Fine mapping and development of DNA markers for the <Emphasis Type="Italic">qPDH1</Emphasis> locus associated with pod dehiscence in soybean

Pod dehiscence (shattering) is a major cause of yield loss in mechanical harvesting of soybeans. To develop useful selection markers, we conducted a high-resolution mapping of a major quantitative trait locus (QTL) controlling pod dehiscence, designated as qPDH1. The progeny of a residual heterozygous line, which was a recombinant inbred line segregating only for the genomic region around qPDH1, was screened for flanking markers to obtain various recombinants in the vicinity of the QTL. Analysis of the relationship between degree of pod dehiscence and graphical genotype of these lines confined the location of qPDH1 to a 134-kb region on chromosome 16 (formerly linkage group J), where ten putative genes were predicted to be present. None of these genes showed significant sequence homology with the Arabidopsis genes that have previously been reported to be associated with pod dehiscence, suggesting the presence of a novel gene and mechanism underlying pod dehiscence in soybean. Sequencing analysis of the parental shattering-resistant and -susceptible cultivars for the candidate genes revealed a high-frequency nucleotide polymorphism in this genomic region between the cultivars. Three markers were developed using insertion/deletion variations in the region. Polymorphism at these marker loci was basically conserved between diverse shattering-resistant and -susceptible cultivars/lines, suggesting the versatility and usefulness of these markers for marker-assisted selection.     

Development of novel telomerase inhibitors based on a bisindole unit

Telomerase is the enzyme that elongates telomere repeat at the ends of a chromosome. As high telomerase activity is observed in most cancer cells, inhibitors of human telomerase have been expected as new chemotherapeutic agents for cancer. We describe here the discovery of novel inhibitors with IC50 values in the submicromolar range. The structure of the novel inhibitors will be useful as a scaffold for construction of the library in the search for telomerase inhibitors.     

Structural Insights into the Low pH Adaptation of a Unique Carboxylesterase from Ferroplasma: ALTERING THE pH OPTIMA OF TWO CARBOXYLESTERASES

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.     

Endomorphin-1, an endogenous mu-opioid receptor-selective agonist, stimulates oxygen consumption in mice.

This study was undertaken to investigate the effect of endogenous mu-receptor-selective peptide endomorphin 1, administered intracerebroventricularly, on oxygen consumption in mice. The intracerebroventricular injection of endomorphin 1 (3-30 nmol) significantly increased oxygen consumption in unrestrained mice. The effect of endomorphin 1 (30 nmol) was significantly antagonized by the simultaneous intraperitoneal administration of naloxone (100 nmol). These results suggest that endomorphin 1 stimulates oxygen consumption, and that the mu-opioid receptor influences energy balance in mice.     

Detection of the integrated feline leukemia viruses in a cat lymphoid tumor cell line by fluorescence in situ hybridization

Feline leukemia virus (FeLV) is a type-C retrovirus associated with lymphoid and hematopoietic malignancies in cats. The FeLV-induced tumors are thought to be caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. This study was undertaken to enumerate and map the acquired proviral insertions in the genome of a feline thymic lymphoma cell line (FT-1) infected with FeLV. Fluorescence in situ hybridization (FISH) combined with tyramide signal amplification was applied on the chromosome specimen of FT-1 cells and normal cat lymphocytes, with an entire FeLV-A genome used as a probe. Specific hybridization signals were detected from only the metaphases of the FT-1 cells, not from those of normal cat lymphocytes. Statistically based on the Poisson's distribution, at least six loci of chromosomal regions, A2p23-p22, B2p15-p14, B4p15-p14, D4q23-q24, E1p14-p13, and E2p13-p12, appeared to be positive for FeLV integration. Consistently, Southern blot hybridization analysis using an FeLV LTR-U3 probe specific for exogenous FeLV showed the integration of at least six FeLV proviral genomes in FT-1 cells. The cytogenetic technique employed here will provide valuable molecular tags to reveal unidentified tumor-associated genes in FeLV-associated tumor cells.     

