RESUMO
This study established a rat model of foreign body-associated urinary tract infection. A spiral polyethylene tube (PT) was placed transurethrally into the bladder without surgical manipulation, followed by transurethral inoculation with Pseudomonas aeruginosa. The persistence of P. aeruginosa in the kidneys and bladder was significantly enhanced by placement of the PT, whereas the bacteria were eliminated rapidly from the urinary tract in the animals without the PT. Scanning electron microscopy revealed a thick biofilm on the surface of the PT from the early stage of infection. Histopathologically, acute pyelonephritis was followed by chronic renal inflammation as well as continuous and sporadic polymorphonuclear leukocyte accumulation and hemorrhage in the pelvis and adjacent tissues, suggesting continuous ascending introduction of the bacteria from the biofilm adhering to the PT. We believe our model simulates the pathophysiology of foreign body-associated urinary tract infection characterized by biofilm formation on the surface of a foreign body.
Assuntos
Modelos Animais de Doenças , Corpos Estranhos , Infecções por Pseudomonas/etiologia , Infecções Urinárias/etiologia , Animais , Bacteriúria/etiologia , Feminino , Rim/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/isolamento & purificação , Ratos , Especificidade da Espécie , Bexiga Urinária/ultraestrutura , Infecções Urinárias/patologiaRESUMO
We examined whether strand identity with respect to DNA replication influences strand bias for 8-oxo-7,8-dihydroguanine (8-oxoG) mutagenesis. The specificity of 8-oxoG mutagenesis was determined in a mutM mutY or a mutT strain carrying the supF gene on one of two vectors that differed only in the orientation of supF with respect to a unique origin of replication. Most of the supF mutations in the mutM mutY strain were base substitutions (67%), predominantly G:C-->T:A transversions (> 64%), while the majority in the mutT strain were base substitutions (> 92%), predominantly A:T-->C:G transversions (> 91%). The distributions of frequently mutated sites of G:C-->T:A and A:T-->C:G transversions in the supF gene in the mutM mutY and mutT strains, respectively, did not differ markedly between the two vectors. These results suggest that gene orientation is not an important determinant of the strand bias of 8-oxoG mutagenesis.
Assuntos
DNA Glicosilases , Replicação do DNA , DNA Bacteriano/biossíntese , Proteínas de Escherichia coli , Escherichia coli/genética , Guanina/análogos & derivados , Guanina/metabolismo , Sequência de Bases , DNA Bacteriano/genética , DNA-Formamidopirimidina Glicosilase , Mutação da Fase de Leitura , Genes Supressores , Dados de Sequência Molecular , Mutagênese , Mutação , N-Glicosil Hidrolases/genética , RNA de Transferência/genética , Deleção de SequênciaRESUMO
Escherichia coli K-12 strain EY5, deficient in oxyR, was constructed to assess the role of oxyR and oxyR-regulated regulon in spontaneous mutagenesis. Mutagenesis was monitored by selecting two forward mutations of colicin B-sensitive to resistance and valine-sensitive to resistance, one base substitution mutation of rifampicin-sensitive to resistance and one reversion of argE3 his-4 to Arg(+) His(+). Deficiency of oxyR did not lead to the enhancement of spontaneous mutation frequencies of the four markers tested. By DNA sequence analysis, we determined 49 colicin B-resistant mutants derived from EY5 and found that 37% were base substitutions, 29% IS element insertions, 20% deletions, and 14% single base frameshifts. Among the base substitutions, G:C-->T:A transversions predominated followed by G:C-->A:T transitions and A:T-->T:A transversions. These spectra were essentially the same as those from oxyR(+) strains. The results indicate that oxyR and oxyR-regulated genes do not play a significant role in the defense against spontaneous mutagenesis.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Membrana/genética , Mutação , Regulon , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Bases , Elementos de DNA Transponíveis , Mutação da Fase de Leitura , Genótipo , Mutação de Sentido Incorreto , Deleção de SequênciaRESUMO
The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS-PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.
Assuntos
Acetiltransferases , Aciltransferases/isolamento & purificação , Escherichia coli/enzimologia , Acetilcoenzima A/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Cinética , Plasmídeos , ortoaminobenzoatos/metabolismoRESUMO
A strain of Streptococcus pneumoniae, when inoculated intranasally in 2 microl of suspension into BALB/c mice preinfected with influenza virus, colonized first in the nose, and several days thereafter also colonized significantly in the trachea and lungs with purulent inflammation. Pneumoccocal colonization was also observed in the noses of normal mice after the same bacterial inoculation, but not apparently in the lower respiratory tract. These results suggest that pneumococcal infection may develop from the upper to the lower respiratory tract as a possible sequence preferentially in influenza virus-infected subjects.
