The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin

Ubiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins.     

Overfeeding during lactation modulates insulin and leptin signaling cascade in rats' hearts

Insulin has been described as a potential mediator of intrinsic responses to the nutritional state in the heart due to its effects on cardiac metabolism, mainly on glucose transport. It has been demonstrated that leptin can act through some components of the insulin-signaling cascade. We investigated the association between overfeeding during lactation and alterations of insulin and leptin signaling in the heart. In summary, we analyzed a feasible cross-talk between insulin and leptin through the study of some key proteins of their cascades in the heart. In order to study the effect of overfeeding on these cascades, Wistar rats were overfed through litter size reduction to only three pups. At 10 and 21 days of life, key proteins such as insulin receptor, leptin receptor, PI3-kinase, JAK2, STAT3, and GLUT4 were measured by Western blotting. Furthermore, the pups' weight and the plasma levels of insulin, leptin and glucose were determined. Overfed animals were overweight, had high insulin and leptin plasma levels, and displayed an activation of insulin and leptin cascade, leading to an increased translocation of GLUT4. We suggest that overfeeding during lactation probably alters cardiac metabolism, through the activation of a modulated cross-talk between leptin and insulin cascades.     

Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142

Human cytomegalovirus (HCMV) employs a variety of strategies to modify or evade the host immune response, and natural killer (NK) cells play a crucial role in controlling cytomegalovirus infections in mice and humans. Activation of NK cells through the receptor NKG2D/DAP10 leads to killing of NKG2D ligand-expressing cells. We have previously shown that HCMV is able to down-regulate the surface expression of some NKG2D ligands, ULBP1, ULBP2, and MICB via the viral glycoprotein UL16. Here, we show that the viral gene product UL142 is able to down-regulate another NKG2D ligand, MICA, leading to protection from NK cytotoxicity. UL142 is not able to affect surface expression of all MICA alleles, however, which may reflect selective pressure on the host to thwart viral immune evasion, further supporting an important role for the MICA-NKG2D interaction in immune surveillance.     

Antioxidant and antiulcer activity of aqueous extract of a polyherbal formulation

The aqueous extract of Hingwashtak churna was evaluated for gastroprotection in rats using the ibuprofen and ethanol induced ulcer models. Efficacy was assessed by determination of mean ulcer size, ulcer number and ulcer index. Oral administration of the aqueous extract (750 mg/kg) significantly protected against gastric lesions by 84.96% and 91.12% as compared to ranititidine (95.54 and 95.2%) in the ibuprofen and alcohol induced ulcer models respectively. The findings suggest that the significant gastroprotective activity could be mediated by its antioxidant activity which was evaluated by using different antioxidant models of screening.     

Development of a high-capacity homogeneous fluorescent assay for the measurement of leukotriene B4

Current immunoassays for the measurement of leukotriene B(4) (LTB(4)) typically utilize an enzyme-linked immunosorbent assay (ELISA) format that requires multiple incubations and washing steps and often expensive immunoassay kits. We have developed a bead-based, mix and read, indirect fluorescence-linked immunosorbent assay utilizing fluorometric microvolume assay technology (FMAT). The assay employs a monoclonal anti-LTB(4) antibody-coated onto goat antimouse antibody coupled polystyrene beads and an AlexaFluor-647-coupled LTB(4) ligand. Because the FMAT measurement is made only in the portion of the well volume containing the settled beads coated with AF647-LTB(4), the free label in the solution is not measured. Similarly, substances present in plasma that interfere with other immunoassays are largely ignored. The assay is robust (Z=0.8; S/N=250) and can be measured in the presence of relatively high concentrations of dimethyl sulfoxide or serum. It is inexpensive (<0.10 dollars/assay) and amenable to robotics and has a sensitivity comparable to that of the most sensitive ELISA assays; the concentration of LTB(4) giving 50% inhibition (IC(50)) was ca. 55pg/ml. Cross-reactivity in the FMAT assay was comparable to that of the ELISA assay with significant cross-reactivity found only with 20-hydroxy LTB(4) and 12-epi LTB(4). Measurements of LTB(4) determined by FMAT were equivalent to those measured by standard ELISA in samples of ionophore-stimulated human neutrophils or whole blood.     

