General and Versatile Autoinhibition of PLC Isozymes

Phospholipase C (PLC) isozymes are directly activated by heterotrimeric G proteins and Ras-like GTPases to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Although PLCs play central roles in myriad signaling cascades, the molecular details of their activation remain poorly understood. As described here, the crystal structure of PLC-β2 illustrates occlusion of the active site by a loop separating the two halves of the catalytic TIM barrel. Removal of this insertion constitutively activates PLC-β2 without ablating its capacity to be further stimulated by classical G protein modulators. Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli

MKLN1 splicing defect in dogs with lethal acrodermatitis

Lethal acrodermatitis (LAD) is a genodermatosis with monogenic autosomal recessive inheritance in Bull Terriers and Miniature Bull Terriers. The LAD phenotype is characterized by poor growth, immune deficiency, and skin lesions, especially at the paws. Utilizing a combination of genome wide association study and haplotype analysis, we mapped the LAD locus to a critical interval of similar to 1.11 Mb on chromosome 14. Whole genome sequencing of an LAD affected dog revealed a splice region variant in the MKLN1 gene that was not present in 191 control genomes (chr14:5,731,405T>G or MKLN/:c.400+3A>C). This variant showed perfect association in a larger combined Bull Terrier/Miniature Bull Terrier cohort of 46 cases and 294 controls. The variant was absent from 462 genetically diverse control dogs of 62 other dog breeds. RT-PCR analysis of skin RNA from an affected and a control dog demonstrated skipping of exon 4 in the MKLN1 transcripts of the LAD affected dog, which leads to a shift in the MKLN1 reading frame. MKLN1 encodes the widely expressed intracellular protein muskelin 1, for which diverse functions in cell adhesion, morphology, spreading, and intracellular transport processes are discussed. While the pathogenesis of LAD remains unclear, our data facilitate genetic testing of Bull Terriers and Miniature Bull Terriers to prevent the unintentional production of LAD affected dogs. This study may provide a starting point to further clarify the elusive physiological role of muskelin 1 in vivo.Peer reviewe

Transport of Pregabalin in Rat Intestine and Caco-2 Monolayers

Purpose . The purpose of this study was to determine if the intestinal transport of pregabalin (isobutyl -γ-aminobutyric acid, isobutyl GAB A), a new anticonvulsant drug, was mediated by amino acid carriers with affinity for large neutral amino acids (LNAA).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41477/1/11095_2004_Article_303539.pd

Murine mucopolysaccharidosis type VII: the impact of therapies on the clinical course and pathology in a murine model of lysosomal storage disease.

Murine mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by a recessively inherited deficiency of the lysosomal enzyme beta-glucuronidase. Affected mice have clinical, biochemical and pathological findings similar to those seen in humans with MPS VII (Sly syndrome), including growth retardation, facial dysmorphism, deafness, behavioural deficits and widespread glycosaminoglycan storage in lysosomes in the viscera, skeleton and brain. This mouse model is a useful tool for the evaluation of the effectiveness and experimental therapies for the MPS disorders. Syngeneic bone marrow transplantation performed in newborn MPS VII animals--before clinical evidence of disease is pronounced--prolongs life, improves hearing and bone growth, and prevents lysosomal storage in many sites, but does not correct the central nervous system disease. Enzyme therapy with beta-glucuronidase from the first days of life does reduce lysosomal storage in the brain in murine MPS VII. The enzyme-replaced mice also have reduced visceral lysosomal storage, impressive normalization of their phenotype and an improved life span. The effectiveness of gene therapy for the treatment of lysosomal storage disease has also been tested using the MPS VII model. When transplanted into MPS VII mice, syngeneic haematopoietic stem cells or mouse skin fibroblasts infected with retrovirus expressing beta-glucuronidase decreased storage, but only in the liver and spleen. Injection of an adenovirus vector expressing beta-glucuronidase into the vitreous of the MPS VII mice reduced storage in the retinal pigment epithelium and corneal endothelium. Intravenous administration of the adenovirus vector transduced with the beta-glucuronidase gene reduced liver and spleen storage and, when instilled into the cerebral ventricles, this viral vector caused beta-glucuronidase production in epithelial cells lining the ventricles. Recently, retroviral vector-corrected MPS VII fibroblasts secreting high levels of beta-glucuronidase were engrafted directly into the brains of adult MPS VII mice with resultant reduction in storage in neurons and glia adjacent to the grafts. Future efforts aimed at prolonging expression of the beta-glucuronidase gene by viral vectors and more precisely directing the therapeutic effect to the skeleton and brain will be important in optimizing treatments for murine MPS VII and extending the results of such therapies to humans with MPS