RESUMEN
We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.
Asunto(s)
ADN/análisis , Músculos/análisis , Tropomiosina/genética , Animales , Secuencia de Bases , Células Cultivadas , Embrión de Pollo , Clonación Molecular , ADN/aislamiento & purificación , Fibroblastos/análisis , Datos de Secuencia Molecular , Peso Molecular , Empalme del ARNRESUMEN
OBJECTIVE: To evaluate human monoclonal antibodies (MAb) for neutralizing activity against primary HIV-1 isolates in peripheral blood mononuclear cells. DESIGN: Neutralization activity data were obtained from 11 laboratories on a coded panel consisting of six human MAb to HIV envelope V3, CD4-binding region or gp41. Hyperimmune globulin against HIV-1 and normal human immunoglobulin G were supplied as controls. Each laboratory received pre-titered virus for use in the studies. METHODS: Each laboratory measured neutralization of the MAb against laboratory strain HIVMN, genomic clone HIVJR-CSF, two subtype B and one subtype D primary isolates. RESULTS: The titers of the centrally supplied virus stocks as determined by re-titration or back-titration varied among laboratories and were generally 10-100-fold less than provided. The neutralizing activity of each MAb varied by as much as a 1000-fold among laboratories. These differences may result from varying sensitivity in neutralization assay protocols and the differing susceptibility of primary cells to infection with HIV-1. CONCLUSIONS: To consolidate the data from multiple laboratories, the neutralization titers were compared by classifying antibodies as neutralizing if the antibody concentration for 50% virus inhibition was < or = 10 micrograms/ml. By this criterion, the CD4-binding region and gp41 MAb neutralized all four subtype B viruses and the subtype D isolate in a few of the laboratories. The V3 MAb neutralized only HIVMN and the closely related HIVJR-CSF viruses.
Asunto(s)
Anticuerpos Monoclonales , Productos del Gen env/inmunología , Anticuerpos Anti-VIH , VIH-1/inmunología , Antígenos CD4/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/clasificación , VIH-1/aislamiento & purificación , Humanos , Técnicas In Vitro , Leucocitos Mononucleares/virología , Pruebas de Neutralización , Fragmentos de Péptidos/inmunologíaRESUMEN
As part of the WHO Network for HIV Isolation and Characterization, we PCR amplified, cloned, and sequenced gp120 and gp160 genes from 12 HIV-1 isolates collected in four WHO-sponsored vaccine evaluation sites (Brazil, Rwanda, Thailand, Uganda). Envelope clones were derived from PBMC-grown isolates obtained from asymptomatic individuals within 2 years of seroconversion. Analysis of their deduced amino acid sequences identified all but one to contain an uninterrupted open reading frame. Transient expression and biological characterization of selected gp160 constructs identified six clones to encode full length and functional envelope glycoproteins. Phylogenetic analysis of their nucleotide sequences revealed that they represent HIV-1 subtypes A, B, C, and E. Since current knowledge of HIV-1 envelope immunobiology is almost exclusively derived from subtype B viruses, these reagents should facilitate future envelope structure, function and antigenicity studies on a broader spectrum of viruses. This should assist in the design and evaluation of effective vaccines against HIV-1.
Asunto(s)
Productos del Gen env/genética , Variación Genética , VIH-1/genética , Precursores de Proteínas/genética , Vacunas contra el SIDA/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Brasil/epidemiología , Clonación Molecular , Cartilla de ADN/genética , ADN Viral/genética , Proteína gp120 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/inmunología , Humanos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Rwanda/epidemiología , Homología de Secuencia de Aminoácido , Tailandia/epidemiología , Uganda/epidemiología , Organización Mundial de la SaludAsunto(s)
Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , Infecciones por VIH/prevención & control , VIH-1/inmunología , Ensayos Clínicos como Asunto , Ensayos Clínicos Fase III como Asunto , Vectores Genéticos , Infecciones por VIH/inmunología , VIH-1/genética , Humanos , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Vacunas de ADN/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, unlike most retroviruses, preassembles core structures in the cytoplasm of infected cells during morphogenesis. We have shown previously (J. Bradac and E. Hunter, 1984, Virology 138, 260-275) that M-PMV virions contain 5 gag-encoded polypeptides. In this report, the translational order of the proteins encoded on the gag and env genes of M-PMV has been determined by pactamycin mapping. The proposed order of the viral proteins on their respective precursors is env: NH2-gp85-gp20-COOH; gag: NH2-p10-pp24/pp16-p12-p27-p14-COOH. Myristic acid-labeled virions contained a single radioactive protein, p10, supporting the mapping of this molecule to the amino terminus. From these studies it is clear that M-PMV contains an unusual additional gag polypeptide, p12, for which no function has been assigned to date. A remarkable similarity exists in the size and organization of M-PMV and mouse mammary tumor virus (MMTV) gag polypeptides; suggesting a common ancestor to these two viruses.
