A continuous fluorescence assay for protein kinase C

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.     

A novel nonsense mutation in the DMP1 gene identified by a genome-wide association study is responsible for inherited rickets in Corriedale sheep

Inherited rickets of Corriedale sheep is characterized by decreased growth rate, thoracic lordosis and angular limb deformities. Previous outcross and backcross studies implicate inheritance as a simple autosomal recessive disorder. A genome wide association study was conducted using the Illumina OvineSNP50 BeadChip on 20 related sheep comprising 17 affected and 3 carriers. A homozygous region of 125 consecutive single-nucleotide polymorphism (SNP) loci was identified in all affected sheep, covering a region of 6 Mb on ovine chromosome 6. Among 35 candidate genes in this region, the dentin matrix protein 1 gene (DMP1) was sequenced to reveal a nonsense mutation 250C/T on exon 6. This mutation introduced a stop codon (R145X) and could truncate C-terminal amino acids. Genotyping by PCR-RFLP for this mutation showed all 17 affected sheep were "T T" genotypes; the 3 carriers were "C T"; 24 phenotypically normal related sheep were either "C T" or "C C"; and 46 unrelated normal control sheep from other breeds were all "C C". The other SNPs in DMP1 were not concordant with the disease and can all be ruled out as candidates. Previous research has shown that mutations in the DMP1 gene are responsible for autosomal recessive hypophosphatemic rickets in humans. Dmp1_knockout mice exhibit rickets phenotypes. We believe the R145X mutation to be responsible for the inherited rickets found in Corriedale sheep. A simple diagnostic test can be designed to identify carriers with the defective "T" allele. Affected sheep could be used as animal models for this form of human rickets, and for further investigation of the role of DMP1 in phosphate homeostasis.     

A profile of carbohydrate metabolites in the fasting northern elephant seal

Northern elephant seals endure prolonged periods of food deprivation at multiple life-history stages and simultaneous with energetically costly activities—including reproduction and development. Most mammals decrease their energy expenditure while fasting, with simultaneous reductions in gluconeogenesis and circulating glucose concentration. Paradoxically, elephant seals maintain high rates of both energy expenditure and gluconeogenesis, and high blood glucose concentrations throughout fasting. We therefore characterized the suite of changes that occur in carbohydrate metabolites during fasting in northern elephant seals. Using a broad-based metabolomics platform we investigated fasting during two states—lactation in adult females and the post-weaning developmental period in pups. A total of 227 metabolites were detected in seal plasma; 31 associated with carbohydrate metabolism were analyzed in the present study. Several compounds showed similar responses during lactation and the post-weaning fast (e.g. glycerol and mesaconate) whereas other compounds displayed quite different abundances between groups (e.g. citrate and pyruvate). This work found that, while the changes that occur with fasting were frequently similar in lactating females and developing pups, the relative abundance of compounds often varied markedly. These differences suggest that the metabolic strategies used to endure prolonged fasts are influenced by life-history or nutrient constraints.     

Determining the Effects of Cattle Grazing Treatments on Yosemite Toads (Anaxyrus [=Bufo] canorus) in Montane Meadows

Amphibians are experiencing a precipitous global decline, and population stability on public lands with multiple uses is a key concern for managers. In the Sierra Nevada Mountains (California, USA), managers have specifically identified livestock grazing as an activity that may negatively affect Yosemite toads due to the potential overlap of grazing with toad habitat. Grazing exclusion from Yosemite toad breeding and rearing areas and/or entire meadows have been proposed as possible management actions to alleviate the possible impact of cattle on this species. The primary objective of this study was to determine if different fencing treatments affect Yosemite toad populations. We specifically examined the effect of three fencing treatments on Yosemite toad breeding pool occupancy, tadpoles, and young of the year (YOY). Our hypothesis was that over the course of treatment implementation (2006 through 2010), Yosemite toad breeding pool occupancy and early life stage densities would increase within two fencing treatments relative to actively grazed meadows due to beneficial changes to habitat quality in the absence of grazing. Our results did not support our hypothesis, and showed no benefit to Yosemite toad presence or early life stages in fenced or partially fenced meadows compared to standard USDA Forest Service grazing levels. We found substantial Yosemite toad variation by both meadow and year. This variation was influenced by meadow wetness, with water table depth significant in both the tadpole and YOY models.