Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry

A protein with a molecular mass of approximately 62.10(3), derived from open reading frame 2 (ORF-2) of the hepatitis E virus (HEV: Burma strain), was expressed in a baculovirus expression vector and purified to homogeneity. The recombinant 62 kDa protein appeared to be a doublet, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tryptic digestion in conjunction with laser desorption mass spectrometry (LD-MS) and sequence analysis of the tryptic peptides indicated that the amino terminus was blocked, although no proteolytic degradation occurred. The determined internal sequences of peptides were in agreement with the predicted ORF-2 protein. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) resolved the doublet proteins into two major components with molecular masses of 56548.5 and 58161.4. Confirmation of the amino terminus of the molecule by LD-MS post-ion decay enabled us to tentatively assign the carboxyl terminus of each species at residues 540 and 525. Sequencing of the intact protein by automated carboxyl terminal sequencing confirmed that the carboxyl terminus was truncated and that the sequence assignment predicted by LC-MS was correct.     

Myocardial uptake and release of lactate after high dose neurolept endotracheal intubation in coronary surgery

To evaluate the relationship between the hemodynamic and ECG variables used in routine surveillance of coronary surgery and myocardial lactate metabolism, 23 middle-aged, male, beta 1-blocked patients about to undergo coronary surgery were monitored before and after endotracheal intubation with high dose (30 micrograms/kg) fentanyl-midazolam anesthesia. The induction of anesthesia was followed by a mean arterial pressure decrease (from 98 +/- 4 to 76 +/- 3 mm Hg) and heart rate increase (from 53 +/- 3 to 66 +/- 2 beats/min). After intubation the hemodynamic variables were stable except for a further, transient increase in heart rate (to 69 +/- 2 beats/min). The myocardial uptake of lactate decreased after intubation, from 48 +/- 5 mumol/min to a lowest level of 24 +/- 3 mumol/min. A lactate release was exhibited in 7/23 patients (30%). No ST-segment changes were observed. The correlation between the myocardial lactate uptake/release and hemodynamic or ECG variables was unimpressive or non-existent (r < or = 0.20). Thus, a reduced uptake and even a release of lactate occurred irrespective of the ST-segment, heart rate, or systemic or pulmonary artery pressures. In conclusion, endotracheal intubation in patients with coronary disease was consistently (17/23 patients) followed by a reduced myocardial uptake of lactate, in spite of high dose neurolept anesthesia and beta 1-blockade. This metabolic event was not consistently related to hemodynamic changes.     

Characterization of a multicatalytic proteinase complex (20S proteasome) from Trypanosoma brucei brucei

African trypanosomes are tsetse-transmitted protozoan parasites that cause sleeping sickness in humans and 'Nagana' in animals. A high relative molecular mass multicatalytic proteinase complex (MCP) was purified and biochemically characterized from the cytosolic fraction of Trypanosoma brucei brucei. The isolation procedure consisted of fractionation of the lysate by high speed centrifugation, chromatography on Q-sepharose molecular sieve filtration on Sephacryl S-300, chromatography on HA-Ultrogel and glycerol density gradient centrifugation (10-40%). The final enzyme preparation yielded a single protein band corresponding to a relative molecular mass of 630 kDa on a non-denaturing polyacrylamide gel. The enzyme hydrolyses a wide range of peptide substrates characteristic of chymotrypsin-like, trypsin-like, peptidylglutamylpeptide-hydrolysing activities determined by fluorogenic peptides, Z-Gly-Gly-Leu-NHMec, Z-Arg-Arg-NHMec and Z-Leu-Leu-Glu-beta NA, respectively. The enzyme was found to have a wide variation in pH optimal activity profile, with optimum activity against Z-Gly-Gly-Leu-NHMec at 7.8, Z-Arg-Arg-NHMec at pH 10.5 and Z-Leu-Leu-Glu-beta NA at pH 8.0, showing that the different activities are distinct. The enzyme hydrolysed oxidized proteins. In addition, the chymotryptic and trypsin-like activities were susceptible to inhibition by peptide aldehyde inhibitors with variable inhibition effects. The study demonstrates the presence of a non-lysosomal proteasome pathway of intracellular protein degradation in the bloodstream form of T. b. brucei. Further, the ability of the enzyme to hydrolyse most oxidized proteins, and the high immunogenicity exhibited suggests a possible involvement of the enzyme in pathogenesis of the disease.     

Quantitation of radiolabeled antibody binding to cells by thin-layer chromatography

Thin layer chromatography (TLC) was used to monitor binding of radiolabeled antibodies to cells. Labeled antibodies were reacted with cells and aliquots chromatographed on serum-blocked, ITLC strips. The cell-antibody complexes remain at the origin and unbound antibody migrates with the solvent front. The antibody binding was estimated from the ratio of radioactivity at the origin compared to the total applied. Separations are completed in about 10 min. This method does not use centrifugation or wash steps, and provides an inexpensive and self-contained system to evaluate radioligand binding. Cell binding assay results using this method are approximately the same as those obtained using bead- or cell-type assays.     

Design and synthesis of new potent C2-symmetric HIV-1 protease inhibitors. Use of L-mannaric acid as a peptidomimetic scaffold

A study on the use of derivatized carbohydrates as C2-symmetric HIV-1 protease inhibitors has been undertaken. L-Mannaric acid (6) was bis-O-benzylated at C-2 and C-5 and subsequently coupled with amino acids and amines to give C2-symmetric products based on C-terminal duplication. Potent HIV protease inhibitors, 28 Ki = 0.4 nM and 43 Ki = 0.2 nM, have been discovered, and two synthetic methodologies have been developed, one whereby these inhibitors can be prepared in just three chemical steps from commercially available materials. A remarkable increase in potency going from IC50 = 5000 nM (23) to IC50 = 15 nM (28) was observed upon exchanging -COOMe for -CONHMe in the inhibitor, resulting in the net addition of one hydrogen bond interaction between each of the two -NH- groups and the HIV protease backbone (Gly 48/148). The X-ray crystal structures of 43 and of 48 have been determined (Figures 5 and 6), revealing the binding mode of these inhibitors which will aid further design.     

Viral hepatitis infection and response to the hepatitis B vaccine in hemodialyzed patients

Data from 219 hemodyalized patients receiving attention in our Hospital and other private centers in our city are shown. Mean age was 46.9 (range: 14-85), and 132 were male; mean time under dialysis was 20 months, and subjects received an average of 5 transfusions per patient year. Serological reactivity to HBs Ag, Anti HBs and IgG anti HBc by ELISA were investigated in all of them, and anti HCV by second generation enzimo-immunoassay (EIA II) in 73 HBe Ag/anti HBe system were determined in HBs Ag positive patients and those reactive to anti HCV (EIA II) were confirmed by LIA (immunoblotting of synthetic peptides LIA-TEK Organos Teknica). Recombinant anti HBV vaccine 40 mcg at 0-1 and six month were received by 81 cases without HBV markers in their sera and a protective response was considered when anti HBs titration of 10 mU/ml or more were obtained two months later. Prevalence for anti HBc and anti HBs were 38.8% respectively and that for HBs Ag was 21% with 78% of them reactive for HBs Ag. True reactivity for anti HCV (confirmed by LIA) was present in 35.6%, but it was 9.7% in our Hospital and 54.8% in private units (p < 0.0002). Anti HBs titration was done in 69/81 patients who received anti HBV vaccine, and a protective response in 49% were obtained; the other 12 patients underwent acute hepatitis B during the vaccination period.(ABSTRACT TRUNCATED AT 250 WORDS)