Isoforms of the DHTKD1-Encoded 2-Oxoadipate Dehydrogenase, Identified in Animal Tissues, Are not Observed upon the Human DHTKD1 Expression in Bacterial or Yeast Systems.

Unlike the OGDH-encoded 2-oxoglutarate dehydrogenase (OGDH), which is an essential enzyme present in all animal tissues, expression of the DHTKD1-encoded isoenzyme, 2-oxoadipate dehydrogenase (OADH), depends on a number of factors, and mutant DHTKD1 phenotypes are rarely manifested. Physiological significance of OADH is also obscured by the fact that both isoenzymes transform 2-oxoglutarate and 2-oxoadipate. By analogy with other members of the 2-oxo acid dehydrogenases family, OADH is assumed to be a component of the multienzyme complex that catalyzes oxidative decarboxylation of 2-oxoadipate. This study aims at molecular characterization of OADH from animal tissues. Phylogenetic analysis of 2-oxo acid dehydrogenases reveals OADH only in animals and Dictyostelium discoideum slime mold, within a common branch with bacterial OGDH. Examination of partially purified animal OADH by immunoblotting and mass spectrometry identifies two OADH isoforms with molecular weights of about 130 and 70 kDa. These isoforms are not observed upon the expression of human DHTKD1 protein in either bacterial or yeast system, where the synthesized OADH is of expected molecular weight (about 100 kDa). Thus, the OADH isoforms present in animal tissues, may result from the animal-specific regulation of the DHTKD1 expression and/or posttranslational modifications of the encoded protein. Mapping of the peptides identified in the OADH preparations, onto the protein structure suggests that the 70-kDa isoform is truncated at the N-terminus, but retains the active site. Since the N-terminal domain of OGDH is required for the formation of the multienzyme complex, it is possible that the 70-kDa isoform catalyzes non-oxidative transformation of dicarboxylic 2-oxo acids that does not require the multienzyme structure. In this case, the ratio of the OADH isoforms in animal tissues may correspond to the ratio between the oxidative and non-oxidative decarboxylation of 2-oxoadipate.

Preoperative fasting - "nihil per os" a difficult myth to break down: a randomized controlled study.

INTRODUCTION: For several years the scientific anaesthesia societies declared a preoperative fast of 6 hours for solid foods and 2 hours for clear liquids before elective surgical interventions to be sufficient. The aim of this study is to identify the extent of the gap that exists between the preoperative fasting time required and that actually encountered in operating rooms. PATIENTS AND METHODS: The safety and clinical applicability of a reduction of the preoperative fasting time was investigated through the use of oral solutions enriched with maltodextrin and their effects on the pre- and postoperative well-being that this may have on patients who are candidates for elective abdominal surgery. The study was conducted in two successive phases (I and II) and patients divided into two groups (A and B). DISCUSSION: Clinical practice is slow to change, in fact, in our study the duration of fasting was an average of 19 hours for solids and 13 hours for liquids. The duration of the fasting did not show differences in the various surgical departments, demonstrating that it is a transversal practice and is not only limited to abdominal surgery in which the utility of fasting would theoretically be greater. Among Group patients A, the fasting time for liquids was about 9 hours. This shows that the time is certainly shorter but not much different when compared to the fasting time for liquids in group B which was on average 14 hours. It is important how difficult it is to achieve good compliance from patients when trying to reduce the time of preoperative fasting based on scientific evidence that is now well established. CONCLUSION: The use of carbohydrate-enriched drinks up to 2 hours after induction of anaesthesia appears to be a safe procedure. The use of these solutions reduces the catabolic response to surgery and contributes to maintaining a pre-operative state of well-being by reducing feelings of hunger and thirst and the state of preoperative anxiety.

The prevalence of dysphonia and dysphagia in patients affected by immunomediated diseases and the role of psychometric tests.

