RESUMO
The present study aims at probing the influence of different substituents of sodium carboxylate salts R-COO-:Na+ in aqueous solutions, with R = H, CH3, C2H5, CH2Cl, CF3, and C6H5. X-ray absorption spectroscopy was used in the oxygen K-edge region to highlight the effect of R on the energy position of the O1s-to-πCOO* resonance of the carboxylate ion. Ab initio static exchange and ΔSCF calculations are performed and confirm the experimental observations. We qualitatively discuss the results on the basis of the polar properties of these groups as well as on the basis of the πCOO* orbital energy in the ground states, the oxygen 1s orbital ionization energy, and the O1s-to-πCOO* resonance energy.
RESUMO
BACKGROUND/AIMS: Oxygen-derived free radicals such as superoxide play an important role in ischemia/reperfusion (IR) injury during and after extensive liver surgery or liver transplantation. Superoxide dismutase (SOD) has protective effects against hepatic IR injury. The effect of native SOD is, however, limited because of rapid elimination from the blood circulation and poor affinity for liver cells. It was reported by our collaborators that a SOD derivative modified with galactose (Gal-SOD) was selectively delivered well to hepatocytes by direct attachment to galactose receptors. In the present study, the efficacy of this agent for attenuating hepatic warm IR injury was investigated using the pig model. METHODOLOGY: After 45-min clamping of the hepatic artery and portal vein, pigs were divided into 3 groups according to the following treatments. Ten milliliters of normal saline in Group 1 (n=5), 10,000 units/kg of native SOD in Group 2 (n=5) and 10,000 units/kg of Gal-SOD in Group 3 (n=5) were given just prior to hepatic reperfusion. Liver function including clearance of total bile acid (TBA) and hyaluronic acid (HA) was investigated. Lipid peroxidase of the liver tissue (LPO) and histological findings were examined. In addition, survival rates of the pigs in each group were evaluated. RESULTS: The survival rates at the 7th day after the operation were 60%, 80%, 100% in Groups 1, 2 and 3, respectively. Liver function tests, clearance of TBA and HA, and LPO levels were significantly improved in Groups 3 over findings in Groups 1 and 2. Congestion of hepatic tissues and vacuolization of hepatocytes in Group 3 were less than those in Groups 1 and 2. These results suggested that oxygen-derived free radicals were scavenged by Gal-SOD and IR injury was attenuated. CONCLUSIONS: A safe and novel agent, Gal-SOD has a protective effect against hepatic warm IR injury.
Assuntos
Antioxidantes/farmacologia , Galactose/farmacologia , Fígado/irrigação sanguínea , Traumatismo por Reperfusão/prevenção & controle , Superóxido Dismutase/farmacologia , Animais , Ácidos e Sais Biliares/sangue , Feminino , Ácido Hialurônico/sangue , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/patologia , Testes de Função Hepática , Proteínas Recombinantes/farmacologia , Traumatismo por Reperfusão/patologia , Relação Estrutura-Atividade , Análise de Sobrevida , Suínos , Resultado do TratamentoRESUMO
When cells of E coli are transferred from 30 to 42 degrees C, the cellular level of sigma 32 (rpoH gene product) increases transiently, resulting in increased transcription of a set of heat shock genes. Both increased synthesis and increased stability of sigma 32 contribute to transient accumulation of sigma 32. Evidence suggests that synthesis of sigma 32 is enhanced primarily at the level of rpoH translation. We have constructed and examined the expression of deletion derivatives of rpoH-lacZ gene fusion at 30 degrees C and after shift to 42 degrees C. It was revealed that two cis-acting sequences within the rpoH coding region are involved in thermal regulation of fusion protein synthesis. One region immediately downstream of the initiation codon is required for high level expression, whereas the other internal region is involved in repression at low temperature. Thus, these regions act as positive and negative cis-elements in thermal regulation of rpoH translation. The rpoH mRNA secondary structure model suggesting an interplay between the two regions has been proposed to account for the temperature-induced sigma 32 synthesis as a primary cellular response to the heat shock stress.
