BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility.

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1muM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.     

Association of single nucleotide polymorphisms of the interleukin-10 promoter gene and susceptibility to primary biliary cirrhosis: immunogenetic differences in Italian and Japanese patients

Several lines of data suggest that genetic factors play an important role in the onset and/or progression of primary biliary cirrhosis (PBC). Since PBC is an autoimmune disease, it is reasoned to assume that genes encoding cytokines may confer susceptibility to disease. Amongst these factors, interleukin-10 (IL-10) has received significant attention. The promoter region of IL-10 gene has three single nucleotide polymorphisms (SNPs) at positions -1082, -819 and -592. To elucidate the association of the three SNPs of IL-10 promoter region with susceptibility of PBC in two different genetic populations, 159 unrelated patients with PBC (94 Italian and 65 Japanese) and 143 local controls (72 Italian and 71 Japanese) were enrolled. SNPs were determined using allele-specific PCR/RFLP. In Italian PBC patients, the frequency of homozygosity for G/G at position -1082 was significantly higher than that of local controls (p < 0.041, OR = 2.44, 95% C.I.; 1.02-5.86). The frequencies of haplotype GCC in PBC patients, possibly linked to higher IL-10 production, were also significant higher than local controls (p < 0.033). However, in Japanese population, there were no significant differences in the three SNPs and haplotypes between PBC patients and controls. Excessive production of IL-10 may play an important role in some populations in modulating the onset of PBC. Further, immunogenetic studies of PBC should take into account ethnic and geographic variations; this makes such studies in heterogeneous population, like the USA, more difficult.     

Lycium barbarum polysaccharide fraction associated with photobiomodulation protects from epithelium thickness and collagen fragmentation in a model of cutaneous photodamage

Lasers in Medical Science - Ultraviolet radiation (UVR) is the major etiologic agent of cutaneous photoaging, and different strategies are used to prevent and treat this condition. The...     

Quality of life outcomes after minimally invasive repair of pectus excavatum utilizing a new set of metallic bars and stabilizers

Background/PurposeThe aim of the study was to evaluate the postoperative quality of life (QoL) of patients who underwent minimally invasive repair of pectus excavatum (MIRPE) with a newly designed bar and bar stabilizers.MethodsWe conducted a prospective randomized study in which patients were operated either with standard perpendicular stabilizers (control group) or with the newly designed oblique stabilizers (intervention group). All patients were evaluated 6 months after the operation with the Pectus Excavatum Evaluation Questionnaire (PEEQ).ResultsThere were 16 patients in the control group and 14 in the intervention group. Mean age was 17 (SD: 3.3, range 14–27) years. There were no demographic differences between groups. Two patients in the control group and one in the intervention group were repaired with two bars instead of one. There was one reoperation in each group. There was a significant difference between the pre- and postoperative scores, in both groups, in the patient body image domain (control group: 9.5 to 3; p < 0.01; intervention group 10 to 3; p < 0.01), as well as in the psychosocial domain (control group: 13.5 to 24, p < 0.01; intervention group: 15 to 24, p < 0.01). With regards to the patients' perception of physical difficulties before and after MIRPE, the difference between pre- and postoperative scores was greater in the intervention group (8 to 12, p < 0.01) than in the control group (10 to 11, p = 0.04). The mean length of stay was 4.5 and 5 days in the intervention group and the control group, respectively.ConclusionOur study showed that patients who underwent MIRPE with the newly designed bars and stabilizers had non-inferior outcomes than patients reported in the literature who underwent MIRPE with standard bars and stabilizers. We found slightly better outcomes in patients in the intervention group compared to the control group, but larger studies will be needed to confirm if those differences are statistically significant.Level of evidenceII     

Percutaneous transluminal coronary angioplasty - Histopathological analysis of nine necropsy cases

Summary From 1982 to 1984 nine of 300 patients undergoing transluminal coronary angioplasty died. The nine coronary arteries and one saphenous aorto-coronary by-pass graft affected by angioplasty were studied by light microscopy. The following types of lesions were found, frequently in association: rupture of the plaque, circumscribed or reaching to the intimal layer or extending beyond it, dissections (fissures) between arterial layers, intra-plaque haemorrhage, plaque emboli and thrombosis. In two cases the therapeutic approach was considered to be clinically and pathologically successful; the patients survived 24 h (case 6) and forty days (case 4). Case 6 which presented recent lesions indicative of success showed, in contrast with the other non-successful cases, rupture affecting not only the initimal layer but also deeper structures of the arterial wall. There were also more extensive fissures. Case 4 which presented late alterations indicative of success showed a plaque fracture whose borders were kept apart by fibrous tissue. In conclusion, we believe that angioplasty allows the re-establishment of arterial blood flow by provoking deep intimal and medial rupture producing a small fissure between the arterial layers and a widening of the lumen; in cases with good late results these alterations cicatrize leaving a wider arterial lumen.     

