Differential requirement for the N-terminal catalytic domain of the DNA polymerase ε p255 subunit in the mitotic cell cycle and the endocycle

In Drosophila, the 255kDa catalytic subunit (dpolεp255) and the 58kDa subunit of DNA polymerase ε (dpolεp58) have been identified. The N-terminus of dpolεp255 carries well-conserved six DNA polymerase subdomains and five 3'→5' exonuclease motifs as observed with Polε in other species. We here examined roles of dpolεp255 during Drosophila development using transgenic fly lines expressing double stranded RNA (dsRNA). Expression of dpolεp255 dsRNA in eye discs induced a small eye phenotype and inhibited DNA synthesis, indicating a role in the G1-S transition and/or S-phase progression of the mitotic cycle. Similarly, expression of dpolεp255 dsRNA in the salivary glands resulted in small size and endoreplication defects, demonstrating a critical role in endocycle progression. In the eye disc, defects induced by knockdown of dpolεp255 were rescued by overexpression of the C-terminal region of dpolεp255, indicating that the function of this non-catalytic domain is conserved between yeast and Drosophila. However, this was not the case for the salivary gland, suggesting that the catalytic N-terminal region is crucial for endoreplication and its defect cannot be complemented by other DNA polymerases. In addition, several genetic interactants with dpolεp255 including genes related to DNA replication such as RFC, DNA primase, DNA polη, Mcm10 and Psf2 and chromatin remodeling such as Iswi were also identified.     

Further Characterization of 5-HT1A Receptors in the Goldfish Retina: Role of Cyclic AMP in the Regulation of the In Vitro Outgrowth of Retinal Explants

The presence of serotonin 5-HT_1A receptors and their physiological role were further characterized in the goldfish retina. The effects of the 5-HT_6/7 receptor antagonists pimozide, fluphenazine and amoxapine, the 5-HT_1A receptor antagonist WAY-100,135, and the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, on the 5-HT_1A receptor agonist [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes, were evaluated. In addition, the effects of serotonin, 8-hydroxy-2-(di-n-propylamino)tetralin, WAY-100,135, the adenylate cyclase inhibitors SQ22536 and MDL12330A, and the cyclic AMP analog 8-bromoadenosine-3:5 cyclic monophosphate were also studied on neuritic outgrowth from retinal explants. WAY-100,135 but not 5-HT_6/7receptor antagonists inhibited [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline decreased [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding sites up to 70%, while receptor turnover was similar to that reported in other tissues. Serotonin and 8-hydroxy-2-(di-n-propylamino)tetralin stimulated cyclic AMP production, both ex vivo and in vitro, and these increases were related to inhibition of neuritic outgrowth. The inhibitory effect was reduced by SQ22536 and by WAY-100,135, and was mimicked by 8-bromoadenosine-3:5cyclic monophosphate. This study supports previous findings about the role of serotonin as a regulator of axonal outgrowth during in vitro regeneration of the goldfish retina and demonstrates that this effect is mediated, at least in part, by 5-HT_1A receptors through a mechanism which involves an increase of cyclic AMP levels.     

p115–SNARE Interactions: A Dynamic Cycle of p115 Binding Monomeric SNARE Motifs and Releasing Assembled Bundles

Tethering factors regulate the targeting of membrane‐enclosed vesicles under the control of Rab GTPases. p115, a golgin family tether, has been shown to participate in multiple stages of ER/Golgi transport. Despite extensive study, the mechanism of action of p115 is poorly understood. SNARE proteins make up the machinery for membrane fusion, and strong evidence shows that function of p115 is directly linked to its interaction with SNAREs. Using a gel filtration binding assay, we have demonstrated that in solution p115 stably interacts with ER/Golgi SNAREs rbet1 and sec22b, but not membrin and syntaxin 5. These binding preferences stemmed from selectivity of p115 for monomeric SNARE motifs as opposed to SNARE oligomers. Soluble monomeric rbet1 can compete off p115 from coat protein II (COPII) vesicles. Furthermore, excess p115 inhibits p115 function in trafficking. We conclude that monomeric SNAREs are a major binding site for p115 on COPII vesicles, and that p115 dissociates from its SNARE partners upon SNAREpin assembly. Our results suggest a model in which p115 forms a mixed p115/SNARE helix bundle with a monomeric SNARE, facilitates the binding activity and/or concentration of the SNARE at prefusion sites and is subsequently ejected as SNARE complex formation and fusion proceed.      