Assuntos
Escherichia coli/genética , Proteínas de Choque Térmico/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Fator sigma/genética , Fatores de Transcrição , Sequência de Bases , Escherichia coli/metabolismo , Proteínas de Choque Térmico/biossíntese , Temperatura Alta , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Proteínas Recombinantes de Fusão/biossíntese , Fator sigma/biossíntese , Transcrição GênicaRESUMO
Nafamostat mesilate (NM), a protease inhibitor, possesses a cytoprotective effect and inhibits the activation of complement. The present study investigated whether NM has any protective effect against injury of porcine hepatocytes by human plasma in a bioartificial liver support system. Porcine hepatocytes were harvested and seeded at a density of 2 x 10(5) cells on a 35-mm collagen-coated plate in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. Twenty-four hours later, the medium was replaced with human plasma with three concentrations of NM between 3.8 x 10(-5) and 3.8 x 10(-4) M and then cultured for 6 h. The viability of porcine hepatocytes, lactate dehydrogenase (LDH) levels, lidocaine clearance, porcine albumin production, and changes in complement (C3) levels were measured. The viability of porcine hepatocytes in human plasma decreased significantly to 37.7 +/- 11.4% of that in DMEM. NM improved the viability of the hepatocytes, lowered the levels of LDH, and increased lidocaine clearance and albumin production in a concentration-dependent manner. The concentrations of C3, the marker of xenogeneic reactions, did not change significantly, indicating that no hyperacute xenogeneic reaction occurred in our series. Together, our results suggested that NM exerts favorable effects on porcine hepatocytes in human plasma through direct effect such as prevention of protease activity in the plasma membrane of porcine hepatocytes rather than inhibition of complement-dependent immunoreactions.
Assuntos
Proteínas Sanguíneas/efeitos adversos , Guanidinas/farmacologia , Fígado Artificial , Fígado/citologia , Inibidores de Proteases/farmacologia , Albuminas/biossíntese , Anestésicos Locais/farmacocinética , Animais , Benzamidinas , Sobrevivência Celular/efeitos dos fármacos , Transplante de Células , Células Cultivadas , Complemento C3/análise , Complemento C3/farmacologia , Feminino , Humanos , L-Lactato Desidrogenase/análise , Lidocaína/farmacocinética , Fígado/enzimologia , Suínos , Transplante HeterólogoRESUMO
We recently encountered a rare case of late-onset biliary leakage after laparoscopic cholecystectomy using laparoscopic coagulating shears (LCS). The patient was a 49-year-old Japanese man who had undergone a laparoscopic cholecystectomy at Hamamatsu Medical Center after a diagnosis of cholecystolithiasis associated with localized adenomyomatosis. The cystic duct and the cystic artery were closed using LCS instead of metal endoclips. The postoperative course was uneventful, and the patient was discharged on the 4th operative day. However, on the 7th day after the surgery, the patient developed severe upper abdominal pain and was readmitted to our center with the diagnosis of a late biliary leakage, which was confirmed by an endoscopic retrograde cholangiogram. We then treated the leak successfully with endoscopic nasobiliary drainage.