Assuntos
Vírus da Influenza A/fisiologia , Pulmão/microbiologia , Nariz/microbiologia , Infecções Pneumocócicas/patologia , Streptococcus pneumoniae/patogenicidade , Traqueia/microbiologia , Animais , Humanos , Influenza Humana/complicações , Camundongos , Camundongos Endogâmicos BALB C , Infecções Pneumocócicas/microbiologia , Superinfecção/microbiologiaRESUMO
A collaborative study was conducted to evaluate whether a replicative DNA synthesis (RDS) test using the rat liver can detect nongenotoxic (Ames-negative) hepatocarcinogens with three or seven daily administrations at dose-levels effective in long-term bioassays. The assay methods were well-validated by the 14 participants. Of six compounds tested, carbon tetrachloride (50 and 100 mg/kg), clofibrate (125 and 250 mg/kg), diethylstilbestrol (0.125 and 0.25 mg/kg) and urethane (100 mg/kg) gave positive results, methyl carbamate (200 and 400 mg/kg) exerted equivocal effects, and D,L-ethionine (125 mg/kg) failed to elevate RDS. These findings suggest that the RDS test can detect many nongenotoxic rat hepatocarcinogens with short-term administration at dose-levels used in long-term bioassays.
Assuntos
Testes de Carcinogenicidade/métodos , Replicação do DNA/efeitos dos fármacos , DNA/biossíntese , Fígado/metabolismo , Testes de Mutagenicidade/métodos , Animais , Antimetabólitos Antineoplásicos/toxicidade , Bromodesoxiuridina/toxicidade , Imuno-Histoquímica , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
The mutagenic and clastogenic effects of N-nitrosodi-n-propylamine (NDPA) in lacZ transgenic mice (MutaMouse) were investigated as a part of the second collaborative study of the transgenic mouse mutation assay by a subgroup of the Mammalian Mutagenesis Study Group, a suborganization of the Environmental Mutagen Society of Japan. Male MutaMouse mice were administered NDPA intraperitoneally at a dose of 250 mg/kg, which is half of the LD(50) of the compound. The clastogenicity of NDPA was examined by the peripheral blood micronucleus test just before and at 24, 48 and 72 h after the treatment. The mutant frequencies in the bone marrow, liver, lung, kidney and urinary bladder were examined by the positive selection method for lacZ kidney. These findings demonstrate that NDPA induces organ-specific mutagenesis with specific expression times, and that the mutagenicity of NDPA in lacZ transgenic mice is consistent with its carcinogenicity.
Assuntos
Carcinógenos/toxicidade , Óperon Lac , Mutagênicos/toxicidade , Nitrosaminas/toxicidade , Animais , Masculino , Camundongos , Camundongos Transgênicos , Testes para Micronúcleos , Mutagênese , Especificidade de ÓrgãosRESUMO
To clarify the process of post-translational modification of L-histidine decarboxylase (HDC), we investigated the conversion of the 74-kDa form of HDC into the 53-kDa form in specialized organella of a rat basophilic/mast cell line (RBL-2H3). With treatment of streptolysin-O, RBL-2H3 cells released approximately 40% of HDC activity accompanied by over 90% of lactate dehydrogenase activity. Only the 74-kDa form of HDC was detected in the leaked fraction by SDS-polyacrylamide gel electrophoresis. The 74-kDa form in the homogenate of pulse-labeled cells was recovered in both the supernatant and particulate fractions, while the 53-kDa form was detected only in the particulate fraction containing marker proteins of microsomes, Golgi, and lysosomal granules. Confocal microscopic observation using double staining immunofluorescence with anti-GST fusion HDC antiserum showed that most of the HDC coexists with protein-disulfide isomerase, a typical marker of the luminal space of the ER. With treatment of digitonin, RBL-2H3 cells released only 74-kDa HDC. Trypsin digestion of digitonin-permeabilized cells resulted in the disappearance of the 74-kDa form but not the 53-kDa form. From these results, it is assumed that the 74-kDa form of HDC, synthesized in the cytosol, is translocated into the lumen of the ER, where it is converted to the 53-kDa form.
Assuntos
Basófilos/enzimologia , Histidina Descarboxilase/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Microscopia Confocal , RatosRESUMO
L-Histidine decarboxylase (HDC) is a dimer consisting of two identical 53 kDa subunits. On the other hand, the size of HDC deduced from its cDNA sequence is around 74 kDa, indicating that the translated 74 kDa form of HDC is subjected to post-translational processing to generate the 53 kDa form. However, modification of the translated 74 kDa form of HDC in histamine-forming cells is unknown. Here we demonstrate that the 74 kDa form is translated in rat basophilic leukemia cells, followed by conversion to the 53 kDa form, and that the 74 kDa form is a short half-life protein because of the degradation mediated by the ubiquitin-proteasome pathway. Degradation of the 74 kDa form was stimulated in the presence of an ATP-generating system, accompanied by ubiquitination, and inhibited by specific proteasome inhibitors such as ZL3H and lactacystin. A significant amount of proteasome activity was detected in RBL-2H3 cells.
