Characterization and comparison of recombinant simian immunodeficiency virus from drill (Mandrillus leucophaeus) and mandrill (Mandrillus sphinx) isolates

Since simian immunodeficiency virus (SIV) was found to be the source of the human AIDS pandemic, a major goal has been to characterize the diversity of SIV strains in the wild and to assess their potential for crossover into humans. In the present study, SIV was isolated from a seropositive drill (Mandrillus leucophaeus) and three seropositive mandrills (Mandrillus sphinx) by using macaque peripheral blood mononuclear cells (PBMC). Full-length sequences were obtained from a drill and mandrill and designated SIVdrl1FAO and SIVmnd5440, respectively. A 182-bp fragment of the pol genes of the two remaining mandrill SIV isolates was also analyzed. Phylogenetic analyses demonstrated that SIVdrl1FAO formed a monophyletic clade with SIVmnd5440 and SIVmndM14, recently designated SIVmnd type 2. Both the SIVdrl and SIVmnd type 2 genomes carried a vpx gene and appeared to share a common ancestor with SIVrcm in the 5' region of the genome and with SIVmndGB1 (type 1) in the 3' region of the genome. A statistically significant recombination breakpoint was detected at the beginning of envelope, suggesting that the viruses were descendents of the same recombinant. Phylogenetic analysis of vpx and vpr genes demonstrated that the vpx genes formed a monophyletic cluster that grouped with vpr from SIVagm. In addition, both SIVdrl1FAO and SIVmnd5440 replicated in human PBMC and therefore could pose a risk of transmission to the human population.     

Identification of regions of Ail required for the invasion and serum resistance phenotypes

Yersinia enterocolitica is an enteric pathogen that has served as a model system for the study of microbial pathogenesis. Numerous virulence gene have been identified both on the virulence plasmid and on the chromosome. One of the chromosomal genes that is highly correlated with virulence is ail, a gene identified along with inv in a screen for Y. enterocolitica genes that could confer an invasive phenotype to Escherichia coli. Ail also promotes serum resistance in both E. coli and Y. enterocolitica. Several virulence factors homologous to Ail have been identified in other pathogens, yet very little is known about what constitutes the functional domain(s) of these proteins. Proteins in this family are predicted to consist of eight transmembrane beta-sheets and four cell surface-exposed loops. We constructed and characterized a number of insertion, deletion and point mutations in the regions of ail predicted to encode the cell surface loops. The results from the analysis of these mutants indicate that cell surface loops one and four do not directly promote invasion or serum resistance, whereas mutations in loop three appear to modulate both phenotypes. Analysis of mutations in loop 2 suggests that this surface-exposed loop contains sequences required for serum resistance and invasion. In addition, a peptide derived from the sequence of loop 2 was able specifically to inhibit Ail-mediated invasion in a dose-dependent manner. These results suggest that Ail directly promotes invasion and that loop 2 contains an active site, perhaps a receptor-binding domain. Analyses of the mutations also suggest that the serum resistance and invasion phenotypes may be separable, because there are numerous mutations that affect one phenotype but not the other.     

Small-scale distribution of interstitial nitrite in freshwater sediment microcosms: the role of nitrate and oxygen availability,and sediment permeability

The spatial distribution of interstitial NO2(-) concentrations was studied in NO3(-)-exposed freshwater sediment microcosms, using pore water extractions as well as ion-selective microsensors. Porewater extractions revealed ecotoxicologically critical NO2(-) concentrations in hypoxic and anoxic sediment layers in which significant NO3(-) consumption took place. In contrast, the use of ion-selective microsensors demonstrated the high capacity of the thin oxic surface layer of the sediments to consume NO2(-) and to produce NO3(-). Two modes of NO3(-) supply to the sediments were compared: In treatments with NO3(-) supply to the overlying water, a subsurface maximum of NO2(-) concentration was observed, coinciding with the site of maximum NO3(-) consumption. When NO3(-) was perfused up through the sediment cores, however, NO2(-) accumulated throughout the entire sediment column. Such spatially extensive NO2(-) accumulations were only observed in sediments poor in organic matter with a relatively high permeability. By manipulating the O2 content of the overlying water, the release of NO2(-) from the sediments could be influenced: In treatments with air-saturated overlying water, the sediments did not release detectable amounts of NO2(-) into the water phase. When kept hypoxic (25% air saturation) instead, significant NO2(-) accumulations were recorded in the overlying water. These findings suggest that in treatments with air-saturated overlying water, NO2(-) that was produced in deeper sediment layers (denitrifying conditions) was completely consumed at the oxic sediment surface (nitrifying conditions) before it could reach the overlying water.     

Cystathionine pathway-dependent cytotoxicities of diethyl maleate and diamide in rat and human hepatoma-derived cell cultures

Glutathione (GSH) plays a role in many toxicologically important metabolic processes. It was previously established that L-buthionine S,R-sulphoximine (BSO), a specific inhibitor of (- glutamylcysteine synthetase, reduces the GSH content more efficiently in rat (Fa32) than in human (HEp-G2) hepatoma-derived cells. We therefore investigated whether the cystathionase inhibitor propargylglycine (PPG) could further decrease the BSO-induced GSH depletion in HEp-G2 cells. The influence of the cystathionine precursors N-acetylmethionine, methionine and homocysteine on the cytotoxicity of diethyl maleate (DEM) and diamide [1,1'-azobis(N,N-dimethylformamide)] was also investigated. PPG reduced the GSH content in both cell lines. A further GSH decrease in HEp-G2 was obtained when using a BSO + PPG combination containing relatively high concentrations of PPG. BSO diminished the toxicity of PPG. Homocysteine was the most efficacious of the tested cystathionine precursors in increasing the GSH content and reducing the cytotoxicity of DEM and diamide in Fa32 and HEp-G2 cells.     