Assuntos
Ductos Biliares/lesões , Colecistectomia Laparoscópica/efeitos adversos , Complicações Pós-Operatórias , Colecistectomia Laparoscópica/instrumentação , Humanos , Masculino , Pessoa de Meia-Idade , Instrumentos CirúrgicosRESUMO
Tissue transfer and clinical effects of imipenem/cilastatin sodium (MK-0787/MK-0791), a new carbapenem antibiotic, were studied and the following results were obtained. Penetrations of MK-0787 into uterine arterial blood and into pelvic dead space exudate were good. When MK-0787/MK-0791 was administered at a dose of 500 mg/500 mg by a 30-minute intravenous drip infusion, the peak level of MK-0787 in uterine arterial blood was 22.2 micrograms/ml, 30 minutes after the completion of the drip infusion. The peak level of MK-0787 in pelvic dead space exudate was 12.9 micrograms/ml at 2 hours and it dropped to 2.6 micrograms/ml at 6 hours. MK-0791 levels were similar to those of MK-0787. Penetrations of MK-0787 into tissues were also good. When MK-0787/MK-0791 was administered at a dose of 500 mg/500 mg by a 30-minute intravenous drip infusion, the level of MK-0787 was 2.2 +/- 1.1 micrograms/g in the oviduct, 2.7 +/- 2.1 micrograms/g in the ovary, 2.5 +/- 1.2 micrograms/g in the endometrium, 3.0 +/- 1.6 micrograms/g in the myometrium, 3.1 +/- 1.9 micrograms/g in the cervix uteri and 3.8 +/- 2.0 micrograms/g in the portio vaginalis at 1 hour after administration. These levels were reduced to halves, respectively, in approximately 2 hours. Four patients with intrauterine infections and 2 with vaginal stump infections were treated with MK-0787/MK-0791 at a daily dose of 1 g/1 g (500 mg/500 mg X 2). Good clinical and bacteriological responses were observed in 5 patients and causative organisms were eradicated in 2 patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Infecções Bacterianas/tratamento farmacológico , Ciclopropanos/administração & dosagem , Doenças dos Genitais Femininos/tratamento farmacológico , Genitália Feminina/metabolismo , Troca Materno-Fetal , Gravidez/metabolismo , Tienamicinas/administração & dosagem , Adulto , Idoso , Cilastatina , Ciclopropanos/metabolismo , Combinação de Medicamentos , Feminino , Humanos , Imipenem , Infusões Intravenosas , Cinética , Pessoa de Meia-Idade , Tienamicinas/metabolismoRESUMO
The Niigata Gynecological Cancer Society has been investigating the role of the second look operation (SLO) in the multi-disciplinary treatment of ovarian cancer, in cooperation with many other institutions, since 1981. One hundred seventy patients who underwent initial treatment for primary common epithelial ovarian cancer were analyzed in the present study. The distribution of clinical stages was as follows: stage I, 60 cases: stage II, 27 cases; stage III, 72 cases; and stage IV, 11 cases. Results; Five-year survival rates were 96% in stage I, 54% in stage II, 26% in stage III and 23% (4-year rate) in stage IV. Survival rates classified by the degree of completion of initial operation were 94% (5-year) in the complete extirpation group, 45% (4-year) in the group with residual tumor within 2 cm, 8% (4.5-year) in the group with residual tumor over 2 cm and 18% (5-year) in the exploratory laparotomy group. There were 89 cases of incomplete initial surgery, and 51 of them underwent SLO after FCAP therapy (Cisplatin + Adriamycin + Cyclophosphamide + 5-fluorouracil). Cancer was macroscopically completely removed in 20 cases (39.2%) by SLO, with the 4.5-year survival rate of 58%. In 25% of the cases with initial exploratory laparotomy group, the residual cancer was completely extirpated. There were 74 cases of complete initial surgery at stage I and stage II. They have been given FCAP therapy with 3 courses and maintenance chemotherapy. SLO was performed in 40 cases without clinical signs of recurrence. Recurrent cancer was noted in 2 cases (5%). On the other hand, 3 cases (12.5%) had recurred in 24 cases without SLO within about 2 years from initial operation. Consequently, SLO in the treatment of ovarian cancer is considered to be effective in the extirpation of residual cancer and in the early detection of cancer recurrence.
Assuntos
Neoplasias Ovarianas/cirurgia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cisplatino/administração & dosagem , Terapia Combinada , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Estudos de Avaliação como Assunto , Feminino , Fluoruracila/administração & dosagem , Humanos , Japão , Estudos Multicêntricos como Assunto , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/cirurgia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , ReoperaçãoRESUMO
Human fibroblast interferon (IFN-beta) was given 13 cases of advanced gynecological cancers. Eight patients, who were clinically evaluable, were reported as follows; Patients consisted of ovarian adenocarcinoma (5), cervical adenocarcinoma (1), endometrial carcinoma (1) and tubal carcinoma (1). Route of administration was intravenous in 5 cases and intratumorous in 3 cases. IFN-beta dose ranged from 2, 650 X 10(4) to 10, 620 X 10(4) units. Clinical effects according to Koyama - Saitoh 's category was progressive disease (PD) in 7 cases and minor response (MR) only in one case who received intratumorous injection for recurrent tumor mass of tubal carcinoma in vaginal stump. Side effects of IFN-beta were chill and fever, fatigue and anorexia, leucocyte--and thrombocyte-- penia and hepatic dysfunction, though they were mild in grade and not dose-limiting factors. No anti-IFN-beta-antibodies were detected in any cases.
