The polymorphism P315L of human toll-like receptor 1 impairs innate immune sensing of microbial cell wall components

As a pattern recognition receptor, TLR1 mediates innate immune responses to a variety of microbial cell wall components including bacterial lipoproteins. We have previously shown that the central region of the extracellular domain of human TLR1, comprising leucine-rich repeat (LRR) motifs 9-12, is required for the sensing of bacterial lipopeptides. In this study, we have investigated three nonsynonymous single nucleotide polymorphisms (SNPs) located in this region of TLR1 by generating these variants and examining receptor function. We have found that a variant of TLR1 based upon the SNP P315L, located in the loop of LRR motif 11 (LRR11), is greatly impaired in mediating responses to lipopeptides and a variety of other bacterial agonists for this receptor. Despite normal cell surface expression, the P315L variant also fails to bind to GD2.F4, a commonly used anti-TLR1 mAb. Although a number of amino acid substitutions at position 315 impair receptor function, the leucine substitution has the strongest deleterious effect. GD2.F4 inhibits agonist-induced activation of TLR1, supporting a crucial role for the loop of LRR11 in receptor function. These results also suggest that the P315L SNP may predispose certain individuals to infectious diseases for which the sensing of microbial cell components by TLR1 is critical to innate immune defense.     

At diabetes-like concentration, glucose down-regulates the placental serotonin transport system in a cell-cycle-dependent manner

Serotonin [5-hydroxytryptamine (5HT)] is a vasoconstrictor that also acts as a developmental signal early in embryogenesis. The 5HT transporter (SERT) on the membranes of the placental trophoblast cells controls 5HT levels in the maternal bloodstream to maintain stable transplacental blood flow and simultaneously provide 5HT to the embryo. The 5HT uptake rate of placental SERT is important for both the mother and the developing embryo. The impact of glucose on the placental SERT system during diabetic pregnancy is not known. The present in vitro study investigated this important issue in human placental choriocarcinoma (JAR) cells that were cultured for 24-96 h in a medium containing either 5.5 (physiologic concentration) or 25 mmol/L D-glucose (diabetic-like concentration). The 5HT uptake rates of the cultured cells were not altered at exogenous D-glucose concentrations in the range of 5.5-15 mmol/L, but were decreased significantly at a diabetic-like concentration (>or=25 mmol/L). To understand better the role of glucose on the placental 5HT system, we first characterized SERT in JAR cells at different cell-cycle phases and then determined the expression levels of SERT on the plasma membrane and in the intracellular pools of JAR cells at the late-S and G2 phases, where the uptake rates were decreased 73% under diabetic-like glucose concentrations. Finally, the importance of self-association of SERT molecules was examined. In JAR cells co-expressing Flag- and myc-tagged SERT, myc-antibody precipitated 70% of Flag-SERT, indicating that a large percentage of SERT proteins exist as oligomers in situ. Under diabetic conditions, myc-antibody no longer precipitated Flag-SERT, suggesting a disruption in the aggregation of SERT molecules. Therefore, we propose that under uncontrolled diabetic conditions, glucose down-regulates 5HT uptake rates of placental SERT by interfering with its functional expression in a cell-cycle-dependent manner.     

Specificity of olfactory receptor interactions with other G protein-coupled receptors

Studies on olfactory receptor (OR) pharmacology have been hindered by the poor plasma membrane localization of most ORs in heterologous cells. We previously reported that association with the beta(2)-adrenergic receptor (beta(2)-AR) facilitates functional expression of the OR M71 at the plasma membrane of HEK-293 cells. In the present study, we examined the specificity of M71 interactions with other G protein-coupled receptors (GPCRs). M71 was co-expressed in HEK-293 cells with 42 distinct GPCRs, and the vast majority of these receptors had no significant effect on M71 surface expression. However, co-expression with three subtypes of purinergic receptor (P2Y(1)R, P2Y(2)R, and A(2A)R) resulted in markedly enhanced plasma membrane localization of M71. Agonist stimulation of M71 co-expressed with P2Y(1)R and P2Y(2)R activated the mitogen-activated protein kinase pathway via coupling of M71 to Galpha(o). We also examined the ability of beta(2)-AR, P2Y(1)R, P2Y(2)R, and A(2A)Rto interact with and regulate ORs beyond M71. We found that co-expression of beta(2)-AR or the purinergic receptors enhanced the surface expression for an M71 subfamily member but not for several other ORs from different subfamilies. In addition, through chimeric receptor studies, we determined that the second transmembrane domain of beta(2)-AR is necessary for beta(2)-AR facilitation of M71 plasma membrane localization. These studies shed light on the specificity of OR interactions with other GPCRs and the mechanisms governing olfactory receptor trafficking.     

