160 research outputs found
Reaction of phenylglyoxal with arginine groups in D-amino acid oxidase from Rhodotorula gracilis
No full textD-Amino-acid oxidase from Rhodotorula gracilis was irreversibly inactivated by phenylglyoxal in a biphasic process. The fast phase was completed in less than 1 min. Its extent was linearly dependent on phenylglyoxal concentration and was not influenced by the presence of FAD or benzoate, a pseudo-substrate. The second phase of inactivation was due to a simple second-order reaction. The presence of FAD exerted only partial protection; the second-order rate constants of inactivation were 8.3 M-1 min-1 for holoprotein and 18.0 M-1 min-1 for apoprotein. The addition of benzoate completely protected against this second phase of inactivation. Efforts to isolate the enzyme modified at a single arginine residue at the end of the fast phase were unsuccessful, but analysis of the enzyme isolated at the end of the slow phase identified an arginine residue, protected by benzoate, that is highly conserved in all D-amino-acid oxidases and corresponds to Arg283 in the pig kidney enzyme. Modification of this residue is directly involved in the inactivation process during the slow phase. This arginine may represent the basic residue ion pairing with the carboxylate group of the substrate or the residue interacting with the flavin N1-C2 = O locus
Studies on the structural and functional aspects of Rhodotorula gracilis D-amino acid oxidase by limited trypsinolysis
No full textThe structure-function relationships of purified Rhodotorula gracilis D-amino acid oxidase (in its holo-, apo- and holo-enzyme-benzoate complex forms) was analysed by digestion with trypsin. In all cases trypsin cleaves this 80 kDa dimeric enzyme at the C-terminal region, since the peptide bonds sensitive to proteinase attack are clustered in this region. Digestion of native enzyme with trypsin produced a nicked and truncated form of 38.3 kDa containing two polypeptides of 34 and 5 kDa starting from Met1 and Ala319 respectively, and with detachment of the Thr306-Arg318 and Glu365-Leu368 peptides. Our results show that this 'core', folded into a compact structure, is catalytically competent. The acquisition of this nicked form was marked by a shift from a dimeric to a monomeric active enzyme, a result never previously obtained. The deleted sequences, Thr306-Arg318 and Glu365-Leu368, are essential for the monomer-monomer interaction, and, in particular, the region encompassing Thr306-Arg318 should play an essential role in the dimerization process. interestingly, the Ser308-Lys321 sequence present in the lost peptide corresponds to a sequence not present in other known D-amino acid oxidases [Faotto, Pollegioni, Ceciliani, Ronchi and Pilone (1995) Biotechnol. Lett. 17, 193-198]. A role of the cleaved-off region for the thermostabilization of the enzyme is also discussed
Limited proteolysis and site directed mutagenesis revealed the origin of microheterogeneity of Rhodotorula gracilis D-amino acid oxidase
No full textSmall bowel obstruction after gastric by-pass: Diagnostic limits and percutaneous drain. A paradigmatic case
No full textBackground: Obesity is showing a constant increase worldwide, making it the major public health problem. Bariatric surgery seems to be the best treatment for these to achieve a good excess weight loss. Gastric by-pass (GBP) is one the most important bariatric procedure, but there are a lot of complications as bowel obstruction for internal hernia, fistula and intussusception. Case report: We present a peculiar case report of a 53-years old woman that underwent to GBP. On the fourth postoperative day, she presented a bowel obstruction and underwent a second surgical intervention for incisional hernia. Another surgical revision was necessary because of dilatation of excluded stomach. Subsequent external drain of excluded stomach solved the paralytic ileum post bowel obstruction. Conclusion: Postoperative occlusion is more complex in bariatric patients, either for variety of etiology, either for choosing best approach to the new anatomy. In addition, post-surgery course may be different, because GBP may determine many modifications on gastrointestinal motility and clinical symptoms. An accurate knowledge of bariatric surgery is mandatory in these situations, obtaining the best management
Manganese recovery from tyrrhenian nodules by hydrometallurgical treatment
