TLR-3 is Present in Human Adipocytes,but Its Signalling is Not Required for Obesity-Induced Inflammation in Adipose Tissue In Vivo

Innate immunity plays a pivotal role in obesity-induced low-grade inflammation originating from adipose tissue. Key receptors of the innate immune system including Toll-like receptors-2 and -4 (TLRs) are triggered by nutrient excess to promote inflammation. The role of other TLRs in this process is largely unknown. In addition to double-stranded viral mRNA, TLR-3 can also recognize mRNA from dying endogenous cells, a process that is frequently observed within obese adipose tissue. Here, we identified profound expression of TLR-3 in adipocytes and investigated its role during diet-induced obesity. Human adipose tissue biopsies (n=80) and an adipocyte cell-line were used to study TLR-3 expression and function. TLR-3-/- and WT animals were exposed to a high-fat diet (HFD) for 16 weeks to induce obesity. Expression of TLR-3 was significantly higher in human adipocytes compared to the non-adipocyte cells part of the adipose tissue. In vitro, TLR-3 expression was induced during differentiation of adipocytes and stimulation of the receptor led to elevated expression of pro-inflammatory cytokines. In vivo, TLR-3 deficiency did not significantly influence HFD-induced obesity, insulin sensitivity or inflammation. In humans, TLR-3 expression in adipose tissue did not correlate with BMI or insulin sensitivity (HOMA-IR). Together, our results demonstrate that TLR-3 is highly expressed in adipocytes and functionally active. However, TLR-3 appears to play a redundant role in obesity-induced inflammation and insulin resistance.     

Genome-wide study of gene variants associated with differential cardiovascular event reduction by pravastatin therapy

Statin therapy reduces the risk of coronary heart disease (CHD), however, the person-to-person variability in response to statin therapy is not well understood. We have investigated the effect of genetic variation on the reduction of CHD events by pravastatin. First, we conducted a genome-wide association study of 682 CHD cases from the Cholesterol and Recurrent Events (CARE) trial and 383 CHD cases from the West of Scotland Coronary Prevention Study (WOSCOPS), two randomized, placebo-controlled studies of pravastatin. In a combined case-only analysis, 79 single nucleotide polymorphisms (SNPs) were associated with differential CHD event reduction by pravastatin according to genotype (P<0.0001), and these SNPs were analyzed in a second stage that included cases as well as non-cases from CARE and WOSCOPS and patients from the PROspective Study of Pravastatin in the Elderly at Risk/PHArmacogenomic study of Statins in the Elderly at risk for cardiovascular disease (PROSPER/PHASE), a randomized placebo controlled study of pravastatin in the elderly. We found that one of these SNPs (rs13279522) was associated with differential CHD event reduction by pravastatin therapy in all 3 studies: P = 0.002 in CARE, P = 0.01 in WOSCOPS, P = 0.002 in PROSPER/PHASE. In a combined analysis of CARE, WOSCOPS, and PROSPER/PHASE, the hazard ratio for CHD when comparing pravastatin with placebo decreased by a factor of 0.63 (95% CI: 0.52 to 0.75) for each extra copy of the minor allele (P = 4.8 × 10(-7)). This SNP is located in DnaJ homolog subfamily C member 5B (DNAJC5B) and merits investigation in additional randomized studies of pravastatin and other statins.     

Sugar-regulated susceptibility of tomato fruit to Colletotrichum and Penicillium requires differential mechanisms of pathogenicity and fruit responses

Colletotrichum gloeosporioides and Penicillium expansum cause postharvest diseases in tropical and deciduous fruit. During colonization, C. gloeosporioides and P. expansum secrete ammonia in hosts with low sugar content (LowSC) and gluconic acid in hosts with high sugar content (HighSC), respectively, as a mechanism to modulate enhanced pathogenicity. We studied the pathogens interactions with tomato lines of similar genetic background but differing in their sugar content. Colletotrichum gloeosporioides showed enhanced colonization of the LowSC line with differential expression response of 15% of its genes including enhanced relative expression of glycosyl hydrolases, glucanase and MFS-transporter genes. Enhanced colonization of P. expansum occurred in the HighSC line, accompanied by an increase in carbohydrate metabolic processes mainly phosphoenolpyruvate carboxykinase, and only 4% of differentially expressed genes. Gene response of the two host lines strongly differed depending on the sugar level. Limited colonization of HighSC line by C. gloeosporioides was accompanied by a marked alteration of gene expression compared the LowSC response to the same pathogen; while colonization by P. expansum resulted in a similar response of the two different hosts. We suggest that this differential pattern of fungal/host responses may be the basis for the differential of host range of both pathogens in nature.     

