Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus leucas) [published erratum appears in Mol Biol Evol 1995 Nov;12(6):1174]

Genetic variation at the Major Histocompatibility Complex locus DQ beta was analyzed in 233 beluga whales (Delphinapterus leucas) from seven populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island, and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the High Arctic beluga population. Variation was assessed by amplification of the exon coding for the peptide binding region via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Five alleles were found across the beluga populations and one in the narwhal. Pairwise comparisons of these alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per site leading to eight amino acid differences, five of which were nonconservative substitutions, centered around positions previously shown to be important for peptide binding. Although the amount of allelic variation is low when compared with terrestrial mammals, the nature of the substitutions in the peptide binding sites indicates an important role for the DQ beta locus in the cellular immune response of beluga whales. Comparisons of allele frequencies among populations show the High Arctic population to be different (P < or = .005) from the other beluga populations surveyed. In these other populations an allele, Dele-DQ beta*0101-2, was found in 98% of the animals, while in the High Arctic it was found in only 52% of the animals. Two other alleles were found at high frequencies in the High Arctic population, one being very similar to the single allele found in narwhal.      

Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.      

Mortality in over 350,000 Insured Swedish Dogs from 1995–2000: II. Breed-Specific Age and Survival Patterns and Relative Risk for Causes of Death

This study continues analysis from a companion paper on over 350,000 insured Swedish dogs up to 10 years of age contributing to more than one million dog-years at risk during 1995–2000. The age patterns for total and diagnostic mortality and for general causes of death (trauma, tumour, locomotor, heart and neurological) are presented for numerous breeds. Survival estimates at five, eight and 10 years of age are calculated. Survival to 10 years of age was 75% or more in Labrador and golden retrievers, miniature and toy poodles and miniature dachshunds and lowest in Irish wolfhounds (91% dead by 10 years). Multivariable analysis was used to estimate the relative risk for general and more specific causes of death between breeds accounting for gender and age effects, including two-way interactions. Older females had tumour as a designated cause of death more often than males in most breeds, but not in the Bernese mountain dog. Information presented in this and the companion paper inform our understanding of the population level burden of disease, and support decision-making at the population and individual level about health promotion efforts and treatment and prognosis of disease events.     

GAPDH mediates nitrosylation of nuclear proteins

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.     

Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases

Inositol (1,4,5) trisphosphate (Ins(1,4,5)P(3)) is a well-known messenger molecule that releases calcium from intracellular stores. Homologues with up to six phosphates have been characterized and recently, homologues with seven or eight phosphate groups, including pyrophosphates, have been identified. These homologues are diphosphoinositol pentakisphosphate (PP-InsP(5)/InsP(7)) and bis(diphospho)inositol tetrakisphosphate (bis-PP-InsP(4)/InsP(8)) [1], the rapid turnover of which [2] is regulated by calcium [2] and adrenergic receptor activity [3]. It has been proposed that the high-energy pyrophosphates might participate in protein phosphorylation [4]. We have purified InsP(6) kinase [5] and PP-InsP(5) kinase [6], both of which display ATP synthase activity, transferring phosphate to ADP. Here, we report the cloning of two mammalian InsP(6) kinases and a yeast InsP(6) kinase. Furthermore, we show that the yeast protein, ArgRIII, is an inositol-polyphosphate kinase that can convert InsP(3) to InsP(4), InsP(5) and InsP(6). We have identified a new family of highly conserved inositol-polyphosphate kinases that contain a newly identified, unique consensus sequence.     

Clozapine: selective labeling of sites resembling 5HT6 serotonin receptors may reflect psychoactive profile.

BACKGROUND: Clozapine, the classic atypical neuroleptic, exerts therapeutic actions in schizophrenic patients unresponsive to most neuroleptics. Clozapine interacts with numerous neurotransmitter receptors, and selective actions at novel subtypes of dopamine and serotonin receptors have been proposed to explain clozapine''s unique psychotropic effects. To identify sites with which clozapine preferentially interacts in a therapeutic setting, we have characterized clozapine binding to brain membranes. MATERIALS AND METHODS: [3H]Clozapine binding was examined in rat brain membranes as well as cloned-expressed 5-HT6 serotonin receptors. RESULTS: [3H]Clozapine binds with low nanomolar affinity to two distinct sites. One reflects muscarinic receptors consistent with the drug''s anticholinergic actions. The drug competition profile of the second site most closely resembles 5HT6 serotonin receptors, though serotonin itself displays low affinity. [3H]Clozapine binding levels are similar in all brain regions examined with no concentration in the corpus striatum. CONCLUSIONS: Besides muscarinic receptors, clozapine primarily labels sites with properties resembling 5HT6 serotonin receptors. If this is also the site with which clozapine principally interacts in intact human brain, it may account for the unique beneficial actions of clozapine and other atypical neuroleptics, and provide a molecular target for developing new, safer, and more effective agents.     

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.     

Live imaging of <Emphasis Type="Italic">Drosophila</Emphasis>gonad formation reveals roles for Six4 in regulating germline and somatic cell migration

Background  

Movement of cells, either as amoeboid individuals or in organised groups, is a key feature of organ formation. Both modes of migration occur during Drosophila embryonic gonad development, which therefore provides a paradigm for understanding the contribution of these processes to organ morphogenesis. Gonads of Drosophila are formed from three distinct cell types: primordial germ cells (PGCs), somatic gonadal precursors (SGPs), and in males, male-specific somatic gonadal precursors (msSGPs). These originate in distinct locations and migrate to associate in two intermingled clusters which then compact to form the spherical primitive gonads. PGC movements are well studied, but much less is known of the migratory events and other interactions undergone by their somatic partners. These appear to move in organised groups like, for example, lateral line cells in zebra fish or Drosophila ovarian border cells.