Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei

Trypanosoma brucei variant surface glycoprotein (VSG) genes are activated either by duplicative (DA) transposition of the gene to a pre-activated expression site or by nonduplicative (NDA) activation of a previously silent telomeric gene. We have obtained a recombinant clone spanning the 5' barren region of the expression linked copy of the duplicated VSG gene 117a. By DNA sequence and hybridization analyses we have identified a pleomorphic family of 14-25 non-VSG genes that lie upstream of both DA and NDA VSG expression sites. These expression site associated genes (ESAGs) encode 1.2 kb poly(A)+ mRNAs that are specifically transcribed from the active VSG expression telomere in mammalian bloodstream stages of T. brucei but, in common with VSG genes, are not transcribed in procyclic culture forms. cDNA and genomic sequences predict open reading frames that are conserved in the two ESAGs examined.     

Evolution of eutherian cytochrome c oxidase subunit II: heterogeneous rates of protein evolution and altered interaction with cytochrome c

Cytochrome c oxidase subunit II (COII), encoded by the mitochondrial genome, exhibits one of the most heterogeneous rates of amino acid replacement among placental mammals. Moreover, it has been demonstrated that cytochrome c oxidase has undergone a structural change in higher primates which has altered its physical interaction with cytochrome c. We collected a large data set of COII sequences from several orders of mammals with emphasis on primates, rodents, and artiodactyls. Using phylogenetic hypotheses based on data independent of the COII gene, we demonstrated that an increased number of amino acid replacements are concentrated among higher primates. Incorporating approximate divergence dates derived from the fossil record, we find that most of the change occurred independently along the New World monkey lineage and in a rapid burst before apes and Old World monkeys diverged. There is some evidence that Old World monkeys have undergone a faster rate of nonsynonymous substitution than have apes. Rates of substitution at four-fold degenerate sites in primates are relatively homogeneous, indicating that the rate heterogeneity is restricted to nondegenerate sites. Excluding the rate acceleration mentioned above, primates, rodents, and artiodactyls have remarkably similar nonsynonymous replacement rates. A different pattern is observed for transversions at four-fold degenerate sites, for which rodents exhibit a higher rate of replacement than do primates and artiodactyls. Finally, we hypothesize specific amino acid replacements which may account for much of the structural difference in cytochrome c oxidase between higher primates and other mammals.      

Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms

Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.      

Veterans’ Perspectives on Interventions to Improve Retention in HIV Care

Poor retention in HIV medical care is associated with increased mortality among patients with HIV/AIDS. Developing new interventions to improve retention in HIV primary care is needed. The Department of Veteran Affairs (VA) is the largest single provider of HIV care in the US. We sought to understand what veterans would want in an intervention to improve retention in VA HIV care. We conducted 18 one-on-one interviews and 15 outpatient focus groups with 46 patients living with HIV infection from the Michael E. DeBakey VAMC (MEDVAMC). Analysis identified three focus areas for improving retention in care: developing an HIV friendly clinic environment, providing mental health and substance use treatment concurrent with HIV care and encouraging peer support from other Veterans with HIV.     

Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain L-2-hydroxy acid oxidase

Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.     

Hypoglycemia induced changes in cholinergic receptor expression in the cerebellum of diabetic rats

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Hypoglycemia and hyperglycemia is found to be associated with central and peripheral nerve system dysfunction. Changes in acetylcholine receptors have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). In the present study we showed the effects of insulin induced hypoglycemia and streptozotocin induced diabetes on the cerebellar cholinergic receptors, GLUT3 and muscle cholinergic activity. Results showed enhanced binding parameters and gene expression of Muscarinic M1, M3 receptor subtypes in cerebellum of diabetic (D) and hypoglycemic group (D + IIH and C + IIH). α7nAchR gene expression showed a significant upregulation in diabetic group and showed further upregulated expression in both D + IIH and C + IIH group. AchE expression significantly upregulated in hypoglycemic and diabetic group. ChAT showed downregulation and GLUT3 expression showed a significant upregulation in D + IIH and C + IIH and diabetic group. AchE activity enhanced in the muscle of hypoglycemic and diabetic rats. Our studies demonstrated a functional disturbance in the neuronal glucose transporter GLUT3 in the cerebellum during insulin induced hypoglycemia in diabetic rats. Altered expression of muscarinic M1, M3 and α7nAchR and increased muscle AchE activity in hypoglycemic rats in cerebellum is suggested to cause cognitive and motor dysfunction. Hypoglycemia induced changes in ChAT and AchE gene expression is suggested to cause impaired acetycholine metabolism in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. The results shows that cerebellar cholinergic neurotransmission is impaired during hyperglycemia and hypoglycemia and the hypoglycemia is causing more prominent imbalance in cholinergic neurotransmission which is suggested to be a cause of cerebellar dysfunction associated with hypoglycemia.     

RNA interference pinpoints regulators of cell size and the cell cycle

Cell-based genome-wide RNA interference screens are being used to address an increasingly broad spectrum of biological questions. In one recent screen, Drosophila cell cultures treated with double-stranded RNA were analyzed by flow cytometry, providing a wealth of new information and identifying 488 regulators of the cell cycle, cell size, and cell death.     

Picrotoxin Blockade of Invertebrate Glutamate-Gated Chloride Channels: Subunit Dependence and Evidence for Binding Within the Pore

Glutamate-gated chloride channels have been described in nematodes, insects, crustaceans, and mollusks. Subunits from the nematode and insect channels have been cloned and are phylogenetically related to the GABA and glycine ligand-gated chloride channels. Ligand-gated chloride channels are blocked with variable potency by the nonselective blocker picrotoxin. The first two subunits of the glutamate-gated chloride channel family, GluClα and GIuClβ, were cloned from the free living nematode Caenorhabditls elegans. In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluClα and GluClβ RNAs were injected individually or coinjected into Xenopus oocytes. The EC₅₀ values for picrotoxin block of homomeric GluClα and GluClβ were 59 μM and 77 nM, respectively. Picrotoxin block of homomeric GluClβ channels was promoted during activation of membrane current with glutamate. In addition, recovery from picrotoxin block was faster during current activation by glutamate. A chimeric channel between the N-terminal extracellular domain of GluClα and the C-terminal membrane-spanning domain of GIuClβ localized the higher affinity picrotoxin binding site to the membrane-spanning domains of GluClβ. A point mutation within the M2 membrane-spanning domain of GluClβ reduced picrotoxin sensitivity >10,000-fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.     

Determination of selectivity and efficacy of fatty acid synthesis inhibitors

Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics. In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development. To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. We demonstrated that the inverse t(1/2) value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity. The Km values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 +/- 0.3 and 10 +/- 2.7 microM in Staphylococcus aureus and Bacillus subtilis, respectively. The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn2+ > Ca2+ > Mn2+ > Mg2+; no inhibition was found with beta-mercaptoethanol or dithiothreitol. We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure. BABX showed IC50 values of 11.4 and 35.3 microg/ml in the S. aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S. aureus and permeable E. coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 microg/ml. Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.