RESUMO
Glutathione transferase A4-4 (GST A4-4) is involved in the detoxication of lipid peroxidation products such as alkenals. The human enzyme has been heterologously expressed in Escherichia coli, but for more extensive characterization of the enzyme the expression level had to be elevated. A clone providing up to 8-fold higher yields was created, by screening an expression library with random silent mutations in the 5' region of the cDNA encoding GST A4-4.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glutationa Transferase/biossíntese , Códon , DNA Complementar/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Glutationa Transferase/genética , Humanos , Mutagênese , Proteínas Recombinantes/biossínteseRESUMO
Estrogens affect longitudinal bone growth through their action on endochondral bone formation. Two estrogen receptors are known, the classical estrogen receptor-alpha (ER alpha), newly demonstrated in human growth plate cartilage, and a recently cloned estrogen receptor-beta (ER beta). The present study aimed to localize a possible expression of ER beta protein in human growth plates. Tissue samples were obtained from tibial and femoral growth plates in four female pubertal patients undergoing epiphyseal surgery. Immunohistochemistry, using two different ER beta-specific antibodies, demonstrated positive staining for ER beta in hypertrophic epiphyseal chondrocytes from all patients. No staining was noted in resting or proliferative chondrocytes. These data suggest that in addition to ER alpha, human epiphyseal chondrocytes also express ER beta. The physiological role of ER beta in the regulation of longitudinal bone growth in humans remains to be elucidated.
Assuntos
Lâmina de Crescimento/química , Receptores de Estrogênio/análise , Adolescente , Especificidade de Anticorpos , Criança , Receptor beta de Estrogênio , Feminino , Humanos , Imuno-Histoquímica , Receptores de Estrogênio/imunologiaRESUMO
The effect of ileal resection and cholecystectomy on bile salt metabolism was studied in female prairie dogs 4 weeks after either a sham laparotomy, cholecystectomy, ileal resection, or cholecystectomy and ileal resection. Bile was collected from a common bile duct cannula at hourly intervals for 12 hours. Pool sizes and synthetic rates of primary and secondary bile salts were determined from washout curves. Cholate, chenodeoxycholate, deoxycholate, and lithocholate levels were determined by gas chromatography from pooled collections of bile. After cholecystectomy and ileal resection, the pool sizes of primary and secondary bile salts were significantly reduced to amounts that were much less than the pool sizes after either procedure alone. Primary bile salt synthesis was significantly increased after combined cholecystectomy and ileal resection, to the same degree as cholecystectomy alone. After the combined procedures, there was a decrease in the proportion of cholate in hepatic bile associated with an increase in chenodeoxycholate, deoxycholate, and lithocholate levels. The data suggest that after the loss of both ileum and gallbladder the bile salt pool sizes are drastically reduced, the synthesis of primary bile salts is increased, and the proportion of secondary bile salts is increased. Cholecystectomy should be avoided, if possible, in patients with ileal resection in order to conserve the bile salt pool and prevent severe fat malabsorption.
Assuntos
Ácidos e Sais Biliares/biossíntese , Colecistectomia , Íleo/cirurgia , Animais , Bile/análise , Bile/metabolismo , Ácidos e Sais Biliares/análise , Ácido Quenodesoxicólico/análise , Ácido Quenodesoxicólico/biossíntese , Ácido Cólico , Ácidos Cólicos/análise , Ácidos Cólicos/biossíntese , Cromatografia Gasosa , Ácido Desoxicólico/análise , Ácido Desoxicólico/biossíntese , Feminino , Ácido Litocólico/análise , Ácido Litocólico/biossíntese , SciuridaeRESUMO
Studies of protein structure provide information about principles of protein design that have come into play in natural evolution. This information can be exploited in the redesign of enzymes for novel functions. The glutathione-binding domain of glutathione transferases has similarities with structures in other glutathione-linked proteins, such as glutathione peroxidases and thioredoxin (glutaredoxin), suggesting divergent evolution from a common ancestral protein fold. In contrast, the binding site for glutathione in human glyoxalase I is located at the interface between the two identical subunits of the protein. Comparison with the homologous, but monomeric, yeast glyoxalase I suggests that new domains have originated through gene duplications, and that the oligomeric structure of the mammalian glyoxalase I has arisen by 'domain swapping'. Recombinant DNA techniques are being used for the redesign of glutathione-linked proteins in attempts to create binding proteins with novel functions and catalysts with tailored specificities. Enzymes with desired properties are selected from libraries of variant structures by use of phage display and functional assays.
