Visceral adiposity and subclinical coronary artery disease in elderly adults: Rancho Bernardo Study

Objective: Despite growing attention to central obesity as a predictor of clinical coronary heart disease (CHD), there are few reports about the association between directly measured visceral obesity and subclinical coronary atherosclerosis in elderly adults. We examined this association in elderly, community‐dwelling adults without clinically recognized CHD. Methods and Procedures: Elderly adults (190 men, BMI 27.2 ± 3.6 kg/m²; 220 women, BMI 25.8 ± 4.6) aged 55–88 years (median 69 years) with no history of CHD or coronary revascularization had an electron beam computed tomography (EBCT) to measure coronary artery calcification score (CACS), an estimate of coronary plaque burden. Visceral and subcutaneous adiposity were assessed by a triple‐slice EBCT scan at the lumbar 4–5 disc level and height, weight, and waist and hip circumferences were measured. Results: In sex‐specific ordinal logistic regression analyses, no measure of obesity or body fat distribution, including BMI, waist‐to‐hip ratio, waist girth, and visceral and subcutaneous fat by EBCT, was significantly associated with CACS before or after adjusting for multiple covariates of CACS (age, smoking, alcohol intake, exercise, pulse pressure, low‐density lipoprotein (LDL)/high‐density lipoprotein (HDL)‐cholesterol ratio, and fasting plasma glucose). Discussion: In elderly adults without clinically recognized CHD, body weight and fat distribution do not predict coronary artery plaque burden. These results raise questions about the value of weight reduction diets for preventing heart disease in elderly survivors without clinical heart disease.     

Effect of UVA fluence rate on indicators of oxidative stress in human dermal fibroblasts

During the course of a day human skin is exposed to solar UV radiation that fluctuates in fluence rate within the UVA (290-315 nm) and UVB (315-400 nm) spectrum. Variables affecting the fluence rate reaching skin cells include differences in UVA and UVB penetrating ability, presence or absence of sunscreens, atmospheric conditions, and season and geographical location where the exposure occurs. Our study determined the effect of UVA fluence rate in solar-simulated (SSR) and tanning-bed radiation (TBR) on four indicators of oxidative stress---protein oxidation, glutathione, heme oxygenase-1, and reactive oxygen species--in human dermal fibroblasts after receiving equivalent UVA and UVB doses. Our results show that the higher UVA fluence rate in TBR increases the level of all four indicators of oxidative stress. In sequential exposures when cells are exposed first to SSR, the lower UVA fluence rate in SSR induces a protective response that protects against oxidative stress following a second exposure to a higher UVA fluence rate. Our studies underscore the important role of UVA fluence rate in determining how human skin cells respond to a given dose of radiation containing both UVA and UVB radiation.     

Species succession and the development of a lacustrine fish community in an ephemeral lake

Here, we present a gillnet survey of Lake Liambezi a 370 km² shallow ephemeral floodplain lake situated in north-eastern Namibia, which is fed irregularly by the upper Zambezi and Kwando Rivers during years of high flooding. The lake dried up in 1985 and, with the exception of sporadic minor annual inundation events, remained dry until 2007. We describe the temporal succession of fish species over an 8 year period from initial inundation 2007 to maturation in 2014. The succession of the fish community did not follow the typical pattern of opportunistic strategists during colonisation, to periodic strategists that are eventually succeeded by equilibrium strategists. Instead, the evolution of the fish community was characterised by three distinct phases. The first phase involved the inundation and colonisation of the lake in 2007, followed by its decline until the floods that filled the lake in 2009. During this phase the lake was colonised by fishes from the adjacent upper Zambezi and Chobe River floodplains. Fish communities predominantly comprised floodplain specialists including the barbs Enteromius paludinosus and Enteromius poechii, the mormyrid Marcusenius altisambesi and catfishes Schilbe intermedius and Clarias gariepinus. The filling of the lake in the March 2009 floods marked the beginning of the second, successional phase. The barbs declined in abundance and the alestid Rhabdalestes maunensis underwent explosive population growth between 2009 and 2010, but populations crashed equally rapidly and were replaced by Brycinus lateralis which, together with S. intermedius went on to dominate the fish community 2011–2014. Larger, slower growing tilapiine cichlids increased steadily in abundance and became the dominant components in a 2700 t y^–1 artisanal fishery that developed on the lake. The fish community in the ephemeral Lake Liambezi is clearly influenced by numerous factors including connectivity, lake level fluctuations, competition and the effects of fishing, which may disrupt typical succession processes in floodplain ecosystems.     

