without a loss of activity and that DNA-binding and Pol-binding activities are correlated with the ability of UL42 to stimulate Pol activity.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Simplexvirus/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Técnicas In Vitro , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes/metabolismo , Reticulócitos , Alinhamento de Sequência , Simplexvirus/enzimologia , Relação Estrutura-Atividade , Proteínas Virais/químicaRESUMO
We have analyzed the effects of mutations in the herpes simplex virus type 1 DNA polymerase (Pol) C-terminal UL42 binding domain on the activity of Pol and its ability to form complexes with and be stimulated by UL42 in vitro. Wild-type Pol expressed in Saccharomyces cerevisiae was both bound and stimulated by UL42 in vitro. C-terminal truncations of 19 and 40 amino acids (aa) did not affect the ability of Pol to be stimulated by UL42 in vitro. This stimulation as well as basal Pol activity in the presence of UL42 was inhibited by polyclonal anti-UL42 antiserum, thus indicating a physical interaction between Pol and UL42. Removal of the C-terminal 59 aa of Pol and internal deletions of 72 aa within the Pol C terminus eliminated stimulation by UL42. None of the truncations or deletions within Pol affected basal polymerase activity. In contrast with their ability to be stimulated by UL42, only wild-type Pol and Pol lacking the C-terminal 19 aa bound UL42 in a coimmunoprecipitation assay. These results demonstrate that a functional UL42 binding domain of Pol is separable from sequences necessary for basal polymerase activity and that the C-terminal 40 aa of Pol appear to contain a region which modulates the stability of the Pol-UL42 interaction.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases , Simplexvirus/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Anticorpos Antivirais , Análise Mutacional de DNA , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Ativação Enzimática , Substâncias Macromoleculares , Dados de Sequência Molecular , Testes de Neutralização , Testes de Precipitina , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Simplexvirus/enzimologia , Relação Estrutura-Atividade , Proteínas Virais/imunologia , Proteínas Virais/farmacologiaRESUMO
The Indian Health Service (IHS) was transferred from the Department of Interior to the Public Health Service in the Department of Health, Education, and Welfare in 1955. At that time, the general health of Indian people substantially lagged behind the rest of the U.S. population. This gap was reflected in mortality rates which were several-fold higher for Indians, or reflected in time; there were decades between the dates when the U.S. population achieved certain lower death rates compared with the dates when similar reductions were achieved by Indians. As a result of preventive health programs, improvements in sanitation, and the development of a number of medical advances, substantial progress has been achieved in improving the health of American Indians and Alaska Natives. Life expectancy of Indians has increased 20 years between 1940 and 1980. From 1955 through 1982, the death rate for Indian infants dropped by 82 percent. Also, the age-adjusted death rate for tuberculosis decreased from 57.9 per 100,000 population in 1955 to 3.3 in 1983. These and other improvements are summarized in this paper.
Assuntos
Inquéritos Epidemiológicos , Indígenas Norte-Americanos , United States Public Health Service , Adolescente , Adulto , Idoso , Criança , Serviços de Saúde da Criança/tendências , Estudos de Avaliação como Assunto , Feminino , Gastroenteropatias/mortalidade , Humanos , Lactente , Mortalidade Infantil , Recém-Nascido , Expectativa de Vida , Serviços de Saúde Materna/tendências , Pessoa de Meia-Idade , Gravidez , Tuberculose/mortalidade , Estados UnidosRESUMO
A survey of cancer incidence among Alaska Natives for the 5-year period 1969-73 revealed fewer cancer cases overall than expected in relation to US rates, but significantly increased risk for certain cancer sites: the nasopharynx in both sexes (with excesses over 15-fold), the liver in males, and the salivary glands, gallbladder, kidney and thyroid in females. Compared with earlier reports, the observations suggest marked changes in cancer incidence among Alaska Natives over the past two decades, with declines in esophageal and invasive cervical cancers, and increases in cancers of the lung, colon and rectum.
Assuntos
Neoplasias/epidemiologia , Adolescente , Adulto , Idoso , Alaska , Povo Asiático , Criança , Pré-Escolar , Feminino , Neoplasias da Vesícula Biliar/epidemiologia , Humanos , Indígenas Norte-Americanos , Lactente , Inuíte , Neoplasias Renais/epidemiologia , Neoplasias Hepáticas/epidemiologia , Masculino , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/epidemiologia , Neoplasias das Glândulas Salivares/epidemiologia , Fatores Sexuais , Neoplasias da Glândula Tireoide/epidemiologia , Estados UnidosRESUMO
Health care for scattered populations is discussed. An optimal system applying current technology is described emphasizing the economic supportability of such a system. This is accomplished by basing the system on health auxiliaries and middle level health personnel who have undergone problem oriented training and who receive regular supervision. The Alaska Native Health Program is described in detail. Several other health care systems in use in sparsely populated areas of the world are reported on. Emergency care as a particular and essential aspect of any health care program for isolated populations is discussed. Finally future trends including current experiments utilizing satellite technology are reported on.