Vascular-homing peptides for targeted drug delivery and molecular imaging: Meeting the clinical challenges

The vasculature of each organ expresses distinct molecular signatures critically influenced by the pathological status. The heterogeneous profile of the vascular beds has been successfully unveiled by the in vivo phage display, a high-throughput tool for mapping normal, diseased, and tumor vasculature. Specific challenges of this growing field are targeted therapies against cancer and cardiovascular diseases, as well as novel bioimaging diagnostic tools. Tumor vasculature-homing peptides have been extensively evaluated in several preclinical and clinical studies both as targeted-therapy and diagnosis. To date, results from several Phase I and II trials have been reported and many other trials are currently ongoing or recruiting patients. In this review, advances in the identification of novel peptide ligands and their corresponding receptors on tumor endothelium through the in vivo phage display technology are discussed. Emphasis is given to recent findings in the clinical setting of vascular-homing peptides selected by in vivo phage display for the treatment of advanced malignancies and their altered vascular beds.     

Laminin alpha5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through beta1 integrin signaling

Laminin alpha chains have unique spatiotemporal expression patterns during development and defining their function is necessary to understand the regulation of epithelial morphogenesis. We investigated the function of laminin alpha5 in mouse submandibular glands (SMGs). Lama5(-/-) SMGs have a striking phenotype: epithelial clefting is delayed, although proliferation occurs; there is decreased FGFR1b and FGFR2b, but no difference in Lama1 expression; later in development, epithelial cell organization and lumen formation are disrupted. In wild-type SMGs alpha5 and alpha1 are present in epithelial clefts but as branching begins alpha5 expression increases while alpha1 decreases. Lama5 siRNA decreased branching, p42 MAPK phosphorylation, and FGFR expression, and branching was rescued by FGF10. FGFR siRNA decreased Lama5 suggesting that FGFR signaling provides positive feedback for Lama5 expression. Anti-beta1 integrin antibodies decreased FGFR and Lama5 expression, suggesting that beta1 integrin signaling provides positive feedback for Lama5 and FGFR expression. Interestingly, the Itga3(-/-):Itga6(-/-) SMGs have a similar phenotype to Lama5(-/-). Our findings suggest that laminin alpha5 controls SMG epithelial morphogenesis through beta1 integrin signaling by regulating FGFR expression, which also reciprocally regulates the expression of Lama5. These data link changes in basement membrane composition during branching morphogenesis with FGFR expression and signaling.     

High resolution methylation analysis of the HoxA5 regulatory region in different somatic tissues of laboratory mouse during development

Homeobox genes encode a group of DNA binding regulatory proteins whose key function occurs in the spatial-temporal organization of genome during embryonic development and differentiation. The role of these Hox genes during ontogenesis makes it an important model for research. HoxA5 is a member of Hox gene family playing a central role during axial body patterning and morphogenesis. DNA modification studies have shown that the function of Hox genes is partly governed by the methylation-mediated gene expression regulation. Therefore the study aimed to investigate the role of epigenetic events in regulation of tissue-specific expression pattern of HoxA5 gene during mammalian development. The methodology adopted were sodium bisulfite genomic DNA sequencing, quantitative real-time PCR and chromatin-immunoprecipitation (ChIP). Methylation profiling of HoxA5 gene promoter shows higher methylation in adult as compared to fetus in various somatic tissues of mouse being highest in adult spleen. However q-PCR results show higher expression during fetal stages being highest in fetal intestine followed by brain, liver and spleen. These results clearly indicate a strict correlation between DNA methylation and tissue-specific gene expression. The findings of chromatin-immunoprecipitation (ChIP) have also reinforced that epigenetic event like DNA methylation plays important role in the regulation of tissue specific expression of HoxA5.     

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.     

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.