No full textThe Tyrrhenian micro-nodules, findable at low depths (200/600 m under s 1) ore a reasonable reserve of manganese. They can be easily and cheaply recovered but do not contain valuable metals as in the case of oceanic nodules. In this work a hydrometallurgical process for manganese recovery from Tyrrhenian nodules is presented. The studied treatment includes the following operations: magnetic enrichment, reducing leaching, leach liquor purification and electrowinning. The study shows that the most important step in the process is the preliminary manganese enrichment. It can be done directly on the platform reducing not only the transport cost, but also the residue mud in the metallurgical plant. The leaching parameters such as status and size of nodules, temperature, composition of leaching solution and S/L ratio have been investigated. After purifying the leach liquor by iron precipitation as ammonium jarosite, the conditions to electrowin manganese have been investigated. The yields of both manganese leaching and iron precipitation together with current efficiency and specific energy consumption in the electrowinning step are given
Enzymatic detection of D-amino acids
No full textD: -Amino acids play several key roles and are widely diffused in living organisms, from bacteria (in which D-alanine is a component of the cell wall) to mammals (where D-serine is involved in glutamatergic neurotransmission in the central nervous system). The study of the biological processes involving D-amino acids and their use as clinical or biotechnological biomarkers requires reliable methods of quantifying them. Although "traditional" analytical techniques have been (and still are) employed for such tasks, enzymatic assays based on enzymes which possess a strict stereospecificity (i.e., that are only active on the D-enantiomers of amino acids) allowed the set-up of low-cost protocols with a high sensitivity and selectivity and suitable for determining the D-amino acid content of complex biological samples. The most exploited enzyme in these assays is D-amino acid oxidase, a flavoenzyme that exclusively oxidizes D-amino acids and possesses with a broad substrate specificity and a high kinetic efficiency
80 m LONG STEEL CABLE-STAYED BRIDGE
No full textV diplomskem delu je prikazana zasnova jeklenega mosta s poševnimi zategami z glavnim razponom 50 m. Most je namenjen mešanemu cestnemu prometu izven naselja. Izdelani so računski modeli z različno vzdolžno razporeditvijo kablov in z različnim načinom sidranja zateg. Po preliminarni analizi teh modelov izberemo najugodnješi model in ga temeljito analiziramo. Izvedena je statična in dinamična analiza izbranega modela ter kontrola mejnih stanj nosilnosti in uporabnosti v skladu z Evrokod standardi.. Za pilone in prekladno konstrukcijo je uporabljeno jeklo S 355, za zatege pa so uporabljeni kabli z vrvmi iz paralelnih žic kvalitete f_(p0,1,k)/f_pk =1520/1770 MPa. Voziščna plošča je armiranobetonska kvalitete C 35/45.civil engineering, steel structures, cable - stayed bridg
Omentopexy with Glubran®2 for reducing complications after laparoscopic sleeve gastrectomy: Results of a randomized controlled study
No full textBackground: Gastric fistulas, bleeding, and strictures are commonly reported after laparoscopic sleeve gastrectomy (LSG), that increase morbidity and hospital stay and may put the patient's life at risk. We report our prospective evaluation of application of synthetic sealant, a modified cyanoacrylate (Glubran®2), on suture rime, associated with omentopexy, to identify results on LSG-related complications. Methods: Patients were enrolled for LSG by two Bariatric Centers, with high-level activity volume. Intraoperative recorded parameters were: operative time, estimated intraoperative bleeding, conversion rate. We prospectively evaluated the presence of early complications after LSG during the follow up period. Overall complications were analyzed. Perioperative data and weight loss were also evaluated. A control group was identified for the study. Results: Group A (treated with omentopexy with Glubran®2) included 96 cases. Control group included 90 consecutive patients. There were no differences among group in terms of age, sex and Body Mass Index (BMI). No patient was lost to follow-up for both groups. Overall complication rate was significantly reduced in Group A. Mean operative time and estimated bleeding did not differ from control group. We observed three postoperative leaks in Group B, while no case in Group A (not statistical significancy). We did not observe any mortality, neither reoperation. Weight loss of the cohort was similar among groups. In our series, no leaks occurred applying omentopexy with Glubran®2. Conclusion: Our experience of omentopexy with a modified cyanoacrylate sealant may lead to a standardized and reproducible approach that can be safeguard for long LSG-suture rime. Trial registration: Retrospective registration on clinicaltrials.gov PRS, with TRN NCT03833232 (14/02/2019)
pLG72 modulates intracellular D-serine levels through its interaction with D-amino acid oxidase - Effect on schizophrenia susceptibility
No full textHuman genes coding for pLG72 and d-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein d-amino acid oxidase (hDAAO). In the brain this oxidizes d-serine, a potent activator of N-methyl-d-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of d-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of d-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility
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