A tree-based algorithm for determining the effects of solvation on the structure of salivary gland tripeptide NH3+-D-PHE-D-GLU-GLY-COO-

A D-enantiomeric analog of the submandibular gland rat-1 tripeptide FEG (Seq: NH(3)(+)-Phe-Glu-Gly-COO(-)) called feG (Seq: NH(3)(+)-D-Phe-D-Glu-Gly-COO(-)) was examined by molecular dynamics simulations in water. Previous in vacuo simulations suggested a conformation consisting predominantly of interactions between the Phe side chain and glutamyl-carboxyl group and a carboxyl/amino termini interaction. The solvated peptide was simulated using two approaches which were compared-a single 400-ns simulation and a "simulation tree." The "tree" approach utilized 45 10-ns simulations with different conformations used as initial structures for given trajectories. We demonstrate that multiple short duration simulations are able to describe the same conformational space as that described by longer simulations. Furthermore, previously described in vacuo interactions were confirmed with amendments: the previously described head-to-tail arrangement of the amino and carboxyl termini, was not observed; the interaction between the glutamyl carboxyl and Phe side chain describes only one of a continuum of conformations present wherein the aromatic residue remains in close proximity to the glutamyl carbonyl group, and also interacts with either of the two available carboxyl groups. Finally, utilizing only two separate 10-ns trajectories, we were able to better describe the conformational space than a single 60-ns trajectory, realizing a threefold decrease in the computational complexity of the problem.     

Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster

Octopamine is a major monoamine in invertebrates and affects many physiological processes ranging from energy metabolism to complex behaviors. Octopamine binds to receptors located on various cell types and activates distinct signal transduction pathways to produce these diverse effects. We previously identified one of the Drosophila octopamine receptors named OAMB that produces increases in cAMP and intracellular Ca2+ upon ligand binding. It is expressed at high levels in the brain. To explore OAMB's physiological roles, we generated deletions in the OAMB locus. The resultant oamb mutants were viable without gross anatomical defects. The oamb females displayed normal courtship and copulation; however, they were impaired in ovulation with many mature eggs retained in their ovaries. RT-PCR, in situ hybridization, and expression of a reporter gene revealed that OAMB was also expressed in the thoracicoabdominal ganglion, the female reproductive system, and mature eggs in the ovary. Moreover, analysis of various alleles pinpointed the requirement for OAMB in the body, but not in the brain, for female fecundity. The novel expression pattern of OAMB and its genetic resource described in this study will help advance our understanding on how the neuromodulatory or endocrine system controls reproductive physiology and behavior.     

Environmental influences on epistatic interactions: viabilities of cytochrome c genotypes in interpopulation crosses

Abstract .The genetic incompatibilities that underlie F₂ hybrid breakdown and reproductive isolation between al-lopatric populations may be susceptible to environmental interactions. Here we show that epistatic interactions between cytochrome c ( CYC ) alleles and mitochondrial DNA (mtDNA) variation are dramatically influenced by environmental temperature in interpopulation hybrids of the copepod Tigriopus californicus . CYC is a nuclear-encoded gene that functionally interacts with electron transport system (ETS) complexes composed in part of mtDNA-encoded proteins. Previous studies have provided evidence for functional coadaptation between CYC and ETS complex IV (cytochrome c oxidase) and for cytoplasmic effects on the fitness of CYC genotype in copepod hybrids. In this study, selection on CYC genotype is shown to continue into advanced generation hybrids (F₂-F₈) increasing the likelihood that CYC itself is involved in the interaction (and not a linked factor). Relative viabilities varied markedly between copepods raised in two different temperature/light regimes. These results suggest that both intrinsic coadaptation and extrinsic selection will influence the outcome of natural hybridizations between populations. Furthermore, the results indicate that the fitness of particular hybrid genotypes depends on additional non-mtDNA encoded genes that interact with CYC.     

The cytosolic chaperonin CCT associates to cytoplasmic microtubular structures during mammalian spermiogenesis and to heterochromatin in germline and somatic cells

Spermiogenesis, the haploid phase of spermatogenesis, is characterised by a dramatic cytodifferentiation of spermatids. The two major steps, nuclear shaping and cytoplasmic reorganisation of the organelles, rely on an extensive remodelling of the microtubule cytoskeleton. Folding of alpha- and beta-tubulin is mediated by the cytoplasmic chaperonin containing TCP-1 (CCT), highly expressed in testis. We studied CCT cellular distribution throughout spermatogenesis by immunofluorescence and immunoelectron microscopy. We unveil two main cytoplasmic localisations for CCT: at the centrosome and at the microtubules of the manchette, a structure unique to male germ cells. Both structures are essential for spermatid differentiation and may require CCT function. Although CCT is essentially cytoplasmic, a few reports suggest that a subset may have a nuclear localisation. We demonstrate that in the nucleus of germline and somatic cells, part of CCT associates to heterochromatin. In interphase cells, CCT seems generally confined to constitutive heterochromatin. Nevertheless, in condensing nucleus of future spermatozoon, it is also associated with chromatin undergoing compaction. Finally, in fully-condensed mitotic chromosomes, CCT is located all along the chromosomes. Our finding that CCT is associated with constitutive heterochromatin and to compacting chromatin raises the possibility that it may be implicated in maintenance and remodelling of heterochromatin.     

A microscope-based screening platform for large-scale functional protein analysis in intact cells

A modular microscope-based screening platform, with applications in large-scale analysis of protein function in intact cells is described. It includes automated sample preparation, image acquisition, data management and analysis, and the genome-wide automated retrieval of bioinformatic information. The modular nature of the system ensures that it is rapidly adaptable to new biological questions or sets of proteins. Two automated functional assays addressing protein secretion and the integrity of the Golgi complex were developed and tested. This shows the potential of the system in large-scale, cell-based functional proteomic projects.     

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.     

An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X(7) receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into various lymphocyte subsets and was decreased to values of approximately 25% of normal. The expression of the P2X(7) receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X(7) carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-gamma up-regulated P2X(7) function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X(7) receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.