The Importance of GLUT3 for De Novo Lipogenesis in Hypoxia-Induced Lipid Loading of Human Macrophages

Atherosclerotic lesions are characterized by lipid-loaded macrophages (foam cells) and hypoxic regions. Although it is well established that foam cells are produced by uptake of cholesterol from oxidized LDL, we previously showed that hypoxia also promotes foam cell formation even in the absence of exogenous lipids. The hypoxia-induced lipid accumulation results from increased triglyceride biosynthesis but the exact mechanism is unknown. Our aim was to investigate the importance of glucose in promoting hypoxia-induced de novo lipid synthesis in human macrophages. In the absence of exogenous lipids, extracellular glucose promoted the accumulation of Oil Red O-stained lipid droplets in human monocyte-derived macrophages in a concentration-dependent manner. Lipid droplet accumulation was higher in macrophages exposed to hypoxia at all assessed concentrations of glucose. Importantly, triglyceride synthesis from glucose was increased in hypoxic macrophages. GLUT3 was highly expressed in macrophage-rich and hypoxic regions of human carotid atherosclerotic plaques and in macrophages isolated from these plaques. In human monocyte-derived macrophages, hypoxia increased expression of both GLUT3 mRNA and protein, and knockdown of GLUT3 with siRNA significantly reduced both glucose uptake and lipid droplet accumulation. In conclusion, we have shown that hypoxia-induced increases in glucose uptake through GLUT3 are important for lipid synthesis in macrophages, and may contribute to foam cell formation in hypoxic regions of atherosclerotic lesions.     

Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo

Several people with Parkinson's disease have been treated with intrastriatal grafts of fetal dopaminergic neurons. Following autopsy, 10-22 years after surgery, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. Numerous studies have attempted to explain these findings in cell and animal models. In cell culture, α-synuclein has been found to transfer from one cell to another, via mechanisms that include exosomal transport and endocytosis, and in certain cases seed aggregation in the recipient cell. In animal models, transfer of α-synuclein from host brain cells to grafted neurons has been shown, but the reported frequency of the event has been relatively low and little is known about the underlying mechanisms as well as the fate of the transferred α-synuclein. We now demonstrate frequent transfer of α-synuclein from a rat brain engineered to overexpress human α-synuclein to grafted dopaminergic neurons. Further, we show that this model can be used to explore mechanisms underlying cell-to-cell transfer of α-synuclein. Thus, we present evidence both for the involvement of endocytosis in α-synuclein uptake in vivo, and for seeding of aggregation of endogenous α-synuclein in the recipient neuron by the transferred α-synuclein. Finally, we show that, at least in a subset of the studied cells, the transmitted α-synuclein is sensitive to proteinase K. Our new model system could be used to test compounds that inhibit cell-to-cell transfer of α-synuclein and therefore might retard progression of Parkinson neuropathology.     

A field test for host fruit odour discrimination and avoidance behaviour for Rhagoletis pomonella flies in the western United States

Prezygotic isolation due to habitat choice is important to many models of speciation-with-gene-flow. Habitat choice is usually thought to occur through positive preferences of organisms for particular environments. However, avoidance of non-natal environments may also play a role in choice and have repercussions for post-zygotic isolation that preference does not. The recent host shift of Rhagoletis pomonella (Diptera: Tephritidae) from downy hawthorn, Crataegus mollis, to introduced apple, Malus domestica, in the eastern United States is a model for speciation-with-gene-flow. However, the fly is also present in the western United States where it was likely introduced via infested apples ≤ 60 years ago. R. pomonella now attacks two additional hawthorns in the west, the native C. douglasii (black hawthorn) and the introduced C. monogyna (English ornamental hawthorn). Flight tunnel tests have shown that western apple-, C. douglasii- and C. monogyna-origin flies all positively orient to fruit volatile blends of their respective natal hosts in flight tunnel assays. Here, we show that these laboratory differences translate to nature through field-trapping studies of flies in the state of Washington. Moreover, western R. pomonella display both positive orientation to their respective natal fruit volatiles and avoidance behaviour (negative orientation) to non-natal volatiles. Our results are consistent with the existence of behaviourally differentiated host races of R. pomonella in the west. In addition, the rapid evolution of avoidance behaviour appears to be a general phenomenon for R. pomonella during host shifts, as the eastern apple and downy hawthorn host races also are antagonized by non-natal fruit volatiles.     

Staphylococcal peptidoglycan initiates an inflammatory response and procoagulant activity in human vascular endothelial cells: a comparison with highly purified lipoteichoic acid and TSST-1

Staphylococcus aureus is one of the most significant pathogens in human sepsis and endocarditis. A hallmark of these endovascular S. aureus infections is that the coagulation system is triggered by a tissue factor (TF)-dependent pathway. This study demonstrates that highly purified S. aureus peptidoglycan, lipoteichoic acid (LTA) and TSST-1 increase TF mRNA and TF surface protein in human umbilical vein endothelial cells (ECs). Concomitantly, peptidoglycan- and LTA-activated ECs express significant TF-dependent procoagulant activity (TF PCA). In addition peptidoglycan, but not LTA or TSST-1, induced surface expression of the EC inflammation markers ICAM-1 and VCAM-1, which supported the adhesion of monocytes to these ECs. During the coculture of peptidoglycan-activated ECs and adherent monocytes, a marked additional increase of TF PCA was observed. Marginal increases in TF PCA were observed in cocultures of monocytes with LTA- or TSST-1-activated ECs. This study defines in particular staphylococcal peptidoglycan, previously known as a potent initiator of TF PCA in monocytes, as also being an activator of a coagulant response in human ECs that is further intensified by the presence of surface-bound monocytes.     

