RESUMO
Vacuolar ATPases (V-ATPases) are multisubunit enzymes that couple the hydrolysis of ATP to the transport of H+ across membranes, and thus acidify several intracellular compartments and some extracellular spaces. Despite the high degree of genetic and pharmacological homogeneity of V-ATPases, cells differentially modulate the lumenal pH of organelles and, in some cells, V-ATPases are selectively targetted to the plasma membrane. Although the mechanisms underlying such differences are not known, the subunit isoform composition of V-ATPases could contribute to altered assembly, targeting or activity. We previously identified an alternatively spliced variant of the chicken A subunit in which a 30 amino acid cassette (A1) containing the Walker consensus sequence for ATP binding is replaced by a 24 amino acid cassette (A2) that lacks this feature. We have examined the ability of chimeric yeast/chicken A subunits containing either the A1 or the A2 cassette to restore the V-ATPase activity of yeast that lack the A subunit. The A1-containing chimeric subunit, but not the chimera that contains the A2 cassette, partially restores the ability of the mutated yeast to grow at neutral pH. Both chimeric proteins are expressed, although at lower levels than the similarly transfected yeast A subunit. The A2-containing subunit fails to associate with the vacuolar membrane or support the assembly of V-ATPase complexes. Thus, the substitution of the A1 sequence by A2 not only removes the Walker nucleotide binding sequence but also compromises the ability of the A subunit to assemble with other V-ATPase subunits.
Assuntos
ATPases Translocadoras de Prótons/química , Splicing de RNA , ATPases Vacuolares Próton-Translocadoras , Trifosfato de Adenosina/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Galinhas , Sequência Consenso , Proteínas Fúngicas/química , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Alinhamento de SequênciaRESUMO
Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the Vo sector, attached to a complex of peripheral membrane proteins, the V1 sector. We have examined the early steps in biosynthesis of the yeast vacuolar ATPase by biosynthetically labeling wild-type and mutant cells for varied pulse and chase times and immunoprecipitating fully and partially assembled complexes under nondenaturing conditions. In wild-type cells, several V1 subunits and the 100-kDa Vo subunit associate within 3-5 min, followed by addition of other Vo subunits with time. Deletion mutants lacking single subunits of the enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells and independent V1 and Vo sectors that form without any apparent V1Vo subunit interaction. Two yeast sec mutants that show a temperature-conditional block in export from the endoplasmic reticulum accumulate a complex containing several V1 subunits and the 100-kDa Vo subunit during incubation at elevated temperature. This complex can assemble with the 17-kDa Vo subunit when the temperature block is reversed. We propose that assembly of the yeast V-ATPase can occur by two different pathways: a concerted assembly pathway involving early interactions between V1 and Vo subunits and an independent assembly pathway requiring full assembly of V1 and Vo sectors before combination of the two sectors. The data suggest that in wild-type cells, assembly occurs predominantly by the concerted assembly pathway, and V-ATPase complexes acquire the full complement of Vo subunits during or after exit from the endoplasmic reticulum.
Assuntos
Bombas de Próton/biossíntese , ATPases Translocadoras de Prótons/biossíntese , ATPases Vacuolares Próton-Translocadoras , Vacúolos/enzimologia , Transporte Biológico , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/biossíntese , Modelos Biológicos , Mutação , Ligação Proteica , Bombas de Próton/genética , ATPases Translocadoras de Prótons/genética , Leveduras/genética , Leveduras/metabolismoRESUMO
PURPOSE: To evaluate the cytotoxicity of immunotoxin 4197X-ricin A (4197X-RA) and its ability to inhibit protein synthesis and human lens epithelial cell (LEC) proliferation on the inner surface of the lens capsule. SETTING: Houston Biotechnology, Inc., The Woodlands, Texas. METHODS: A cell culture system was established using human LECs as a model for the proliferation of remnant LECs that occurs during posterior capsule opacification (PCO) after extracapsular cataract extraction. The LEC culture system was also used in vitro for testing compounds that might inhibit this process in vivo. Human LECs were cultured on the surface of the original lens capsule fixed to collagen. Variability was reduced by dissecting each lens capsule into equivalent halves and exposing the segments to immunotoxin 4197X-RA. RESULTS: Protein synthesis and LEC proliferation were almost completely inhibited at relatively low 4197X-RA concentrations after short exposure. The inhibitory effects persisted up to 3 weeks after withdrawal of the immunotoxin and after several media exchanges. CONCLUSION: Immunotoxin 4197X-RA may help prevent PCO after primary cataract surgery.