Ion-mediated flow changes suppressed by minimal calcium presence in xylem sap in Chrysanthemum and Prunus laurocerasus

After the discovery of ion-mediated changes in xylem hydraulic resistance a few years ago, a number of research papers were published that related ion-mediated flow changes in the xylem to various aspects of whole plant functioning and evolutionary diversification of vascular cells. Ion-mediated changes in xylem hydraulic resistance are commonly quantified as the percentile change in hydraulic resistance, relative to the hydraulic resistance measured using a reference fluid, usually (ultra) pure deionized water. In this research the impact was investigated of the complete absence of all ions in deionized water compared with reference fluids containing a minimal amount of free calcium on the quantification of ion-mediated flow changes in stem segments of Chrysanthemum (Dendranthemaxgrandiflorum Tzvelev) and Prunus L. (Prunus laurocerasus L.). The addition of 10 mM KCl to deionized water significantly increased flow rate in Chrysanthemum (17-24%) and Prunus L. (16%). The addition of 1 mM CaCl(2) to the reference fluid reduced this KCl-mediated increase in flow rate to 1-2% in both species. 1 mM Ca(2+) is within the lower range of Ca(2+)-concentrations normally measured in xylem sap of many plant species, and three times lower than the original Ca(2+)-concentration measured in the xylem sap of Chrysanthemum plants used for the present measurements. The present results indicate that the complete removal of cations from the xylem fluid with deionized water causes the major part of the ion-mediated flow change previously reported in the xylem of plants. It is concluded that the use of deionized water as a reference fluid should be avoided. Earlier proposed relationships between ion-mediated changes and water flow in xylem of plants should be re-evaluated if they were based on deionized water as the reference fluid.     

Phosphorylation of microtubule-associated protein STOP by calmodulin kinase II

STOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity.     

Multiple consequences of mutating two conserved beta-bridge forming residues in the translocation cycle of a neuronal glutamate transporter

Glutamate transporters remove this neurotransmitter from the synapse in an electrogenic process. After sodium-coupled glutamate translocation, the cycle is completed by obligatory outward translocation of potassium. In the crystal structure of an archaeal homologue, two conserved residues form a beta-bridge, which points away from the binding pocket. In the neuronal glutamate transporter EAAC1, the equivalent residues are asparagine 366 and aspartate 368. Substitution mutants N366Q and D368E, but not N366D and D368N, show glutamate-induced inwardly rectifying steady-state currents, but their apparent substrate affinity is dramatically decreased. Such currents, which reflect electrogenic net uptake of substrate are not observed with the reciprocal double mutant N366D/D368N. Remarkably, the double mutant exhibits slow substrate-induced voltage-dependent capacitative transient currents. These currents apparently reflect the reversible sodium-coupled glutamate translocation step, because the interaction of the double mutant with potassium is largely impaired. Moreover, when the analogous double mutant in the glutamate transporter GLT-1 is reconstituted into liposomes, a slow exchange of radioactive and unlabeled acidic amino acids is observed. Our results suggest that it is the interaction of asparagine 366 and aspartate 368 that is important during the glutamate translocation step. On the other hand, the side chains of these residues themselves are required for the subsequent potassium relocation step.     

Living in nonbreeding groups: an alternative strategy for maturing gorillas

The one-male reproductive strategy implies that maturing males are temporarily excluded from reproduction. In gorillas, these excluded males live either solitarily or in nonbreeding groups (NBGs) that are devoid of adult females. The dynamics of NBGs are not well known. In this study, which was conducted on a gorilla population (Gorilla gorilla gorilla) of 377 individuals that visited the Lokoué clearing in the Republic of Congo, we detail how the NBGs formed, and analyze their dynamics according to age-sex classes, the relatedness of members, and the origin and destination of transferring individuals. We discuss the potential benefits gained by individuals living in these groups. The NBGs included mainly immature males, most of which appeared to have migrated voluntarily from their natal groups. Some individuals (including juvenile females) came from disbanded breeding groups (BGs). Migrants preferentially joined NBGs that included a silverback male. Their dispersal patterns were not determined by their degree of relatedness, but they tended to associate with related silverbacks. In this way, the migrants could enhance their protection against predators and gain experience with different environmental conditions. By tolerating and protecting offspring, aging silverbacks could enhance their inclusive fitness. Finally, young and healthy silverbacks could increase their likelihood of forming a future BG when unrelated females joined them.     

Botrytis cinerea virulence is drastically reduced after disruption of chitin synthase class III gene (Bcchs3a)

Botrytis cinerea is an important phytopathogenic fungus requiring new methods of control. Chitin biosynthesis, which involves seven classes of chitin synthases, could be an attractive target. A fragment encoding one of the class III enzymes was used to disrupt the corresponding Bcchs3a gene in the B. cinerea genome. The resulting mutant exhibited a 39% reduction in its chitin content and an 89% reduction in its in vitro chitin synthase activity, compared with the wild-type strain. Bcchs3a mutant was not affected in its growth in liquid medium, neither in its production of sclerotia, micro- and macroconidia. In contrast, the mutant Bcchs3a was severely impaired in its growth on solid medium. Counterbalancing this defect in radial growth, Bcchs3a mutant presented a large increase in hyphal ramification, resulting in an enhanced aerial growth. Observations by different techniques of microscopy revealed a thick extracellular matrix around the hyphal tips. Moreover, Bcchs3a mutant had a largely reduced virulence on Vitis vinifera and Arabidopsis thaliana leaves.