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1.
Vaccine ; 18(15): 1522-30, 2000 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-10618550

RESUMEN

Plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) was complexed with asialoorosomucoid conjugated to poly-L-lysine. Following its intravenous injection into BALB/c mice, this complex was targeted to the liver. Liver cells expressing gD-1 were detected immunohistochemically through day 6 post-immunization, while gD-1 DNA was detectable through 14 days post-immunization. Decline of gD-1 expression and detectable gD-1 DNA in the liver correlated with influx of T cells, predominantly CD4(+). The ASOR-poly-L-lysine DNA carrier system promotes hepatic expression of gD-1 and may be useful in vaccination against herpes simplex virus type-1.


Asunto(s)
Herpesvirus Humano 1/inmunología , Hígado/metabolismo , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/genética , Vacunas Virales/inmunología , Animales , Asialoglicoproteínas/administración & dosificación , Femenino , Inmunización , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Orosomucoide/administración & dosificación , Orosomucoide/análogos & derivados , Polilisina/administración & dosificación , Proteínas del Envoltorio Viral/análisis , Proteínas del Envoltorio Viral/inmunología
2.
Vaccine ; 17(9-10): 1091-9, 1999 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-10195619

RESUMEN

DNA molecules complexed with an asialoglycoprotein-polycation conjugate, consisting of asialoorosomucoid (ASOR) coupled to poly-L-lysine, can enter hepatocytes which bear receptors for ASOR. We used this receptor-mediated DNA delivery system to deliver plasmid DNA encoding glycoprotein D (gD) of herpes simplex virus type 1 to ASOR-positive cells. Maximum expression of gD protein was seen at 3 days after injection of this preparation in approximately 13% of cells from BALB/c mice [hepatocytes from mice injected intravenously (i.v.) or peritoneal exudate cells from mice injected intraperitoneally (i.p.)]. In comparison with mice injected with either the plasmid vector alone or the gD-containing plasmid uncomplexed to ASOR, mice immunized with gD-containing plasmid complexed with ASOR-poly-L-lysine induced marked antigen-specific CTL responses. BALB/c mice immunized with gD-DNA developed a T-cell-mediated CTL response against target cells expressing gD and MHC class II glycoproteins, but not against cells expressing only gD and MHC class I molecules. In C3H mice, gD-DNA induced a T-cell-mediated CTL response against target cells expressing gD and class I MHC molecules. Serum anti-gD antibody in low titers were produced in both strains of mice. DNA complexed with ASOR-poly-L-lysine induced CTL responses in mice.


Asunto(s)
Anticuerpos Antivirales/biosíntesis , Asialoglicoproteínas/inmunología , Hemaglutininas Virales/inmunología , Orosomucoide/análogos & derivados , Polilisina/inmunología , Simplexvirus/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunación/métodos , Proteínas del Envoltorio Viral/inmunología , Animales , Receptor de Asialoglicoproteína , Asialoglicoproteínas/metabolismo , Antígenos CD4/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Hígado/citología , Hígado/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Orosomucoide/inmunología , Orosomucoide/metabolismo , Plásmidos , Polilisina/metabolismo , Receptores de Superficie Celular/metabolismo , Transfección , Proteínas del Envoltorio Viral/biosíntesis
3.
Infect Immun ; 66(11): 5082-8, 1998 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9784507

RESUMEN

Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-gamma) and interleukin-12 than did sham-injected controls. The majority of IFN-gamma production appeared to be CD8(+) T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-gamma. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to "bystander" fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-gamma decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo.


