Alternative promoter usage of the membrane glycoprotein CD36

Background  

The premature aging and cancer-prone Werner and Bloom syndromes are caused by defects in the RecQ helicase enzymes WRN and BLM, respectively. Recently, both WRN and BLM (as well as several other RecQ members) have been shown to possess a strand annealing activity in addition to the requisite DNA unwinding activity. Since an annealing function would appear to directly oppose the action of a helicase, we have examined in this study the dynamic equilibrium between unwinding and annealing mediated by either WRN or BLM.     

siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2h in medium containing either 1.0mM [(15)NH(4)(+)] or 100μM [(15)N]glutamate. For those exposed to [(15)N]glutamate an additional 100μM was added after 1h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The (15)N incorporation from [(15)NH(4)(+)] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [(15)N]glutamate, the reduced GDH activity led to an increased (15)N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.     

Factors associated with inadequate colorectal cancer screening with flexible sigmoidoscopy

Background and study aim: Inadequate colorectal cancer screening wastes limited endoscopic resources. We examined patients factors associated with inadequate flexible sigmoidoscopy (FSG) screening at baseline screening and repeat screening 3–5 years later in 10 geographically-dispersed screening centers participating in the ongoing Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Methods: A total of 64,554 participants (aged 55–74) completed baseline questionnaires and underwent FSG at baseline. Of these, 39,385 participants returned for repeat screening. We used logistic regression models to assess factors that are associated with inadequate FSG (defined as a study in which the depth of insertion of FSG was <50 cm or visual inspection was limited to <90% of the mucosal surface but without detection of a polyp or mass). Results: Of 7084 (11%) participants with inadequate FSG at baseline, 6496 (91.7%) had <50 cm depth of insertion (75.3% due to patient discomfort) and 500 (7.1%) participants had adequate depth of insertion but suboptimal bowel preparation. Compared to 55–59 year age group, advancing age in 5-year increments (odds ratios (OR) from 1.08 to 1.51) and female sex (OR = 2.40; 95% confidence interval (CI): 2.27–2.54) were associated with inadequate FSG. Obesity (BMI >30 kg/m²) was associated with reduced odds (OR = 0.67; 95% CI: 0.62–0.72). Inadequate FSG screening at baseline was associated with inadequate FSG at repeat screening (OR = 6.24; 95% CI: 5.78–6.75). Conclusions: Sedation should be considered for patients with inadequate FSG or an alternative colorectal cancer screening method should be recommended.     

Transient Inability to Manage Proteobacteria Promotes Chronic Gut Inflammation in TLR5-Deficient Mice

Colitis results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both environmental and genetic influencing factors. Flagellin receptor TLR5-deficient mice (T5KO) display elevated intestinal proinflammatory gene expression and colitis with incomplete penetrance, providing a genetically sensitized system to study the contribution of microbiota to driving colitis. Both colitic and noncolitic T5KO exhibited transiently unstable microbiotas, with lasting differences in colitic T5KO, while their noncolitic siblings stabilized their microbiotas to resemble wild-type mice. Transient high levels of proteobacteria, especially enterobacteria species including E.?coli, observed in close proximity to the gut epithelium were a striking feature of colitic microbiota. A Crohn's disease-associated E.?coli strain induced chronic colitis in T5KO, which persisted well after the exogenously introduced bacterial species had been eliminated. Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring proteobacteria can drive and/or instigate chronic colitis.     

Effects of aging and reproduction on protein quality control in soma and gametes of Drosophila melanogaster

In organisms with a soma-germ demarcation, the germline must be 'preserved' such that harmful damage is not transmitted to the offspring. Keeping the progeny free of damage may be achieved by gametes enjoying elevated, and/or more functional, homeostatic maintenance systems. This possibility was approached here by testing whether the soma and maturating oocytes (eggs) dissected from female Drosophila melanogaster in reproductive ages display differential capacities for protein quality control and whether these capacities change during aging and mating. Eggs exhibited a high capacity to prevent protein aggregation, strong capacity for 26S proteasome-dependent degradation and reduced levels of oxidatively damaged (carbonylated) proteins compared to the soma. The capacity to prevent protein aggregation was not affected in either soma or eggs by age and/or mating, while the 26S proteasome capacity declined in the soma but was maintained in the eggs of aged females. However, the levels of carbonylated proteins increased with age in both soma and eggs, and this increase was more pronounced in females allowed to mate continuously. Furthermore, the levels of carbonylated proteins in the eggs of mated flies correlated negatively with the propensity of the eggs to develop into an adult fly. In young flies, mating caused a decrease in 26S proteasome capacity and an increase in protein carbonylation in the soma, but not in the eggs. These results are in line with trade-off theories of aging where aging is considered a consequence of investment in reproduction over somatic maintenance.     

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.     

Fast renewal of the distal colonic mucus layers by the surface goblet cells as measured by in vivo labeling of mucin glycoproteins

The enormous bacterial load and mechanical forces in colon create a special requirement for protection of the epithelium. In the distal colon, this problem is largely solved by separation of the bacteria from the epithelium by a firmly attached inner mucus layer. In addition, an outer mucus layer entraps bacteria to be cleared by distal transport. The mucus layers contain a network of Muc2 mucins as the main structural component. Here, the renewal rate of the inner protective mucus layer was studied as well as the production and secretion of Muc2 mucin in the distal colon. This was performed by intraperitoneal injection of N-azidoacetyl-galactosamine (GalNAz) that was in vivo incorporated during biosynthesis of O-glycosylated glycoproteins. The only gel-forming mucin produced in the colon is the Muc2 mucin and as it carries numerous O-glycans, the granulae of the goblet cells producing Muc2 mucin were intensely stained. The GalNAz-labeled glycoproteins were first observed in the Golgi apparatus of most cells. Goblet cells in the luminal surface epithelium had the fastest biosynthesis of Muc2 and secreted material already three hours after labeling. This secreted GalNAz-labeled Muc2 mucin formed the inner mucus layer. The goblet cells along the crypt epithelium accumulated labeled mucin vesicles for a longer period and secretion of labeled Muc2 mucin was first observed after 6 to 8 h. This study reveals a fast turnover (1 h) of the inner mucus layer in the distal colon mediated by goblet cells of the luminal surface epithelium.