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T Caskey

Publications and source records attributed to T Caskey.

13 recordsLinked to original sources
PubMed2001

UCP3 expressed in yeast is primarily localized in extramitochondrial particles.

Previously it was concluded (1) that, differently from UCP1, on expression in Saccharomyces cerevisiae, UCP3, and UCP3 short (UCP3s) are in a deranged state, allowing for unregulated uncoupling. Here we show that the bulk of UCP3 and UCP3s is in extramitochondrial aggregates whether expressed with high or medium expression vectors. The evidence is based on the insolubility of most UCP3 and UCP3s in nonionic detergents such as Triton X100, in contrast to UCP1. Using very high expression vector, macroscopic evidence for extramitochondrial UCP3 containing particles is a viscous white sediment surrounding the mitochondrial fraction which contains UCP3 as inclusion body type aggregate. Together with the previous data it is concluded that uncoupling due to small amounts of incorporated, deranged, and nucleotide insensitive UCP3 prevents incorporation of the bulk of UCP3 into mitochondria. This finding also provides a simple and stringent assay for the state of heterologously expressed in mitochondrial membrane proteins.

Carrier Proteins↗
PubMed2000

The bulk of UCP3 expressed in yeast cells is incompetent for a nucleotide regulated H+ transport.

The impact of uncoupling protein (UCP) 1, UCP3 and UCP3s expressed in yeast on oxidative phosphorylation, membrane potential and H+ transport is determined. Intracellular ATP synthesis is inhibited by UCP3, much more than by UCP1, while similar levels of UCP3 and UCP1 exist in the mitochondrial fractions. Measurements of membrane potential and H+ efflux in isolated mitochondria show that, different from UCP1, with UCP3 and UCP3s there is a priori a preponderant uncoupling not inhibited by GDP. The results are interpreted to show that UCP3 and UCP3s in yeast mitochondria are in a deranged state causing uncontrolled uncoupling, which does not represent their physiological function.

Animals↗
PubMed1999

Regulation of UCP3 by nucleotides is different from regulation of UCP1.

UCP3 is an isoform of UCP1, expressed primarily in skeletal muscle. Functional properties of UCP3 are still largely unknown. Here, we report about the expression of UCP3 and of UCP1 in inclusion bodies of Escherichia coli. On solubilization and reconstitution into proteoliposomes, both UCP3 and UCP1 transport Cl- at rates equal to the reconstituted native UCP1. Cl- transport is inhibited by low concentrations of ATP, ADP, GTP and GDP. However, no H+ transport activity is found possibly due to the lack of a cofactor presents in UCP from mitochondria. The specificity of inhibition by nucleoside tri- and diphosphate is different between UCP1 and UCP3. UCP1 is more sensitive to tri- than diphosphate whereas in UCP3, the gradient is reverse. These results show a new paradigm for the regulation of thermogenesis at various tissues by the ATP/ADP ratio. In brown adipose tissue, the thermogenesis is correlated with a low ATP/ADP whereas in skeletal muscle, non-shivering thermogenesis is active at a high ATP/ADP ratio, i.e. in the resting state.

Adipose Tissue↗
PubMed1999

Cloning of a novel G-protein-coupled receptor GPR 51 resembling GABAB receptors expressed predominantly in nervous tissues and mapped proximal to the hereditary sensory neuropathy type 1 locus on chromosome 9.

Query of the expressed sequence tag database with the rat metabotropic GABABR1A receptor amino acid sequence using the TFASTA algorithm revealed two partial cDNA fragments whose sequence information was then used to isolate by PCR a novel full-length human cDNA encoding a putative G-protein-coupled receptor (GPCR), termed GPR 51. Sequence analysis revealed that it encoded a protein of 941 amino acids, similar in size and homology to GABAB receptors followed by metabotropic glutamate receptors but not other GPCRs. GPR 51 expressed in COS-1 cells showed no specific binding for [3H](+)baclofen and when expressed in Xenopus oocyte and Xenopus melanophore functional assays showed no activity to GABA, (-)baclofen, and glutamic acid. Northern blot analysis and in situ hybridization revealed that GPR 51 transcripts were predominantly expressed in the central nervous system with highest abundance in the cortex, thalamus, hippocampus, amygdala, cerebellum, and spinal cord. In contrast, GPR 51 receptor transcripts were almost not detected in the peripheral tissues. Gene GPR 51 was localized by radiation hybrid mapping to chromosome 9, 4.81 cR from the WI-8684 marker, and proximal to the hereditary sensory neuropathy type 1 locus.

Amino Acid Sequence↗
PubMed1992

Two hot spots of recombination in the DMD gene correlate with the deletion prone regions.

Genetic mapping has indicated that meiotic recombination occurs about 4 time more frequently in the dystrophin gene than expected on the basis of its length. To detect where recombinations occur within the gene, we have studied the CEPH families panel using highly polymorphic microsatellite markers located at the ends of the gene or flanking the major deletion hot spot in intron 44. We found a major hot spot of recombination between markers STR44 and STR50(1), i.e., between exons 44 and 51. Within this hot spot, a peak of recombination was located in the large intron 44. A second minor recombination prone region was found between DXS 206, (XJ, in the large intron 7) and the 5' end of the DMD gene. The distribution of the recombination events in the gene of healthy individuals was very similar to that of deletion breakpoints in DMD/BMD patients, suggesting that the two phenomenon may share a common mechanism. These results should also improve efficiency and accuracy of linkage analysis applied to carrier detection and prenatal diagnosis. In particular, if markers located at the very 3' end of the gene are not informative, the highly polymorphic ones located between exons 50 and 60 can be used instead of presently available extragenic markers, with a very low risk of diagnostic error due to recombination.

Dystrophin↗
PubMed1970

Peptide chain termination. VI. Purification and site of action of S.

PEPTIDE CHAIN TERMINATION IS A RESULT OF AT LEAST TWO EVENTS: terminator codon recognition and hydrolysis of peptidyl tRNA. A protein factor S, isolated from the supernatant of Escherichia coli B, stimulates fMet release. Factor S lowers the K(m) for terminator trinucleotides without altering the V(max) of release and therefore acts at terminator codon recognition. The S protein differs from initiation factors, elongation factor G, several forms of elongation factor T and release factors. The importance of the 2% Tu content in purified S is unresolved.

Bacterial Proteins↗
PubMed1969

Peptide chain termination. 3. Stimulation of in vitro termination.

Throughout extensive purification, the release factors R(1) and R(2) each behave as a single molecular species with alternate codon recognition (R(1), UAA or UAG; R(2), UAA or UGA). The release of f[(3)H]methionine from f[(3)H]-Met-tRNA.AUG.ribosome complex requires R factor and terminator codon and does not appear to require tRNA or transfer factors T and G. Purification of the components of the release assay has enabled identification of a protein factor S in the 55-80 per cent ammonium sulfate fraction of E. coli B supernatant fraction which stimulates the rate but not the extent of release dependent upon R factor and appropriate termination codon. The S factor has properties similar to T, but further purification is required to determine the nature and function of S in peptide chain termination.

Amino Acid Sequence↗