Food intake in rhesus monkeys following central administration of orexins

Orexin A has been reported to stimulate food intake in rats while orexin B does not. The purpose of this study was to determine the role of orexin A or orexin B administration on food intake in adult, male rhesus monkeys. Food intake was measured at 2 and 8 h after the morning feeding following central injections of vehicle, orexin A (10, or 20 microg) or orexin B (10, 30, or 100 microg). When compared to vehicle injections, the 10 and 20 microg doses of orexin A decreased food intake at 2 h post-dose by 45% and 64%, respectively. Eight-hour food intake was decreased at only the 20 microg orexin A dose. Orexin B at all doses and time points did not alter food intake when compared to vehicle. These results indicate that orexin A exhibits anorectic activity while orexin B does not affect food intake in the rhesus monkey.     

Comparative Analysis of the Complete Plastid Genome Sequence of the Red Alga Gracilaria tenuistipitata var. liui Provides Insights into the Evolution of Rhodoplasts and Their Relationship to Other Plastids

We sequenced to completion the circular plastid genome of the red alga Gracilaria tenuistipitata var. liui. This is the first plastid genome sequence from the subclass Florideophycidae (Rhodophyta). The genome is composed of 183,883 bp and contains 238 predicted genes, including a single copy of the ribosomal RNA operon. Comparisons with the plastid genome of Porphyra pupurea reveal strong conservation of gene content and order, but we found major genomic rearrangements and the presence of coding regions that are specific to Gracilaria. Phylogenetic analysis of a data set of 41 concatenated proteins from 23 plastid and two cyanobacterial genomes support red algal plastid monophyly and a specific evolutionary relationship between the Florideophycidae and the Bangiales. Gracilaria maintains a surprisingly ancient gene content in its plastid genome and, together with other Rhodophyta, contains the most complete repertoire of plastid genes known in photosynthetic eukaryotes.Supplementary material () is available for this article.[Reviewing Editor: Dr. W. Ford Doolittle]     

Purification of plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata

We report a straightforward protocol for isolating plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata. Plastids were purified using differential centrifugation and 2-step sucrose density gradients. We found that 10% polyethylene glycol 4000 was essential for maintaining plastid integrity prior to lysis. Plastid DNA isolated directly from the purified rhodoplasts was sufficiently pure for restriction endonuclease fragment analyses. Database comparisons of sequences generated randomly from a shotgun genomic library indicated that plastid DNA was 89% pure following ultracentrifugation in isopycnic cesium chloride equilibrium gradients. The protocol yields 30–50 μg of plastid DNA per 100 g of fresh algal tissue.     

An islet-cell protein tyrosine phosphatase is a likely precursor to the 37-kDa autoantigen in type 1 diabetes: human and macaque sequences, tissue distribution, unique and shared epitopes, and predictive autoantibodies.

BACKGROUND: We sought to identify novel islet-cell autoantigens to better understand the pathogenesis, prediction, and immunotherapy of type 1 diabetes. MATERIALS AND METHODS: Macaque and human islet cDNA libraries expressed in mammalian cells were screened with human diabetes sera. A positive clone was sequenced directly and after 5' rapid amplification of cDNA ends (RACE). Northern blotting and in situ hybridization revealed the tissue distribution of the corresponding protein. Antigen, expressed by in vitro translation, and tryptic peptides were analyzed by SDS-PAGE. For the immunoprecipitations, 183 diabetic, 60 prediabetic, and 91 control sera were used. Truncated antigens were used in immunoprecipitations for epitope mapping. Recombinant antigen expressed in transfected fibroblasts was used in competition assays. RESULTS: Sequencing yielded an 111-kDa, 1,013 amino acid, transmembrane protein (M1851) containing consensus protein tyrosine phosphatase (PTPase) sequence. M1851 was 77% identical in the intracellular domain, but only 31% identical extracellularly, to the islet-cell autoantigen ICA512. mRNA localized to brain, prostate, pancreatic islets, and adrenal medulla. After limited trypsinization, the in vitro translated antigen was 37 kDa. M1851 was recognized by 47% of prediabetes sera, 31% of new diabetes sera, but only 1% of healthy controls. Only 1/73 sera binding M1851 failed to bind ICA512, whereas 42/114 binding ICA512 did not bind M1851. M1851 reactivity was not fully displaced by ICA512 in 24/49 sera. Removing the C-terminal 27, 80, or 160 amino acids of M1851 decreased reactivity by 70%, 90%, and 100%, respectively. CONCLUSIONS: This new islet-cell PTPase is likely to be the precursor to the 37-kDa tryptic fragment antigen. It is structurally related to ICA512 but has distinct diabetes autoantibody epitopes located at the C terminus.     

Membrane glycoprotein biosynthesis: Changes in levels of glycosyl transferases in fibroblasts transformed by oncogenic viruses

Fibroblasts transformed by oncogenic viruses were found to have greater enzymic activities of four membrane glycoprotein:glycosyl transferases on a cell or protein basis then two non-transformed fibroblastic lines. These enzymes are responsible for the synthesis of membrane glycoproteins; each of the four transferases studied, the polypeptide:N-acetylgalactosaminyl, glycoprotein:galactosyl, fetuin:fucosyl and PSM:fucosyl transferases, was more than twice as active in the transformed cell lines using both endogenous and added receptor. The most pronounced differences occurred with the doubly (SV-PY-3T3) transformed fibroblasts in all cases; with the N-acetylgalactosaminyl and galactosyl transferases the increase was 8–16 fold over the non-transformed cells. It was demonstrated that these results do not arise from a changed level of glycosidase activities.     

A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1

The 2.9 A crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a beta strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.