Unique mutational patterns in the envelope alpha 2 amphipathic helix and acquisition of length in gp120 hypervariable domains are associated with resistance to autologous neutralization of subtype C human immunodeficiency virus type 1

Autologous neutralizing antibodies (NAb) against human immunodeficiency virus type 1 generate viral escape variants; however, the mechanisms of escape are not clearly defined. In a previous study, we determined the susceptibilities of 48 donor and 25 recipient envelope (Env) glycoproteins from five subtype C heterosexual transmission pairs to NAb in donor plasma by using a virus pseudotyping assay, thereby providing an ideal setting to probe the determinants of susceptibility to neutralization. In the present study, acquisition of length in the Env gp120 hypervariable domains was shown to correlate with resistance to NAb in donor plasma (P = 0.01; Kendall's tau test) but not in heterologous plasma. Sequence divergence in the gp120 V1-to-V4 region also correlated with resistance to donor (P = 0.0002) and heterologous (P = 0.001) NAb. A mutual information analysis suggested possible associations of nine amino acid positions in V1 to V4 with NAb resistance to the donor's antibodies, and five of these were located within an 18-residue amphipathic helix (alpha2) located on the gp120 outer domain. High nonsynonymous-to-synonymous substitution (dN/dS) ratios, indicative of positive selection, were also found at these five positions in subtype C sequences in the database. Nevertheless, exchange of the entire alpha2 helix between resistant donor Envs and sensitive recipient Envs did not alter the NAb phenotype. The combined mutual information and dN/dS analyses suggest that unique mutational patterns in alpha2 and insertions in the V1-to-V4 region are associated with NAb resistance during subtype C infection but that the selected positions within the alpha2 helix must be linked to still other changes in Env to confer antibody escape. These findings suggest that subtype C viruses utilize mutations in the alpha2 helix for efficient viral replication and immune avoidance.     

The 2'-O-ribose methyltransferase for cap 1 of spliced leader RNA and U1 small nuclear RNA in Trypanosoma brucei

mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.     

Novel benzodifuran analogs as potent 5-HT2A receptor agonists with ocular hypotensive activity

A series of 8-substituted benzodifuran analogs was prepared and evaluated for 5-HT(2A) receptor binding and activation. Several compounds containing ether and ester functionality were found to be potent agonists. Topical ocular administration of 5, 18, and 25 effectively reduced intra-ocular pressure in the hypertensive cynomolgus monkey eye in the range of 25-37%.     

1alpha,25-Dihydroxyvitamin D3 enhances proliferation of rat prostate cancer cells in the presence of living bone

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) is known to inhibit prostate cancer cells in vitro. Its effects on proliferation in the presence of living bone have not been reported, but are especially relevant since much of the morbidity and mortality associated with prostate cancer is due to metastatic bone disease. We investigated the effect of 1alpha,25(OH)(2)D(3) on MatLyLu-beta(2) cells (MatLyLu cells), a rat prostate cancer line, co-cultured in transwells with living rat calvaria. Cultures of MatLyLu cells with living calvaria treated with 1alpha,25(OH)(2)D(3) exhibited a statistically significant increase in proliferation (range 1.4 to 1.7-fold; p<0.05). Cultures of MatLylu cells alone, with spleen cells, muscle tissue, or with living or inactivated calvarial bone showed no differences in proliferation. To investigate the mechanism for enhanced proliferation, Galardin, a matrix metalloproteinase (MMP) inhibitor, or pamidronate, an antiresorptive agent, was added. Enhanced proliferation was prevented by either agent, but not to an equal extent. The presence of 1alpha,25(OH)(2)D(3) may lead to proteolytic release or activation of growth factors from bone. These results may explain the variability in reports on the in vivo effects of Vitamin D and suggest a potential concern in using Vitamin D or its analogs alone in patients with metastatic prostate cancer.     

Atropine pretreatment enhances airway hyperreactivity in antigen-challenged guinea pigs through an eosinophil-dependent mechanism

Airway hyperreactivity in antigen-challenged animals is mediated by eosinophil major basic protein (MBP) that blocks inhibitory M(2) muscarinic receptors on parasympathetic nerves, increasing acetylcholine release onto M(3) muscarinic receptors on airway smooth muscle. Acutely, anticholinergics block hyperreactivity in antigen-challenged animals and reverse asthma exacerbations in the human, but are less effective in chronic asthma. We tested whether atropine, given before antigen challenge, affected hyperreactivity, M(2) receptor function, eosinophil accumulation, and activation. Sensitized guinea pigs received atropine (1 mg/kg ip) 1 h before challenge and 6 h later. Twenty-four hours after challenge, animals were anesthetized, vagotomized, paralyzed, and ventilated. Airway reactivity to electrical stimulation of the vagi and to intravenous acetylcholine was not altered by atropine pretreatment in nonsensitized animals, indicating that atropine was no longer blocking postjunctional muscarinic receptors. Antigen challenge induced airway hyperreactivity to vagal stimulation that was significantly potentiated by atropine pretreatment. Bronchoconstriction induced by acetylcholine was not changed by antigen challenge or by atropine pretreatment. M(2) receptor function was lost in challenged animals but protected by atropine pretreatment. Eosinophils in bronchoalveolar lavage and within airway tissues were significantly increased by challenge but significantly reduced by atropine pretreatment. However, extracellular MBP in challenged airways was significantly increased by atropine pretreatment, which may account for reduced eosinophils. Depleting eosinophils with antibody to IL-5 before challenge prevented hyperreactivity and significantly reduced MBP in airways of atropine-pretreated animals. Thus atropine pretreatment potentiated airway hyperreactivity by increasing eosinophil activation and degranulation. These data suggest that anticholinergics enhance eosinophil interactions with airway nerves.     

