The role of the tryptophyl residue in the hypotensive peptide xenopsin

The role of the Trp⁶ residue in the biological activity of the hypotensive peptide xenopsin (<Glu-Gly-Lys-Arg-Pro-Trp-Ile-Leu-OH) was investigated. This residue was satisfactorily reduced to 2,3-dihydro-Trp on treatment with excess pyridine-borane in trifluoroacetic acid without any detectable change in other parts of the molecule. The analogous peptide, (Lys², Gly³) xenopsin, was also reduced in a similar manner. Both reduction products were purified by gel filtration and characterized by UV absorption, amino acid composition, and structural analysis.The reduced peptides were assayed on the fundus strip of isolated rat stomach and were found to possess less than 1 percent of the activity of the original peptides. Although each of the reduced analogs had an indoline substituted for an indole in the tryptophyl residue, their biological activity was virtually lost. This suggests that the tryptophyl residue of xenopsin is crucial for its biological activity.     

Possible incorporation of phosphoserine into globin readthrough protein via bovine opal suppressor phosphoseryl-tRNA

Suppressor [32P]phosphoseryl-tRNA, prepared using bovine seryl-tRNA synthetase and ATP:seryl-tRNA phosphotransferase, was mixed with rabbit reticulocyte lysates containing endogenous hemoglobin mRNA having the termination codon UGA (opal). The chromatographic pattern of the lysate on Sephacryl S-200 showed that the radioactivity of [32P]phosphate in the hot trichloroacetic acid-precipitate (phosphoprotein) was eluted at the position between mature hemoglobin and globin subunits. The phosphoprotein, obtained by chromatography on S-200, moved to the position corresponding to that of globin readthrough protein on SDS-PAGE. The analyses of the hydrolyzate of the phosphoprotein showed the presence of phosphoserine in the protein. These results suggest that animal opal suppressor tRNA functions in vitro to transfer phosphoserine to the position of the termination codon UGA (opal) on mRNA.     

Immunocytochemical detection of triphosphoinositide in vestibular hair cells

Triphosphoinositide (TPI), an aminoglycoside receptor and a possible regulator of cationic permeation through its ability to bind with Ca++, was localized by the protein-A gold technique in vestibular sensory epithelia using an antibody highly specific to TPI. TPI was detected on the stereocilia, kinocilia, and cuticular plate of hair cells, and in the reticular membrane of supporting cells. The cilia of hair cells are damaged by aminoglycosides at a relatively early stage of toxicity. Ca++-regulated bioactivity in this area is probably involved.     

Behavioral teratogenic effect of methylmercury and d-amphetamine: meta-analysis and power analysis of data from the Collaborative Behavioral Teratology Study of National Center for Toxicological Research

The data of the Collaborative Behavioral Teratology Study (CBTS) were analyzed. Population-effect sizes were estimated, in terms of confidence intervals, by meta-analysis. A treatment effect of methylmercury was clearly observed and a dose-response effect was also observed. On the other hand, no clear treatment effect was observed in the d-amphetamine study. The effect of auditory startle habituation was very large, although it was limited to the high-dose group in the methylmercury study. The effects observed for physical landmarks were, as a whole, somewhat larger than those for behavioral measures. There is no clear evidence for sex-related differences except for activity measures. A curious treatment effect that was not referred to in the CBTS report was observed: eye-opening day and incisor-eruption day were hastened by the treatment of methylmercury. Power analysis results showed that obtaining a statistically significant effect for these compounds is very difficult, suggesting that even though published papers reported the significant effect of these compounds there might have been many unpublished experiments that obtained nonsignificant effects. If one uses these compounds as positive controls, a very large sample size will be needed. No highly sensitive test that is reproducible across compounds or dose-levels was observed among tests employed in the CBTS.     

Ligh- and electron-microscopic immunocytochemistry of somatotropes in the anterior pituitary gland of European ferret,Mustela putorius furo

Light-microscopic immunocytochemistry of ferret anterior pituitary revealed the localization of somatotropes in the pars distalis, but no immunoreactive cells were detected in the pars tuberalis. Ultrastructural studies by superimposition immunocytochemistry and immuno-electron microscopy, clucidated the morphological heterogeneity of these somatotropic cells. They were classified into 2 subtypes on the basis of size of the secretory granules. Type-I cells with small granules (mean diameter, 192 nm), were considered to be the immature somatotrop, while Type-II cells, with comparatively larger secretory granules (mean diameter, 257 nm), were considered to be the matured form of Type-I cells and the typical somatotropic cell-type, and were much more predominant than the Type-I cells. The fact that Type-II cells had a distinct Golgi zone and many mitochondria, while in Type-I cells the intracellular organelles were generally less developed, supports this suggestion. In addition to these two extreme subtypes, several intermediate forms were also encountered that may represent different transitional phases during the conversion of Type I to Type II. Protein A-gold immuno-electron microscopy illustrated the specific localization of growth hormone over the granules, with no labelling over any other cytoplasmic organelles of the 2 somatotrope subtypes.     

Binding sites of an aminoglycoside in the cochlea examined by immunocytochemistry

Summary To localize the binding sites of aminoglycosides in the cochlea, immunocytochemistry was used with the antibody to gentamicin and the protein-A/gold complex. We found that the main binding sites were the stereocilia, the cuticular plates of hair cells, the head plates of Deiters' cells, cell filaments and the cones of pillar cells, tectorial membranes, basilar membranes, the matrix of the spiral limbus, plasma membranes, mitochondria, and the chromatin of various kinds of cells. Triphosphoinositide and acidic glycosaminoglycans are the two most likely candidates for the cause of binding activity.     

Modification of Male Courtship Motivation by Olfactory Habituation via the GABAA Receptor in Drosophila melanogaster

A male-specific component, 11-cis-vaccenyl acetate (cVA) works as an anti-aphrodisiac pheromone in Drosophila melanogaster. The presence of cVA on a male suppresses the courtship motivation of other males and contributes to suppression of male-male homosexual courtship, while the absence of cVA on a female stimulates the sexual motivation of nearby males and enhances the male-female interaction. However, little is known how a male distinguishes the presence or absence of cVA on a target fly from either self-produced cVA or secondhand cVA from other males in the vicinity. In this study, we demonstrate that male flies have keen sensitivity to cVA; therefore, the presence of another male in the area reduces courtship toward a female. This reduced level of sexual motivation, however, could be overcome by pretest odor exposure via olfactory habituation to cVA. Real-time imaging of cVA-responsive sensory neurons using the neural activity sensor revealed that prolonged exposure to cVA decreased the levels of cVA responses in the primary olfactory center. Pharmacological and genetic screening revealed that signal transduction via GABA_A receptors contributed to this olfactory habituation. We also found that the habituation experience increased the copulation success of wild-type males in a group. In contrast, transgenic males, in which GABA input in a small subset of local neurons was blocked by RNAi, failed to acquire the sexual advantage conferred by habituation. Thus, we illustrate a novel phenomenon in which olfactory habituation positively affects sexual capability in a competitive environment.     

Annexin A1 Preferentially Predicts Poor Prognosis of Basal-Like Breast Cancer Patients by Activating mTOR-S6 Signaling

IntroductionAnnexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s).MethodsBy mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways.ResultsOur in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1.ConclusionsOur data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.     

Experimental Approach Reveals the Role of alx1 in the Evolution of the Echinoderm Larval Skeleton

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.