Lipid Binding Defects and Perturbed Synaptogenic Activity of a Collybistin R290H Mutant That Causes Epilepsy and Intellectual Disability

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects—or synaptopathies—are at the basis of many neurological and psychiatric disorders. In key areas of the mammalian brain, such as the hippocampus or the basolateral amygdala, the clustering of the scaffolding protein Gephyrin and of γ-aminobutyric acid type A receptors at inhibitory neuronal synapses is critically dependent upon the brain-specific guanine nucleotide exchange factor Collybistin (Cb). Accordingly, it was discovered recently that an R290H missense mutation in the diffuse B-cell lymphoma homology domain of Cb, which carries the guanine nucleotide exchange factor activity, leads to epilepsy and intellectual disability in human patients. In the present study, we determined the mechanism by which the Cb^R290H mutation perturbs inhibitory synapse formation and causes brain dysfunction. Based on a combination of biochemical, cell biological, and molecular dynamics simulation approaches, we demonstrate that the R290H mutation alters the strength of intramolecular interactions between the diffuse B-cell lymphoma homology domain and the pleckstrin homology domain of Cb. This defect reduces the phosphatidylinositol 3-phosphate binding affinity of Cb, which limits its normal synaptogenic activity. Our data indicate that impairment of the membrane lipid binding activity of Cb and a consequent defect in inhibitory synapse maturation represent a likely molecular pathomechanism of epilepsy and mental retardation in humans.     

A validated model of passive skeletal muscle to predict force and intramuscular pressure

The passive properties of skeletal muscle are often overlooked in muscle studies, yet they play a key role in tissue function in vivo. Studies analyzing and modeling muscle passive properties, while not uncommon, have never investigated the role of fluid content within the tissue. Additionally, intramuscular pressure (IMP) has been shown to correlate with muscle force in vivo and could be used to predict muscle force in the clinic. In this study, a novel model of skeletal muscle was developed and validated to predict both muscle stress and IMP under passive conditions for the New Zealand White Rabbit tibialis anterior. This model is the first to include fluid content within the tissue and uses whole muscle geometry. A nonlinear optimization scheme was highly effective at fitting model stress output to experimental stress data (normalized mean square error or NMSE fit value of 0.993) and validation showed very good agreement to experimental data (NMSE fit values of 0.955 and 0.860 for IMP and stress, respectively). While future work to include muscle activation would broaden the physiological application of this model, the passive implementation could be used to guide surgeries where passive muscle is stretched.     

The Link between Knowledge,Attitudes and Practices in Relation to Atmospheric Haze Pollution in Peninsular Malaysia

Transboundary haze episodes caused by seasonal forest fires have become a recurrent phenomenon in Southeast Asia, with serious environmental, economic, and public health implications. Here we present a cross-sectional survey conducted among people in Kuala Lumpur and surrounds to assess the links between knowledge, attitudes, and practices in relation to the transboundary haze episodes. Of 305 respondents, 125 were amateur athletes participating in a duathlon event and the remainder were surveyed in an inner-city shopping mall. Across the whole sample, people who possessed more factual information about the haze phenomenon showed significantly higher levels of concern. Duathletes were more knowledgeable than non-duathletes and also more concerned about the negative effects of haze, especially on health. For all people who regularly practice outdoor sports (including people interviewed at the shopping mall), higher levels of knowledge and concerned attitudes translated into a greater likelihood of engaging in protective practices, such as cancelling their outdoor training sessions, while those with greater knowledge were more likely to check the relevant air pollution index on a daily basis. Our results indicate that the provision of accurate and timely information about air quality to residents will translate into beneficial practices, at least among particularly exposed individuals, such as amateur athletes who regularly practice outdoor sports.     

Cloning and restriction fragment length polymorphism analysis of a cDNA for swine PIT-1, a gene controlling growth hormone expression*

A partial swine cDNA which encodes the functional domain of PIT-1 was isolated by the polymerse chain reaction (PCR). The swine PIT-1 cDNA clone is 95% identical at the protein level to the rat Pit-1 gene. Thus, Pit-l's known function in control of rat growth hormone and prolactin expression is likely to be conserved in swine. This swine cDNA clone was used to investigate genetic variability at PIT-1 in several American and Chinese breeds. Polymorphic BamIII fragments were found in pure-bred Meishan animals (n= 13), but only monomorphic fragments in five American breeds (n= 36).     

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.