This cross-sectional investigation examined the prevalence and severity of dysphonia, globus pharyngeus, and dysphagia in patients affected by immunomediated (IM) diseases. Seventy subjects were administered the Voice Handicap Index (VHI) (scale 0-4), Glasgow-Edinburgh Throat Scale (GETS) (scale from 0 to 7) for globus pharyngeus assessment, and modified Swallowing Outcomes After Revised Laryngectomy (SOAL) (scale 0, 1, 2) to test swallowing symptoms. VHI: the mean percentage of answers with a score greater than 1 (corresponding to a frequency of situation's occurrence "sometimes," "almost always," or "always") was 25.7, 26.7, and 44.1% for functional, emotional, and physical groups of sub-items respectively. GETS: the mean percentage of answers with a score ≥ 3 was 60.85%, significantly higher if compared with that of answers with a score < 3 (40.14%). The mean percentages of answers with a score 0-2, 3-4, and 5-7 were 40.1, 16.7, and 43.7% respectively. SOAL: a mean of 57.9% of answers gained a symptomatic score (1 ["a little"] or 2 ["a lot"]) and 41.9%, the score 0. The difference was statistically significant (p < 0.05). The first two most recurrent items with a score 2 ("a lot") were "Do you have a problem swallowing dry food?" (46%) and "Do you have a problem swallowing solid food?" (36%). The study represents the first to describe the globus pharyngeus symptoms in IM population. Moreover, it allows to confirm the recurrence of dysphonia and dysphagia in this type of patients. Particularly, it has been demonstrated that the alteration of swallowing function is related to solid and dry food. The self-assessment questionnaires proved as a useful tool to early detection of dysfunctions in order to avoid further deterioration of quality of life and to prevent serious life-threatening complications.

Enzymatic debridement: is HA-collagenase the right synergy? Randomized double-blind controlled clinical trial in venous leg ulcers.

OBJECTIVE: The aim of this study is to evaluate the efficacy and safety of a new ointment containing Hyaluronic Acid and collagenase from non-pathogenic Vibrio alginolyticus. PATIENTS AND METHODS: Double blind, multicenter, controlled clinical trial (no. ISRCTN71239043) conducted to demonstrate the superiority of Hyaluronic Acid-Collagenase applied once a day over placebo in mean reduction of devitalized/fibrinous/slough tissue after 15 days of treatment. 113 patients with venous ulcers were enrolled and randomized to receive active treatment therapy or vehicle preparation. Both arms also received compression therapy. Subjects were assessed at baseline and at 4 different clinical study visits up to a maximum of 30 days. Outcome measures included mean percentage debridement evaluated by digital planimetry, pain during change of dressing measured on a visual analogue scale and adverse event assessment for tolerance. RESULTS: After 15 days the debridement rate in the active group was 67.5% compared to 59% in the placebo group (p = 0.0436). A significantly higher number of patients in the treatment group achieved 100% debridement by day 15 (p = 0.0025) than in the control group, and a higher percentage also demonstrated complete debridement at every other time point. Pain perception was similar in both groups with low levels during medication. No differences in tolerance were observed between groups. CONCLUSIONS: Chronic venous ulcers treated with this novel compound of Hyaluronic Acid and collagenase resulted in a significantly higher debridement rate at Day 15 vs. the control group. Hyaluronic Acid-Collagenase was well tolerated and a low degree of pain was perceived during dressing change. The preparation of 0.2% of Hyaluronic acid-collagenase shows significant benefits in the management of chronic ulcers.

Treatment of coronary bifurcation lesions with bioresorbable vascular scaffolds.

AIM: Aim of the study was to report on the feasibility and early safety and efficacy of Absorb everolimus-eluting bioresorbable vascular scaffold (BVS) for coronary artery bifurcations (CABs) treatment at a single high-volume center. METHODS: All patients treated with Absorb implantation at our institution from March 2013 to March 2014 were enrolled in the prospective, single-center, ongoing, all-comers registry, which has the main purpose of evaluating the safety and efficacy of Absorb implantation in unselected patients treated in daily practice. In-hospital and at follow-up clinical outcomes of 46 patients undergoing treatment with Absorb in 46 CAB lesions were reported in the present study. RESULTS: Treated CAB lesions involved the left main in 13.0% of cases, the left anterior descending artery in 65.0%, the left circumflex in 19.6%, and the right coronary artery in 2.2%. Bifurcations were classified as true (47.8%) and non-true (52.2%) according to the Medina classification. The provisional and the two-BVS techniques were used in 78.3% and 21.7%, respectively. Two-BVS techniques included: mini-crush 13.0%; reverse culotte 2.2%; T-stenting 2.2%; and V-stenting 4.3%. No in-hospital adverse events occurred. At 6 months no adverse events occurred. Only one case of target lesion revascularization was observed at day 227. No stent thrombosis occurred during follow-up. CONCLUSION: Our preliminary experience suggested that CABs treatment with Absorb is feasible and associated with promising immediate and short-term clinical outcomes. However, larger studies with long-term follow-up are needed to adequately address the safety and efficacy of BVS use in CABs.

