Pharmacological comparison of a recombinant CB1 cannabinoid receptor with its G(alpha 16) fusion product

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB(1) agonist-mediated signal transduction via an engineered G(alpha 16) system is different than that of the G(i/o) coupling normally preferred by the CB(1) receptor, we transfected the human recombinant CB(1) receptor (hCB(1)) or a fusion protein comprising the hCB(1) receptor and G(alpha 16) (hCB(1)-G(alpha 16)) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB(1)-G(alpha 16) fusion protein was found to be similar to that for hCB(1): HU 210 > CP 55,940 > or = SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [(35)S]GTP gamma S binding or inhibited forskolin-stimulated cAMP, presumably by coupling to G(i/o), in cells expressing hCB(1) but not hCB(1)-G(alpha 16). However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCB(1)-G(alpha 16) but not hCB(1). These data demonstrate that ligand affinities for the hCB(1) receptor are not affected by fusion to the G(alpha 16) subunit. Furthermore, there is essentially no difference in the function of the hCB(1) receptor when coupled to G(i/o) or G (alpha 16).     

Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein

AAA proteins remodel other proteins to affect a multitude of biological processes. Their power to remodel substrates must lie in their capacity to couple substrate binding to conformational changes via cycles of nucleotide binding and hydrolysis, but these relationships have not yet been deciphered for any member. We report that when one AAA protein, Hsp104, engages polypeptide at the C-terminal peptide-binding region, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2) drives a conformational change in the middle region. This, in turn, drives ATP hydrolysis in the N-terminal ATPase domain (NBD1). This interdomain communication pathway can be blocked by mutation in the middle region or bypassed by antibodies that bind there, demonstrating the crucial role this region plays in transducing signals from one end of the molecule to the other.     

An ontology driven architecture for derived representations of macromolecular structure

SUMMARY: An object metamodel based on a standard scientific ontology has been developed and used to generate a CORBA interface, an SQL schema and an XML representation for macromolecular structure (MMS) data. In addition to the interface and schema definitions, the metamodel was also used to generate the core elements of a CORBA reference server and a JDBC database loader. The Java source code which implements this metamodel, the CORBA server, database loader and XML converter along with detailed documentation and code examples are available as part of the OpenMMS toolkit. AVAILABILITY: http://openmms.sdsc.edu CONTACT: dsg@sdsc.edu     

KNAT1 and ERECTA regulate inflorescence architecture in Arabidopsis

Plant architecture is dictated by morphogenetic factors that specify the number and symmetry of lateral organs as well as their positions relative to the primary axis. Mutants defective in the patterning of leaves and floral organs have provided new insights on the signaling pathways involved, but there is comparatively little information regarding aspects of the patterning of stems, which play a dominant role in architecture. To this end, we have characterized five alleles of the brevipedicellus mutant of Arabidopsis, which exhibits reduced internode and pedicel lengths, bends at nodes, and downward-oriented flowers and siliques. Bends in stems correlate with a loss of chlorenchyma tissue at the node adjacent to lateral organs and in the abaxial regions of pedicels. A stripe of achlorophyllous tissue extends basipetally from each node and is positioned over the vasculature that services the corresponding lateral organ. Map-based cloning and complementation studies revealed that a null mutation in the KNAT1 homeobox gene is responsible for these pleiotropic phenotypes. Our observation that wild-type Arabidopsis plants also downregulate chlorenchyma development adjacent to lateral organs leads us to propose that KNAT1 and ERECTA are required to restrict the action of an asymmetrically localized, vasculature-associated chlorenchyma repressor at the nodes. Our data indicate that it is feasible to alter the architecture of ornamental and crop plants by manipulating these genetically defined pathways.     

Biparental care in house sparrows: negotiation or sealed bid?

We explored the responses of monogamous house sparrow parentsto deviations in their mates' contributions to nestling provisioning.Following 1-2 days of baseline measurement of parental fooddelivery rates, we applied small lead fishing weights to thetail feathers of either male or female parents. Weighting hadmuch greater immediate impact on male parental care than on female care, but the handicapping had little long-term effecton either male or female provisioning behavior. When parentalperformance of handicapped males was most impaired, their matesdid not show significant increases in parental care as compensation,nor did females mated to handicapped males reduce their provisioningas their mates recovered from weighting. Similarly, males matedto handicapped females did not respond to their partners' recovery with declines in their own efforts; paradoxically, these malesshowed a sustained elevation of provisioning throughout thepost-treatment interval, despite no significant reduction inprovisioning by weighted females. The apparent insensitivityof both males and females to changes in their mates' parentalbehavior, and the ineffectiveness of current partner behaviorat predicting an individual's provisioning effort, fail toconform to assumptions of biparental care models that requirefacultative responses to partner deviations in effort. Instead,the remarkable consistency of each individual's behavior supportsthe notion of "sealed bids" and suggests that variation innestling provisioning is largely attributable to factors thatare independent of the mate's current behavior, such as differencesin individual quality.     

A fusion protein system for the recombinant production of short disulfide-containing peptides

A recombinant fusion protein system for the production, oxidation, and purification of short peptides containing a single disulfide bond is described. The peptides are initially expressed in Escherichia coli as a fusion to an engineered mutant of the N-terminal SH2 domain of the intracellular phosphatase, SHP-2. This small protein domain confers several important properties which facilitate the production of disulfide-containing peptides: (i) it is expressed at high levels in E. coli; (ii) it can be purified via a hexahistidine tag and reverse-phase HPLC; (iii) it contains no endogenous cysteine residues, allowing the formation of an intrapeptide disulfide bond while still attached to the fusion partner; (iv) it is highly soluble in native buffers, facilitating the production of very hydrophobic peptides and the direct use of fusion products in biochemical assays; (v) it contains a unique methionine residue at the junction of the peptide and fusion partner to facilitate peptide cleavage by treatment with cyanogen bromide (CNBr). This method is useful for producing peptides, which are otherwise difficult to prepare through traditional chemical synthesis approaches, and this has been demonstrated by preparing a number of hydrophobic disulfide-containing peptides derived from phage-display libraries.     

The effects of high-dose glutamine ingestion on weightlifting performance

The purpose of this study was to determine if high-dose glutamine ingestion affected weightlifting performance. In a double-blind, placebo-controlled, crossover study, 6 resistance-trained men (mean +/- SE: age, 21.5 +/- 0.3 years; weight, 76.5 +/- 2.8 kg(-1)) performed weightlifting exercises after the ingestion of glutamine or glycine (0.3 g x kg(-1)) mixed with calorie-free fruit juice or placebo (calorie-free fruit juice only). Each subject underwent each of the 3 treatments in a randomized order. One hour after ingestion, subjects performed 4 total sets of exercise to momentary muscular failure (2 sets of leg presses at 200% of body weight, 2 sets of bench presses at 100% of body weight). There were no differences in the average number of maximal repetitions performed in the leg press or bench press exercises among the 3 groups. These data indicate that the short-term ingestion of glutamine does not enhance weightlifting performance in resistance-trained men.