Role of arginine 285 in the active site of Rhodotorula gracilis D-amino acid oxidase. A site-directed mutagenesis study.

Arg(285), one of the very few conserved residues in the active site of d-amino acid oxidases, has been mutated to lysine, glutamine, aspartate, and alanine in the enzyme from the yeast Rhodotorula gracilis (RgDAAO). The mutated proteins are all catalytically competent. Mutations of Arg(285) result in an increase ( approximately 300-fold) of K(m) for the d-amino acid and in a large decrease ( approximately 500-fold) of turnover number. Stopped-flow analysis shows that the decrease in turnover is paralleled by a similar decrease in the rate of flavin reduction (k(2)), the latter still being the rate-limiting step of the reaction. In agreement with data from the protein crystal structure, loss of the guanidinium group of Arg(285) in the mutated DAAOs drastically reduces the binding of several carboxylic acids (e.g. benzoate). These results highlight the importance of this active site residue in the precise substrate orientation, a main factor in this redox reaction. Furthermore, Arg(285) DAAO mutants have spectral properties similar to those of the wild-type enzyme, but show a low degree of stabilization of the flavin semiquinone and a change in the redox properties of the free enzyme. From this, we can unexpectedly conclude that Arg(285) in the free enzyme form is involved in the stabilization of the negative charge on the N(1)-C(2)=O locus of the isoalloxazine ring of the flavin. We also suggest that the residue undergoes a conformational change in order to bind the carboxylate portion of the substrate/ligand in the complexed enzyme.

Divided vertical banded gastroplasty either for correction or as a first-choice operation.

BACKGROUND: Staple-line disruption may occur after vertical banded gastroplasty (VBG). METHODS: Since May 1996, the authors have performed the gastric restrictive procedure divided VBG, as described by MacLean, as a first-choice operation and not only as correction for staple-line breakdown. Divided VBG was done in 111 patients, 32 as correction after staple-line disruption and 79 as a first choice, 29 of them by hand-assisted laparoscopy with the dexterity pneumosleeve. RESULTS: Weight loss and nutritional status have been very satisfactory. Operating time for the standardized operation has been no longer than 60 minutes. CONCLUSION: Divided VBG, especially if done by hand-assisted laparoscopy using the dexterity pneumosleeve, is a valid restrictive procedure.

Overexpression in Escherichia coli of a recombinant chimeric Rhodotorula gracilis d-amino acid oxidase.

This paper reports a novel expression system constructed to maximize the production in Escherichia coli of d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). We produced a recombinant plasmid by the insertion of the cDNA encoding for the RgDAAO into the multiple cloning site of the expression vector pT7.7 (pT7-DAAO), downstream of the T7 RNA polymerase binding site. The pT7-DAAO, which encodes a fully active fusion protein with six additional residues at the N-terminus of DAAO, was used to transform the BL21(DE3) and BL21(DE3)pLysS E. coli cells. In the latter host and under optimal IPTG induction conditions, soluble and active chimeric DAAO was expressed in these cells up to 930 U/g of cell (and a fermentation yield of 2300 U/liter of fermentation broth), with a specific activity of 8.8 U/mg protein. RgDAAO represents approximately 8% of the total soluble protein content of the cell.

Biliopancreatic diversion with transitory gastroplasty preserving duodenal bulb: 3 years experience.

BACKGROUND: The authors have performed 521 bariatric surgery operations (319 restrictive procedures and 202 malabsorptive procedures). METHODS: During the last few years we have introduced an evolution of biliopancreatic diversion (BPD): BPD with transitory gastroplasty, preserving the duodenal bulb (53 cases). From a technical point of view, the operation consists of a BPD, coupled with a gastroplasty which is transitory due to the use of a polydioxanone (PDS) band. In the last few cases, instead of a VBG (with PDS band) in order to make the operation completely reversible without any suture on the stomach, we made a gastric pouch by banding with PDS calibrated with the same tube as for the Lap-band (20 cc). We maintained completely the duodenal bulb (5 cm from the pylorus), making an end-to-side duodeno-ileal isoperistaltic anastomosis. RESULTS: With this anastomosis, only 2% of patients developed an anastomotic ulcer. With this new procedure, results have been good in terms of weight loss (similar to that of BPD-AHS) and in nutritional complications. No patient has had hypoalbuminemia, diarrhea or halitosis. CONCLUSION: BPD with temporary gastric restriction has provided satisfactory results.

[Digitalis therapy in decompensation and in heart rhythm disturbances in the elderly. Personal experience]. / La terapia digitalica nello scompenso e nelle turbe del ritmo cardiaco nell'anziano. Esperienza personale.

Mistaken drug administration often complicates the unanswered questions regarding the correct use of digitalis. Clinical data and radioimmunological measurements were used to evaluate in the elderly the effects of daily administration and those of traditional administration of the drug which provides for suspension for two days a week.

[Etiologic determination of chronic liver diseases]. / Contributo allo studio della individuazione eziologica delle epatopatie croniche.

Sixty patients with chronic liver conditions (mostly cirrhotic) were examined for HBV markers. The percentage of markers identified, far higher than in samples of healthy subjects, suggests that HBV either alone or in association with other causes may be implicated in the pathogenesis of these conditions.