Feasibility study on the utilization of boron neutron capture therapy (BNCT) in a rat model of diffuse lung metastases.

In order for boron neutron capture therapy (BNCT) to be eligible for application in lung tumour disease, three fundamental criteria must be fulfilled: there must be selective uptake of boron in the tumour cells with respect to surrounding healthy tissue, biological effectiveness of the radiation therapy and minimal damage or collateral effects of the irradiation on the surrounding tissues. In this study, we evaluated the biological effectiveness of BNCT by in vitro irradiation of rat colon-carcinoma cells previously incubated in boron-enriched medium. One part of these cells was re-cultured in vitro while the other was inoculated via the inferior vena cava to induce pulmonary metastases in a rat model. We observed a post-irradiation in vitro cell viability of 0.05% after 8 days of cell culture. At 4 months follow-up, all animal subjects in the treatment group that received irradiated boron-containing cells were alive. No animal survived beyond 1 month in the control group that received non-treated cells (p<0.001 Kaplan-Meier). These preliminary findings strongly suggest that BNCT has a significant lethal effect on tumour cells and post irradiation surviving cells lose their malignant capabilities in vivo. This radio-therapeutic potential warrants the investigation of in vivo BNCT for lung tumour metastases.

Surgical repair of the distal biceps brachii tendon: clinical and isokinetic long-term follow-up.

Anatomical reinsertion of the avulsed distal biceps tendon is the recommended treatment, but the results are hampered by complications. The purpose of this study is to show the results of patients surgically treated with a non-anatomical reinsertion of this tendon. From 1972 to 2006, 26 non-professional athletic patients were surgically treated by suture of the tendon on the brachialis muscle tendon. At follow-up 23/26 patients underwent clinical and isokinetic evaluation. At a medium follow-up of 84 months, patients provided satisfactory subjective and objective clinical results. Flexion was restored in all patients, while a 10 degrees supination deficit was found in two patients. Dynamometric tests showed satisfactory results both regarding Maximum Strength Power and Endurance tests. Reinsertion of the distal biceps tendon on the brachialis tendon can be considered, in a long-term follow-up, a safe and effective procedure, with low complication rate.

Long term results of unprotected left main percutaneous coronary intervention with DES versus BMS.

AIM: Stenosis in the unprotected left main coronary artery (ULMCA) is considered a standard indication for surgical revascularization. Some studies have demonstrated that stenting of the ULMCA is safe and feasible in selected patients. Drug eluting stents (DES) have been shown to be superior to bare metal stents (BMS) in reducing restenosis and major adverse cardiac events (MACE) both in-hospital and at follow-up after treatment of ULMCA disease. Several studies showed that the mid-term prognosis of patients with left main stenting is good, but most of them are limited by small populations and the availability of mid-term results. Thus, we sought to evaluate the very long term impact of DES vs BMS in a large cohort of patients undergoing stent implantation for ULMCA disease in our center. METHODS: Between June 2002 and June 2008 a total of 354 consecutive patients with ULMCA stenosis were treated with percutaneous coronary intervention with BMS (53 patients) or DES (301 patients) implantation. A multivariable adjustment was provided in order to account for baseline differences between groups. RESULTS: The average clinical follow-up was 551+/-512 days. Overall, MACE rate was significantly lower in the DES group (16.6% vs 26.4%, P=0.02). The beneficial effect was driven by a reduction of death (6.0% vs 9.4%, P=0.11), MI (2.7% vs 3.8%, P=0.33) and target vessel revascularization after DES implantation (9.0 % vs 15.1%, P=0.11). After correcting for independent predictors of adverse events, the adjusted hazard ratios (HRs) for the risk of mortality and myocardial infarction after DES implantation relative to BMS implantation were 0.99 (95% CIs 0.30-3.21, P=0.98) and 0.59 (95% CIs 0.01-3.45, P=0.56), respectively. The adjusted HR for two-year MACE was 0.50 (95 CIs 0.25-1.02), P=0.056, mainly driven by a statistical significant reduction of TVR (HR 0.30 [95 CIs 0.11-0.82], P=0.018]. CONCLUSIONS: Patients presenting with ULMCA disease, who are treated with DES have a significant reduction in the rate of target lesion revascularization with no increased risk of death or myocardial infarction.

Activity profile of elite goalkeepers during football match-play.