Asunto(s)
Genes Virales , Biosíntesis de Péptidos , Biosíntesis de Proteínas , Precursores de Proteínas/genética , Retroviridae/genética , Proteínas Virales/biosíntesis , Animales , Macaca mulatta , Pactamicina/farmacología , Precursores de Proteínas/biosíntesis , Retroviridae/metabolismo , Proteínas Virales/genéticaRESUMEN
The effect of the monovalent carboxylic ionophore monensin on the biosynthesis, intracellular transport, and surface expression of the glycoproteins of Mason-Pfizer monkey virus was examined. Cells treated with monensin at concentrations of 10(-7) or 10(-6) M continued to synthesize virus particles, which from electron microscopic studies appeared to bud normally from the plasma membrane of the cells. However, the particles released had an altered buoyant density in sucrose gradients and were noninfectious. These noninfectious virions had a normal complement of non-glycosylated polypeptides but showed a significantly reduced amount of glycosylated proteins. The gp70 and gp20 polypeptides appeared to be completely absent, and a heterogeneous, higher-molecular-weight protein was observed on the virions instead. Studies on intracellular protein synthesis indicated that the precursor (Pr86env) to gp70 and gp20 is synthesized normally but is not cleaved to the mature proteins. Immunofluorescence studies showed, however, that the uncleaved molecule is expressed on the cell surface. In this system, therefore, Mason-Pfizer monkey virus glycoprotein migration appears to occur in the presence of monensin, whereas the cleavage and insertion of the glycoproteins into virions are inhibited.
Asunto(s)
Furanos/farmacología , Glicoproteínas/genética , Monensina/farmacología , Retroviridae/genética , Proteínas Virales/genética , Animales , Línea Celular , Embrión de Mamíferos , Femenino , Humanos , Riñón , Macaca mulatta , Neoplasias Mamarias Experimentales/microbiología , Neoplasias Mamarias Experimentales/ultraestructura , Microscopía Electrónica , Embarazo , Retroviridae/efectos de los fármacos , Retroviridae/ultraestructura , Virión/aislamiento & purificaciónRESUMEN
The molecular characterization of the proviral DNA genome of Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, is described. An analysis of unintegrated viral DNAs present in acutely infected cells revealed open and closed circular molecules and linear species. The size of the M-PMV linear proviral DNA is determined to be 8.1 kbp in length. A preliminary screening of restriction enzymes indicated that many of those commonly used for cloning (EcoRI, SalI, ClaI, XhoI) did not cut the provirus. Digestion of a mixture of linear and circular forms of unintegrated DNA with HindIII produced a set of restriction fragments 2.3-3 kbp in length. These subgenomic fragments where cloned into the bacterial plasmid pAT153, and two classes of M-PMV subgenomic clones isolated. The first of these contained fragments that spanned the ends of the linear genome and presumably were derived from circular proviruses. Six of the seven clones in this class contained a single long terminal repeat (LTR), represented by pMP6, while the seventh, pMP9, contains two LTRs. Digestion of the latter clone with an enzyme that cleaves once within the LTR allowed the length of the M-PMV LTR to be determined as 350 bp. Both the LTR containing clones and the second class of subgenomic clones have been used in developing a detailed restriction map of the M-PMV proviral DNA and in orienting it with regard to transcription of viral RNA. Thus, pMP6/pMP9 contain sequences from the LTR-gag region of the genome and the second class of subclones (represented by pMP1) span the env-coding region. No clones containing the pol-coding region have been isolated. In order to determine the nature of M-PMV-related endogenous sequences in the chromosomal DNA of Old World primates, EcoRI-digested primate DNA was hybridized at low stringency to the subgenomic clones and then washed under conditions of low, moderate, and high stringencies. Multiple sequences closely related to the LTR-gag region of the M-PMV genome, were detected. Sequences more distantly related to the env region were also found in Old World monkeys. Ape and human DNAs were shown to contain sequences related to the LTR-gag region of the M-PMV genome, but were only weakly detectable at low stringency.
