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.     

Immunoglobulin heavy chain Diversity genes rearrangement pattern indicates that MALT-type gastric lymphoma B cells have undergone an antigen selection process

Gastric MALT lymphoma usually develops from chronic gastritis, the vast majority of which (>90%) is associated with Helicobacter pylori infection. We sequenced the third complementarity determining region (CDR3) of immunoglobulin heavy chain genes in 19 gastric MALT lymphoma clones to determine the pattern of variable (V), diversity (D) and joining (J) gene utilization during immunoglobulin gene rearrangement.
DNA was extracted from paraffin-embedded sections and the rearranged CDR3 regions were amplified using a semi-nested polymerase chain reaction (with primers complementary to the conserved framework-three segment of the variable region [FR3A] and J regions). The DNA used for cloning and sequencing was obtained after purification of monoclonal bands excised from polyacrylamide gels. The N-D-N region specific to each clone was compared with known germline D sequences.
Similarly to that observed in normal and leukaemic B cells, our series of gastric MALT lymphomas showed apparent preferential utilization of genes from the DXP family. In two cases no similarity between the CDR3 nucleotide sequences of the neoplastic clones and the known germline D sequences could be found. In 10/19 analysed alleles the lymphoma B-cell clones appeared to contain two D gene segments (D-D recombination), a rare occurrence in normal individuals but one which has been described as a significant event in the determination of idiotype expression and antigen-binding affinity. Remarkably, despite the use of different D and J segments, the resultant amino acid sequences matched in two patients, suggesting the presence of a common selecting antigen.
The observed pattern of D gene rearrangement suggests that MALT lymphoma B-cell clones have undergone antigen selection, which seems to indicate that the antigen stimulation plays a pivotal role in the development of the lymphoma.     

Separation of aceclofenac and diclofenac in human plasma by free zone capillary electrophoresis using N-methyl-D-glucamine as an effective electrolyte additive.

Aceclofenac (A) and diclofenac (D) are effective non-steroidal anti-inflammatory drugs (NSAIDs) derived from the phenylacetic acid with pronounced antirheumatic, anti-inflammatory, analgesic and antipyretic properties. Our work proposes a new, fast-free zone capillary electrophoresis method for the simultaneous determination of aceclofenac and diclofenac in human plasma. The effect of increasing concentrations of N-methyl-D-glucamine organic base on borate run buffer was investigated. A good separation was achieved using a 40 cm x 75 microm uncoated silica capillary, 300 mmol/l sodium borate buffer, 200 mmol/l N-methyl-D-glucamine, pH 8.9, in about 3 min. Moreover, the plasma sample pre-treatment procedure was examined: acidic precipitants such as trichloroacetic acid (TCA), metaphosphoric acid (MPA), perchloric acid (PCA) or 5-sulphosalicylic acid (SSA) cause a total loss of analytes while acetonitrile allows a recovery of 97-98% of both compounds. Its simplicity and rapidity and the low analysis costs demonstrate that our method is a reliable and efficient mean for the comprehensive determination of aceclofenac and diclofenac in human plasma when pharmacokinetics studies are required.     

Arthroscopic Latissimus Dorsi Transfer

The patient is placed in lateral decubitus. A 6-cm incision made in the axilla allows access to the latissimus dorsi tendon and its neurovascular pedicle. Holding the arm in internal rotation, the surgeon detaches sharply the tendon off the humeral shaft and then reinforces it with wrapping sutures. Pulling the free limbs of the sutures exposes the under surface of the muscle and helps to identify the neurovascular pedicle. Special lighting retractors suited for a large diameter scope are helpful. Mobilization is completed when 2 cm of the tendon crosses the posterior edge of the acromion. The standard lateral portal is used for visualization. A silicon drain tube stiffened by a Wissinger rod is advanced from the posterior portal under direct visualization in the space between teres minor and deltoid, exiting in the auxiliary incision. A suture loop passed down the tube retrieves the tendon sutures out the posterior portal. These are then moved out the anterior portal, thus pulling the tendon over the tuberosity. The first anchor is inserted at the anterior aspect of the greater tuberosity, close to the articular cartilage and long head of the biceps tendon. Two to 3 anchors are inserted fixing the tendon to the tuberosity until it is stable.     