Copper-induced LDL peroxidation: interrelated dependencies of the kinetics on the concentrations of copper, hydroperoxides and tocopherol

Excessive uptake of oxidized low density lipoprotein plays a role in the onset of atherosclerosis. Lipid-associated antioxidants, the most abundant of which is tocopherol (vitamin E), are therefore believed to have anti-atherogenic properties. By contrast, hydroperoxides enhance the peroxidation of low density lipoprotein. We demonstrate that none of these compounds markedly affect the maximal rate of oxidation of low density lipoprotein, whereas the lag preceding rapid oxidation is prolonged by tocopherol but shortened by hydroperoxides. The corresponding 'prolongation' and 'shortening' can be compensated by each other in low density lipoprotein preparations enriched with both these compounds. The dependence of the balance between the effects of tocopherol and hydroperoxides on the copper concentration indicates that the antioxidative effect of vitamin E increases with the oxidative stress.     

The internal aqueous volume of small unilamellar vesicles changes at the phase transition temperature of the phospholipid

Changes in the fluorescence of partially self-quenched 5(6)-carboxyfluorescein trapped within the internal aqueous compartment of small unilamellar dipalmitoylphosphatidylcholine vesicles indicate that the trapped volume of these vesicles decreases when the phospholipid undergoes the liquid crystalline to gel state transition. This volume change is completely reversible and is not caused by vesicle-vesicle fusion. Furthermore, this decrease in volume of the internal aqueous compartment may be attributed to a change in vesicle shape upon undergoing the phase transition.     

Three-dimensional images of choanoflagellate loricae

Choanoflagellates are unicellular filter-feeding protozoa distributed universally in aquatic habitats. Cells are ovoid in shape with a single anterior flagellum encircled by a funnel-shaped collar of microvilli. Movement of the flagellum creates water currents from which food particles are entrapped on the outer surface of the collar and ingested by pseudopodia. One group of marine choanoflagellates has evolved an elaborate basket-like exoskeleton, the lorica, comprising two layers of siliceous costae made up of costal strips. A computer graphic model has been developed for generating three-dimensional images of choanoflagellate loricae based on a universal set of 'rules' derived from electron microscopical observations. This model has proved seminal in understanding how complex costal patterns can be assembled in a single continuous movement. The lorica, which provides a rigid framework around the cell, is multifunctional. It resists the locomotory forces generated by flagellar movement, directs and enhances water flow over the collar and, for planktonic species, contributes towards maintaining cells in suspension. Since the functional morphology of choanoflagellate cells is so effective and has been highly conserved within the group, the ecological and evolutionary radiation of choanoflagellates is almost entirely dependent on the ability of the external coverings, particularly the lorica, to diversify.     

Modeling the influence of superoxide dismutase on superoxide and nitric oxide interactions,including reversible inhibition of oxygen consumption

A mathematical mass transport model was constructed in cylindrical geometry to follow coupled biochemical reactions and diffusion of oxygen, nitric oxide, superoxide, peroxynitrite, hydrogen peroxide, nitrite, and nitrate around a blood vessel. Computer simulations were performed for a 50 microm internal diameter arteriole to characterize mass transport in five concentric regions (blood, plasma layer, endothelium, vascular wall, perivascular tissue). Steady state gradients in nitric oxide, oxygen partial pressure, superoxide, and peroxynitrite, and associated production of hydrogen peroxide, nitrite, and nitrate were predicted for varying superoxide production rates, superoxide dismutase concentrations, and other physiological conditions. The model quantifies how competition between superoxide scavenging by nitric oxide and superoxide dismutase catalyzed removal varies spatially. Reversible inhibition of oxygen consumption by nitric oxide, which causes increased tissue oxygenation at deeper locations, was also included in the model. The mass transport model provides insight into complex interactions between reactive oxygen and nitrogen species in blood and tissue, and provides an objective way to evaluate the relative influence of different biochemical pathways on these interactions.     

Cloning and comparative sequence analysis of the gene coding for isopenicillin N synthase in Streptomyces

Summary The genes coding for isopenicillin N synthase (IPNS) in Streptomyces jumonjinensis and S. lipmanii were isolated from recombinant phage lambda libraries using the S. clavuligerus IPNS gene as a heterologous probe. The S. jumonjinensis IPNS gene has an open reading frame coding for 329 amino acids, identical in size to that of the previously cloned S. clavuligerus IPNS gene. A partial nucleotide sequence was also determined for the S. lipmanii IPNS gene. Comparison of the predicted amino acid sequences of all three streptomycete IPNS proteins shows that they exhibit more than 70% similarity, close to that found in comparisons among fungal IPNS proteins and significantly greater than that found, approximately 60%, between Streptomyces and fungal IPNS proteins. We conclude that procaryotic and eucaryotic IPNS genes are subgroups of a single family of microbial IPNS genes. Hybridization probes prepared from IPNS genes of the above streptomycete species were used to detect analogous genes in eight other strains that included both penicillin and cephalosporin producers and non-producers. Each producer strain responded with all three probes implying the presence of an IPNS gene. Surprisingly, several non-producer strains also responded with one or two of the probes. Our results suggest that IPNS-related genes may be more prevalent in Streptomyces than previously believed.