Bacillus subtilis ResA is a thiol-disulfide oxidoreductase involved in cytochrome c synthesis

Covalent attachment of heme to apocytochromes c in bacteria occurs on the outside of the cytoplasmic membrane and requires two reduced cysteinyls at the heme binding site. A constructed ResA-deficient Bacillus subtilis strain was found to lack c-type cytochromes. Cytochrome c synthesis was restored in the mutant by: (i) in trans expression of resA; (ii) deficiency in BdbD, a thiol-disulfide oxidoreductase that catalyzes formation of an intramolecular disulfide bond in apocytochrome c after transfer of the polypeptide across the cytoplasmic membrane; or (iii) by addition of the reductant dithiothreitol to the growth medium. In vivo studies of ResA showed that it is membrane-associated with its thioredoxin-like domain on the outside of the cytoplasmic membrane. Analysis of a soluble form of the protein revealed two redox reactive cysteine residues with a midpoint potential of about -340 mV at pH 7. We conclude that ResA, probably together with another thiol-disulfide oxidoreductase, CcdA, is required for the reduction of the cysteinyls in the heme binding site of apocytochrome c.     

Design, synthesis, and biological evaluation of HIV/FIV protease inhibitors incorporating a conformationally constrained macrocycle with a small P3' residue

A series of norstatine-based HIV/FIV protease inhibitors incorporating a 15-membered macrocycle as a mimic of the tripeptide (Ala-Val-Phe), a motif with a small P3' residue elective against the FIV protease and the drug-resistant HIV proteases, has been synthesized. It was found that the macrocycle is important to the overall activity of the inhibitors. Certain inhibitors were developed expressing low nanomolar inhibitory activity against the HIV/FIV proteases and they are also effective against some drug-resistant as well as TL3-resistant HIV proteases.     

Structure-activity studies of FIV and HIV protease inhibitors containing allophenylnorstatine

The interaction of P1 and P3 side chains with the combining S1 and S3 hydrophobic subsites of HIV and FIV proteases has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1', Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most effective inhibitor against FIV protease, and it contains Phe at P3, but no P3' residue. VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule.     

Nitric oxide production in the hypoxic lung

Nitric oxide (NO) is a potent vasodilator and inhibitor of vascular remodeling. Reduced NO production has been implicated in the pathophysiology of pulmonary hypertension, with endothelial NO synthase (NOS) knockout mice showing an increased risk for pulmonary hypertension. Because molecular oxygen (O2) is an essential substrate for NO synthesis by the NOSs and biochemical studies using purified NOS isoforms have estimated the Michaelis-Menten constant values for O2 to be in the physiological range, it has been suggested that O2 substrate limitation may limit NO production in various pathophysiological conditions including hypoxia. This review summarizes numerous studies of the effects of acute and chronic hypoxia on NO production in the lungs of humans and animals as well as in cultured vascular cells. In addition, the effects of hypoxia on NOS expression and posttranslational regulation of NOS activity by other proteins are also discussed. Most studies found that hypoxia limits NO synthesis even when NOS expression is increased.     

Synaptic pattern of sex complements and sperm head malformation in X-autosome translocation carrier bulls

Testicular activity and semen characteristics of bulls carrying an X-autosome translocation t(Xp +;23q-) revealed all stages of spermatogenesis although their semen consisted of few and, exclusively, of malformed spermatozoa. Chromosome painting on metaphase spreads of their mother and synaptonemal complex analysis on these and normal bulls were carried out to test whether the location and meiotic pairing behaviour of the rearranged segments could have contributed to the sperm head malformation and oligospermia in our X-autosome translocation (X-AT) carrier bulls. Spermatocytes of X-AT carriers displayed the rearranged chromosomes in a univalent-trivalent association, with 23q- always remaining as a univalent and Xp + in synapsis with normal chromosome 23 and the Y chromosome. Chromosome painting studies to test whether the total absence of meiocytes showing a quadrivalent is due to the non-reciprocal nature of this translocation, identified Xp sequence homology with the distal end of 23q- confirming its relocation to the terminal segment of 23q-. Our synaptonemal complex analyses also confirmed that the bovine pseudo-autosomal region (PAR) is at the distal ends of Xq and Yp and further revealed that over 85% of spermatocytes of X-AT carriers (and up to 13% of spermatocytes of normal bulls) sustain a Y-axis break adjacent to the PAR. Although the exact cause of a Y-axis break in bovine spermatocytes is not known at present, we believe that the break and possible loss of Yq in such high proportions of spermatocytes of X-AT carriers could have contributed to the sperm head malformation and oligospermia in our X-AT carrier bulls.     

MHC-I-restricted presentation of HIV-1 virion antigens without viral replication

Dendritic cells and macrophages can process extracellular antigens for presentation by MHC-I molecules. This exogenous pathway may have a crucial role in the activation of CD8+ cytotoxic T lymphocytes during human viral infections. We show here that HIV-1 epitopes derived from incoming virions are presented through the exogenous MHC-I pathway in primary human dendritic cells, and to a lower extent in macrophages, leading to cytotoxic T-lymphocyte activation in the absence of viral protein synthesis. Exogenous antigen presentation required adequate virus-receptor interactions and fusion of viral and cellular membranes. These results provide new insights into how anti-HIV cytotoxic T lymphocytes can be activated and have implications for anti-HIV vaccine design.     

Azoospermie et microinjection

In cases of azoospermia, testicular biopsy combined with cryopreservation of spermatozoa allows ICSI to be performed under good conditions. In this study, the authors present their results by emphasizing three major aspects:

- Retrieval of testicular spermatozoa by open biopsy or percutaneous needle aspiration: 40 patients with obstructive azoospermia underwent epididymal or testicular retrieval by open biopsy and 37 by percutaneous needle aspiration. All biopsies were positive. 133 patients with nonobstructive azoospermia underwent percutaneous needle aspiration and spermatozoa were successfully retrieved from 50 patients (38%).

- The freezing process was performed with a cryoprotective medium devoid of egg yolk after dilaceration of the testicular tissue using two sterile glass slides. No significant difference in the outcome of the ICSI procedure was observed between fresh and frozen-thawed spermatozoa. In cases of obstructive azoospermia, 13 pregnancies out of 41 ICSI cycles (31%) were obtained with the use of fresh testicular or epididymal spermatozoa and 24 pregnancies out of 115 ICSI cycles (20%) were obtained with the use of cryopreserved spermatozoa. In cases of non-obstructive azoospermia, 6 pregnancies out of 31 ICSI cycles (19%) were obtained with the use of fresh testicular spermatozoa and 12 pregnancies out of 33 ICSI cycles (36%) were obtained with the use of frozen-thawed spermatozoa.

- After the freezing-thawing process, the percentage motile testicular spermatozoa is very low (about 4%), with a weakly shaking motility making selection of live spermatozoa very long and difficult. The addition of pentoxifylline (3 mM) significantly increases this motility within 15 minutes, as 30% of spermatozoa have a progressive motility. Selection of viable motile spermatozoa is therefore easier and more rapid. Fertilization and pregnancy rates are comparable to those generally reported. No malformation was observed on 51 live births.