Y chromosome lineage- and village-specific genes on chromosomes 1p22 and 6q27 control visceral leishmaniasis in Sudan

Familial clustering and ethnic differences suggest that visceral leishmaniasis caused by Leishmania donovani is under genetic control. A recent genome scan provided evidence for a major susceptibility gene on Chromosome 22q12 in the Aringa ethnic group in Sudan. We now report a genome-wide scan using 69 families with 173 affected relatives from two villages occupied by the related Masalit ethnic group. A primary ten-centimorgan scan followed by refined mapping provided evidence for major loci at 1p22 (LOD score 5.65; nominal p = 1.72 × 10⁻⁷; empirical p < 1 × 10⁻⁵; λ_S = 5.1) and 6q27 (LOD score 3.74; nominal p = 1.68 × 10⁻⁵; empirical p < 1 × 10⁻⁴; λ_S = 2.3) that were Y chromosome–lineage and village-specific. Neither village supported a visceral leishmaniasis susceptibility gene on 22q12. The results suggest strong lineage-specific genes due to founder effect and consanguinity in these recently immigrant populations. These chance events in ethnically uniform African populations provide a powerful resource in the search for genes and mechanisms that regulate this complex disease.     

Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes

All primate lentiviruses known to date contain one or two open reading frames with homology to the human immunodeficiency virus type 1 (HIV-1) vpr gene. HIV-1 vpr encodes a 96-amino-acid protein with multiple functions in the viral life cycle. These functions include modulation of the viral replication kinetics, transactivation of the long terminal repeat, participation in the nuclear import of preintegration complexes, induction of G2 arrest, and induction of apoptosis. The simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagm) contains a vpr homologue, which encodes a 118-amino-acid protein. SIVagm vpr is structurally and functionally related to HIV-1 vpr. The present study focuses on how three specific functions (transactivation, induction of G2 arrest, and induction of apoptosis) are related to one another at a functional level, for HIV-1 and SIVagm vpr. While our study supports previous reports demonstrating a causal relationship between induction of G2 arrest and transactivation for HIV-1 vpr, we demonstrate that the same is not true for SIVagm vpr. Transactivation by SIVagm vpr is independent of cell cycle perturbation. In addition, we show that induction of G2 arrest is necessary for the induction of apoptosis by HIV-1 vpr but that the induction of apoptosis by SIVagm vpr is cell cycle independent. Finally, while SIVagm vpr retains its transactivation function in human cells, it is unable to induce G2 arrest or apoptosis in such cells, suggesting that the cytopathic effects of SIVagm vpr are species specific. Taken together, our results suggest that while the multiple functions of vpr are conserved between HIV-1 and SIVagm, the mechanisms leading to the execution of such functions are divergent.     

Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in prokaryotic and eukaryotic systems: comparison with the recombinant enzyme purified from Escherichia coli.

Sulfation, catalyzed by members of the sulfotransferase enzyme family, is a major metabolic pathway which modulates the biological activity of numerous endogenous and xenobiotic chemicals. A number of these enzymes have been expressed in prokaryotic and eukaryotic systems to produce protein for biochemical and physical characterization. However, the effective use of heterologous expression systems to produce recombinant enzymes for such purposes depends upon the expressed protein faithfully representing the "native" protein. For human sulfotransferases, little attention has been paid to this despite the widespread use of recombinant enzymes. Here we have validated a number of heterologous expression systems for producing the human dopamine-metabolizing sulfotransferase SULT1A3, including Escherichia coli, Saccharomyces cerevisiae, COS-7, and V79 cells, by comparison of Km values of the recombinant enzyme in cell extracts with enzyme present in human platelets and with recombinant enzyme purified to homogeneity following E. coli expression. This is the first report of heterologous expression of a cytosolic sulfotransferase in yeast. Expression of SULT1A3 was achieved in all cell types, and the Km for dopamine under the conditions applied was approximately 1 microM in all heterologous systems studied, which compared favorably with the value determined with human platelets. We also determined the subunit and native molecular weights of the purified recombinant enzyme by SDS-PAGE, electrospray ionization mass spectrometry, dynamic light scattering, and sedimentation analysis. The enzyme purified following expression in E. coli existed as a homodimer with Mr approximately 68,000 as determined by light scattering and sedimentation analysis. Mass spectrometry revealed two species with experimentally determined masses of 34,272 and 34,348 which correspond to the native protein with either one or two 2-mercaptoethanol adducts. We conclude that the enzyme expressed in prokaryotic and eukaryotic heterologous systems, and also purified from E. coli, equates to that which is found in human tissue preparations.     