8-Hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors

A series of potent novel 8-hydroxy-3,4-dihydropyrrolo[1,2-a]pyrazine-1(2H)-one HIV-1 integrase inhibitors was identified. These compounds inhibited the strand transfer process of HIV-1 integrase and viral replication in cells. Compound 12 is active against replication of HIV-1 in cell culture with a CIC(95) of 0.31microM. Further SAR exploration led to the preparation of pseudosymmetrical tricyclic pyrrolopyrazine inhibitors 23 and 24 with further improvement in antiviral activity.     

Fine mapping versus replication in whole-genome association studies

Association replication studies have a poor track record and, even when successful, often claim association with different markers, alleles, and phenotypes than those reported in the primary study. It is unknown whether these outcomes reflect genuine associations or false-positive results. A greater understanding of these observations is essential for genomewide association (GWA) studies, since they have the potential to identify multiple new associations that that will require external validation. Theoretically, a repeat association with precisely the same variant in an independent sample is the gold standard for replication, but testing additional variants is commonplace in replication studies. Finding different associated SNPs within the same gene or region as that originally identified is often reported as confirmatory evidence. Here, we compare the probability of replicating a gene or region under two commonly used marker-selection strategies: an "exact" approach that involves only the originally significant markers and a "local" approach that involves both the originally significant markers and others in the same region. When a region of high intermarker linkage disequilibrium is tested to replicate an initial finding that is only weak association with disease, the local approach is a good strategy. Otherwise, the most powerful and efficient strategy for replication involves testing only the initially identified variants. Association with a marker other than that originally identified can occur frequently, even in the presence of real effects in a low-powered replication study, and instances of such association increase as the number of included variants increases. Our results provide a basis for the design and interpretation of GWA replication studies and point to the importance of a clear distinction between fine mapping and replication after GWA.     

Bacterial Nonhomologous End Joining Requires Teamwork

All living organisms must repair DNA double-stranded breaks (DSBs) in order to survive. Many bacteria rely on nonhomologous end joining (NHEJ) when only a single copy of the genome is available and maintain NHEJ pathways with a minimum of two proteins. In this issue, Bhattarai and colleagues identify additional factors that can work together to aid in survival of stationary-phase cells with chromosomal breaks.     

Control of dynamic foot-ground interactions in male and female soccer athletes: Females exhibit reduced dexterity and higher limb stiffness during landing

Controlling dynamic interactions between the lower limb and ground is important for skilled locomotion and may influence injury risk in athletes. It is well known that female athletes sustain anterior cruciate ligament (ACL) tears at higher rates than male athletes, and exhibit lower extremity biomechanics thought to increase injury risk during sport maneuvers. The purpose of this study was to examine whether lower extremity dexterity (LED) – the ability to dynamically control endpoint force magnitude and direction as quantified by compressing an unstable spring with the lower limb at submaximal forces – is a potential contributing factor to the “at-risk” movement behavior exhibited by female athletes. We tested this hypothesis by comparing LED-test performance and single-limb drop jump biomechanics between 14 female and 14 male high school soccer players. We found that female athletes exhibited reduced LED-test performance (p=0.001) and higher limb stiffness during landing (p=0.008) calculated on average within 51 ms of foot contact. Females also exhibited higher coactivation at the ankle (p=0.001) and knee (p=0.02) before landing. No sex differences in sagittal plane joint angles and center of mass velocity at foot contact were observed. Collectively, our results raise the possibility that the higher leg stiffness observed in females during landing is an anticipatory behavior due in part to reduced lower extremity dexterity. The reduced lower extremity dexterity and compensatory stiffening strategy may contribute to the heightened risk of ACL injury in this population.     

Oxidative damage in colon and mammary tissue of the HFE-knockout mouse

The HFE mutation is common and, when homozygous, can lead to a morbid accumulation of body iron and the disease hereditary hemochromatosis. Heterozygotes compose 10-15% of the European-American population, and have evidence of elevated body iron compared to homozygous normal people. Dietary iron content was hypothesized to interact with the HFE genotype to influence oxidative damage in mammary and colon tissue. Two groups of HFE-knockout mice were fed a standard iron diet (300 ppm) or a low iron diet (30 ppm). There was a significantly elevated concentration of malondialdehyde (by HPLC) in mammary (305 pmol/g vs. 166, p =.04) and colon (349 pmol/g vs. 226, p =.02) tissue among those mice on the standard iron diet compared to those on the low iron diet. These results suggest that dietary modification may affect the course of iron overload from HFE mutations.