Glycogenin protein and mRNA expression in response to changing glycogen concentration in exercise and recovery

Glycogenin (GN-1) is essential for the formation of a glycogen granule; however, rarely has it been studied when glycogen concentration changes in exercise and recovery. It is unclear whether GN-1 is degraded or is liberated and exists as apoprotein (apo)-GN-1 (unglycosylated). To examine this, we measured GN-1 protein and mRNA level at rest, at exhaustion (EXH), and during 5 h of recovery in which the rate of glycogen restoration was influenced by carbohydrate (CHO) provision. Ten males cycled (65% VO2 max) to volitional EXH (117.8 +/- 4.2 min) on two separate occasions. Subjects were administered carbohydrate (CHO; 1 g.kg(-1).h(-1) Gatorlode) or water [placebo (PL)] during 5 h of recovery. Muscle biopsies were taken at rest, at EXH, and following 30, 60, 120, and 300 min of recovery. At EXH, total glycogen concentration was reduced (P < 0.05). However, GN-1 protein and mRNA content did not change. By 5 h of recovery, glycogen was resynthesized to approximately 60% of rest in the CHO trial and remained unchanged in the PL trial. GN-1 protein and mRNA level did not increase during recovery in either trial. We observed modest amounts of apo-GN-1 at EXH, suggesting complete degradation of some granules. These data suggest that GN-1 is conserved, possibly as very small, or nascent, granules when glycogen concentration is low. This would provide the ability to rapidly restore glycogen during early recovery.     

Structure of the antiviral assembly inhibitor CAP-1 complex with the HIV-1 CA protein

The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CA^N) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CA^N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N′-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.     

The crystal structure of mouse Exo70 reveals unique features of the mammalian exocyst

The exocyst is a eukaryotic tethering complex necessary for the fusion of exocytic vesicles with the plasma membrane. Its function in vivo is tightly regulated by interactions with multiple small GTPases. Exo70, one of the eight subunits of the exocyst, is important for the localization of the exocyst to the plasma membrane. It interacts with TC10 and Rho3 GTPases in mammals and yeast, respectively, and has been shown recently to bind to the actin-polymerization complex Arp2/3. Here, we present the crystal structure of Mus musculus Exo70 at 2.25 A resolution. Exo70 is composed of alpha-helices in a series of right-handed helix-turn-helix motifs organized into a long rod of length 170 A and width 35 A. Although the alpha-helical organization of this molecule is similar to that in Saccharomyces cerevisiae Exo70, major structural differences are observed on the surface of the molecule, at the domain boundaries, and in various loop structures. In particular, the C-terminal domain of M. musculus Exo70 adopts a new orientation relative to the N-terminal half not seen in S. cerevisiae Exo70 structures. Given the low level of sequence conservation within Exo70, this structure provides new insights into our understanding of many species-specific functions of the exocyst.     

Shorebird predation of horseshoe crab eggs in Delaware Bay: species contrasts and availability constraints

1. Functional responses -- the relationship between resource intake rate and resource abundance -- are widely used in explaining predator-prey interactions yet many studies indicate that resource availability is crucial in dictating intake rates. 2. For time-stressed migrant birds refuelling at passage sites, correct decisions concerning patch use are crucial as they determine fattening rates and an individual's future survival and reproduction. Measuring availability alongside abundance is essential if spatial and temporal patterns of foraging are to be explained. 3. A suite of shorebird species stage in Delaware Bay where they consume horseshoe crab Limulus polyphemus eggs. Several factors including spawning activity and weather give rise to marked spatial and temporal variation in the abundance and availability of eggs. We undertook field experiments to determine and contrast the intake rates of shorebird species pecking for surface and probing for buried eggs. 4. Whether eggs were presented on the sand surface or buried, we demonstrate strong aggregative responses and rapid depletion (up to 80%). Depletion was greater at deeper depths when more eggs were present. No consistent give-up densities were found. Type II functional responses were found for surface eggs and buried eggs, with peck success twice as high in the former. Maximum intake rates of surface eggs were up to 83% higher than those of buried eggs. 5. Caution is needed when applying functional responses predicted on the basis of morphology. Our expectation of a positive relationship between body size and intake rate was not fully supported. The smallest species, semipalmated sandpiper, had the lowest intake rate but the largest species, red knot, achieved only the same intake rate as the mid-sized dunlin. 6. These functional responses indicate that probing is rarely more profitable than pecking. Currently, few beaches provide egg densities sufficient for efficient probing. Areas where eggs are deposited on the sand surface are critical for successful foraging and ongoing migration. This may be especially true for red knot, which have higher energetic demands owing to their larger body size yet appear to have depressed intake rates because they consume smaller prey than their body size should permit.     

Inhibition of S. aureus alpha-hemolysin and B. anthracis lethal toxin by beta-cyclodextrin derivatives

Many pathogens utilize the formation of transmembrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors, which makes them attractive targets for the discovery of new therapeutic agents. Our research is based on the idea that compounds designed to block the pores can inhibit the action of virulence factors, and that the chances to find high affinity blocking agents increase if they have the same symmetry as the target pore. Recently, we demonstrated that derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. To test the broader applicability of this approach, we sought beta-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcus aureus alpha-hemolysin (alpha-HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of beta-cyclodextrin that inhibited the activity of alpha-HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by alpha-HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapeutics against various pathogens that utilize pore-forming proteins as virulence factors.     

In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor

Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells, via transactivation of the EGF receptor (EGFR). H. pylori activation of EGFR may be relevant to epithelial hyperproliferation and gastric carcinogenesis. The aim of this study was to develop an 'In-Cell Western' (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling, to enable the role of the EGFR in H. pylori-induced phosphorylation of ERK in epithelial cells to be ascertained. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. H. pylori strains both with, and without a cag PAI, and Helicobacter felis, significantly increased pERK levels in A431 cells. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phosphorylation in A431 and AGS cells. A significantly lower reduction was observed with cag+ strains in A431 but not AGS cells. The cag PAI was not necessary for EGFR signal transactivation. These data suggest that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.     

A novel role for catalase B in the maintenance of fungal cell-wall integrity during host invasion in the rice blast fungus Magnaporthe grisea

Asexual spores of the rice blast fungus germinate to produce a specialized and melanized infection structure, the appressorium, which is pivotal to successful plant penetration. To investigate whether Magnaporthe grisea counteracts the toxic burst of H2O2 localized beneath the site of attempted invasion, we examined the temporal expression of five candidate antioxidant genes. Of these, the putatively secreted large subunit catalase CATB gene was 600-fold up-regulated in vivo, coincident with penetration, and moderately up-regulated in vitro, in response to exogenous H2O2. Targeted gene replacement of CATB led to compromised pathogen fitness; the catB mutant displayed paler pigmentation and accelerated hyphal growth but lower biomass, poorer sporulation, fragile conidia and appressoria, and impaired melanization. The catB mutant was severely less pathogenic than Guy 11 on barley and rice, and its infectivity was further reduced on exposure to H2O2. The wild-type phenotype was restored by the reintroduction of CATB into the catB mutant We found no evidence to support a role for CATB in detoxification of the host-derived H2O2 at the site of penetration. Instead, we demonstrated that CATB plays a part in strengthening the fungal wall, a role of particular importance during forceful entry into the host.