Starch Binding Domain-containing Protein 1/Genethonin 1 Is a Novel Participant in Glycogen Metabolism

Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin-producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.     

Studies of enzyme polymorphisms in the Kamuela population of Drosophila mercatorum. II. Evaluation of glycolytic intermediates

A simple and effective cryogenic procedure for the extraction of glycolytic intermediates from whole Drosophila has been developed. This procedure gives consistent results when a measure (µM/liter/OD₂₆₀) is adopted which corrects for differences in extraction efficiency. Using this measure and a homozygous strain of D. mercatorum, there are no significant differences among extracts for the levels of any of the 15 glycolytic intermediate or energy molecules considered. The profile of means is consistent across experimental designs and instrument types. Coefficients of variation are well below 50% for most variables. The methodology presented has the statistical power to detect a mean change of 10 to 50% using an experimental design which requires as few as 32 observations. The estimated energy charge for resting Drosophila from these studies is the expected value of 0.86.     

Trichomonas gallinae in columbiform birds from the Galapagos Islands

Domestic pigeons were introduced into the Galapagos Islands in 1972 or 1973. There is a high prevalence of Trichomonas gallinae among them and some evidence of canker. Trichomonas gallinae can be found also in endemic Galapagos doves in the vicinity of Puerto Ayora on Santa Cruz Island. Doves examined on pigeon-free islands were not found infected.     

The apparent absence of serotonin-mediated vascular thermogenesis in perfused rat hindlimb may result from vascular shunting

Vasoconstriction by norepinephrine, angiotensin II and vasopressin in the constant-flow perfused rat hindlimb is associated with increased oxygen uptake and has given rise to the concept of vascular thermogenesis. In the present study serotonin (5-hydroxytryptamine, 5HT) was found to inhibit oxygen uptake by up to 40% in a dose dependent manner whilst inducing vasoconstriction in this model, whereas norepinephrine increased oxygen consumption by up to 100% during vasoconstriction. This contrasted with the perfused isolated rat mesenteric artery arcade in which serotonin stimulated oxygen uptake by up to 130% in association with vasoconstriction in a dose dependent manner similar to the previously described norepinephrine induced vascular thermogenesis in this arterial preparation. In both perfusion systems, changes in pressure and oxygen uptake mediated by serotonin were completely blocked by ketanserin. These results and evidence from dye washout studies suggest that serotonin-mediated vascular thermogenesis, if it occurs in the constant-flow hindlimb, is masked by vascular shunting.     

The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle

Two monoclonal antibodies, 1F5 and B1, directed against the CD20 (Bp35) antigen were found to have both stimulatory and inhibitory effects on B cells. 1F5, but not B1, induces small resting tonsillar B cells and prolymphocytic leukemia cells to enlarge, to rapidly increase their RNA synthesis, and to become responsive to growth factors present in mixed lymphocyte reaction supernatants. In addition, 1F5 induces a moderate increase in thymidine uptake, which is accompanied by enhanced viability of the cells, but not by any increase in total cell number or by any detectable entry into S phase or mitosis. Taken together, these observations suggest that 1F5 can initiate transition from the G0 to the G1 phase of the cell cycle. The fact that all the changes observed can be inhibited by low concentrations (I50 = 50 ng/ml) of cyclosporin A is further evidence that 1F5 is involved at an early stage of B cell activation. Because both 1F5 and B1 belong to the IgG2a subclass, differences in their activities are likely to reflect their different epitope specificities. Although only 1F5 had stimulatory activity, both 1F5 and B1 strongly inhibited B cell differentiation to immunoglobulin secretion. Possible explanations for the dual activities of 1F5 and implications for the role of the CD20 antigen in B cell differentiation are discussed.