Role of actin in regulated exocytosis and compensatory membrane retrieval: insights from an old acquaintance

This review summarizes new insights into the role of the actin cytoskeleton in exocytosis and compensatory membrane retrieval from mammalian regulated secretory cells. Data from our lab and others now indicate that the actin cytoskeleton is involved in exocytosis both as a negative regulator of membrane fusion under resting conditions and as a facilitator of movement of secretory granules to their site of fusion with the apical plasmalemma. Coating of docked secretory granules with actin filaments correlates with the dissociation of secretory-granule-associated rab3D, pointing out a novel role for rab proteins in modulating the actin cytoskeleton during regulated exocytosis. Compensatory membrane retrieval following regulated exocytosis is also critically dependent on the actin cytoskeleton both in initiating the formation of clathrin-coated retrieval vesicles and subsequent trafficking back into the cell. We propose that insertion of secretory granule membrane into the plasmalemma initiates a trigger for membrane retrieval, possibly by exposing sites where proteins involved in compensatory membrane retrieval are assembled. The results summarized in this review were derived primarily from investigations on the pancreatic acinar cell, an old friend who is providing modern wisdom not attainable in other simpler systems.     

Predictors of mortality of patients with acute respiratory failure secondary to chronic obstructive pulmonary disease admitted to an intensive care unit: A one year study

Background

Patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) commonly require hospitalization and admission to intensive care unit (ICU). It is useful to identify patients at the time of admission who are likely to have poor outcome. This study was carried out to define the predictors of mortality in patients with acute exacerbation of COPD and to device a scoring system using the baseline physiological variables for prognosticating these patients.

Methods

Eighty-two patients with acute respiratory failure secondary to COPD admitted to medical ICU over a one-year period were included. Clinical and demographic profile at the time of admission to ICU including APACHE II score and Glasgow coma scale were recorded at the time of admission to ICU. In addition, acid base disorders, renal functions, liver functions and serum albumin, were recorded at the time of presentation. Primary outcome measure was hospital mortality.

Results

Invasive ventilation was required in 69 patients (84.1%). Fifty-two patients survived to hospital discharge (63.4%). APACHE II score at the time of admission to ICU {odds ratio (95 % CI): 1.32 (1.138–1.532); p < 0.001} and serum albumin (done within 24 hours of admission) {odds ratio (95 % CI): 0.114 (0.03-0.432); p = 0.001}. An equation, constructed using the adjusted odds ratio for the two parameters, had an area under the ROC curve of 91.3%. For the choice of cut-off, sensitivity, specificity, positive and negative predictive value for predicting outcome was 90%, 86.5%, 79.4% and 93.7%.

Conclusion

APACHE II score at admission and SA levels with in 24 hrs after admission are independent predictors of mortality for patients with COPD admitted to ICU. The equation derived from these two parameters is useful for predicting outcome of these patients.     

Ultrastructure of the mature spermatozoa of caecilians (Amphibia: Gymnophiona)

The spermatozoa of Gymnophiona show the following autapomorphies: 1) penetration of the distal centriole by the axial fiber; 2) presence of an acrosomal baseplate; 3) presence of an acrosome seat (flattened apical end of nucleus); and 4) absence of juxta-axonemal fibers. The wide separation of the plasma membrane bounding the undulating membrane is here also considered to be apomorphic. Three plesiomorphic spermatozoal characters are recognized that are not seen in other Amphibia but occur in basal amniotes: 1) presence of mitochondria with a delicate array of concentric cristae (concentric cristae of salamander spermatozoa differ in lacking the delicate array); 2) presence of peripheral dense fibers associated with the triplets of the distal centriole; and 3) presence of a simple annulus (a highly modified, elongate annulus is present in salamander sperm). The presence of an endonuclear canal containing a perforatorium is a plesiomorphic feature of caecilian spermatozoa that is shared with urodeles, some basal anurans, sarcopterygian fish, and some amniotes. Spermatozoal synapomorphies are identified for 1) the Uraeotyphlidae and Ichthyophiidae, and 2) the Caeciliidae and Typhlonectidae, suggesting that the members of each pair of families are more closely related to each other than to other caecilians. Although caecilian spermatozoa exhibit the clear amphibian synapomorphy of the unilateral location of the undulating membrane and its axial fiber, they have no apomorphic characters that suggest a closer relationship to either the Urodela or Anura.     

Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 cells

Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatment-induced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulse-chase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.