Asunto(s)
Antígenos Bacterianos/farmacología , Bacteriólisis/inmunología , Citocinas/metabolismo , Enterotoxinas/inmunología , Enterotoxinas/farmacología , Animales , Linfocitos T CD8-positivos/inmunología , Comunicación Celular/inmunología , Línea Celular , Técnicas de Cocultivo , Pruebas Inmunológicas de Citotoxicidad , Femenino , Fibroblastos , Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/inmunología , Staphylococcus aureus/inmunología , Streptococcus mutans/inmunología , Superantígenos/inmunología
4.
J Immunol Methods ; 211(1-2): 147-58, 1998 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-9617839

RESUMEN

We have developed a novel co-culture system in which murine splenocytes are cultured with live bacteria in the presence of a bacteriostatic antibiotic. Superantigens, like staphylococcal enterotoxin B (SEB) are important factors in bacterial pathogenicity. Research has shown that superantigens affect numerous immune cell types, either directly or indirectly, yet their involvement in pathogenic mechanisms remains poorly defined. In these studies, we utilize the co-culture system to study how superantigen pretreatment affects interferon-gamma (IFN-gamma) production by splenocytes co-cultured with gram-positive bacteria. Streptococcus mutans, S. sanguis and Bacillus subtilis were tested for susceptibility to a panel of antibiotics. Spectinomycin was found to maintain a bacteriostatic state of approximately 10(5) bacteria ml(-1) at optimal concentrations for each bacterial strain. Co-culturing splenocytes with bacteria did not affect splenocyte viability and cultured splenocytes responded to mitogenic stimulation as expected. Two days after SEB pretreatment, isolated splenocytes cultured with either Streptococcus species produced 10-15 times more IFN-gamma than splenocytes from sham-injected controls; however, no differences in CD4+ or CD8+ T cell populations appeared in cultures with or without bacteria. Splenocytes isolated four days after SEB treatment did not produce significant amounts of IFN-gamma in co-culture. Co-cultures containing live bacteria produced four times more IFN-gamma than cultures containing heat-killed bacteria. Splenocytes depleted of natural killer (NK) cells prior to SEB treatment produced 25% less IFN-gamma after 20 h co-culturing with S. mutans. T lymphocytes were identified to be the major producer of IFN-gamma at this time point by intracellular cytokine staining. Apparently SEB exposure primes a response to live bacteria and the response is evident two days after initial exposure. The in vitro co-culture system allows us to observe host responses to bacteria in the context of the multicellular interdependent immune response. With this assay we can more closely 'mimic' in vivo events, particularly immune cell interactions in microfloral environments, to study how the pathogenic effects of superantigens alter this response.


Asunto(s)
Bacillus subtilis/inmunología , Técnicas de Cultivo de Célula/métodos , Enterotoxinas/inmunología , Interferón gamma/biosíntesis , Bazo/inmunología , Streptococcus mutans/inmunología , Streptococcus sanguis/inmunología , Superantígenos/inmunología , Animales , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/crecimiento & desarrollo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Fraccionamiento Celular , Supervivencia Celular , Células Cultivadas , Enterotoxinas/farmacología , Femenino , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Coloración y Etiquetado , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/crecimiento & desarrollo , Streptococcus sanguis/efectos de los fármacos , Streptococcus sanguis/crecimiento & desarrollo , Superantígenos/farmacología , Factores de Tiempo
5.
Can J Microbiol ; 44(1): 91-4, 1998 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9522453

RESUMEN

The putative 4.5S RNA of Haemophilus influenzae was identified in the genome by computer analysis, amplified by the polymerase chain reaction, and cloned. We have determined that this putative 4.5S RNA will complement an Escherichia coli strain conditionally defective in 4.5S RNA production. The predicted secondary structures of the molecules were quite similar, but Northern analysis showed that the H. influenzae RNA was slightly larger than the E. coli RNA. The H. influenzae gene encoding this RNA is the functional homolog of the ffs gene in E. coli.