Cyclosporine as prophylaxis for graft-versus-host disease: a randomized study in patients undergoing marrow transplantation for acute nonlymphoblastic leukemia

Seventy-five patients, 13 to 49 years of age, with acute nonlymphoblastic leukemia in first remission were treated with cyclophosphamide, fractionated total body irradiation, and marrow transplantation from an HLA-identical sibling and randomized to receive either cyclosporine (CSP) (n = 36) or methotrexate (MTX) (n = 39) as prophylaxis for graft-v-host disease (GVHD). All patients engrafted, and 22 who were given CSP and 21 who were given MTX, are alive at 20 to 47 (median, 35) months (P = .5). Engraftment as assessed by granulocyte recovery (P less than .0005) and platelet transfusion requirement (P = .01) was faster in patients on CSP. Twelve patients (33%) on CSP and 22 (56%) on MTX developed acute GVHD of grades II through IV (P = .07) and 15 of 30 on CSP and 14 of 32 on MTX that were at risk developed chronic GVHD. The most frequent causes of death were interstitial pneumonitis and marrow relapse of leukemia, which occurred with similar frequency in both groups. Beneficial effects observed in patients on CSP included less severe mucositis and shorter duration of hospitalization; adverse effects included renal function impairment and hypertension. These data confirm that CSP is a useful immunosuppressant in patients undergoing marrow transplantation but fail to show a significant improvement in survival as compared with the standard regimen of MTX.     

Adenocarcinoma arising from a strictureplasty site in Crohn's disease

The occurrence of small-bowel cancer in Crohn's disease (CD) is a rare event. The risk seems to be greatest in patients with long-standing disease. Strictureplasty has proved to be a valuable alternative in the management of Crohn's strictures of the small-bowel. Critics and proponents of strictureplasty for selected patients with smallbowel Crohn's disease have voiced their concerns about cancer risk in the strictured or strictureplasty site. To date, there has been no clear or detailed report of such an occurrence. The authors report the first case of small-bowel adenocarcinoma arising at the site of a previous strictureplasty. In this patient, biopsies of the strictures at the original operation confirmed CD and excluded both cancer and dysplasia. Malignancy occurred seven years later at a strictureplasty site. The main clinical sign associated with the adenocarcinoma was severe, persistent anemia. The authors conclude that the risk of adenocarcinoma developing at the site of a previous strictureplasty for CD, although small, is real.     

Phase I trial of interleukin-2 after unmodified HLA-matched sibling bone marrow transplantation for children with acute leukemia

Allogeneic bone marrow transplantation (BMT) for advanced acute leukemia is associated with a high risk of relapse. It is postulated that interleukin-2 (IL-2) administered after BMT might induce or amplify a graft-versus-leukemia effect and thereby reduce the relapse rate. To identify an IL-2 regimen for testing this hypothesis, a phase I trial of IL-2 (Roche) was performed in children in complete remission (CR) without active graft-versus-host disease (GVHD) off immunosuppressive agents after unmodified allogeneic matched-sibling BMT for acute leukemia beyond first remission. Beginning a median of 68 days after BMT, 17 patients received escalating doses of induction IL-2 (0.9, 3.0, or 6.0 x 10(6) IU/m2/d representing levels I, II, and III) for 5 days by continuous intravenous infusion (CIV). After 6 days of rest, they received maintenance IL-2 (0.9 x 10(6) IU/m2/d) for 10 days by CIV infusion. Levels I and II were well-tolerated, but, of 6 patients at level III, 1 developed pulmonary infiltrates, 1 developed hypotension (both resolved), and 1 died of bacterial sepsis and acute respiratory distress syndrome. Grade II acute GVHD developed in 1 patient at level I and 1 at level III. The maximum tolerated dose of induction IL-2 was level II. IL-2 induced lymphocytosis, with an increase in CD56+ and CD8+ cells. Ten patients remain in CR at 5+ to 67+ months. Thus, a regimen of IL-2 has been identified that did not induce a high incidence of acute GVHD when administered to children after unmodified allogeneic BMT. Its clinical activity will be assessed in a phase II trial.