Assuntos
Imunotoxinas/farmacologia , Cápsula do Cristalino/citologia , Ricina , Anticorpos Monoclonais , Técnicas de Cultura de Células , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalinas/antagonistas & inibidores , Cristalinas/biossíntese , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Seguimentos , Humanos , Cápsula do Cristalino/efeitos dos fármacos , Cápsula do Cristalino/metabolismoRESUMO
Neonatal pigs were inoculated with porcine enterotoxigenic Escherichia coli 431, which carries genes for K99 fimbriae and STaP enterotoxin. Colonies of strain 431 were recovered from feces of pigs for up to 17 days after inoculation and tested for hybridization with gene probes for K99 and STaP. Variants of strain 431 that did not hybridize with the probes were considered to have lost the genes. Variants were recovered from 10 of 13 suckling pigs that survived the infection. Only 0.4% of the isolates recovered during the first 2 days after inoculation were variants. Of the isolates recovered 3 to 5 days after inoculation, 20 to 36% were variants. Variant colonies were detected more frequently among pigs in some litters than in others. The litter with the highest number of variant-shedding pigs had the dam with the highest titer of K99 antibody in her colostrum. Variants also occurred in colostrum-deprived, artificially reared pigs. However, the number of variants detected was lower and they occurred later in the course of the infection in colostrum-deprived pigs than in suckling pigs. More variants were detected and they were detected earlier in colostrum-deprived pigs fed anti-K99 monoclonal antibody than in controls fed anti-K88 monoclonal antibody. Loss of STaP appeared to be secondary to loss of K99 in that some variants lacked only K99 (K99- STaP+) and some lacked both genes (K99- STaP-), but none was of the K99+ STaP- type. Our results confirmed reports of gene loss from enterotoxigenic E. coli during infection. They are consistent with the hypothesis that variants emerge under in vivo selection pressure of K99 antibody and with the speculation that gene loss may be an important component of protection in vaccinated populations. However, the emergence of variants did not appear to play a major role in the recovery of individual pigs from clinical disease.
Assuntos
Toxinas Bacterianas/fisiologia , Enterotoxinas/fisiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/patogenicidade , Fímbrias Bacterianas/fisiologia , Animais , Animais Lactentes/microbiologia , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/análise , Antígenos de Bactérias/imunologia , Colostro/imunologia , Escherichia coli/genética , Proteínas de Escherichia coli , Fezes/microbiologia , Imunização , Imunização Passiva , Mutação , SuínosRESUMO
The guinea-pig model of cytomegalovirus (CMV) infection was used in continuing studies of experimental CMV vaccines for prevention of congenital CMV infection. Low-passage guinea-pig cytomegalovirus (GPCMV) vaccine, which has been shown to be effective in preventing transplacental CMV transmission, was tested for reactivation during pregnancy. In Hartley strain guinea pigs intraperitoneally given 10(5.5) 50% tissue culture infective doses (TCID50) of low-passage vaccine, 27 (41%) of 66 throat swabs obtained during subsequent pregnancies showed CMV, in contrast to 28 (24%) of 115 throat swabs obtained from nonpregnant control animals. High-passage GPCMV vaccine (approximately 5 X 10(3) TCID50) did not cause acute viremia, detectable generalized infection, or GPCMV reactivation during subsequent pregnancies. Immune pregnant animals and their fetuses were protected against generalized CMV infection when challenged with virulent GPCMV. In contrast, nonimmune pregnant controls developed generalized maternal, placental, and fetal infection. Experimental low-dose, high-passage GPCMV vaccine can protect against transplacental CMV transmission without apparent vaccine reactivation during pregnancy.
Assuntos
Infecções por Citomegalovirus/transmissão , Citomegalovirus/imunologia , Doenças Fetais/prevenção & controle , Placenta/microbiologia , Complicações Infecciosas na Gravidez/prevenção & controle , Vacinas Virais , Animais , Anticorpos Antivirais/biossíntese , Citomegalovirus/crescimento & desenvolvimento , Infecções por Citomegalovirus/prevenção & controle , Feminino , Cobaias , Hipersensibilidade Tardia , Troca Materno-Fetal , Gravidez , Vacinação , Viremia , Ativação ViralRESUMO
The pathogenesis and prevention of generalized infection and interstitial pneumonia due to cytomegalovirus (CMV) in inbred strain 2 guinea pigs were investigated. Eighteen animals inoculated with approximately 10(7) 50% tissue culture infective doses of virulent salivary gland-passaged guinea pig CMV (GPCMV-SG) developed generalized, viremic CMV infections, and 44% of the animals died. Most (68%) cocultivated tissues showed CMV infection, including lungs, which demonstrated severe bilateral interstitial pneumonia. Guinea pigs inoculated with live tissue culture-passaged GPCMV (GPCMV-TC) vaccine developed mild, nonlethal, generalized CMV infections and minimal histopathologic changes in infected tissues, including lungs. After challenge of 19 GPCMV-TC-vaccinated animals with GPCMV-SG, viremia was detectable in two (11%); CMV was detected in 15% of cocultivated tissues and rarely in lungs. Although vaccinated animals showed histologic evidence of interstitial pneumonia after challenge, none died. Strain 2 guinea pigs are useful for studies of pathogenesis and prevention of generalized CMV infection, particularly CMV-associated interstitial pneumonia.
Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Fibrose Pulmonar/imunologia , Vacinação , Vacinas Virais , Animais , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/microbiologia , Feminino , Cobaias , Imunidade , Pulmão/microbiologia , Masculino , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/microbiologiaRESUMO
Live guinea pig cytomegalovirus (CMV) vaccine was prepared after 11 serial passages in tissue culture; noninfectious envelope antigen vaccine was prepared by n-octyl glucoside treatment of CMV-derived dense bodies and virions. Hartley strain guinea pigs immunized with either vaccine were compared with guinea pigs inoculated with virulent, salivary gland-passaged CMV (approximating natural infection), with passively immunized animals, and with nonimmune controls. All vaccinated animals had neutralizing antibodies to CMV. After challenge with virulent CMV, animals previously inoculated with either tissue culture-passaged or virulent CMV were protected against acute viremia and death; pregnant animals previously inoculated with live CMV vaccine had lower incidences of viremia and generalized maternal and fetal infection. Envelope antigen-vaccinated and passively immunized pregnant animals showed acute viremia after similar challenge with virulent virus; however, infection was less generalized than that in control animals, and CMV was not isolated from the fetuses of these vaccinated mothers.