Genetic basis for the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis

Mycobacteria produce two unusual polymethylated polysaccharides, the 6-O-methylglucosyl-containing lipopolysaccharides (MGLP) and the 3-O-methylmannose polysaccharides, which have been shown to regulate fatty acid biosynthesis in vitro. A cluster of genes dedicated to the synthesis of MGLP was identified in Mycobacterium tuberculosis and Mycobacterium smegmatis. Overexpression of the putative glycosyltransferase gene Rv3032 in M. smegmatis greatly stimulated MGLP production, whereas the targeted disruption of Rv3032 in M. tuberculosis and that of the putative methyltransferase gene MSMEG2349 in M. smegmatis resulted in a dramatic reduction in the amounts of MGLP synthesized and in the accumulation of precursors of these molecules. Disruption of Rv3032 also led to a significant decrease in the glycogen content of the tubercle bacillus, indicating that the product of this gene is likely to be involved in the elongation of more than one alpha-(1-->4)-glucan in this bacterium. Results thus suggest that Rv3032 encodes the alpha-(1-->4)-glucosyltransferase responsible for the elongation of MGLP, whereas MSMEG2349 encodes the O-methyltransferase required for the 6-O-methylation of these compounds.     

Epidermal growth factor receptor and notch pathways participate in the tumor suppressor function of gamma-secretase

Gamma-secretase, a unique aspartyl protease, is required for the regulated intramembrane proteolysis of Notch and APP, pathways that are implicated, respectively, in the pathogenesis of cancer and Alzheimer disease. However, the mechanism whereby reduction of gamma-secretase causes tumors such as squamous cell carcinoma (SCC) remains poorly understood. Here, we demonstrate that gamma-secretase functions in epithelia as a tumor suppressor in an enzyme activity-dependent manner. Notch signaling is down-regulated and epidermal growth factor receptor (EGFR) is activated in SCC caused by genetic reduction of gamma-secretase. Moreover, the level of EGFR is inversely correlated with the level of gamma-secretase in fibroblasts, suggesting that the up-regulation of EGFR stimulates hyperproliferation in epithelia of mice with genetic reduction of gamma-secretase. Supporting this notion is our finding that the proliferative response of fibroblasts lacking gamma-secretase activity is more sensitive when challenged by either EGF or an inhibitor of EGFR as ompared with wild type cells. Interestingly, the up-regulation of EGFR is independent of Notch signaling, suggesting that the EGFR pathway functions in parallel with Notch in the tumorigenesis of SCC. Collectively, our results establish a novel mechanism linking the EGFR pathway to the tumor suppressor role of gamma-secretase and that mice with genetic reduction of gamma-secretase represent an excellent rodent model for clarifying pathogenesis of SCC and for testing therapeutic strategy to ameliorate this type of human cancer.     

Metabolic syndrome in Yup'ik Eskimos: the Center for Alaska Native Health Research (CANHR) Study

Objective: This study investigated the prevalence of metabolic syndrome and its defining components among Yup'ik Eskimos. Research Methods and Procedures: A cross‐sectional study design that included 710 adult Yup'ik Eskimos ≥18 years of age residing in 8 communities in Southwest Alaska. The prevalence of metabolic syndrome was determined using the recently updated Adult Treatment Panel III criteria. Results: The prevalence of metabolic syndrome in this study cohort was 14.7%, and varied by sex with 8.6% of the men and 19.8% of the women having metabolic syndrome. This is lower than the prevalence of 23.9% in the general U.S. adult population. The most common metabolic syndrome components/risk factors were increased waist circumference and elevated blood glucose. High‐density lipoprotein (HDL) cholesterol levels in Yup'ik Eskimos were significantly higher, and triglycerides lower than levels reported in National Health and Nutritional Examination III. Discussion: Compared with other populations, metabolic syndrome is relatively uncommon in Yup'ik Eskimos. The higher prevalence among Yup'ik women is primarily explained by their large waist circumference, suggesting central body fat accumulation. Further increases in metabolic syndrome risk factors among Yup'ik Eskimos could lead to increases in the prevalence of type 2 diabetes and cardiovascular disease, once rare in this population.     

Early energy deficit in Huntington disease: identification of a plasma biomarker traceable during disease progression

Huntington disease (HD) is a fatal neurodegenerative disorder, with no effective treatment. The pathogenic mechanisms underlying HD has not been elucidated, but weight loss, associated with chorea and cognitive decline, is a characteristic feature of the disease that is accessible to investigation. We, therefore, performed a multiparametric study exploring body weight and the mechanisms of its loss in 32 presymptomatic carriers and HD patients in the early stages of the disease, compared to 21 controls. We combined this study with a multivariate statistical analysis of plasma components quantified by proton nuclear magnetic resonance ((1)H NMR) spectroscopy. We report evidence of an early hypermetabolic state in HD. Weight loss was observed in the HD group even in presymptomatic carriers, although their caloric intake was higher than that of controls. Inflammatory processes and primary hormonal dysfunction were excluded. (1)H NMR spectroscopy on plasma did, however, distinguish HD patients at different stages of the disease and presymptomatic carriers from controls. This distinction was attributable to low levels of the branched chain amino acids (BCAA), valine, leucine and isoleucine. BCAA levels were correlated with weight loss and, importantly, with disease progression and abnormal triplet repeat expansion size in the HD1 gene. Levels of IGF1, which is regulated by BCAA, were also significantly lower in the HD group. Therefore, early weight loss in HD is associated with a systemic metabolic defect, and BCAA levels may be used as a biomarker, indicative of disease onset and early progression. The decreased plasma levels of BCAA may correspond to a critical need for Krebs cycle energy substrates in the brain that increased metabolism in the periphery is trying to provide.     