A solid-phase approach towards the synthesis of PDE5 inhibitors

PDE5 inhibitors based upon the xanthine scaffold 8 have been expediently synthesized using a solid-phase route. Attachment of the xanthine scaffold 8 using the Rink chloride linker 4 and N-1 functionalization using Mitsunobu chemistry is described. A library of compounds was produced in multi-milligram quantities with excellent purities and acceptable yields. The compounds were tested for their PDE5 inhibitory activity.     

Discordant protein and mRNA expression in lung adenocarcinomas

The relationship between gene expression measured at the mRNA level and the corresponding protein level is not well characterized in human cancer. In this study, we compared mRNA and protein expression for a cohort of genes in the same lung adenocarcinomas. The abundance of 165 protein spots representing 98 individual genes was analyzed in 76 lung adenocarcinomas and nine non-neoplastic lung tissues using two-dimensional polyacrylamide gel electrophoresis. Specific polypeptides were identified using matrix-assisted laser desorption/ionization mass spectrometry. For the same 85 samples, mRNA levels were determined using oligonucleotide microarrays, allowing a comparative analysis of mRNA and protein expression among the 165 protein spots. Twenty-eight of the 165 protein spots (17%) or 21 of 98 genes (21.4%) had a statistically significant correlation between protein and mRNA expression (r > 0.2445; p < 0.05); however, among all 165 proteins the correlation coefficient values (r) ranged from -0.467 to 0.442. Correlation coefficient values were not related to protein abundance. Further, no significant correlation between mRNA and protein expression was found (r = -0.025) if the average levels of mRNA or protein among all samples were applied across the 165 protein spots (98 genes). The mRNA/protein correlation coefficient also varied among proteins with multiple isoforms, indicating potentially separate isoform-specific mechanisms for the regulation of protein abundance. Among the 21 genes with a significant correlation between mRNA and protein, five genes differed significantly between stage I and stage III lung adenocarcinomas. Using a quantitative analysis of mRNA and protein expression within the same lung adenocarcinomas, we showed that only a subset of the proteins exhibited a significant correlation with mRNA abundance.     

A New Formulation of Calcitriol (DN-101) for High-Dose Pulse Administration in Prostate Cancer Therapy

Although the antineoplastic activity of calcitriol in prostate cancer has been known for many years, the agent's use in oncology has been prevented because of the occurrence of hypercalcemia with daily administration. High-dose pulse administration of calcitriol has the potential to improve the therapeutic index of calcitriol. Results of a phase II study of calcitriol and docetaxel (Taxotere(R)) suggest that this combination may have utility in androgen-independent prostate cancer (AIPC). DN-101, a high-dose (15 mug) formulation of calcitriol suitable for use in oncology, is now being tested in a randomized trial (AIPC Study of Calcitriol Enhancing Taxotere). This formulation of calcitriol could become an important new tool for improving the efficacy of docetaxel in the treatment of AIPC and would join the ranks of other nuclear receptor ligands in cancer treatment. Investigations of DN-101 in the treatment of a broad range of tumor types and in combination with a variety of agents are an exciting new area of research.     

Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model

Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties. LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations. MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1. In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability. Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.     

HDL modification by secretory phospholipase A(2) promotes scavenger receptor class B type I interaction and accelerates HDL catabolism

During inflammatory states plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) are reduced. Secretory group IIa phospholipase A(2) (sPLA(2)) is a cytokine-induced acute-phase enzyme associated with HDL. Transgenic mice overexpressing sPLA(2) have reduced HDL levels. Studies were performed to define the mechanism for the HDL reduction in these mice. HDL isolated from sPLA(2) transgenic mice have a significantly lower phospholipid content and greater triglyceride content. In autologous clearance studies, (125)I-labeled HDL from sPLA(2) transgenic mice was catabolized significantly faster than HDL from control mice (4.24 +/- 1.16 vs. 2.84 +/- 0.1 pools per day, P < 0.008). In both sPLA(2) transgenic and control mice, the cholesteryl ester component of HDL was more rapidly catabolized than the protein component, indicating a selective uptake mechanism. In vitro studies using CHO cells transfected with scavenger receptor class B type I (SR-BI) showed that sPLA(2)-modified HDL was nearly twice as efficient as a substrate for cholesteryl ester transfer. These data were confirmed in in vivo selective uptake experiments using adenoviral vector overexpression of SR-BI. In these studies, increased hepatic selective uptake was associated with increased (125)I-labeled apolipoprotein uptake in the kidney.We conclude that during inflammation sPLA(2) hydrolysis of HDL phospholipids alters the lipid composition of the particle, allowing for more efficient SR-BI-mediated selective cholesteryl ester uptake. This enhanced SR-BI activity generates HDL remnants that are preferentially catabolized in the kidney.