Assuntos
Adenocarcinoma/terapia , Cistadenocarcinoma/terapia , Interferon Tipo I/uso terapêutico , Neoplasias Ovarianas/terapia , Neoplasias Uterinas/terapia , Adenocarcinoma/patologia , Adulto , Idoso , Cistadenocarcinoma/patologia , Endometriose/patologia , Endometriose/terapia , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/terapia , Neoplasias Uterinas/patologiaRESUMO
Antitumor effects of UFT, tegafur (FT-207), cisplatin (CDDP) and the combination of UFT with CDDP on a human ovarian cancer xenograft in nude mice and the concentration of 5-FU in the tumor tissue and major organs were studied. UFT (48.6mg/kg/day X 20) or tegafur (15.0mg/kg/day X 20) was daily administrated orally, and CDDP (5mg/kg/day X 3) was administrated intraperitoneally at an 7-day interval. The inhibition rates of the tumor growths were 49.6% with UFT, -2.3% with tegafur and 17.7% with CDDP, respectively. In the combination of UFT with CDDP, severe side effect were observed. The concentration of 5-FU in UFT-treated group was higher than tegafur group: about 2 times in the tumor, 5 times in the liver, 9 times in the kidney and 4 times in the spleen, respectively. In the combination of UFT with CDDP, the concentration of 5-FU in major organs, especially in the kidney, in nude mice that died at 10 day after drug administration were higher than in those of UFT. These findings indicate that UFT increases the intratumoral concentration of 5-FU to elicit better antitumor effect and also the concentration of 5-FU in various normal organs after long time administration.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Fluoruracila/análise , Neoplasias Ovarianas/tratamento farmacológico , Animais , Cisplatino/farmacocinética , Cisplatino/uso terapêutico , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Ovarianas/metabolismo , Tegafur/análise , Tegafur/farmacocinética , Tegafur/uso terapêutico , Distribuição Tecidual , Uracila/farmacocinética , Uracila/uso terapêuticoAssuntos
Translocação Bacteriana/efeitos dos fármacos , Dipeptídeos/farmacologia , Mucosa Intestinal/transplante , Intestino Delgado/transplante , Nutrição Parenteral Total/métodos , Transplante Autólogo/fisiologia , Animais , Sobrevivência de Enxerto , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/fisiologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/fisiologia , Albumina Sérica/análise , Suínos , Fatores de Tempo , Transplante HeterotópicoAssuntos
Parada Cardíaca , Transplante de Fígado/patologia , Transplante de Fígado/fisiologia , Fígado , Soluções para Preservação de Órgãos , Adenosina , Alopurinol , Animais , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Endotelina-1/sangue , Feminino , Glutationa , Ácido Hialurônico/sangue , Insulina , Preservação de Órgãos , Cloreto de Potássio , Rafinose , Suínos , Coleta de Tecidos e ÓrgãosAssuntos
Criopreservação/veterinária , Cães/fisiologia , Estro/fisiologia , Inseminação Artificial/veterinária , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Criopreservação/métodos , Feminino , Inseminação Artificial/métodos , Hormônio Luteinizante/sangue , Masculino , GravidezRESUMO
BACKGROUND AND PURPOSE: High-attenuation areas (HDAs) called pseudo-subarachnoid hemorrhages (SAHs) may develop in some patients resuscitated from cardiopulmonary arrest (CPA), though no hemorrhage has occurred. We investigated the imaging characteristics and clinical significance of this phenomenon. MATERIALS AND METHODS: CT images of consecutive patients resuscitated from nontraumatic CPA were reviewed and classified into cases with pseudo-SAH (pseudo-SAH[+] group, n = 9), those without pseudo-SAH (pseudo-SAH[-] group, n = 28), and those with true SAH (SAH-CPA group, n = 8). Typical patients with SAH (SAH group, n = 13) and 20 healthy individuals were also extracted as control groups. The degree of brain edema was scored visually as none, mild, or severe, and the CT values of the HDAs and brain parenchyma were measured. These parameters were compared among the groups. We also compared the prognosis between the pseudo-SAH(+) and pseudo-SAH(-) groups. RESULTS: On CT, pseudo-SAH was associated with severe brain edema, whereas there was mild or no edema without pseudo-SAH. The CT values of the HDAs in the pseudo-SAH(+) group were significantly lower than those of the CPA-SAH and SAH groups (P < .0001). The brain parenchyma of the pseudo-SAH(+) group had the lowest CT values among all of the groups (P < .0001). The prognosis of the pseudo-SAH(+) group was significantly poorer than that of the pseudo-SAH(-) group in terms of both clinical outcome (P = .02) and survival (P = .046). CONCLUSION: The findings of pseudo-SAH have several imaging characteristics differing from SAH and predict a poor prognosis. This provides important information that can be used for deciding treatment strategies.