Asunto(s)
Haemophilus influenzae/genética , ARN Bacteriano/genética , Secuencia de Aminoácidos , Escherichia coli/genética , Prueba de Complementación Genética , Genoma Bacteriano , Datos de Secuencia Molecular , Conformación de Ácido Nucleico
6.
FEMS Microbiol Lett ; 153(2): 387-92, 1997 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-9271867

RESUMEN

A small RNA sequence identified in an rRNA-tRNA cluster from the thermophilic Bacillus sp. strain PS3 was examined. An oligonucleotide probe specific for the RNA bound to multiple restriction fragments in Bacillus sp. strain PS3 DNA, thus several copies of this sequence occur in its genome. Similar findings were observed using DNA from B. subtilis, B. stearothermophilus, Escherichia coli, Staphylococcus aureus, Haemophilus influenzae and Thermus thermophilus. This sequence apparently is widespread in the eubacteria. Northern analysis of RNA from sporulating Bacillus sp. strain PS3 and B. subtilis cells revealed RNA species homologous to the probe in both bacteria. Expression of the small RNA in B. subtilis depended on sigma H.


Asunto(s)
Bacillus subtilis/genética , Bacillus/genética , Regulación Bacteriana de la Expresión Génica/fisiología , ARN Bacteriano/genética , Esporas Bacterianas/genética , Bacillus/fisiología , Bacillus subtilis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , ADN Bacteriano/análisis , Datos de Secuencia Molecular , Mutación , ARN Ribosómico 23S/genética , ARN de Transferencia de Asparagina/genética , Factor sigma/genética , Factor sigma/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología
7.
Protein Expr Purif ; 6(5): 707-15, 1995 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-8535166

RESUMEN

The thermophilic bacterium, Bacillus PS3, was grown in a vigorously aerated nutrient broth at 65 degrees C with 100 mM glutamic acid serving as a supplemental carbon and nitrogen source. These growth conditions resulted in membranes highly enriched in cytochrome c oxidase (COX) [23.32 +/- 4.32 nmol heme a/g of cells (n = 5)], which is nearly a threefold higher concentration of COX (heme caa3-type) than previously reported for this organism. A new high-yield purification of COX was performed by extracting the bacterial membranes with Triton X-100 (7 mg/mg protein), followed by ion-exchange fast liquid protein chromatography using a QAE (trimethyl ammonium) resin with subsequent hydroxyapatite chromatography and ammonium sulfate fractionation. This purification regime resulted in a 16% yield of cytochrome c oxidase with 20 mg of pure caa3-type COX (13 nmol heme a/mg protein) isolated from 100 g of cells. SDS-PAGE showed that the isolated enzyme had four subunits with apparent Mr of 68, 38, 23, and 13 kDa. In addition, a new 34-kDa peptide was also detected in this preparation, which may represent the ORF1 gene product for this organism. Subunit II (Mr = 38 kDa) of the isolated enzyme was shown to contain covalently bound heme c by using both heme-staining of SDS-PAGE and immunoreactivity with an anti-cytochrome c antibody. The purified enzyme also exhibited high electron transfer activity (340s-1) when assayed at pH 6.5 in the presence of the nonionic detergent, beta-dodecyl maltoside.


Asunto(s)
Bacillus/enzimología , Cromatografía Liquida/métodos , Complejo IV de Transporte de Electrones/aislamiento & purificación , Bacillus/citología , Bacillus/crecimiento & desarrollo , División Celular , Membrana Celular/química , Grupo Citocromo c/metabolismo , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Hemo/química , Microbiología Industrial/métodos , Oxidación-Reducción , Espectrofotometría Atómica
10.
Microb Pathog ; 10(6): 493-9, 1991 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-1665537

RESUMEN

In this study, the 100 kb plasmid of Salmonella typhimurium, which is known to contribute to the pathogenicity of the organism, was tagged with the transposon Tn5 to define regions of the plasmid contributing to overall virulence. Eleven randomly selected vir::Tn5 plasmids carried by the plasmid-free S. typhimurium strain WS1321 were physically mapped and then examined in mice for subcutaneous LD50 value, ability to induce splenomegaly, and ability to grow to high numbers in the spleens of infected mice. Nine strains were found to be virulence-attenuated and showed varied levels of growth in the spleens of subcutaneously infected BALB/c mice. Eight of these nine strains carried Tn5 insertions which lie outside the previously defined virulence region. These studies corroborate the findings of other investigators as well as defining novel regions of the 100 kb virulence plasmid involved in the pathogenicity of this organism.