Assuntos
Evolução Molecular Direcionada , Enzimas/química , Enzimas/metabolismo , Glutationa/metabolismo , Engenharia de Proteínas , Sítios de Ligação/genética , Desenho de Fármacos , Enzimas/genética , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Lactoilglutationa Liase/química , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Família Multigênica , Dobramento de ProteínaRESUMO
Plasma 1-norgestrel (1-Ng) levels after administration of a common oral contraceptive in fertile women (mean age 29 +/- 5 yrs) with mild ulcerative colitis before or after proctocolectomy with a conventional ileostomy or a continent ileostomy reservoir were determined and compared to those of a group of healthy volunteers serving as controls. Before operation, peak and 10-hour levels of 1-Ng were not statistically different from control. Levels in patients with either type of ileostomy were slightly lower than controls, but were only statistically significantly lower in patients with continent ileostomy (p less than 0.05). In a subgroup of patients studied both before and after proctocolectomy with construction of a continent ileostomy, there was a slight reduction in peak and 10-hour levels compared to control. After administration of the pill directly into the reservoir, significant levels of 1-Ng in plasma were found with peak and 10-hour levels approximately one-half of those achieved after oral administration. Although the material is small, it suggests that patients with mild ulcerative colitis and with small ileal resections (8.8 +/- 8 cm) can use combined contraceptive pills containing 1-norgestrel with confidence. Caution should be exercised, however, in prescribing "mini-pills" to patients after proctocolectomy and ileal resection.
Assuntos
Colectomia , Colite Ulcerativa/sangue , Ileostomia , Absorção Intestinal , Norgestrel/sangue , Adulto , Colite Ulcerativa/cirurgia , Anticoncepcionais Orais Combinados/sangue , Feminino , Humanos , Cinética , Levanogestrel , Complicações Pós-Operatórias/sangueAssuntos
Doenças do Colo/cirurgia , Incontinência Fecal/prevenção & controle , Ileostomia/métodos , Doenças Retais/cirurgia , Colectomia , Colite Ulcerativa/cirurgia , Neoplasias do Colo/cirurgia , Doença de Crohn/cirurgia , Seguimentos , Humanos , Pólipos Intestinais/cirurgia , Cuidados Pós-Operatórios/métodos , Complicações Pós-Operatórias/prevenção & controle , Neoplasias Retais/cirurgia , Reoperação , Técnicas de SuturaRESUMO
Previous reports show evidence that perforation of the sigmoid colon with diffuse peritonitis, when conventionally treated (i.e. by suture, drainage, and proximal defunctioning colostomy) is associated with a high mortality. Recent investigations, however, indicate that the mortality can be lowered considerably if acute resection or exteriorization of the affected segment of the bowel is performed. Fifteen patients with perforated lesions in the sigmoid colon associated with diffuse peritonitis were treated with drainage and right transverse colostomy during the 10-year period 1963-72 with five deaths. In comparison, during the last years, ten patients with perforation and diffuse peritonitis were treated with acute resection or exteriorization of the affected segment of the bowel without any mortality. Though the two patient series are not completely comparable, the results imply that removal or exteriorization of the affected bowel is to be preferred in the surgical treatment of most perforated lesions in the sigmoid colon, even if the perforation is associated with diffuse peritonitis.
Assuntos
Colo Sigmoide/cirurgia , Perfuração Intestinal/cirurgia , Adulto , Idoso , Doenças do Colo/cirurgia , Colostomia/métodos , Colostomia/mortalidade , Drenagem , Humanos , Perfuração Intestinal/complicações , Perfuração Intestinal/mortalidade , Pessoa de Meia-Idade , Peritonite/etiologia , Peritonite/mortalidade , SuturasRESUMO
Net transport of water and electrolytes was studied in ileal segments which had been exposed to urine during three months in seven cats. During surgery, a 5 cm long ileal segment was interposed between the ureters and the urinary bladder. At the same time a control segment (5 cm) was indicated with silver clips. After three months, the net transport of water and electrolytes was studied using a continuous perfusion technique with an isotonic and with a hypertonic, urine-like perfusate. Microscopic examination of the mucosa showed a significant reduction of villus height in the transposed segments compared to the controls (p less than 0.05). With the isotonic perfusate there was a net absorption of water in the control segments, and a net secretion in the transposed segments, the difference being statistically significant (p less than 0.05). When the hypertonic perfusate was used, a net secretion occurred in both control and transposed segments. Net transport of electrolytes (Na+, K+, Cl-) was in the same direction as water transport in both control and transposed segments. The results indicate that there does not seem to be any risk of water or electrolyte reabsorption by intestinal mucosa which has been exposed to urine.