AIM: The purpose of this study was to analyse the activities of a goalkeeper during a match and to identify the distances covered at different velocities between the first and second halves. METHODS: Sixty-two goalkeepers belonging to 28 teams in the English Premier League were monitored over 109 matches using the Prozone(R) system. All values are averages and standard deviations. Pearson product moment correlation was used to examine selected bivariate correlation. To analyze the differences in averages, the paired Student t-test was used. The analyses were performed using SPSS (v.13.0; SPSS, Inc. Chicago, IL). The significance level was set at P<0.05. RESULTS: Mean total distance covered by the goalkeeper during the match was 5 611+/-613 m. There were no differences between distances covered in the first and second halves. The distance covered at high-intensity was 56+/-34 m, while the distance covered sprinting was 11+/-12 m. The average number of high speed actions was 10+/-6, with a total range between 0 and 40. The goalkeeper walked during 73% of the match, while spending just 2% moving at high-intensity. CONCLUSION: The goalkeeper's physical activity was not as great as that of the field players, but the high-intensity actions carried out will be very decisive in the final result of the match.

Proteases and chronic leg ulcers.

AIM: The aim of this study was to compare the effect of cadexomer on reducing wound surface area of leg ulcers compared to that obtained in a group patients whose ulcers were treated by compression therapy. METHODS: For each ulcer group, wound surface area was calculated at day 0 and after 28 days of treatment: this allowed to calculate the average wound surface area reduction, the percent reduction in wound size, as well as the weekly wound size reduction index. RESULTS: In the cadexomer-treated ulcers the total wound area reduction was 9.67 cm(2)/week, with a weekly wound size reduction index per patient of 0.96 cm(2); in the controls (compression therapy-treated patients) the total wound area reduction was 6.11 cm(2)/week, with a weekly reduction index per patient of 0.61 cm(2). At the end of treatment, in the group of patients whose ulcers were treated with cadexomer ointment the average wound size reduction was 43%, whereas in the control-treated patient group the average wound size reduction was 28%. CONCLUSION: These data suggest that cadexomer can play an important role in the healing of chronic leg ulcers.

Treatment of anastomotic stenoses of peripheral bypass grafts with cutting balloon angioplasty.

PURPOSE: The aim of this study was to assess the efficacy of cutting-balloon angioplasty (CBA) in the treatment of anastomotic stenoses of peripheral arterial bypass grafts. MATERIALS AND METHODS: Seventeen patients (12 men and five women; age range 54-79 years, mean age 66.5) with stenosis or occlusion at the proximal or distal anastomoses of peripheral bypass grafts were treated with CBA. The diagnosis of stenosis was based on clinical and colour-Doppler ultrasound findings and confirmed by angiography with measurement of the intraluminal transstenotic pressure gradients. The diameter of the selected cutting balloon was 1-mm smaller than the vessel distal to the anastomosis and, in the event of suboptimal outcome, the procedure was completed with repeat dilatation with a larger standard balloon (+1 mm). RESULTS: Technical success was obtained in 100% of cases. In three patients, CBA was performed after locoregional thrombolysis. No patient required stent placement or emergency surgery due to the presence of residual stenosis, suboptimal outcome or dissection. No complication occurred either during or after the procedure. During a mean follow-up period of 10.4 months (range 5-21 months), two restenoses developed at 9 and 7 months, which were treated with the same technique; in one patient with recurrent bypass occlusion at 5 months, a new bypass was created surgically owing to contraindications for locoregional thrombolysis. Cumulative primary patency at 12 and 18 months was 82.35%, whereas the two cases of restenoses treated with repeat CBA underwent further follow-up at 10 and 7 months, respectively. CONCLUSIONS: Our data confirm the efficacy of CBA in the treatment of anastomotic stenoses of peripheral arterial bypass grafts.

Comparison between intravenous iloprost and vasoactive drugs in limb ischemia IIB severe. A retrospective analysis.