New aspects of sphingosine 1-phosphate signaling in mammalian cells

Sphingosine 1-phosphate (S1P) is a bioactive lipid phosphate that binds to cell surface G-protein-coupled receptors (GPCR), but also can elicit intracellular actions. The role of S1P in cancer has been an area of significant interest and we have focused our research on two aspects that are of importance with respect to cancer. First, we have investigated how cross talk between S1P and growth factors might affect the pathophysiology of cancer cells. In this regard, we have demonstrated that S1P receptors function to re-programme the spatial signaling specificity of receptor tyrosine kinases and vice versa to modulate cell responses. Second, we have investigated spatial/temporal aspects of intracellular S1P signaling and how this might be de-regulated in cancer. This has involved studies on: (i) the interaction of sphingosine kinase 1 (which catalyses the phosphorylation of sphingosine to produce S1P) and phospholipase D in the Golgi apparatus linked to regulation of cell survival and (ii) the novel regulatory interaction between sphingosine kinase 1 and 2 and centrosomal S1P₅ receptor linked to the regulation of mitosis in mammalian cells including MDA-MB-231 breast cancer cells. Therefore, we have focused on novel aspects of spatial and temporal S1P signaling that might enable this bioactive lipid phosphate to exhibit normal and aberrant function in health and disease respectively.     

The sphingosine kinase 1 inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole induces proteasomal degradation of sphingosine kinase 1 in mammalian cells

Sphingosine kinase 1 (SK1) is an enzyme that catalyzes the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) induces the proteasomal degradation of SK1 in human pulmonary artery smooth muscle cells, androgen-sensitive LNCaP prostate cancer cells, MCF-7 and MCF-7 HER2 breast cancer cells and that this is likely mediated by ceramide as a consequence of catalytic inhibition of SK1 by SKi. Moreover, SK1 is polyubiquitinated under basal conditions, and SKi appears to increase the degradation of SK1 by activating the proteasome. In addition, the proteasomal degradation of SK1a and SK1b in androgen-sensitive LNCaP cells is associated with the induction of apoptosis. However, SK1b in LNCaP-AI cells (androgen-independent) is less sensitive to SKi-induced proteasomal degradation and these cells are resistant to SKi-induced apoptosis, thereby implicating the ubiquitin-proteasomal degradation of SK1 as an important mechanism controlling cell survival.     

Antiangiogenic activity of 2-deoxy-D-glucose

Background

During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated.

Methodology/Principal Findings

In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DG''s ability to interfere with endothelial N-linked glycosylation. 2-DG''s effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DG''s effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LH_BETAT_AG transgenic retinoblastoma model.

Conclusions/Significance

In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.     

Msh2 Acts in Medium-Spiny Striatal Neurons as an Enhancer of CAG Instability and Mutant Huntingtin Phenotypes in Huntington's Disease Knock-In Mice

The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.     

Reconstitution of KCNE1 into lipid bilayers: comparing the structural, dynamic, and activity differences in micelle and vesicle environments

KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.     

Comparative efficacy of a secretory phospholipase A<Subscript>2</Subscript> inhibitor with conventional anti-inflammatory agents in a rat model of antigen-induced arthritis

Introduction  

Previously, secretory phospholipase A₂ (sPLA₂) inhibition has been used as an adjunct to conventional rheumatoid arthritis therapy in human clinical trials without significant improvement of arthritic pathology. In this study, we compared the efficacy of a potent and orally active group IIa secretory phospholipase A₂ inhibitor (sPLA₂I) to conventional anti-arthritic agents; infliximab, leflunomide and prednisolone, in a rat model of antigen-induced arthritis.