Assuntos
Eletrólitos/metabolismo , Íleo/metabolismo , Derivação Urinária , Água/metabolismo , Animais , Gatos , Íleo/patologia , Íleo/cirurgia , Modelos Biológicos , Perfusão , Complicações Pós-Operatórias , Bexiga Urinária/cirurgiaRESUMO
The D1 reaction center protein in photosystem II (PSII) has a high turnover rate due to light-induced inactivation of the redox components. We have studied the reactivation kinetics of the redox components of PSII after strong illumination and compared these kinetics with the turnover of the D1 protein and translation kinetics of the plastid-encoded PSII core proteins in Chlamydomonas reinhardtii cells. Repair of PSII was to a large extent dependent on protein translation. During the first hours of repair, D1 translation was highly accelerated as compared to the other PSII core proteins. By addition of protein synthesis inhibitors during the recovery process, it was found that the time from protein synthesis to full reassembly and reactivation of the individual PSII complexes was about 55 +/- 10 min. Inactivation and reactivation of the redox components in PSII were followed by electron spin resonance and electron transport measurements. Combining the data shows that reactivation of the individual components proceeded together or shortly after one another. Thus, no accumulation of any partially active reactivation intermediate occurred. We conclude that the rate-limiting step of the repair cycle of PSII lies in the degradation and synthesis of the PSII reaction center proteins. Once stable synthesis of the PSII core proteins is achieved, reactivation of the redox components occurs very quickly.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Animais , Divisão Celular , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/efeitos da radiação , Clorofila/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Cinética , Luz , Complexos de Proteínas Captadores de Luz , Oxirredução , Oxigênio/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/biossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/isolamento & purificação , Complexo de Proteína do Fotossistema II , Biossíntese de Proteínas , Fatores de TempoRESUMO
The evolution of proteins for novel functions involves point mutations and recombinations of domains or structural segments. Mimicking this process by rational design in vitro is still a major challenge. The present report demonstrates that the active site of the enzyme glutathione transferase (GST) A1-1 can be tailored for high catalytic efficiency with alkenals. The result is a >3,000-fold change in substrate selectivity involving a noteworthy change in preferred catalyzed reaction from aromatic nucleophilic substitution to Michael addition. The hydrophobic substrate binding pocket of GST A1-1 is formed by three structural modules, which were redesigned sequentially with four point mutations and the exchange of a helical segment. The substitutions were made to mimic first-sphere interactions with a substrate in GST A4-4, which naturally has high activity with alkenals. These substrates are toxic lipid peroxidation products of pathophysiological significance, and glutathione conjugation is a route of their inactivation. The final product of the sequential redesign of GST A1-1, mutant GIMFhelix, had a 300-fold increase in catalytic efficiency with nonenal and a >10 times decreased activity with 1-chloro-2,4-dinitrobenzene. In absolute values, GIMFhelix is more efficient than wild-type GST A4-4 with some alkenal substrates, with a k(cat)/K(m) value of 1.5 +/- 0. 1 10(6) M(-1) small middle dots(-1) for nonenal. The pKa value of the active-site Tyr-9 of GIMFhelix is 7.3 +/- 0.1, approaching the unusually low value of GST A4-4. Thus, rational redesign of the active-site region of an enzyme may be sufficient for the generation of efficient catalysts with altered chemical mechanism and novel selectivity.
Assuntos
Alcenos/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Peroxidação de Lipídeos , Engenharia de Proteínas , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Glutationa Transferase/genética , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/genética , Mutação Puntual/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato , Tirosina/genética , Tirosina/metabolismoRESUMO
New water absorption and changes in villous height were studied in ileal reservoirs and in ileal segments transposed to the colon in the cat. A continuous perfusion technique was used to estimate net water absorption. The villous height was reduced both in ileal reservoirs and in the transposed segments compared with normal ileum. The serosal area of the reservoir was significantly increased compared with the initial segment used in construction of the reservoir. The reservoir showed a reduced net water absorption rate per unit serosal area compared with a control segment, but when the absorption of the whole reservoir was compared with that of a control segment of the same initial length, there was no significant change. In the transposed segments no significant change in net water absorption could be revealed. Data from both series of experiments showed a positive correlation between net water absorption per unit serosal area and villous height. Blood flow registration in ileal segments transposed to the colon showed a blood flow of the same magnitude in the transposed segment as in the control segment.