Body Fat and Fat‐Free Mass and All‐Cause Mortality

Objective: To investigate whether the association between BMI and all‐cause mortality could be disentangled into opposite effects of body fat and fat‐free mass (FFM). Research Methods and Procedures: All‐cause mortality was studied in the Danish follow‐up study “Diet, Cancer and Health” with 27, 178 men and 29, 875 women 50 to 64 years old recruited from 1993 to 1997. By the end of year 2001, the median follow‐up was 5.8 years, and 1851 had died. Body composition was assessed by bioelectrical impedance. Cox regression models were used to estimate the relationships among body fat mass index (body fat mass divided by height squared), FFM index (FFM divided by height squared), and mortality. All analyses were adjusted for smoking habits. Results: Men and women showed similar associations. J‐shaped associations were found between body fat mass index and mortality adjusted for FFM and smoking. The mortality rate ratios in the upper part of body fat mass were 1.12 per kg/m² (95% confidence interval: 1.07, 1.18) in men and 1.06 per kg/m² (95% confidence interval: 1.02, 1.10) in women. Reversed J‐shaped associations were found between FFM index and mortality with a tendency to level off for high values of FFM. Discussion: Our findings suggest that BMI represents joint but opposite associations of body fat and FFM with mortality. Both high body fat and low FFM are independent predictors of all‐cause mortality.     

Functional interaction of caveolin-1 with Bruton's tyrosine kinase and Bmx.

Bruton's tyrosine kinase (Btk), a member of the Tec family of protein-tyrosine kinases, has been shown to be crucial for B cell development, differentiation, and signaling. Mutations in the Btk gene lead to X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. Using a co-transfection approach, we present evidence here that Btk interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. In addition, we found that native Bmx, another member of the Tec family kinases, is associated with endogenous caveolin-1 in primary human umbilical vein endothelial cells. Second, in transient transfection assays, expression of caveolin-1 leads to a substantial reduction in the in vivo tyrosine phosphorylation of both Btk and its constitutively active form, E41K. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82--101) functionally suppressed the autokinase activity of purified recombinant Btk protein. Third, we demonstrate that mouse splenic B-lymphocytes express substantial amounts of caveolin-1. Interestingly, caveolin-1 was found to be constitutively phosphorylated on tyrosine 14 in these cells. The expression of caveolin-1 in B-lymphocytes and its interaction with Btk may have implications not only for B cell activation and signaling, but also for antigen presentation.     

Pyrosequencing as a Method for Grouping of Listeria monocytogenes Strains on the Basis of Single-Nucleotide Polymorphisms in the inlB Gene

By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.     

Pyrosequencing as a method for grouping of Listeria monocytogenes strains on the basis of single-nucleotide polymorphisms in the inlB gene.

By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.     

Leaf-atmosphere NH(3) exchange of white clover (Trifolium repens L.) in relation to mineral N nutrition and symbiotic N(2) fixation.

Plant-atmosphere NH(3) exchange was studied in white clover (Trifolium repens L. cv. Seminole) growing in nutrient solution containing 0 (N(2) based), 0.5 (low N) or 4.5 (high N) mM NO(3)(-). The aim was to show whether the NH(3) exchange potential is influenced by the proportion of N(2) fixation relative to NO(3)(-) supply. During the treatment, inhibition of N(2) fixation by NO(3)(-) was followed by in situ determination of total nitrogenase activity (TNA), and stomatal NH(3) compensation points (chi(NH(3))) were calculated on the basis of apoplastic NH4(+) concentration ([NH4(+)]) and pH. Whole-plant NH(3) exchange, transpiration and net CO(2) exchange were continuously recorded with a controlled cuvette system. Although shoot total N concentration increased with the level of mineral N application, tissue and apoplastic [NH4(+)] as well as chi(NH(3)) were equal in the three treatments. In NH(3)-free air, net NH(3) emission rates of <1 nmol m(-2) s(-1) were observed in both high-N and N(2)-based plants. When plants were supplied with air containing 40 nmol mol(-1) NH(3), the resulting net NH(3) uptake was higher in plants which acquired N exclusively from symbiotic N(2) fixation, compared to NO(3)(-) grown plants. The results indicate that symbiotic N(2) fixation and mineral N acquisition in white clover are balanced with respect to the NH4(+) pool leading to equal chi(NH(3)) in plants growing with or without NO(3)(-). At atmospheric NH(3) concentrations exceeding chi(NH(3)), the NH(3) uptake rate is controlled by the N demand of the plants.