Asunto(s)
Mutagénesis , Plásmidos/genética , Salmonella typhimurium/genética , Esplenomegalia/microbiología , Virulencia/genética , Animales , Clonación Molecular , Elementos Transponibles de ADN/genética , Femenino , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos BALB C/microbiología , Mapeo Restrictivo , Salmonella typhimurium/crecimiento & desarrollo , Transformación Genética
11.
Biotechniques ; 10(4): 446, 448, 450, 1991 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-1907833

RESUMEN

A method is described for the separation of DNA fragments by ultracentrifugation through a sodium chloride step gradient. The gradients are quickly and easily prepared and require a five- to six-hour centrifugation. Fractionated samples of DNA may be directly examined by agarose gel electrophoresis, then further analyzed by Southern transfer and hybridization. A simple ethanol precipitation followed by several ethanol washes yields fragments that ligate efficiently to vector DNA. The method has been applied to separate chromosomal DNA restriction fragments as well as bacteriophage lambda arms from insert DNA.


Asunto(s)
Centrifugación por Gradiente de Densidad/métodos , ADN Bacteriano/aislamiento & purificación , Cloruro de Sodio , Bacillus subtilis/genética , Southern Blotting
12.
J Gen Microbiol ; 133(10): 2937-44, 1987 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-3449602

RESUMEN

Bacillus amyloliquefaciens strain H is lysogenic for a large temperate phage we call H2. H2 has a polyhedral head 85 nm in diameter and a tail of about 17 x 434 nm. H2 lysogenizes Bacillus subtilis between the tyrA and metB genes, and gives specialized transduction of metB and, at lower frequencies, of ilvD and ilvA. The phage carries a thymidylate synthase gene and converts thymine auxotrophs of B. subtilis to prototrophy. The H2 genome is a linear DNA molecule about 129 kb in length. DNA extracted from phage particles grown in B. subtilis is not cut by the restriction endonucleases HaeIII, Fnu4HI, Bsp1286I, and BamHI; the latter enzyme is produced by B. amyloliquefaciens strain H. The prophage in lysogenic B. subtilis cells can be cut by these enzymes. We have isolated H2 mutants that carry the transposon Tn917, or a mutation resulting in clear-plaque morphology, or both.


Asunto(s)
Bacillus/metabolismo , Bacteriófagos/aislamiento & purificación , Bacteriófagos/genética , ADN Viral , Lisogenia , Transducción Genética
13.
J Bioenerg Biomembr ; 19(2): 143-66, 1987 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-2884216

RESUMEN

Mammalian mitochondrial cytochrome c oxidase catalyzes the transfer of electrons from ferrocytochrome c to molecular oxygen in the respiratory chain, while conserving the energy released during its electron transfer reactions by the vectorial movement of protons across the inner membrane of the mitochondrion. The protein domain that translocates the protons across the membrane is currently unknown. Recent research efforts have investigated the role of one of the transmembrane subunits of the enzyme (III, Mr 29,884) in the vectorial proton translocation reaction. The data that favor subunit III as integral in vectorial proton translocation as well as the data that support a more peripheral role for subunit III in proton translocation are reviewed. Possible experimental approaches to clarify this issue are presented and a general model discussed.


Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/enzimología , ATPasas de Translocación de Protón/metabolismo , Animales , Transporte Biológico Activo , Bovinos , Sistema Libre de Células , Quimotripsina/metabolismo , Complejo IV de Transporte de Electrones/aislamiento & purificación , Concentración de Iones de Hidrógeno , Membranas Intracelulares/fisiología , Sustancias Macromoleculares , Potenciales de la Membrana , Proteínas de la Membrana/metabolismo , Proteolípidos/metabolismo , Ratas
14.
FEBS Lett ; 214(1): 75-80, 1987 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-3032681

RESUMEN

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.