Severe hypercholesterolemia,impaired fat tolerance,and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.     

Cloning and expression of a novel lysophospholipase which structurally resembles lecithin cholesterol acyltransferase

Lecithin cholesterol acyltransferase (LCAT) is the key enzyme in the esterification of plasma cholesterol and in the reverse cholesterol transport on high-density lipoprotein (HDL). We have found a novel LCAT-related gene among differentially expressed cDNA fragments between two types of foam cells derived from THP-1 cells, which are different in cholesterol efflux ability, using a subtractive PCR technique. The deduced 412-amino-acid sequence has 49% amino acid sequence similarity with human LCAT. In contrast to the liver-specific expression of LCAT, mRNA expression of the gene was observed mainly in peripheral tissues including kidney, placenta, pancreas, testis, spleen, heart, and skeletal muscle. The protein exists in human plasma and is probably associated with HDL. Moreover, we discovered that the recombinant protein hydrolyzed lysophosphatidylcholine (lysoPC), a proatherogenic lipid, to glycerophosphorylcholine and a free fatty acid. We have therefore named this novel enzyme LCAT-like lysophospholipase (LLPL), through which a new catabolic pathway for lysoPC on lipoproteins could be elucidated.     

Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38)

A novel neuropeptide with 38 residues (PACAP38) was isolated from ovine hypothalamic tissues using the pituitary adenylate cyclase activation in rat pituitary cell cultures as a parameter of the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567-574, 1989). From the side fractions obtained during the purification of PACAP38, a shorter form peptide with 27 residues corresponding to the N-terminal 27 amino acids of PACAP38 and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP27 showed a biological activity of adenylate cyclase stimulation comparable to PACAP38. Moreover PACAP27 which shows a considerable homology with vasoactive intestinal polypeptide (VIP) demonstrated a similar vasodepressor activity as VIP, but the adenylate cyclase stimulating activity was about 1000 times greater than VIP.     

Preparation, structures and properties of oxalate-bridged binuclear iron(III) complex: [(acac)2Fe(μ-ox)Fe(acac)2] and [(acac)2Fe(μ-ox)Fe(acac)2]·CH2ClCH2Cl

An oxalate-bridged binuclear iron(III) complex, [(acac)₂Fe(μ-ox)Fe(acac)₂], (acac⁻=acetylacetonate anion and ox²⁻=oxalate anion) was prepared. The complex crystallized as two types of crystals under different conditions: one had 1,2-dichloroethane as a solvent molecule of crystallization 2, the other did not 1. Both compounds have been characterized by X-ray crystallography, infrared spectroscopy, and thermogravimetric analysis. Compound 1 has also been characterized by UV-Vis and ¹H NMR spectroscopies, mass spectrometry, and electrochemistry. In both crystals, each iron(III) is coordinated in an octahedral arrangement by the oxygen atoms of an oxalate-bridging ligand and four oxygen atoms belonging to peripheral acac ligands in an octahedral arrangement. The intermetallic distance of Fe?Fe is 5.4368(9) Å in 1 and 5.438(2) Å in 2. Two iron(III) ions in each crystal are bridged by the oxalate and both lie in the oxalate-plane. The results of thermal analyses imply that the thermal stability of 2 is lower than that of 1. Cyclic voltammograms of 1 in acetonitrile and dichloromethane at low temperature showed two consecutive, quasi-Nernstian, one-electron reduction steps corresponding to the reduction of Fe^III-Fe^III to Fe^III-Fe^II followed by the reduction of Fe^III-Fe^II to Fe^II-Fe^II. The electrochemical comproportionation constants (K_c) of the equilibrium (Fe^III-Fe^III) + (Fe^II-Fe^II) ? 2(Fe^III-Fe^II) are 10^8.9 in acetonitrile medium and 10^8.5 in dichloromethane, respectively. The considerably large K_c values indicate that the main factor contributing to the stabilization of the Fe^III-Fe^II mixed-valence state is electronic delocalization through the oxalate-bridge.