Isolation and identification of lateral bud growth inhibitor,indole-3-aldehyde,involved in apical dominance of pea seedlings

A lateral bud growth inhibitor was isolated from etiolated pea seedlings and identified as indole-3-aldehyde. The indole-3-aldehyde content was significantly higher in the diffusates from explants with apical bud and indole-3-acetic acid treated decapitated explants, in which apical dominance is maintained, than in those from decapitated ones releasing apical dominance. When the indole-3-aldehyde was applied to the cut surface of etiolated decapitated plants or directly to the lateral buds, it inhibited outgrowth of the latter. These results suggest that indole-3-aldehyde plays an important role as a lateral bud growth inhibitor in apical dominance of pea seedlings.     

Nuclear translocation of plk1 mediated by its bipartite nuclear localization signal

Polo-like kinase 1 (Plk1), a mammalian ortholog of Drosophila Polo, is a serine-threonine protein kinase implicated in the regulation of multiple aspects of mitosis. The protein level, activity, and localization of Plk1 change during the cell cycle, and its proper subcellular localization is thought to be crucial for its function. Although localization of Plk1 to the centrosome has been established, nuclear localization or nucleocytoplasmic translocation of Plk1 has not been fully addressed. Here we show that Plk1 accumulates in both the nucleus and the cytoplasm in addition to its localization to the centrosome during S and G(2) phases. Our results identify a conserved region in the kinase domain of Plk1 (residues 134-146) as a functional bipartite nuclear localization signal (NLS) sequence that regulates nuclear translocation of Plk1. The identified NLS is necessary and sufficient for directing nuclear localization of Plk1. This bipartite NLS has an unusually short spacer sequence between two clusters of basic amino acids but is sensitive to RanQ69L, a dominant negative form of Ran, similar to ordinary bipartite NLS. Remarkably, the expression of an NLS-disrupted mutant of Plk1 during S phase was found to arrest the cells in G(2) phase. These results suggest that the bipartite NLS-dependent nuclear localization of Plk1 before mitosis is important for ensuring normal cell cycle progression.     

A genome-wide survey of the genes for planar polarity signaling or convergent extension-related genes in Ciona intestinalis and phylogenetic comparisons of evolutionary conserved signaling components

Non-canonical Wnt signals similar to planar cell polarity (PCP) signaling in the fly control convergent extension (CE) of the dorsal mesoderm during gastrulation in vertebrates. Using the Ciona complete genome sequence and EST sequence data, we present here an initial and exhaustive search in non-vertebrate chordates, Ciona intestinalis for the family members as well as homologs or orthologs that are involved in PCP/CE signaling cascades. We clarified 7 cardinal gene families, including the MAPK, STE20 group kinase, Rho small GTPase, STAT, Glypican, Fz and Wnt gene families, as well as gene homologs or orthologs for known PCP/CE signaling components with their phylogenetic nature. As a result, we characterized 62 Ciona component genes. Among them, 59 genes were novel and functional genes which were supported by EST expressions and 15 genes belonged to PCP/CE component orthologs of other organisms or common ancestor genes. Moreover, from the phylogenetic point of view, we compared these components genome-widely with the PCP signaling components of fly and the CE signaling components of vertebrates. We then discovered not only that ascidians contain the basic ancestral signaling pathway components in chordates but also that several signaling components have not found in ascidian, indicating that ascidian CE pathway might have several gaps from vertebrate CE pathway. The present study provides an initial step for the subsequent analysis of CE in the non-vertebrate chordates, ascidians. In addition, this phylogenetic approach will help to facilitate understanding of the relationship between fly PCP signaling and the vertebrate CE pathway.     

Growth and Reproduction of Double-Ended Pipefish, Syngnathoides biaculeatus, in Moreton Bay, Queensland, Australia

Life-history characteristics of the double-ended pipefish, Syngnathoides biaculeatus (Bloch), were investigated to determine growth rate, degree of sexual dimorphism, size at maturity, and reproductive biology. Growth rates of wild juveniles and adults calculated from monthly progression of length-frequency modes ranged from 0.8mmd^–1 (fish lengths 120–145mm standard length (SL)) in summer to 0.2mmd^–1 in winter (185–200mm SL). Growth of laboratory-reared juveniles up to 63d old was greater, ranging from 0.8 to 2.3mmd^-1. The von Bertalanffy growth constant K was estimated at 0.0076d^{- 1}, or 2.8year^–1. Morphological differentiation between the sexes based upon abdominal pattern was possible for fish larger than 120mm SL, with females possessing a zigzag pattern on the abdomen. The association between this pattern and sex was confirmed by histological gonad analysis. Males were significantly longer than females during four of seven seasons examined, and a 1:1 sex ratio was determined for all seasons except autumn when the ratio was female biased. The breeding season was marked by the appearance of pregnant males between October and April, and during courtship both species exhibited increased pigmentation. The minimum paternal size at maturity was 185mm, the maximum length recorded 260mm. Clutch size ranged between 60 and 200 eggs, with a mean of 153. Ovaries had a sequential pattern of egg development, resulting in egg batches that approximated the number of eggs carried by brooding males. Additionally, all eggs in a brood were at the same developmental stage. This suggests that one female provides all of the eggs for one male per breeding event in a monogamous mating system.     

A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.     

Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats

BACKGROUND: Hepatocyte nuclear factor-4alpha (HNF4alpha; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4alpha causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4alpha in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4alpha protein, due, in part, to the limited availability of human HNF4alpha-specific antibodies. RESULTS: Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4alpha fusion proteins as the immunizing agent. The mouse anti-human HNF4alpha monoclonal antibody (K9218) generated against human HNF4alpha1/alpha2/alpha3 amino acids 3-49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4alpha proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4alpha protein, but HEK293 showed no expression of HNF4alpha protein. Nuclear-specific localization of the HNF4alpha protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4alpha protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4alpha protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4alpha in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4alpha mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis. CONCLUSION: These results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4alpha and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4alpha isoforms in humans and in several other mammalian species.     

Effects of dietary vitamin E and selenium on arginase activity in the liver, kidneys, and heart of rats treated with high doses of glucocorticoid

The effects of dietary intake of vitamin E and selenium on arginase activity in the liver, kidneys, and heart of rats treated with high doses of prednisolone were investigated. Rats were divided into five groups. Groups 3, 4, and 5 received a daily supplement in their drinking water of vitamin E, Se, and a combination of vitamin E and Se, respectively, for 30 days. For 3 days subsequently, the control group (group 1) was given a placebo, and the remaining four groups were injected intramuscularly with prednisolone. The tissue samples were collected from each group at 4, 8, 12, 24, and 48 h after the last administration of prednisolone. In the group treated with prednisolone alone, arginase activity in the liver was found to have increased at all the time periods, whereas it had decreased significantly in the heart at 48 h. Arginase activity in the kidneys was not affected by prednisolone. Compared to the control and prednisolone groups, arginase activity in the kidneys and heart of the vitamin E- and Se-supplemented groups was found to be significantly increased at all time periods, however, no difference was seen in the combination group. Arginase activity in the liver of the vitamin E-supplemented group was found to have decreased at all time periods, however, in the Se group compared to the prednisolone group it had reduced at 24 and 48 h only. In the combination group compared to the prednisolone group, liver arginase activity increased constantly up to 12 h returning to normal values at 48 h. Vitamin E and Se in combination may prevent the changes in arginase activity in various tissues caused by prednisolone.     

NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif

Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.