Limited role of aortic size in the genesis of acute type A aortic dissection

Objective: Increased dimension of the aortic root and proximal aorta is considered a significant risk factor for catastrophic events that involve the ascending aorta. The objective of this study was to determine the possible correlation between pre-dissection aortic diameter and the occurrence of Stanford type A aortic dissection. Methods: Samples of dissected ascending aortas were obtained from 220 patients at the time of their operation. Two groups were identified: patients with connective tissue disorders (Group 1, n = 94) and those without (Group 2, n = 126). Measurements of the true (intimal) lumen were conducted and extrapolated as reliable approximation of pre-dissection aortic diameter. The possible association of intimal diameter with anthropometric and demographic data was analyzed. Results: Median aortic diameter was, respectively, 41.8 and 41.3 mm for patients with and without connective tissue disorders (41.4 mm for the entire cohort). Data analysis indicated that 57% of patients had aortic diameter above 40 mm, while patients with frank aneurysm accounted only for 10%; this proportion was higher in Group 1 compared to Group 2 (17.2% vs 4.7%). Poor or no correlation was demonstrated between aortic size and any of the anthropometric or demographic variables essayed. Significant subgroup differences were found among patients with a history of cigarette smoking, hypertension, diabetes, chronic renal insufficiency, and bicuspid aortic valve. Conclusion: Although aortic diameter remains a strong indication for preventive surgery in patients with inherited connective tissue disorders, acute aortic dissection occurs rarely in the setting of true ascending aortic aneurysms, and despite normal or near-normal aortic size in more than one-third of subjects. Dissection superimposing on small aortic diameters can be regarded as an expression of substantial functional tissue susceptibility to aortic catastrophic events.     

The clinical phenotypes of juvenile bipolar disorder: toward a validation of the episodic-chronic-distinction.

BACKGROUND: Recent research has addressed the issue of subtyping juvenile bipolar disorder (JBD). Accordingly, we set out to find out, in a naturalistic sample of bipolar children and adolescents with mania and mixed mania, whether the most useful subtyping should be based on clinical features (elated vs. irritable) or course (episodic vs. chronic). METHODS: We studied 136 patients, 81 male patients (59.6%) and 55 female patients (40.4%), mean age 13.5 +/- 2.9 years, meeting the DSM-IV diagnosis of bipolar disorder, assessed by a structured clinical interview (Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version [K-SADS-PL]). RESULTS: Regarding course, 77 patients (56.6%) had an episodic course and 59 patients (43.4%) had a chronic course. Patients with chronic course were significantly younger, had an earlier onset of JBD, and presented a more frequent comorbidity with disruptive behavior disorders. According to the prevalent mood disturbance, 75 patients (55.1%) showed an elated and 61 patients (44.9%) showed an irritable mood. Elated mood was more frequent in patients with episodic course, whereas irritable mood was more frequent in the patients with chronic course. CONCLUSIONS: These findings suggest that chronic versus episodic course may be a putative differential feature. Further validation of such a distinction would require prospective studies, temperament evaluation, gender and neurobiologic approaches, and differential psychopharmacologic assignment and response.     

Hypophysiotrophic neurons are capable of altering the ratio of co-packaged neurohormones.

Neuronal dense-core vesicles provide a mechanism whereby peptide messengers are secreted in discrete quanta. Here we report on the capacity of rat hypophysiotrophic corticotropin releasing factor-41 neurons to alter the peptide content as well as the size of dense-core vesicles after removal of glucocorticoid negative feedback by adrenalectomy. We demonstrate, using quantitative immunoelectron microscopy, that long-term adrenalectomy induces a progressive increase in the ratio of vasopressin to corticotropin releasing factor-41-immunoreactive sites in the dense-core vesicle compartment. The intravesicular concentration of vasopressin appeared to be the variable parameter while that of the corticotropin releasing factor-41 remained stable at all survival times after adrenalectomy. Moreover, observations for up to 5 weeks indicate that adrenalectomy results in a progressive increase in the mean volume of dense-core vesicles to about three times normal. These results suggest that the quantal size and the composition of dense-core vesicles are subject to long-term modulation. The capacity of corticotropin releasing factor-41 neurons to alter dense-core vesicles could enhance or diminish the efficacy of the hypothalamohypophyseal communication underlying physiological adaptation to stress, as well as pathological changes.     

Human immunodeficiency virus isolates from asymptomatic homosexual men and from AIDS patients have distinct biologic and genetic properties.

Human immunodeficiency virus type 1 (HIV-1) isolates from asymptomatic homosexual men and AIDS patients were compared for their in vitro biologic and genetic properties. Most of the HIV-1 isolates from asymptomatic men, but not from AIDS patients, failed to infect CD4+ H9 cells and phytohemagglutinin-stimulated peripheral blood lymphocytes. In a longitudinal study, serial HIV-1 isolates obtained from men who seroconverted to HIV-1 and later developed AIDS were able to infect H9 cells. In contrast, longitudinal isolates from men who remained asymptomatic did not infect H9 cells. HIV-1 isolates from AIDS patients in general exhibited increased production of intracellular viral DNA, RNA, and protein as compared to isolates from asymptomatic men. Cells infected with HIV-1 isolates from asymptomatic men produced very little gp120, p24, and p55 proteins as compared to those from AIDS patients. The overall restriction patterns of HindIII, Sac-1, Pst-1, EcoR1, and BamH1 were very similar between HIV-1 isolates from asymptomatic men and those from AIDS patients. However, the restriction endonuclease pattern of BglII was quite distinct for isolates from asymptomatic men as compared to AIDS patients. Preliminary studies mapped a unique BglII site in the gag region of most of the isolates from asymptomatic men, approximately 2.0 kb from the 5' end. Thus, HIV-1 isolates from asymptomatic subjects and from AIDS patients have distinct biologic and genetic properties which may be related to the various clinical outcomes of HIV-1 infection.