Improved heterologous expression of human glutathione transferase A4-4 by random silent mutagenesis of codons in the 5' region.

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.

Redesign of substrate-selectivity determining modules of glutathione transferase A1-1 installs high catalytic efficiency with toxic alkenal products of lipid peroxidation.

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.

Demonstration of estrogen receptor-beta immunoreactivity in human growth plate cartilage.

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.

An evolutionary approach to the design of glutathione-linked enzymes.

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.

Synthesis of reaction center proteins and reactivation of redox components during repair of photosystem II after light-induced inactivation.

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.

Long-term follow-up of patients with continent ileostomies.

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.

Total body water and total body potassium in ileostomy patients before and after conversion to the continent ileostomy.

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.

Gastroduodenal and ileal polyps in patients treated surgically for familial polyposis coli with proctocolectomy and continent ileostomy.

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.

Water and electrolyte absorption in ileal segments surgically transposed to the urinary bladder in the cat.

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.

Changes in bile salt composition after cholecystectomy and ileal resection.

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.

Absorption of an oral contraceptive gestagen in ulcerative colitis before and after proctocolectomy and construction of a continent ileostomy.

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.

The effects of ileal resection with cholecystectomy on bile salt metabolism in the prairie dog.

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.

Vitamin B12 in plasma in patients with continent ileostomy and long observation time.

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.

Indomethacin and the gastric mucosal blood flow changes of sepsis.

Recent evidence suggests that sepsis results in increased gastric mucosal blood flow (GMBF). To investigate the possible role of prostaglandins in mediating this response, the GMBF was measured in the fundus, corpus, and antrum of pig stomachs with and without pretreatment with indomethacin, an inhibitor of prostaglandin synthesis, before and after the induction of bacteremia. The studies were done in 22 piglets (seven sepsis controls, seven indomethacin controls and eight experimental [indomethacin pretreated sepsis] ). Sepsis was produced in piglets by bolus intravenous injection of 10(9) live Escherichia coli followed by an infusion of 10(9) E. coli/hr. Cardiac output (C.O.) was measured by thermodilution. GMBF was measured by microsphere trapping. Following sacrifice, hyperemia was noted in the sepsis group but not in the other groups. GMBF was determined by standard techniques (expressed as ml/min/100 gm tissue). There were significant (p less than 0.05) increases in gastric mucosal blood flow to the fundus (+47%), corpus (+50%), and antrum (+101%) at 15 minutes following the onset of E. coli infusion. At 135 minutes, the increase was only significant in the antrum. GMBF, however, did not change in the indomethacin control or indomethacin pretreated sepsis groups. These data demonstrate GMBF in the stomach following sepsis. The changes were not present in the indomethacin control or in the indomethacin pretreated sepsis groups. Since indomethacin is an inhibitor of prostaglandin synthesis, the results suggest that the GMBF may be a prostaglandin mediated response.

Urinary diversion via a continent ileal reservoir: clinical results in 12 patients.

Urinary diversion via a continent ileal reservoir has been performed in 12 patients. An isolated ileal reservoir was constructed using the technique described for patients with a continent ileostomy. The ureters were implanted into an afferent segment provided with a reflux-preventing nipple valve. There were few operative complications and no operative mortality. Late complications involving malfunction of the nipple valves occurred in 8 patients and were corrected surgically. Postoperative followup presently is between 9 months and 6 1/2 years. Two patients have died: 1 in an accident and 1 of metastatic bladder carcinoma. The remaining 10 patients are continent and without reflux to the upper urinary tract. The reservoir generally is emptied by intermittent self-catheterization between 3 to 6 times daily. The volume capacity of the reservoir is more than 500 ml. Urinary cultures have been constantly negative in 7 patients and the contents of the reservoir more or less permanently contained bacteria in 5. Dilatation of the upper urinary tracts, progressive renal deterioration or metabolic disturbances have not been encountered. All patients are satisfied with this type of urinary diversion, especially those who have undergone other types of diversion previously.

Total body water and total body potassium in patients with continent ileostomies.

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.

Sexual adjustment in ileostomy patients before and after conversion to continent ileostomy.

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.