Asunto(s)
Clonación Molecular , Genes Virales , Retroviridae/genética , Animales , Línea Celular , ADN/análisis , Enzimas de Restricción del ADN , Escherichia coli/genética , Humanos , Riñón/embriología , Macaca mulatta , Neoplasias Mamarias Experimentales , Plásmidos , ARN Viral/genética , ARN Viral/aislamiento & purificaciónRESUMEN
A collaborative study was organized to identify monoclonal antibodies (MAbs) that may be broadly and potently neutralizing for a panel of human immunodeficiency virus type 1 (HIV-1) low-passaged adult and pediatric primary isolates in peripheral blood mononuclear cells. Five laboratories evaluated a coded panel of seven human MAbs to HIV-1 subtype B envelope V3, CD4 binding region, gp41, and other conformationally sensitive determinants. Each laboratory measured neutralizing activity of the MAbs against the laboratory isolate HIV(MN) and a panel of 9 subtype B primary isolates. Antibodies were classified as suitable candidates for future clinical studies if they could neutralize at least half of the 9 primary isolates at a concentration of < or = 25 microg/mL for 90% viral inhibition. The study identified three MAbs that met stated performance criteria: IgG1b12, 2G12, and 2F5. These results may provide a rationale for examining the clinical efficacy, either singly or in combination, of the three MAbs.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Anti-VIH , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Seropositividad para VIH/inmunología , VIH-1/clasificación , VIH-1/aislamiento & purificación , Pruebas de Neutralización/normas , Síndrome de Inmunodeficiencia Adquirida/sangre , Síndrome de Inmunodeficiencia Adquirida/inmunología , Adulto , Secuencia de Aminoácidos , Anticuerpos Monoclonales/sangre , Especificidad de Anticuerpos , Antígenos CD/fisiología , Antígenos CD4/fisiología , Niño , Ensayos Clínicos como Asunto/métodos , Epítopos/análisis , Anticuerpos Anti-VIH/sangre , Proteínas gp160 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/química , Seropositividad para VIH/sangre , VIH-1/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Datos de Secuencia Molecular , Sensibilidad y EspecificidadRESUMEN
We prospectively evaluated serum aminoglycoside (AMG) concentrations in 120 patients who received gentamicin or tobramycin for serious gram-negative bacillary disease. AMG serum concentrations were assayed by microbiological and radioimmunoassay techniques. Correlation between the two assay methods was good. When AMG doses were based on total body weight, there was no significant correlation between AMG dosage administered and serum concentrations in patients with either normal or abnormal renal function. The use of ideal body weight for calculation of AMG dosage improved this correlation significantly except in hemodialysis patients. AMG-induced nephrotoxicity occurred in 13 patients. No significant association was noted between the occurrence of toxicity and the specific AMG given or with other commonly recognized risk factors. Among study groups, peak AMG serum concentrations failed to exceed the minimal inhibitory concentration of the infecting organism in 17 to 33% of the cases. Serum inhibitory levels of greater than or equal to 1:8 were not associated with improved survival. There was no significant difference in mortality between the gentamicin- and tobramycin-treated groups. We advise base-line serum AMG levels in seriously ill patients with gram-negative bacillary disease and additional bacteriological studies in selected situations.
Asunto(s)
Antibacterianos/sangre , Infecciones Bacterianas/tratamiento farmacológico , Aminoglicósidos/administración & dosificación , Aminoglicósidos/sangre , Aminoglicósidos/uso terapéutico , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/mortalidad , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Strain improvement of agriculturally important animals will require efficient techniques for gene delivery, the ability to regulate the expression of the newly introduced genes and, most important, the identification of genes whose appropriate expression could cause improvement of the animal. We have developed a series of avian retroviral vectors that can be used to introduce new genetic information into the germ line of chickens, for which transgenics cannot be created by direct microinjection of DNA into fertilized eggs. We have identified a 220-bp segment of the chicken skeletal muscle alpha-actin gene that can cause other genes to be expressed specifically in striated muscle. This chicken promoter shows correct tissue specificity in transgenic mice and presumably could be used in other mammalian species. The skeletal muscle alpha-actin promoter has been inserted into the avian retroviral vectors and the promoter is functional in cultured cells infected by these retroviral vectors. The tissue specificity of the expression of the skeletal muscle alpha-actin promoter carried by the retroviral vectors will soon be tested in vivo. We are studying two types of genes that might be useful in strain improvement; genes that could produce dominant resistance to infection by pathogenic viruses, and genes that could play critical roles in muscle development. Expression of the envelope glycoprotein of retroviruses can specifically block the cellular receptor that viruses use to infect a susceptible cell. Expression of the avian leukosis virus subgroup A envelope in transgenic chickens prevents infection by pathogenic viruses of the same subgroup. We are attempting to block reticuloendotheliosis virus infection by expressing the reticuloendotheliosis envelope glycoprotein. We have shown that we can block infection in cultured cells, and we are now creating retroviral vectors for experiments in vivo. We have also begun to study the cellular homologue of the ski oncogene, which has been shown to stimulate the differentiation of quail myoblasts in vitro. Biologically active cDNAs have been isolated; we have now begun to analyse the effects of expressing the c-ski proteins in the whole animal.