Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells

Background  

Rex1/Zfp42 has been extensively used as a marker for the undifferentiated state of pluripotent stem cells. However, its function in pluripotent stem cells including embryonic stem (ES) cells remained unclear although its involvement in visceral endoderm differentiation in F9 embryonal carcinoma (EC) cells was reported.     

Biochemical and structural characterization of (South)-methanocarbathymidine that specifically inhibits growth of herpes simplex virus type 1 thymidine kinase-transduced osteosarcoma cells

Two analogs of the natural nucleoside dT featuring a pseudosugar with fixed conformation in place of the deoxyribosyl residue (carbathymidine analogs) were biochemically and structurally characterized for their acceptance by both human cytosolic thymidine kinase isoenzyme 1 (hTK1) and herpes simplex virus type 1 thymidine kinase (HSV1 TK) and subsequently tested in cell proliferation assays. 3'-exo-Methanocarbathymidine ((South)-methanocarbathymidine (S)-MCT), which is a substrate for HSV1 TK, specifically inhibited growth of HSV1 TK-transduced human osteosarcoma cells with an IC(50) value in the range of 15 microM without significant toxicity toward both hTK1-negative (TK(-)) and non-transduced cells. 2'-exo-Methanocarbathymidine ((North)-methanocarbathymidine (N)-MCT), which is a weak substrate for hTK1 and a substantial one for HSV1 TK, induced a specific growth inhibition in HSV1 TK-transfected cells comparable to that of (S)-MCT and ganciclovir. A growth inhibition activity was also observed with (N)-MCT and ganciclovir in non-transduced cells in a cell line-dependent manner, whereas TK(-) cells were not affected. The presented 1.95-A crystal structure of the complex (S)-MCT.HSV1 TK explains both the more favorable binding affinity and catalytic turnover of (S)-MCT for HSV1 TK over the North analog. Additionally the plasticity of the active site of the enzyme is addressed by comparing the binding of (North)- and (South)-carbathymidine analogs. The presented study of these two potent candidate prodrugs for HSV1 TK gene-directed enzyme prodrug therapy suggests that (S)-MCT may be even safer to use than its North counterpart (N)-MCT.     

Canine-derived cosmid probes containing microsatellites can be used in physical mapping of Arctic fox (Alopex lagopus) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides) genomes

Rapid development of the canine marker genome map facilitates genome mapping of other Canidae species. In this study we present chromosomal localization of 18 canine-derived cosmid probes containing microsatellites in the arctic fox (Alopex lagopus) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides) genomes by the use of fluorescence in situ hybridization (FISH). The chromosome localizations in the arctic fox are in general agreement with data obtained from comparative genome maps of the dog and the fox. However, our studies showed that the order of the loci on some chromosomes was changed during karyotype evolution. Therefore, we suggest that small intrachromosomal rearrangements took place.     

Modulatory role of food, feeding regime and physical exercise on body weight and insulin resistance

Energy intake and expenditure is a highly conserved and well-controlled system with a bias toward energy intake. In times of abundant food supply, individuals tend to overeat and in consequence to increase body weight, sometimes to the point of clinical obesity. Obesity is a disease that is not only characterized by enormous body weight but also by rising morbidity for diabetes type II and cardiovascular complications. To better understand the critical factors contributing to obesity we performed the present study in which the effects of energy expenditure and energy intake were examined with respect to body weight, localization of fat and insulin resistance in normal Wistar rats. It was found that a diet rich in fat and carbohydrates similar to "fast food" (cafeteria diet) has pronounced implication in the development of obesity, leading to significant body weight gain, fat deposition and also insulin resistance. Furthermore, an irregularly presented cafeteria diet (yoyo diet) has similar effects on body weight and fat deposition. However, these rats were not resistant to insulin, but showed an increased insulin secretion in response to glucose. When rats were fed with a specified high fat/carbohydrate diet (10% fat, 56.7% carbohydrate) ad lib or at the beginning of their activity phase they were able to detect the energy content of the food and compensate this by a lower intake. They, however, failed to compensate when food was given in the resting phase and gained more body weight as controls. Exercise, even of short duration, was able to keep rats on lower body weight and reduced fat deposition. Thus, inappropriate food intake with different levels of energy content is able to induce obesity in normal rats with additional metabolic changes that can be also observed in humans.     

Canine 5S rRNA: nucleotide sequence and chromosomal assignment of its gene cluster in four canid species

The purpose of this study was to determine the nucleotide sequence of canine 5S rRNA and use this information to develop a molecular probe to assign the gene locus to chromosomes of the dog and three other related canid species using fluorescence in situ hybridization. The nucleotide sequence of canine liver 5S rRNA is 120 base pairs long and identical to the 5S rRNA nucleotide sequence of all other mammalian species investigated so far. A single 5S rRNA gene cluster was localized pericentromerically on chromosomes of four canid species: dog 4q1.3, red fox 4q1.3, blue fox 3q1.3 and Chinese raccoon dog 8q1.3. Chromosome arms carrying the 5S rRNA gene cluster showed striking similarities in their QFQ banding patterns, suggesting high conservation of these chromosome arms among the four species studied. The chromosomal assignments of 5S rRNA genes are among the first gene mapping results for the blue fox and the Chinese raccoon dog, and are in accordance with published data on comparative chromosome maps from human, dog, red fox, blue fox and raccoon dogs.     

Remarkable similarities between yeast and mammalian protein phosphatases

Protein phosphatase activities in extracts of the yeast Saccharomyces cerevisiae showed remarkable similarities to the mammalian type 1, type 2A and type 2C enzymes. Similarities included their substrate specificities, including selectivity for the alpha-and beta-subunits of muscle phosphorylase kinase, sensitivity to okadaic acid and to mammalian inhibitor 1 and inhibitor 2, and requirement for divalent cations. The results suggest that the function and regulation of these enzymes has been highly conserved during evolution and indicate that the improved procedure for identifying and quantitating protein phosphatases [(1989) FEBS Lett. 250,000,000] may be applicable to all eukaryotic cells.     

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.