Assuntos
Colo/fisiologia , Íleo/fisiologia , Absorção Intestinal , Água/metabolismo , Equilíbrio Ácido-Base , Animais , Gatos , Colo/citologia , Feminino , Íleo/citologia , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Masculino , Perfusão , Fluxo Sanguíneo RegionalRESUMO
The adverse effects of ileal resection on bile salt metabolism may be aggravated by cholecystectomy. Female prairie dogs had either sham laparotomy, cholecystectomy, distal 50% small bowel resection, or cholecystectomy and distal 50% small bowel resection. After 4 weeks the common bile duct was cannulated and bile collected for up to 12 hr. Bile salt pool size and synthetic rate were measured from the washout curve. Bile salt, phospholipid, and cholesterol concentrations were determined. Bile salt pool size was reduced after cholecystectomy and after ileal resection. Cholecystectomy plus ileal resection further lowered the pool size, increased the synthetic rate, and increased the proportion of secondary bile salts. Cholecystectomy increased the synthesis and the concentration of bile salts in hepatic bile and altered the proportions of biliary lipids. Ileal resection decreased the concentration of hepatic bile salts with the formation of noncholesterol stones. The drastic reduction in pool size with the combined operation might be expected to lead to fat malabsorption. The higher concentration of bile salts after the combined operation compared with ileal resection alone reflects a possible beneficial effect of cholecystectomy.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colecistectomia , Íleo/cirurgia , Animais , Ácidos e Sais Biliares/análise , Feminino , Lipídeos/análise , Fígado/análise , SciuridaeRESUMO
Plasma cobalamins (vitamin B12) were determined by a microbiological method in 235 patients with continent ileostomies and postoperative observation times of 3-13 years (mean, 6 years). The influence of the reservoir on the vitamin B12 values could not be evaluated in 22 patients (9%)--because of prophylactic treatment in 6%, subnormal B12 values before the operation in 1%, and 'treatment' of various neurological symptoms not caused by vitamin B12 deficiency in 2%. Fourteen (7%) of the remaining 213 patients had developed subnormal plasma levels of vitamin B12 and another 14 patients (7%) had 'borderline' values (130-200 pmol/l). The median time interval between reservoir operation and the development of subnormal values was 7.5 years (range, 3-11 years). A small-bowel resection had been added to the proctocolectomy in 11 out of 14 patients with subnormal values and in 8 out of 14 patients with borderline values. Subnormal or borderline values were seen in 27% of patients with Crohn's disease and in 12% of patients with ulcerative colitis. No patient had anaemia or neurological symptoms caused by B12 deficiency. The study shows that most patients with continent ileostomies do not develop B12 deficiency, and there is therefore no need for general prophylaxis. Since at least 7% developed subnormal values, the plasma levels of vitamin B12 should, however, be followed up regularly in all patients with continent ileostomies.
Assuntos
Ileostomia , Vitamina B 12/sangue , Adolescente , Adulto , Idoso , Feminino , Humanos , Ileostomia/efeitos adversos , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/sangue , Fatores de Tempo , Vitamina B 12/administração & dosagem , Deficiência de Vitamina B 12/etiologia , Deficiência de Vitamina B 12/prevenção & controleRESUMO
Total body water and total body potassium were studied in 14 patients submitted to proctocolectomy six to 10 years after they had been provided with a continent ileostomy. Total body water was determined with an isotope dilution technique using tritiated water as a tracer. Total body potassium was determined by counting the gamma radiation from the naturally present nuclid 40K in a total body counter. The results were compared with 'normal values' produced by multiple regression analysis of data from 476 healthy controls which had been published earlier from the same laboratory. The investigations showed no signs of water or potassium depletion in patients with continent ileostomies.
Assuntos
Água Corporal/análise , Ileostomia , Potássio/análise , Adulto , Estatura , Peso Corporal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Técnica de Diluição de Radioisótopos , Valores de Referência , Trítio , Contagem Corporal TotalRESUMO
In a prospective study of postoperative septic complications, young patients suffering mainly from inflammatory bowel disease were subjects to elective surgery. The bacterial concentration in the operative field was determined by an irrigation technique. When at least one of two irrigation fluids yielded greater than or equal to 50 CFU/ml of E. coli or S. aureus the risk of postoperative infection was highly increased (p less than 0.0001, Fisher's exact test).
