Laminar shear stress inhibits lipid peroxidation induced by high glucose plus arachidonic acid in endothelial cells

Elevated blood glucose and free fatty acids induce oxidative stress associated with the incidence of cardiovascular disease. In contrast, laminar shear stress (LSS) plays a critical role in maintaining vascular health. The present study examined the mechanism for the antioxidant effect of LSS attenuating the oxidative stress induced by high glucose (HG) and arachidonic acid (AA) in human umbilical vein endothelial cells. HG and AA synergistically decreased cell viability and increased glutathione (GSH) oxidation and lipid peroxidation. The lipid peroxidation was markedly prevented by LSS as well as tetrahydrobiopterin (BH4) and GSH. LSS increased BH4 and GSH contents, and expression of GTP cyclohydrolase-1 and glutamylcysteine ligase (GCL) involved in their biosynthesis. Inhibition of GCL activity by DL-buthionine-(S,R)-sulfoximine and small-interfering RNA-mediated knockdown of GCL lessened the antioxidant effect of LSS. Therefore, it is suggested that LSS enhances antioxidant capacity of endothelial cells and thereby attenuates the oxidative stress caused by cardiovascular risk factors.     

Human cytidine deaminase APOBEC3H restricts HIV-1 replication

The human genome encodes seven APOBEC3 (A3) cytidine deaminases with potential antiretroviral activity: A3A, A3B, A3C, A3DE, A3F, A3G, and A3H. A3G was the first identified to block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3F, A3B, and A3DE were shown later to have similar activities. HIV-1 produces a protein called Vif that is able to neutralize the antiretroviral activities of A3DE, A3F, and A3G, but not A3B. Only the antiretroviral activity of A3H remains to be defined due to its poor expression in cell culture. Here, we studied the mechanism impairing A3H expression. When primate A3H sequences were compared, a premature termination codon was identified on the fifth exon of the human and chimpanzee A3H genes, which significantly decreased their protein expression. It causes a 29-residue deletion from the C terminus, and this truncation did not reduce human A3H protein stability. However, the mRNA levels of the truncated gene were significantly decreased. Human A3H protein expression could be restored to a normal level either by repairing this truncation or through expression from a vector containing an intron from human cytomegalovirus. Once expression was optimized, human A3H could reduce HIV-1 infectivity up to 150-fold. Importantly, HIV-1 Vif failed to neutralize A3H activity. Nevertheless, extensive sequence analysis could not detect any significant levels of G-to-A mutation in the HIV-1 genome by human A3H. Thus, A3H inhibits HIV-1 replication potently by a cytidine deamination-independent mechanism, and optimizing A3H expression in vivo should represent a novel therapeutic strategy for HIV-1 treatment.     

Characterization of seed oils from fresh Bokbunja (Rubus coreanus Miq.) and wine processing waste

The physicochemical characteristics, fatty acid (FA) profile, and triacylglyceride (TAG) composition of seed oils from fresh Bokbunja (Rubus coreanus Miq.) fruits and traditional Bokbunja wine processing waste were determined in this study. Oil contents of the fresh seeds and the seeds from wine processing waste were similar, accounting for about 18% of dry weight. The free fatty acid (FFA) content between the two seed oils was significantly different (0.50% for fresh seed oil and 73.14% for wine seed oil). Iodine, conjugated diene, saponification values, and unsaponifiable matter were very similar in the oil samples, but the specific extinction coefficients at 232 and 270 nm of wine seed oil were higher than those of fresh seed oil. Linoleic (C18:2, 50.45-53.18%, L) and linolenic (C18:3, 29.36-33.25%, Ln) acids were the dominant FAs in the two seed oils, whereas oleic (C18:1, 7.32-8.04%, O), palmitic (C16:0, 1.55-1.65%, P), and stearic (C18:0, 0.65-0.68%, S) acids were the minor FAs. LLL, OLL, LLLn, OOL, LLnLn, and OOO were the abundant TAGs, representing >90% of the oils.     