Assuntos
Reanimação Cardiopulmonar/efeitos adversos , Parada Cardíaca/complicações , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/etiologia , Tomografia Computadorizada por Raios X/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Parada Cardíaca/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The purpose of this study was to examine the effect of time for mating and gestation length on reproductive efficiency in dogs. Groups of eight, six and six beagle bitches were mated with a total of three sires on days 3, 5 and 7, respectively, after the luteinizing hormone (LH) surge. All the bitches whelped successfully. The gestation lengths (the intervals from the LH surge to the whelping) were 65.1 +/- 1.9, 65.5 +/- 1.9 and 68.0 +/- 1.8 days, respectively. This length of mating 7 days after the LH surge was significantly longer than that of mating 3 and 5 days after the LH surge (p < 0.05). The litter sizes were 5.5 +/- 2.2, 6.2 +/- 1.9 and 4.8 +/- 2.1, respectively and there was no significant difference between any of the groups. In conclusion, mating 3-7 days after a bitch's LH surge results in a good reproductive efficiency and the intervals from the LH surge to the whelping of mating 7 days after the LH surge shows a longer day significantly in comparison with mating 3 and 5 days after the LH surge.
Assuntos
Cães/sangue , Cães/fisiologia , Hormônio Luteinizante/sangue , Prenhez/fisiologia , Reprodução/fisiologia , Animais , Feminino , Idade Gestacional , Inseminação Artificial/veterinária , Tamanho da Ninhada de Vivíparos , Gravidez , Prenhez/sangue , Fatores de TempoRESUMO
Twenty-four patients of cervical cancer and 15 of ovarian cancer were studied to ascertain the presence of tumor-specific immune-response by in vivo and in vitro immunoassays. Soluble extracts of cell membranes as antigens were prepared from surgically obtained tumor tissues and normal tissues by 3M-Kcl method. Skin test and in vitro assays, such as Macrophage Migration Inhibition test, Leucocyte Adherence Inhibition assay and lymphocyte blastogenesis, were performed using these extracted antigens. 1. In skin test with autochthonous tumor extracts, positive reaction was revealed in 12.5% of cervical cancer cases and in 13.3% of ovarian cancer cases. There was no positive reaction in skin test with normal tissue extracts and allogeneic tumor extracts. 2. In in vitro assays, the percentage of positive was 30 to 40% in cervical cancer and 50 to 60% in ovarian cancer. It was observed that the patients with positive skin test tended to show also positive reaction in in vitro assays. 3. The positive percentage of non-specific skin tests (PHA, DNCB, PPD and SK-SD) was higher in the positive cases than in the negative in the tumor-specific skin test. Consequently, it was suggested that there were tumor-specific antigens in tumor tissues of cervical cancer or ovarian cancer and immune response of patients to these antigens might depend on individual-specificity.