Assuntos
Bactérias/isolamento & purificação , Intestinos/cirurgia , Infecção da Ferida Cirúrgica/epidemiologia , Adolescente , Adulto , Idoso , Líquido Ascítico/microbiologia , Bacteroides/isolamento & purificação , Colite Ulcerativa/cirurgia , Doença de Crohn/cirurgia , Escherichia coli/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Risco , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Questionnaires were sent to 48 ileostomy patients to evaluate the effects on sexual life of an ileostomate before and after conversion to a continent ileostomy. Forty-two patients (88 per cent) returned their questionnaires. Ninety-eight per cent of the patients had sometimes felt embarrassed, uncertain or inhibited by the conventional stoma, whereas only 24 per cent had these reactions after conversion to a continent ileostomy. About 80 per cent of the patients thought that the conventional ileostomy disturbed their sexual life because of the risk of leakage, odor, noise or such inhibitory influence to the continent ileostomy. Four men (31 per cent) and 15 women (52 per cent) reported that their sexual activity was reduced or absent when they had their conventional ileostomy. After conversion to a continent ileostomy all but one man and one woman considered their sexual activity to be normal. One-third of the patients claimed that the partner was embarrassed by the conventional ileostomy, but no partner admitted a negative reaction towards the continent ileostomy. Conversion to a continent ileostomy improved the quality of sexual life in about 85 per cent of both men and women.
Assuntos
Ileostomia/psicologia , Comportamento Sexual , Ajustamento Social , Adulto , Idoso , Emoções , Feminino , Humanos , Ileostomia/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sexo , Disfunções Sexuais Fisiológicas/etiologia , Inquéritos e QuestionáriosRESUMO
Eighteen patients, who previously had been treated surgically for familial polyposis coli with proctocolectomy and a continent ileostomy were re-investigated with endoscopy and X-ray for gastric, duodenal and ileal polyps. Gastric and/or duodenal polyps were found in 6 patients and ileal polyps in 2. Altogether upper GI-polyps were found in 7 patients (39%). Most polyps were true adenomas. In one patient with large gastric adenomas, the severe dysplasia called for a gastric resection. It is obvious that familial polyposis may affect the whole gastro-intestinal tract, therefore necessitating regular surveillance of the upper GI-canal as well as the colon and rectum in patients with this hereditary affliction.
Assuntos
Polipose Adenomatosa do Colo/cirurgia , Neoplasias Duodenais/etiologia , Neoplasias do Íleo/etiologia , Pólipos Intestinais/etiologia , Pólipos/etiologia , Neoplasias Gástricas/etiologia , Polipose Adenomatosa do Colo/complicações , Adolescente , Adulto , Idoso , Colectomia/métodos , Neoplasias Duodenais/patologia , Feminino , Seguimentos , Humanos , Neoplasias do Íleo/patologia , Ileostomia/métodos , Pólipos Intestinais/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/patologiaRESUMO
Total body water (TBW) and total body potassium (TBK) were studied in 40 ileostomists before (with conventional ileostomy) and one year after conversion to a continent ileostomy. Each patient acted as his own control. Total body water was determined by using an isotope dilution technique and TBK by counting the gamma radiation from the naturally present nuclide 40K in a whole body counter. Measured values of TBW and TBK were compared intraindividually (conventional versus continent ileostomy) and also with 'normal values' obtained from the same laboratory and based on a multiple regression analysis of data from 476 healthy controls. There was no evidence of water or potassium depletion in ileostomy patients, neither before nor after construction of the continent ileostomy.
Assuntos
Água Corporal/análise , Ileostomia , Potássio/análise , Adulto , Feminino , Humanos , Ileostomia/métodos , Masculino , Pessoa de Meia-Idade , Radioisótopos de Potássio , TrítioRESUMO
Thirty-six patients were evaluated after having continent ileostomies for 16 to 20 years. Follow-up included clinical examination, response to a questionnaire, blood tests, and radiologic studies of the gallbladder. In addition, reservoir biopsies from 15 patients were examined. The patients were in excellent general health, all but one having gained weight postoperatively. There was no increased risk for gallstone formation or for urinary tract stone development. Although 11 of the 36 patients had reservoirs that were not provided with a valve, 92 percent of all patients were continent. Working capacity was normal in most patients and no harmful effects of the reservoir were revealed by morphologic or biochemical studies. In conclusion, no deleterious long-term effects were found as a result of the continent ileostomy.