Asunto(s)
ADN Bacteriano/genética , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Animales , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Secuencia de Bases , Bovinos , Especificidad de la Especie , Thermus/genética
15.
J Bacteriol ; 157(2): 428-34, 1984 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-6319359

RESUMEN

Thymine auxotrophs of Bacillus subtilis strains lysogenic for temperate bacteriophage SP beta c2 were transformed to prototrophy by DNA from related phage phi 3T. During transformation, the phi 3T-encoded thymidylate synthetase gene, thyP3, became integrated into the extreme right end of the SP beta c2 prophage near the bacterial citK gene. Upon heat induction, the transformed B. subtilis cells released SP beta c2T phages that could lysogenize thymine auxotrophs and convert them to prototrophy. Comparison of restriction endonuclease fragments of DNAs from SP beta c2 and SP beta c2T phages revealed that the latter contained a large region of deletion and substitution near the center of the chromosome. This region included the phage attachment site on the SP beta c2 genome.


Asunto(s)
Bacillus subtilis/genética , Bacteriófagos/genética , Genes Virales , Genes , Metiltransferasas/genética , Timidilato Sintasa/genética , Secuencia de Bases , Enzimas de Restricción del ADN , Genotipo , Peso Molecular , Hibridación de Ácido Nucleico , Plásmidos , Especificidad de la Especie , Timina/metabolismo
16.
Gene ; 19(2): 235-8, 1982 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-6293933

RESUMEN

Six different restriction endonucleases were used to generate restriction fragment maps of the genome of the temperate Bacillus subtilis phage SP beta. AvaI and SalI each had six target sites in the phage DNA, AvaII had three, BamHI had seven, PstI had twenty, and SacI had sixteen. Restriction analysis and heteroduplex analysis were used to locate a 10-kb region of DNA that is deleted in the clear-plaque mutant, SP beta cl. The deletion lay approx. 50 kb from the left end of the 126-kb phage genome.


Asunto(s)
Bacillus subtilis/genética , Bacteriófagos/genética , Genes Virales , Secuencia de Bases , Enzimas de Restricción del ADN , Genes Bacterianos , Microscopía Electrónica , Peso Molecular
17.
J Bacteriol ; 150(3): 1274-9, 1982 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-6804441

RESUMEN

Specialized transducing SP beta particles were found that carried the Bacillus subtilis genes lying to the left of the prophage attachment site. Three classes of transducing particles were differentiated, depending upon whether they carried ilvA only, thyB and ilvA, or ilvD, thyB, and ilvA. Lysates prepared by the induction of strains that carried both a transducing phage and a plaque-forming phage contained the two particles in a ratio of about 1:3,000. When the transducing particles were used to transduce a phage-sensitive auxotrophic strain to prototrophy, some of the transductants carried only the transducing phage genomes which, by themselves, were defective. One putative nondefective transducing phage (for ilvA only) is also described. SP beta can mediate specialized transduction even in the absence of the major (recE) bacterial recombination system.


Asunto(s)
Bacillus subtilis/genética , Bacteriófagos/genética , Genes Bacterianos , Transducción Genética , Sitios de Ligazón Microbiológica , Isoleucina/genética , Leucina/genética , Lisogenia , Recombinación Genética , Nucleótidos de Timina/genética , Valina/genética
18.
Mol Gen Genet ; 182(3): 514-5, 1981.
Artículo en Inglés | MEDLINE | ID: mdl-6272067

RESUMEN

The restriction fragment patterns of two mutants forms of the temperate Bacillus subtilis bacteriophage SP beta have been examined. The DNA of a heat-inducible mutant, SP beta c2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SP beta c+ DNA. The DNA of a clear-plaque mutant, SP beta c1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA. Neither SP beta c1 nor SP beta c2 DNA is cleaved by the endonuclease HaeIII.


Asunto(s)
Bacillus subtilis/genética , Bacteriófagos/genética , Deleción Cromosómica , Enzimas de Restricción del ADN/metabolismo , ADN Viral/genética , Peso Molecular , Mutación , Transducción Genética
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