Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).     

I-Region genetic restrictions imposed upon the recognition of KLH by murine T-cell clones

Data presented in this paper show that the recognition of keyhole limpet hemocyanin by murine T-cell clones is restricted by products of the I region. These data have been obtained by genetic mapping studies as well as by the use of monoclonal la-specific antibodies which inhibit the ability of antigen-presenting cells to effectively present antigen to such T-cell clones. Use of heterozygous antigen-presenting cells derived from crosses between B6.C-H-2 ^bm12 and B10.A(4R) mice have allowed us to show that both trans-complementing I-A products are used for restriction of recognition of KLH. These data were confirmed using monoclonal Ia antibodies to inhibition recognition of KLH by the same T-cell clones. Thus, we have shown that there exist hybrid molecules formed by free combinatorial association of products encoded within the I-A subregion which restrict the recognition of soluble antigen. Additionally, we have shown that the molecule formed by complementation between the alpha chain encoded within the I-E region and a beta chain encoded within the A region (A_e) can function effectively in presenting KLH to certain murine T-cell clones. These results support the hypothesis that the recognition of individual antigenic epitopes within large multideterminant antigens is under the control of Ir genes.     

Autophagy and mitochondrial remodelling in mouse mesenchymal stromal cells challenged with Staphylococcus epidermidis

The bone marrow stroma constitutes the marrow‐blood barrier, which sustains immunochemical homoeostasis and protection of the haematopoietic tissue in sequelae of systemic bacterial infections. Under these conditions, the bone marrow stromal cells affected by circulating bacterial pathogens shall elicit the adaptive stress‐response mechanisms to maintain integrity of the barrier. The objective of this communication was to demonstrate (i) that in vitro challenge of mesenchymal stromal cells, i.e. colony‐forming unit fibroblasts (CFU‐F), with Staphylococcus epidermidis can activate the autophagy pathway to execute antibacterial defence response, and (ii) that homoeostatic shift because of the bacteria‐induced stress includes the mitochondrial remodelling and sequestration of compromised organelles via mitophagy. Implication of Drp1 and PINK1–PARK2‐dependent mechanisms in the mitophagy turnover of the aberrant mitochondria in mesenchymal stromal cells is investigated and discussed.     

Demographic Clusters Identified within the Northern Gulf of Mexico Common Bottlenose Dolphin (Tursiops truncates) Unusual Mortality Event: January 2010 - June 2013

A multi-year unusual mortality event (UME) involving primarily common bottlenose dolphins (Tursiops truncates) was declared in the northern Gulf of Mexico (GoM) with an initial start date of February 2010 and remains ongoing as of August 2014. To examine potential changing characteristics of the UME over time, we compared the number and demographics of dolphin strandings from January 2010 through June 2013 across the entire GoM as well as against baseline (1990-2009) GoM stranding patterns. Years 2010 and 2011 had the highest annual number of stranded dolphins since Louisiana’s record began, and 2011 was one of the years with the highest strandings for both Mississippi and Alabama. Statewide, annual numbers of stranded dolphins were not elevated for GoM coasts of Florida or Texas during the UME period. Demographic, spatial, and temporal clusters identified within this UME included increased strandings in northern coastal Louisiana and Mississippi (March-May 2010); Barataria Bay, Louisiana (August 2010-December 2011); Mississippi and Alabama (2011, including a high prevalence and number of stranded perinates); and multiple GoM states during early 2013. While the causes of the GoM UME have not been determined, the location and magnitude of dolphin strandings during and the year following the 2010 Deepwater Horizon oil spill, including the Barataria Bay cluster from August 2010 to December 2011, overlap in time and space with locations that received heavy and prolonged oiling. There are, however, multiple known causes of previous GoM dolphin UMEs, including brevetoxicosis and dolphin morbillivirus. Additionally, increased dolphin strandings occurred in northern Louisiana and Mississippi before the Deepwater Horizon oil spill. Identification of spatial, temporal, and demographic clusters within the UME suggest that this mortality event may involve different contributing factors varying by location, time, and bottlenose dolphin populations that will be better discerned by incorporating diagnostic information, including histopathology.     

Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes

n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures.