Modeling the Structural Consequences of \u3cem\u3eBEST1\u3c/em\u3e Missense Mutations

Mutations in the bestrophin-1 gene (BEST1) are an important cause of inherited retinal disorders. Hitherto, over 100 unique allelic variants have been linked to the human BEST1 (hBEST1), and associated with disease phenotypes, broadly termed as bestrophinopathies. A spontaneous animal model recapitulating BEST1-related phenotypes, canine multifocal retinopathy (cmr), is caused by mutations in the canine gene ortholog (cBEST1). We have recently characterized molecular consequences of cmr, demonstrating defective protein trafficking as a result of G161D (cmr2) mutation. To further investigate the pathological effects of BEST1 missense mutations, canine and human peptide fragments derived from the protein sequence have been studied in silico as models for early events in the protein folding. The results showed that G161D as well as I201T substitutions cause severe conformational changes in the structure of bestrophin-1, suggesting protein misfolding as an underlying disease mechanism. The comparative modeling studies expand our insights into BEST1 pathogenesis

Relationship between psychological and biological factors and physical activity and exercise behaviour in Filipino students

The aim of the present study was threefold. Firstly, it investigated whether a general measure or specific measure of motivational orientation was better in describing the relationship between motivation and exercise behaviour. Secondly, it examined the relationship between the four most popular indirect methods of body composition assessment and physical activity and exercise patterns. Thirdly, the interaction between motivation and body composition on physical activity and exercise behaviour was explored in a sample of 275 Filipino male and female students. Males were found to have higher levels of exercise whereas females had higher levels of physical activity. Furthermore, general self-motivation together with body weight and percentage body fat were found to be the best predictor of exercise behaviour whereas the tension/pressure subscale of the ‘Intrinsic Motivation Inventory’ (IMI) was the best predictor of levels of physical activity. However, significant gender differences were observed. That is, for the males only self-motivation and for the females only body weight and BMI predicted exercise behaviour. Also, tension/pressure predicted physical activity levels for the females but not the males. No inverse relationship was found between the four body composition measures and exercise and physical activity behaviour. The results support the notion that the psychobiological approach might be particularly relevant for high intensity exercise situations but also highlights some important gender differences. Finally, the results of this study emphasise the need for more cross-cultural research

Differential effects of natural palm oil, chemically- and enzymatically-modified palm oil on weight gain, blood lipid metabolites and fat deposition in a pediatric pig model

<p>Abstract</p> <p>Background</p> <p>Increasing prevalence of obesity and overweight in the Western world, continue to be a major health threat and is responsible for increased health care costs. Dietary intervention studies show a strong positive association between saturated fat intake and the development of obesity and cardiovascular disease. This study investigated the effect of positional distribution of palmitic acid (Sn-1, 2 & 3) of palm oil on cardiovascular health and development of obesity, using weaner pigs as a model for young children.</p> <p>Methods</p> <p>Male and female weaner piglets were randomly allocated to 4 dietary treatment groups: 1) pork lard (LRD); 2) natural palm olein (NPO); 3) chemically inter-esterified PO (CPO) and 4) enzymatically inter-esterified PO (EnPO) as the fat source. Diets were formulated with 11% lard or with palm olein in order to provide 31% of digestible energy from fat in the diet and were balanced for cholesterol, protein and energy across treatments.</p> <p>Results</p> <p>From 8 weeks onwards, pigs on EnPO diet gained (P < 0.05) more weight than all other groups. Feed conversion efficiency (feed to gain) over the 12 week experimental period did not vary between treatment groups. Plasma LDL-C content and LDL-C/HDL-C ratio in pigs fed natural PO tended to be lower compared to all other diets. The natural PO lowered (P < 0.02) the plasma triglyceride (TG) content relative to the lard or EnPO diets, but was not different from the CPO diet. The natural PO diet was associated with lower (P < 0.05) saturated fat levels in subcutaneous adipose tissue than the CPO and EnPO diets that had lower saturated fat levels than the lard diet. Female pigs had lower lean and higher fat and fat:lean ratio in the body compared with male pigs. No difference in weight gain or blood lipid parameters was observed between sexes.</p> <p>Conclusions</p> <p>The observations on plasma TG, muscle and adipose tissue saturated fatty acid contents and back fat (subcutaneous) thickness suggest that natural palm oil may reduce deposition of body fat. In addition, dietary supplementation with natural palm oil containing palmitic acid at different positions in meat producing animals may lead to the production of meat and meat products with lower saturated fats. An increase in fat content and a decrease in lean content in female pigs resulted in an increased body fat:lean ratio but gender had no effect on blood lipid parameters or insulin concentrations.</p

Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results

Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results

Imaging of the Muscle-Bone Relationship

Muscle can be assessed by imaging techniques according to its size (as thickness, area, volume, or alternatively, as a mass) and architecture (fiber length and pennation angle), with values used as an anthropometric measure or a surrogate for force production. Similarly, the size of the bone (as area or volume) can be imaged using MRI or pQCT, although typically bone mineral mass is reported. Bone imaging measures of mineral density, size, and geometry can also be combined to calculate bone’s structural strength—measures being highly predictive of bone’s failure load ex vivo. Imaging of muscle-bone relationships can, hence, be accomplished through a number of approaches by adoption and comparison of these different muscle and bone parameters, dependent on the research question under investigation. These approaches have revealed evidence of direct, mechanical muscle-bone interactions independent of allometric associations. They have led to important information on bone mechanoadaptation and the influence of muscular action on bone, in addition to influences of age, gender, exercise, and disuse on muscle-bone relationships. Such analyses have also produced promising diagnostic tools for clinical use, such as identification of primary, disuse-induced, and secondary osteoporosis and estimation of bone safety factors. Standardization of muscle-bone imaging methods is required to permit more reliable comparisons between studies and differing imaging modes, and in particular to aid adoption of these methods into widespread clinical practice

Activation by bitter substances of a cationic channel in membrane patches excised from the bullfrog taste receptor cell

The response to bitter-tasting substances was recorded in outside-out membrane patches excised from the taste receptor cell of the bullfrog fungiform papilla.Application of a bitter-tasting substance, quinine or denatonium, induced channel openings under conditions in which none of the second messenger candidates or their precursors (e.g. cyclic nucleotide, inositol 1,4,5-trisphosphate, Ca2+, ATP and GTP) were present on either side of the membrane. The response could be recorded > 10 min after excision of the patch membrane. These data suggest that the channel was directly gated by the bitter-tasting substances.No change in response was detected upon addition to the cytoplasmic side of either GDPβS (1 mM) or GTPγS (1 mM), suggesting that the G protein cascade has no direct relation to response generation.The quinine-induced current was dose dependent. The lowest effective concentration was approximately 0.1 mM, and the saturating concentration was near 1 mM. The dose-response curve was fitted by the Hill equation with a K½ of 0.52 mM and a Hill coefficient of 3.8.The single channel conductance measured in 120 mM NaCl solution was 10 pS. The channel was cation selective, and the ratio of the permeabilities for Na+, K+ and Cs+ (PNa:PK:PCs) was 1:0.48:0.39. The unitary conductance was dependent on the extracellular Ca2+ concentration ([Ca2+]o); 9.2 pS in a nominally Ca2+-free solution, and 4.5 pS in 1.8 mM [Ca2+]o.The dose dependence, the ion selectivity and the dependence of the unitary conductance on [Ca2+]o were almost identical to those of the quinine-induced whole-cell current reported previously, indicating that the channel activity observed in the excised membrane is the basis of the whole-cell current.The present observations suggest the new possibility that the cationic channel directly gated by bitter substances is involved in the bitter taste transduction mechanism