Prevalence of Chlamydia trachomatis in human immunodeficiency virus-infected women in Cuba

To determine the prevalence rates and serovar distribution of Chlamydia trachomatis cervical infections in Cuban women, two different groups were selected. Group I consisted of 60 human immunodeficiency virus (HIV-1) seropositive women from different regions of Cuba and group II of 60 randomly selected women HIV seronegative and apparently healthy. C. trachomatis was detected in cervical scrapes by mean of nested polymerase chain reaction (PCR) specific for major out membrane protein. The overall prevalence rate of C. trachomatis in cervical scrapes determined by nested PCR was 10% in group I and the estimated prevalence was 6.6% for group II; 83.3% of HIV seropositive women with C. trachomatis infection reported history of pelvic inflammatory disease followed by cervicitis (50%). The control group C. trachomatis-infected women referred a history of cervicitis in 75% of cases. Other reports in the latter group included infertility and pelvic inflamatory disease in 50%. The present study is the first report of C. trachomatis prevalence in Cuba. It showed that there was not significantly difference in the prevalence rate of C. trachomatis between both groups.     

EGCG attenuates AMPA-induced intracellular calcium increase in hippocampal neurons

We have investigated the protective effect of (-)-epigallocatechin gallate (EGCG) on alpha-amino-3-hydroxy-5-methyl-4-isoxazolo propionate (AMPA)-induced toxicity in cultured rat hippocampal neurons. Treatment of 24 h AMPA (10 microM) reduced the neuronal viability in both survival neuron counting and MTT reduction assay compared with control, with increase in cellular concentrations of hydrogen peroxide and malondialdehyde. These responses to AMPA were significantly blocked by co-treatments with EGCG (10 microM), which effect was very similar to the protective ability of a known antioxidant catalase (2000 U/ml). AMPA (50 microM) elicited the increase in intracellular calcium concentration ([Ca(2+)]i) on which EGCG significantly attenuated both peak amplitude and sustained nature of that [Ca(2+)]i increase in a dose-dependent manner. These data suggest that EGCG has a neuroprotective effect against AMPA through inhibition of AMPA-induced [Ca(2+)]i increase and consequent attenuation of reactive oxygen species production and lipid peroxidation as an antioxidant and a radical scavenger.     

Signaling via histamine receptors in cat duodenal smooth muscle cells

Histamine produced concentration-dependent contractions in cat duodenal smooth muscle cells that were obtained by enzymatic digestion of smooth muscle with collagenase F. Pyrilamine, an H1 receptor antagonist, inhibited the contractile response while famotidine, an H2 receptor antagonist, augmented it. In cells with selectively preserved H1 receptors, produced by pretreatment with pyrilamine followed by inactivation of all unprotected receptors with N-ethylmaleimide, histamine-induced contraction was significantly augmented as compared with control cells. Pertussis toxin (PTX) had no effect on contraction, suggesting that the H1 receptor is coupled to a PTX-insensitive G protein. Gi2, Gi3, Go, Gs, and Gq subunits were present in cat duodenum, and histamine-induced contraction was inhibited by Gq antibody after cell permeabilization. Neomycin, a PLC inhibitor, inhibited the histamine-induced cell contraction, but not rhoCMB, a PLD inhibitor, or DEDA, a PLA2 inhibitor. Heparin, an IP3 receptor inhibitor, inhibited contraction whereas chelerythrine, a PKC inhibitor, had no effect. We conclude that histamine-induced contraction in cat duodenal smooth muscle cells is mediated by H1 receptors coupled to a PTX-insensitive Gq protein and results in activation of phosphatidylinositol-specific phospholipase C (PI-PLC).     

Host-seeking behavior of Ixodes pacificus (Acari: Ixodidae) nymphs in relation to environmental parameters in dense-woodland and woodland-grass habitats