Plasticity of the intrinsic period of the human circadian timing system

Human expeditions to Mars will require adaptation to the 24.65-h Martian solar day-night cycle (sol), which is outside the range of entrainment of the human circadian pacemaker under lighting intensities to which astronauts are typically exposed. Failure to entrain the circadian time-keeping system to the desired rest-activity cycle disturbs sleep and impairs cognitive function. Furthermore, differences between the intrinsic circadian period and Earth's 24-h light-dark cycle underlie human circadian rhythm sleep disorders, such as advanced sleep phase disorder and non-24-hour sleep-wake disorders. Therefore, first, we tested whether exposure to a model-based lighting regimen would entrain the human circadian pacemaker at a normal phase angle to the 24.65-h Martian sol and to the 23.5-h day length often required of astronauts during short duration space exploration. Second, we tested here whether such prior entrainment to non-24-h light-dark cycles would lead to subsequent modification of the intrinsic period of the human circadian timing system. Here we show that exposure to moderately bright light ( approximately 450 lux; approximately 1.2 W/m(2)) for the second or first half of the scheduled wake episode is effective for entraining individuals to the 24.65-h Martian sol and a 23.5-h day length, respectively. Estimations of the circadian periods of plasma melatonin, plasma cortisol, and core body temperature rhythms collected under forced desynchrony protocols revealed that the intrinsic circadian period of the human circadian pacemaker was significantly longer following entrainment to the Martian sol as compared to following entrainment to the 23.5-h day. The latter finding of after-effects of entrainment reveals for the first time plasticity of the period of the human circadian timing system. Both findings have important implications for the treatment of circadian rhythm sleep disorders and human space exploration.     

A unique role for nonmuscle myosin heavy chain IIA in regulation of epithelial apical junctions

The integrity and function of the epithelial barrier is dependent on the apical junctional complex (AJC) composed of tight and adherens junctions and regulated by the underlying actin filaments. A major F-actin motor, myosin II, was previously implicated in regulation of the AJC, however direct evidence of the involvement of myosin II in AJC dynamics are lacking and the molecular identity of the myosin II motor that regulates formation and disassembly of apical junctions in mammalian epithelia is unknown. We investigated the role of nonmuscle myosin II (NMMII) heavy chain isoforms, A, B, and C in regulation of epithelial AJC dynamics and function. Expression of the three NMMII isoforms was observed in model intestinal epithelial cell lines, where all isoforms accumulated within the perijunctional F-actin belt. siRNA-mediated downregulation of NMMIIA, but not NMMIIB or NMMIIC expression in SK-CO15 colonic epithelial cells resulted in profound changes of cell morphology and cell-cell adhesions. These changes included acquisition of a fibroblast-like cell shape, defective paracellular barrier, and substantial attenuation of the assembly and disassembly of both adherens and tight junctions. Impaired assembly of the AJC observed after NMMIIA knock-down involved dramatic disorganization of perijunctional actin filaments. These findings provide the first direct non-pharmacological evidence of myosin II-dependent regulation of AJC dynamics in mammalian epithelia and highlight a unique role of NMMIIA in junctional biogenesis.     

Computer-aided lead optimization: improved small-molecule inhibitor of the zinc endopeptidase of botulinum neurotoxin serotype A

Optimization of a serotype-selective, small-molecule inhibitor of botulinum neurotoxin serotype A (BoNTA) endopeptidase is a formidable challenge because the enzyme-substrate interface is unusually large and the endopeptidase itself is a large, zinc-binding protein with a complex fold that is difficult to simulate computationally. We conducted multiple molecular dynamics simulations of the endopeptidase in complex with a previously described inhibitor (K(i) (app) of 7+/-2.4 microM) using the cationic dummy atom approach. Based on our computational results, we hypothesized that introducing a hydroxyl group to the inhibitor could improve its potency. Synthesis and testing of the hydroxyl-containing analog as a BoNTA endopeptidase inhibitor showed a twofold improvement in inhibitory potency (K(i) (app) of 3.8+/-0.8 microM) with a relatively small increase in molecular weight (16 Da). The results offer an improved template for further optimization of BoNTA endopeptidase inhibitors and demonstrate the effectiveness of the cationic dummy atom approach in the design and optimization of zinc protease inhibitors.     

Partial characterization of the proteome of the mouse striatum

Many diseases of the mammalian CNS, including Parkinson's (PD) and Lesch Nyhan disease (LND), are associated with programmatic neurodegeneration or dysfunction of dopaminergic neurons in the mesencephalon, the nigrostriatal pathway, and its projections in the striatum [1-4]. Proteomic studies on brain tissue of both animal models and human PD patients have provided evidence for dysfunction and damage of many pathways, including oxidative stress-related damage, ubiquitin-proteasome dysfunction, mitochondrial energy metabolism deficiencies, and synaptic function [5-11]. To date no such proteomic studies have been reported in the related and rare basal ganglia disorder LND, a developmental rather than a neurodegenerative neurological disorder caused by deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) that regulates a major step in the purine salvage pathway [12]. Many studies have demonstrated that the both human LND patients and a mouse knockout model of HPRT deficiency have significantly reduced levels and uptake of dopamine in the striatum [4, 13-16] that is likely to be the principal cause of the CNS disorder. The precise molecular and cellular mechanisms that underlie this neurotransmitter defect are unknown.