cGMP-dependent protein kinase I interacts with TRIM39R, a novel Rpp21 domain-containing TRIM protein

Nitric oxide modulates vascular smooth muscle cell (SMC) cytoskeletal kinetics and phenotype, in part, by stimulating cGMP-dependent protein kinase I (PKGI). To identify molecular targets of PKGI, an interaction trap screen in yeast was performed using a cDNA encoding the catalytic region of PKGI and a human lung cDNA library. We identified a cDNA that encodes a putative PKGI-interactor that is a novel variant of TRIM39, a member of the really interesting new gene (RING) finger family of proteins. Although this TRIM39 variant encodes the NH(2)-terminal RING finger (RF), B-box, and coiled-coil (RBBC) domains of TRIM39, instead of a complete COOH-terminal B30.2 domain, this TRIM39 isoform contains the COOH-terminal portion of Rpp21, a component of RNase P. RT-PCR demonstrated that the TRIM39 variant, which we refer to as TRIM39R, is transcribed in the human fetal lung and in rat pulmonary artery SMC. Indirect immunofluorescence using an antibody generated against the conserved domains of TRIM39 and TRIM39R revealed the proteins in speckled intranuclear structures in human acute monocytic leukemia (THP-1) and human epidermal carcinoma line (HEp-2) cells. PKGI phosphorylated a typical PKGI/PKA phosphorylation domain in a conserved region of TRIM39 and TRIM39R. Additional studies demonstrated that PKGI interacts with both isoforms of TRIM39 in yeast cells and phosphorylates both isoforms of TRIM39 in human cell lines. Although PKGI has been observed to interact with proteins that regulate cytoskeletal function and gene expression, this investigation shows for the first time that PKGI interacts with tripartite motif (TRIM) proteins, which, through diverse molecular pathways, are often observed to regulate important aspects of cellular homeostasis.     

Paraoxonases are associated with intestinal inflammatory diseases and intracellularly localized to the endoplasmic reticulum

We demonstrated previously that the paraoxonase (PON1/2/3) genes and proteins are expressed in human intestinal biopsies and in Caco-2 cells. The current study aims were to explore whether PON1/2/3 expression is different in inflammatory bowel diseases (IBD) or celiac disease compared to healthy controls, and to explore the intracellular localization of PON1/2/3. Our results showed that significantly fewer biopsies expressed PON1 and PON3 in the duodenum of celiac patients (PON1, P<0.0001; PON3, P=0.03), in the terminal ileum of Crohn's patients (PON1, P=0.001; PON3, P=0.008), and in the colon of UC patients (PON1, P=0.02; PON3, P=0.06) compared to controls. Since all three disorders share markedly elevated inflammatory mediators we explored the PON1/2/3 mRNA expression on cytokine stimulation. No changes were observed in Caco-2 and HT29 cells. Immunofluorescence experiments localized PON1/2/3 exclusively to the endoplasmic reticulum (ER) in both CaCo-2 and HT29 cells. These results demonstrate for the first time a novel relationship between PON1 and PON3 expression and several inflammatory gastrointestinal disorders. Together with the localization of PON1/2/3 enzymes to the ER, it may be suggested that PON1/2/3 may have extracellular functions as part of the host response in IBD and celiac disease.     

tRNA‐dependent alanylation of diacylglycerol and phosphatidylglycerol in Corynebacterium glutamicum

Aminoacyl‐phosphatidylglycerol synthases (aaPGSs) are membrane proteins that utilize aminoacylated tRNAs to modify membrane lipids with amino acids. Aminoacylation of membrane lipids alters the biochemical properties of the cytoplasmic membrane and enables bacteria to adapt to changes in environmental conditions. aaPGSs utilize alanine, lysine and arginine as modifying amino acids, and the primary lipid recipients have heretofore been defined as phosphatidylglycerol (PG) and cardiolipin. Here we identify a new pathway for lipid aminoacylation, conserved in many Actinobacteria, which results in formation of Ala‐PG and a novel alanylated lipid, Alanyl‐diacylglycerol (Ala‐DAG). Ala‐DAG formation in Corynebacterium glutamicum is dependent on the activity of an aaPGS homolog, whereas formation of Ala‐PG requires the same enzyme acting in concert with a putative esterase encoded upstream. The presence of alanylated lipids is sufficient to enhance the bacterial fitness of C. glutamicum cultured in the presence of certain antimicrobial agents, and elucidation of this system expands the known repertoire of membrane lipids acting as substrates for amino acid modification in bacterial cells.