Assuntos
Antígenos de Neoplasias/análise , Epitopos/análise , Neoplasias Ovarianas/imunologia , Neoplasias do Colo do Útero/imunologia , Adolescente , Adulto , Idoso , Inibição de Migração Celular , Feminino , Humanos , Teste de Inibição de Aderência Leucocítica , Ativação Linfocitária , Macrófagos/imunologia , Pessoa de Meia-Idade , Testes Cutâneos , Extratos de Tecidos/imunologiaRESUMO
When Escherichia coli cells are transferred from 30 degrees C to 42 degrees C, transcription from specific promoters recognized by RNA polymerase containing sigma 32 (the rpoH gene product) is transiently activated, resulting in induction of heat shock proteins. Transcription from heat shock promoters is activated by an increased cellular concentration of sigma 32 due to enhanced synthesis and stabilization. We have constructed and examined the expression of mutant derivatives (deletions and base substitutions) of rpoH-lacZ gene fusion. Synthesis of a sigma 32-beta-galactosidase fusion protein was found to be regulated at the translational level involving two distinct 5'-proximal rpoH coding regions. A small region immediately downstream of the initiation codon is required for potentially high-level expression, whereas a much larger internal region is required for thermal regulation--namely, repression at low temperature or nonstress conditions. The two mRNA regions act as positive and negative cis elements, respectively, in controlling rpoH translation. We propose that an interplay between these RNA regions involving secondary structure formation is important in regulating translation initiation and that transient disruption of secondary structure represents a primary step of the heat shock response.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Proteínas de Choque Térmico/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Fator sigma/genética , Fatores de Transcrição , Sequência de Bases , Deleção Cromossômica , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Choque Térmico/biossíntese , Temperatura Alta , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , Fator sigma/biossíntese , Software , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Induction of heat shock proteins following transfer of E. coli cells from 30 degrees C to 42 degrees C depends on rapid accumulation of sigma 32, a minor sigma factor specifically required for transcription of heat shock genes. The synthesis of sigma 32 is induced by enhancing translation of its mRNA transcribed from the rpoH (htpR) gene. We previously showed that the translational control of rpoH-lacZ gene fusion is mediated by two cis-acting rpoH coding regions presumably involving mRNA secondary structure. To further examine this model, we constructed and analyzed a set of gene fusions carrying base substitution(s) or internal deletions within rpoH, including constitutive mutations predicted to destroy the mRNA secondary structure and compensatory second-site mutations that may restore the secondary structure. The results demonstrate that base pairings between the translation initiation region of some 20 nucleotides and part of the internal complementary sequences are critical for maintaining repression during steady-state growth and for modulating heat-induced synthesis of sigma 32-beta-galactosidase fusion protein upon temperature upshift. Furthermore, some of the compensatory mutations resulted in super-repressed (non-inducible) phenotypes, suggesting that the heat induction depends on a specific nucleotide sequence(s) as well as the mRNA secondary structure within the 5'-proximal regulatory segment of rpoH coding region.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , RNA Mensageiro/química , Fator sigma/genética , Fatores de Transcrição , Proteínas de Bactérias/genética , Sequência de Bases , Temperatura Alta , Ligação de Hidrogênio , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão , Relação Estrutura-AtividadeRESUMO
Induction of heat shock proteins in Escherichia coli is caused by a transient increase in the cellular level of sigma 32 (the rpoH gene product), a protein required for transcription of heat shock genes. Both increased synthesis and stabilization of sigma 32 contribute to the increase in sigma 32. We previously showed that heat-induced translation of sigma 32-beta-galactosidase fusion protein encoded by an rpoH-lacZ gene fusion was mediated by an mRNA secondary structure formed between two 5'-proximal segments (A and B) of rpoH coding sequence spanning some 200 nt. We now report that a portion of the sigma 32 polypeptide that corresponds to further downstream (designated region C) is involved in the DnaK-mediated negative control resulting in the shutoff of heat-induced synthesis and degradation of fusion protein. Gene fusions carrying the 5' half (433 nt) or more of the rpoH coding sequence exhibited normal shutoff of synthesis, and the fusion proteins produced were very unstable, like authentic sigma 32; both the shutoff of synthesis and the instability of protein were markedly affected by the dnaK and dnaJ mutations. In contrast, gene fusions carrying < or = 364 nt (lacking region C) and a fusion carrying most of the rpoH sequence but with a frameshift mutation specifically affecting region C exhibited little or no shutoff and produced stable proteins. These results indicate that a distinct segment of sigma 32 plays a critical role in the negative feedback control of sigma 32. The control may be exerted during or after completion of sigma 32 synthesis mediated by interaction between nascent or mature sigma 32 and DnaK/DnaJ proteins.