Assuntos
Cisteína Endopeptidases/metabolismo , Histidina Descarboxilase/metabolismo , Mastócitos/enzimologia , Complexos Multienzimáticos/metabolismo , Ubiquitinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Leupeptinas/farmacologia , Peso Molecular , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma , Processamento de Proteína Pós-Traducional , RatosRESUMO
Lactobacillus plantarum phage phi gle has two consecutive cell lysis genes hol-lys (Oki et al., 1996b). In the present study, functional and structural properties of the hol protein (Hol) were characterized in Escherichia coli. Electron microscopic examinations showed that hol under plac in E. coli XL1-Blue injured the inner membrane to yield empty ghost cells with the bulk of the cell wall undisturbed. Northern blot analysis indicated that hol-lys genes under plac were co-transcribed, although the amount of hol transcript was larger than that of lys, ceasing via an apparently rho-independent terminator just downstream of hol. However, deletion and/or fusion experiments suggested that: (1) the N-terminal half of phi gle Hol composed of three putative transmembrane domains may be responsible for interaction with membrane; (2) the N-terminal end (five amino acids) seems nonessential; and (3) the C-terminal half containing charged amino acids appears to be involved in proper hol function. These results suggest that phi gle Hol is a member of the lambdoid holin family, but divergent in several properties from lambda holin.
Assuntos
Bacteriófagos/genética , Escherichia coli/virologia , Lactobacillus/virologia , Proteínas de Membrana/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Bacteriólise , Sequência de Bases , Membrana Celular/ultraestrutura , DNA Viral/química , Genes Virais/genética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA Viral/análise , Proteínas Recombinantes de Fusão , Mapeamento por Restrição , Proteínas Virais/fisiologia , Proteínas Estruturais Virais/genéticaRESUMO
The response of megakaryocytes and cytokines to the administration of romurtide, a synthetic muramyl dipeptide derivative, was investigated in monkeys with myelosuppression by carboplatin-treatment. Romurtide increased the number of megakaryocytes and promoted the shift of megakaryocytes towards high ploidy class indicative of the promotion of the proliferation and maturation of megakaryocytes. The serum levels of interleukin-6, stem cell factor, and erythropoietin elevated significantly before the enhanced response of megakaryocytes induced by romurtide was observed. Romurtide also enhanced production of colony-stimulating factors (CSFs), such as granulocyte-CSF, macrophage-CSF, and granulocyte-macrophage CSF by monkey mononuclear cells. The stimulating effect of romurtide on the production of those cytokines and CSFs is likely to be responsible for the subsequent promotion of the proliferation and maturation of marrow megakaryocytes.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adjuvantes Imunológicos/uso terapêutico , Doenças da Medula Óssea/tratamento farmacológico , Medula Óssea/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Megacariócitos/efeitos dos fármacos , Acetilmuramil-Alanil-Isoglutamina/uso terapêutico , Animais , Contagem de Células Sanguíneas/efeitos dos fármacos , Doenças da Medula Óssea/induzido quimicamente , Carboplatina , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Fatores Estimuladores de Colônias/biossíntese , Citocinas/fisiologia , Eritropoetina/sangue , Feminino , Interleucina-6/sangue , Leucócitos Mononucleares/metabolismo , Macaca fascicularis , Megacariócitos/citologia , Ploidias , Fator de Células-Tronco/sangueRESUMO
The pathogenesis of infection in mice with herpes simplex virus type 1 (HSV-1) strain 7401H was studied. Mice immunosuppressed by intraperitoneal injection of cyclophosphamide were inoculated cutaneously into the flank with the virus and developed severe zosteriform skin lesions. All of them died within 2 weeks after the infection, while most of the normal mice survived the viral infection with healing of the lesions. In the gastrointestinal tract of the immunosuppressed mice, macroscopic abnormalities were frequently observed, and infectious viruses were detected on days 7 to 9. The viruses were also detectable in the dorsal root ganglia and the spinal cord of thoracolumbar area on days 5 to 7, and in the celiac plexus on day 7. However, no viruses were detected in the blood. Immunohistological examination of the gastrointestinal tract revealed that the viral antigens were localized in Auerbach's myenteric plexus. These results suggest that HSV-1 inoculated into the flank skin invaded the gastrointestinal tract via the nervous system, which gastrointestinal involvement might possibly have caused the death of the mice.