AIM: Peripheral arterial vascular disease is caused by atherosclerosis. The severity of peripheral arterial disease is closely associated with the risk of myocardial infarction, ischemic stroke, and death from vascular causes. Approximately 1/3 of patients with peripheral disease have typical claudication. The goals of treatment for patients with claudication are to relieve their exertional symptoms, increase walking capacity, and globally improve their quality of life. In the peripheral arterial disease IIB stage, with claudication < 100 meters, no useful medical therapy is available. Aim of this investigation is to analyse, in a retrospective way, two groups of patients treated with vasodilator drugs or iloprost. METHODS: Data from 99 patients were registered: 79 patients were treated regularly with iloprost, administered for 10 days, 4 times a year. The 2nd group of 20 patients was treated with buflomedil 600 mg/die or pentoxifilline 1200 mg/die. The end point was the pain-free walking distance (PFWD) evaluated with treadmill test at basal conditions and at 3, 6, 12, 18 months. RESULTS: Treatment groups showed no difference as for occurrence of by-pass surgery or endovascular procedures during follow-up. Improvement in PFWD was significantly more evident in patients treated with iloprost as compared with the control (P = 0.02). Even considering the PFWD variations at any follow-up step vs basal value, we always observed a statistically significant difference with iloprost, P < 0.0001. CONCLUSIONS: The study suggests that iloprost may be effective in improving walking distance in severe IIB stage peripheral arterial vascular disease.

l-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase. A subgroup of strictly related enzymes specialized for different functions.

Serine hydroxymethyltransferase (SHMT) is a member of the fold type I family of vitamin B6-dependent enzymes, a group of evolutionarily related proteins that share the same overall fold. The reaction catalysed by SHMT, the transfer of Cbeta of serine to tetrahydropteroylglutamate (H4PteGlu), represents in the cell an important link between the breakdown of amino acids and the metabolism of folates. In the absence of H4PteGlu and when presented with appropriate substrate analogues, SHMT shows a broad range of reaction specificity, being able to catalyse at appreciable rates retroaldol cleavage, racemase, aminotransferase and decarboxylase reactions. This apparent lack of specificity is probably a consequence of the particular catalytic apparatus evolved by SHMT. An interesting question is whether other fold type I members that normally catalyse the reactions which for SHMT could be considered as 'forced errors', may be close relatives of this enzyme and have a catalytic apparatus with the same basic features. As shown in this study, l-threonine aldolase from Escherichia coli is able to catalyse the same range of reactions catalysed by SHMT, with the exception of the serine hydroxymethyltransferase reaction. This observation strongly suggests that SHMT and l-threonine aldolase are closely related enzymes specialized for different functions. An evolutionary analysis of the fold type I enzymes revealed that SHMT and l-threonine aldolase may actually belong to a subgroup of closely related proteins; fungal alanine racemase, an extremely close relative of l-threonine aldolase, also appears to be a member of the same subgroup. The construction of three-dimensional homology models of l-threonine aldolase from E. coli and alanine racemase from Cochliobolus carbonum, and their comparison with the SHMT crystal structure, indicated how the tetrahydrofolate binding site might have evolved and offered a starting point for further investigations.

Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay.

An enzymatic method for determination of B6 vitamers is presented. In this method pyridoxal 5'-phosphate is used to activate aposerine hydroxymethyltransferase to form the catalytically active holoenzyme. The active serine hydroxymethyltransferase, and two other enzymes that form a metabolic cycle, convert serine to glycine and CO2 with the concomitant production of two equivalents of NADPH. The rate of the cycle is directly proportional to the amount of active holoserine hydroxymethyltransferase, which is a measure of the amount of pyridoxal 5'-phosphate in the original sample. The cycle operates about 50 times per minute giving a 100-fold enhancement of NADPH production with respect to original pyridoxal 5'-phosphate content. Other B6 vitamers are converted to pyridoxal 5'-phosphate by a preincubation with a combination of pyridoxal kinase and pyridoxine 5'-phosphate oxidase. A complete analysis of B6 vitamers can be completed in less than 1 h and the assay is linear in the 2- to 50-pmol range of pyridoxal 5'-phosphate. The method is applied to the determination of the B6 vitamer pools in extracts of Escherichia coli. The results show that the pool of pyridoxal 5'-phosphate that is not bound to proteins is large enough to account for product inhibition of both pyridoxal kinase and pyridoxine 5'-phosphate oxidase.

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution.

Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E. coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution. One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN. A protein conformational change has occurred that partially closes the active site. The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN. When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site. A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent.

Enzymatic determination of homocysteine in cell extracts.