In the far-western United States, the bacteria that cause Lyme disease (Borrelia burgdorferi, Bb) and human granulocytic anaplasmosis (Anaplasma phagocytophilum, Ap) are transmitted by the western black-legged tick (Ixodes pacificus). In a dense woodland, human behaviors involving contact with wood were recently found to pose greater risk for encountering I. pacificus nymphs than behaviors entailing exclusive exposure to leaf litter. A four-year follow-up study was undertaken in the same woodland and, as a comparison area in one year only, in a nearby woodland-grass habitat to explore the biotic and abiotic factors that might elevate human exposure to host-seeking nymphs. Nymphs were active in the dense woodland throughout the daytime, but no consistent pattern of activity was observed with respect to time of day, temperature, or relative humidity. Significantly more nymphs were collected from the southern aspects of dense-woodland trunks than from other aspects, and more nymphs quested at a height of < or = 1 m vs 1-2 m aboveground. The prevalence of bacterial infection in ticks from the dense woodland was highly variable among years, with maxima of 22.6% and 42.9% for Bb, and 15.6% and 1.8% for Ap, in nymphs from logs and trunks, respectively. The mean densities of nymphs, and of Bb- or Ap-infected nymphs, were typically higher on logs and trunks than in adjoining leaf litter or grass in both habitats. The acarologic risk of encountering an infected nymph on dense-woodland logs or trunks was 2.8 to 11 times higher for Bb than for Ap in two of three years, and it was usually higher in dense woodland than in woodland-grass for both agents. Coinfections were rare (0.27%, n = 369 nymphs from both habitats). Individuals having prolonged contact with logs or trunks in spring would be well advised to employ personal protective measures to minimize exposure to I. pacificus nymphs and their attendant bacterial zoonotic agents.     

Protelomerase uses a topoisomerase IB/Y-recombinase type mechanism to generate DNA hairpin ends

Protelomerases are enzymes responsible for the generation of closed hairpin ends in linear DNA. It is proposed that they use a breaking-and-rejoin type mechanism to affect DNA rearrangement on specific DNA sequences. In doing so, one strand turns around and becomes the complementary strand. Using the purified enzyme from the Escherichia coli phage N15 and the Klebsiella phage phiKO2 and synthetic oligonucleotide substrates, we directly demonstrate the location where the cutting/re-ligation occurs. We identified a pair of transient staggered cleavages six base-pairs apart centered around the axis of dyad symmetry of the target site. Two molecules of the protelomerase form a pair of protein-linked DNA intermediates at each 3' end of the cleaved openings leaving a 5'-OH. Then, in a process not yet clearly defined, the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends. The formation of 3'-covalent DNA-protein intermediates is a hallmark of the topoisomerase IB type reaction, and we have thus shown experimentally that protelomerase is a member of the tyrosine-recombinase superfamily. In addition, by introducing single nicks in the substrates as perturbation, we found that the integrity of the nucleotide chain 4 bp away from the cutting site as well as this nucleotide's complementary location on the stem if the strands were to fold into a cruciform structure are required for activity, suggesting that these locations may be important substrate-protein contacts. We determined that N15 and phiKO2 protelomerases are monomers in solution and two molecules are needed to interact with the substrate to form two closed hairpin products. The target sites of protelomerases invariably consist of inverted repeats. Comparative studies using the related target sites of different protelomerases suggest that these proteins may require both sequence-specific and structure (possibly cruciform)-specific recognition for activity.     

Acarologic risk of exposure to Borrelia burgdorferi spirochaetes: long-term evaluations in north-western California, with implications for Lyme borreliosis risk-assessment models

Over a 5-year period (1997-2001) the population densities of Ixodes pacificus Cooley & Kohls (Acari: Ixodidae) nymphs infected with spirochaetes of Borrelia burgdorferi sensu lato (s.l.) were evaluated in areas of 2000 ha at two localities (CHR, nine sites; HREC, seven sites) 25 km apart in Mendocino County, north-western California. The 5-year median density of infected nymphs was significantly higher at CHR than at HREC (0.51 vs. 0.09 per 100 m(2) and site-specific yearly densities exceeding one infected nymph per 100 m2 were 10-fold more likely to occur at CHR than at HREC. The importance of long-term data in acarologic risk assessment was demonstrated by significantly higher median yearly densities of infected nymphs at CHR from 1997 to 1999, whereas both areas had similar densities during 2000-2001. Overall, the causative agent of Lyme borreliosis in North America, B. burgdorferi Johnson et al. sensu stricto (s.s.) accounted for 76% of 46 genetically characterized B. burgdorferi s.l. infections from I. pacificus nymphs. Tremendous variability in acarologic risk was recorded within both areas: yearly densities of infected nymphs varied 11-97-fold between sites at CHR and 8-30-fold at HREC. Part of this variation could be explained by environmental traits, most notably deer usage. However, correlations between environmental factors and density of infected nymphs (for CHR and HREC combined) did not necessarily apply when these areas were considered separately. Thus, a Lyme borreliosis ecology model developed in one of these areas needs testing in the other area.