Assuntos
Gastroenteropatias/microbiologia , Herpes Simples/microbiologia , Hospedeiro Imunocomprometido , Dermatopatias/microbiologia , Animais , Antígenos Virais/análise , Ciclofosfamida , Feminino , Gastroenteropatias/imunologia , Herpes Simples/imunologia , Herpes Simples/mortalidade , Herpes Simples/patologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/isolamento & purificação , Camundongos , Sistema Nervoso/microbiologia , Pele/microbiologia , Dermatopatias/imunologia , Vísceras/patologiaRESUMO
A micronucleus assay using mouse peripheral blood and supravital staining with acridine orange (AO) was validated by two laboratories on triethylenemelamine-treated mice. Dose- and time-dependent increases in micronucleated peripheral reticulocytes were observed. This new method can be used as an alternative to the conventional bone marrow micronucleus assay.
Assuntos
Mutagênicos/toxicidade , Reticulócitos/efeitos dos fármacos , Trietilenomelamina/toxicidade , Laranja de Acridina , Animais , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos , Testes para Micronúcleos/métodos , Mitomicina/toxicidadeRESUMO
Eighty-four cases of inguinal hernioplasty by Bassini's modified technique were performed using 1% lidocaine in regional infiltration block. The report analyzes the use of local anesthesia in the treatment of inguinal hernias. All patients had small reducible and non-recurrent inguinal hernia. Twenty-six compensated high risk patients were operated in the hospital and dismissed in the second post-operative day, whereas the others were operated as outpatients and dismissed two hours after the surgical procedure. The rate of complication was low, but pain was a common complaint. In general, there was a good acceptance of this kind of anesthesia. The authors concluded that local anesthesia is a valuable method for the treatment of inguinal hernia.
Assuntos
Anestesia Local , Hérnia Inguinal/cirurgia , Bloqueio Nervoso , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Métodos , Pessoa de Meia-IdadeRESUMO
Six of seven children with fulminant hepatitis (FH) were treated with a combination of twice daily plasmapheresis and intensive conservative therapy including special amino acid solution, glucagon-insulin therapy, dexamethasone, and so on. The remaining child was treated with intensive conservative therapy only because his condition was not so severe, in spite of being diagnosed as having fulminant hepatitis. Although five patients with biopsy-documented bridging hepatic necrosis or confluent hepatic necrosis in the acute phase made recoveries, the remaining two with massive hepatic necrosis died (the overall survival rate was 71%). The prognostic factors were considered to be the degree and pattern of liver cell necrosis, the degree of coma, and the etiology. The combination of twice daily plasmapheresis and intensive conservative therapy was effective for these pediatric patients with fulminant hepatitis, except those with massive hepatic necrosis.
Assuntos
Encefalopatia Hepática/terapia , Hepatite/terapia , Plasmaferese/métodos , Doença Aguda , Criança , Pré-Escolar , Terapia Combinada , Estudos de Avaliação como Assunto , Feminino , Encefalopatia Hepática/mortalidade , Hepatite/mortalidade , Humanos , Lactente , Masculino , Nutrição Parenteral , Taxa de SobrevidaRESUMO
Retinoblastoma (Rb) occurs in hereditary, non-hereditary, and chromosomal deletion forms and the locus for the Rb gene (Rb-1) is closely linked to the locus for esterase D (ESD) assigned to the chromosome 13q14.11. We describe a patient who was predicted to have Rb from the genetic analysis of the chromosome and ESD phenotype. Furthermore, the gene for lymphocyte cytosol polypeptide with molecular weight of 64,000 (LCP1: McKusick catalogue No. 15343, 1983) was assigned to chromosome 13 by deletion mapping. A 3-month-old female had many characteristics of chromosome 13q-syndrome, including dolichocephaly, epicanthus, ptosis, depressed nasal bridge, micrognathia, short webbed neck, and short fifth fingers with clinodactyly and single crease. The karyotype of the patient was 46,XX,del(13) (q14.1-q32), though both the parents had normal karyotypes. As expected, the phenotype of ESD derived from one of the parents, the father in this case, was not detected in peripheral blood lymphocytes by two-dimensional gel electrophoresis (two-DE), indicating that ESD from the father was deleted in the abnormal chromosome 13. The possibility of paternity was calculated to be 0.996 based on the data using 22 genetic markers. Bilateral retinoblastomas could be diagnosed by ophthalmologic examinations before the manifestation of any clinical signs of the tumor and immediately intensive care was taken. In addition, the phenotype of LCP1 derived from the father was not expressed in the lymphocyte proteins from the patient. These data indicate that the gene for LCP1 (LCP1) is located in the region q14.1-q32 of chromosome 13 and may be a useful genetic marker for preclinical diagnosis of Rb.