Determination of homocysteine levels in cells and serum is important because high homocysteine is a risk factor for cardiovascular disease. The currently used methods for homocysteine analysis either are time consuming or rely on the use of expensive equipment. Described in this study is an enzymatic assay that determines levels of homocysteine in multiple samples in less than 30 min at levels from 5 to 50 pmol using only a spectrophotometer. The reproducibility of the assay is consistent with the other methods currently used. A second assay, that is about 5-fold more sensitive, follows the enzymatic catalyzed solvent exchange of protons on glycine, which requires a scintillation counter. Both the spectrophotometric and the radiometric methods are based on the conversion of 5-methyltetrahydrofolate to tetrahydrofolate by methionine synthase. The tetrahydrofolate is formed in stoichiometric amounts to the homocysteine in the sample. In the spectrophotometric method the tetrahydrofolate is used at catalytic levels by three enzymes to form a metabolic cycle that generates NADPH from NADP(+). In the radiometric assay tetrahydrofolate is required for the enzymatic exchange of the pro 2S proton of glycine with solvent. L-Cysteine, at levels more than 30-fold higher than the upper level of homocysteine used in these assays, does not give any measurable response.

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with FMN at 1.8 A resolution.

BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino acid metabolism. The enzyme oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the 4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently bound to each subunit. A high degree of sequence homology among the 15 known members of the PNPOx family suggests that all members of this group have similar three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli has been determined to 1.8 A resolution. The monomeric subunit folds into an eight-stranded beta sheet surrounded by five alpha-helical structures. Two monomers related by a twofold axis interact extensively along one-half of each monomer to form the dimer. There are two clefts at the dimer interface that are symmetry-related and extend from the top to the bottom of the dimer. An FMN cofactor that makes interactions with both subunits is located in each of these two clefts. CONCLUSIONS: The structure is quite similar to the recently deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also, remarkably, shares a common structural fold with the FMN-binding protein from Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E. coli PNPOx structure permits predictions to be made about residues involved in substrate binding and catalysis. These predictions provide testable hypotheses, which can be answered by making site-directed mutants.

Plasma levels of E-selectin in normolipemic and hyperlipemic arteriopathic patients after vasoactive and lipid-lowering treatment.

BACKGROUND: The authors studied the plasma levels of E-selectin in a group of arteriopathic patients, before and after vasoactive and lipid-lowering treatment. METHODS: The series consisted of 73 subjects (53 males, 20 females, aged 54 +/- 9 suffering from occlusive peripheral arteriopathy; 21 subjects with total cholesterol (TC) below 200 mg/dl were considered as normolipemics (group A); 24 subjects with TC between 200 and 240 mg/dl, mild hypercholesterolemics (group B); 18 with TC above 240 mg/dl, severe hypercholesterolemic (group C); 10 subjects who had high triglyceride values (above 200 mg/dl), (group D); 12 normal controls were also considered. All patients underwent a vasoactive treatment for 15 days; group B also underwent a standard hypolipidic diet (phase I NCEP, lipid 30% die) groups C and D underwent the same diet associated respectively with simvastatin (200 mg/die) and bezafibrate (400 mg/die). For each sample of plasma before and after treatment the determination of levels of E-selectin was carried out by an immunoenzymatic method (kit ELISA Amersham). RESULTS: In groups A-B-C-D a reduction of the plasma levels of E-selectin was found, which was significant (p < 0.05), for group C and D, compared to controls. In groups A-B-D significant changes of E-selectin were not found after treatment; in group C the difference between the values after treatment and at baseline was significant (p < 0.05). CONCLUSIONS: The reduction of the E-selectin plasma levels was proportional to the values of total cholesterol and triglycerides; the chronicity and the dyslipidemia may be responsible for the basal diminished biosynthetic endothelial function; in the severe hypercholesterolemics the lipid-lowering treatment caused a significant decrease of E-selectin, due to a probable reduced endothelial irritation dependent on the hyperlipemic stress.

Structure of human erythrocyte catalase.

Catalase (E.C. 1.11.1.6) was purified from human erythrocytes and crystallized in three different forms: orthorhombic, hexagonal and tetragonal. The structure of the orthorhombic crystal form of human erythrocyte catalase (HEC), with space group P2(1)2(1)2(1) and unit-cell parameters a = 84.9, b = 141.7, c = 232.5 A, was determined and refined with 2.75 A resolution data. Non-crystallographic symmetry restraints were employed and the resulting R value and R(free) were 0.206 and 0.272, respectively. The overall structure and arrangement of HEC molecules in the orthorhombic unit cell were very similar to those of bovine liver catalase (BLC). However, no NADPH was observed in the HEC crystal and a water was bound to the active-site residue His75. Conserved lattice interactions suggested a common growth mechanism for the orthorhombic crystals of HEC and BLC.