Characterization of pNiXa,a serpin of Xenopus laevis oocytes and embryos,and its histidine-rich,Ni(II)-binding domain

A Ni(II)-binding serpin, pNiXA, is abundant in Xenopus oocytes and embryos. Kinetic assays show that purified pNiXa strongly inhibits bovine α-chymotrypsin (K₁ = 3 mM), weakly inhibits porcine elastase (K₁ = 0.5 μM), and does not inhibit bovine trypsin. The reversible, slow-binding inhibition of α-chymotrypsin by pNiXa is unaffected by Ni(II). Ovochymase in egg exudates is inhibited by pNiXa, but to a limited extent, even at high pNiXa concentrations. An octadecapeptide that models the His-rich domain (-HRHRHEQQGHHDSAKHGH-) of pNiXa forms six-coordinate, octahedral Ni(II)-complexes when the N-terminus is acetylated, and a square-planar Ni(II)-complex when the N-terminus is unblocked. Spectroscopy reveals two distinct types of octahedral Ni(II)-coordination to the N-acetylated octadecapeptide, involving, respectively, 3–4 and 5–6 imidazole nitrogens; the octadecapeptide undergoes partial, reversible precipitation in pH-and Ni(II)-dependent fashion, suggesting an insoluble, Ni(II)-coupled (Hx)_n-dimer. Such (Hx)_n-peptide interaction is confirmed by an enzyme-linked biotin-avidin assay with N-biotin-KHRHRHE-amide and N-acetyl-KHRHRHE-resin beads, which become coupled after adding Ni(II) or Zn(II). H₂O₂ oxidation of 2′-deoxyguanosine to mutagenic 8-hydroxy-2′deoxyguanosine is enhanced by the octahedral Ni(II)-octadecapeptide complex, although the effect is more intense with the square-planar Ni(II) octadecapeptide complex. Immunoperoxidase staining of whole mounts wish pNiXa antibody shows that pNiXa is distributed throughout gastrula-stage embryos and is localized during organogenesis in the brain, eye, spinal cord, myotomes, craniofacial tissues, and other sites of Ni(II) induced anomalies. Patterns of pNiXa staining are similar in controls and Ni(II)-exposed embryos. Binding of Ni(II) to pNiXa may cause embryotoxicity by enhancing oxidative reactions that produce tissue injury and genotoxicity. Although the natural target proteinases for pNiXa inhibition have not been established, pNiXa may be an important regulator of proteolysis during embryonic development. © 1996 Wiley-Liss, Inc.     

Isolation and some properties of dioxygenase and co-oxidase activities of adult human liver cytosolic lipoxygenase

The evidence presented here constitutes the first report on the occurrence of lipoxygenase (LO) activity in the adult human liver. LO activity was isolated free of hemoglobin from the whole liver cytosol by affinity chromatography using a concanavalin-A sepharose 4B column, and some properties of its dioxygenase and co-oxidase activities were examined. High-pressure liquid chromatography (HPLC) analyses of arachidonic acid metabolites suggested the presence of 5-, 12-, and 15-LO activities in the human liver. Linoleic acid was converted into 13-hydroperoxyoctadecadienoic acid. The dioxygenase activity with a V_max value of 1.74 μmoles/min/mg protein and a K_m value of 0.48 mM was noted in the presence of different concentrations of linoleic acid at pH 10. The activity was markedly stimulated by the presence of calcium, ATP, hydrogen peroxide, and KCl in the assay medium. Under optimum conditions, all the xenobiotics tested were co-oxidized by the enzyme preparations in the presence of linoleic acid. Kinetic data obtained for benzidine oxidation yielded a K_m value of 0.53 mM and a V_max value of 90.9 nmoles/min/mg protein. At present, the significance of these findings in in vivo toxicity of benzidine is unknown. The linoleic acid-dependent dioxygenase and co-oxidase activities were thermolabile and inhibited by micromolar concentrations of several classical LO inhibitors, further confirming the involvement of LO in these reactions. © 1997 John Wiley & Sons, Inc.     

Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice

Heat shock proteins (HSPs) are highly conserved among all organisms from prokaryotes to eukaryotes. In mice, the HSP genes Hsp70.1 and Hsp70.3 are induced by both endogenous and exogenous stressors, such as heat and toxicants. In order to determine whether such proteins specifically influence genomic instability, mice deficient for Hsp70.1 and Hsp70.3 (Hsp70.1/3(-/-) mice) were generated by gene targeting. Mouse embryonic fibroblasts (MEFs) prepared from Hsp70.1/3(-/-) mice did not synthesize Hsp70.1 or Hsp70.3 after heat-induced stress. While the Hsp70.1/3(-/-) mutant mice were fertile, their cells displayed genomic instability that was enhanced by heat treatment. Cells from Hsp70.1/3(-/-) mice also display a higher frequency of chromosome end-to-end associations than do control Hsp70.1/3(+/+) cells. To determine whether observed genomic instability was related to defective chromosome repair, Hsp70.1/3(-/-) and Hsp70.1/3(+/+) fibroblasts were treated with ionizing radiation (IR) alone or heat and IR. Exposure to IR led to more residual chromosome aberrations, radioresistant DNA synthesis (a hallmark of genomic instability), increased cell killing, and enhanced IR-induced oncogenic transformation in Hsp70.1/3(-/-) cells. Heat treatment prior to IR exposure enhanced cell killing, S-phase-specific chromosome damage, and the frequency of transformants in Hsp70.1/3(-/-) cells in comparison to Hsp70.1/3(+/+) cells. Both in vivo and in vitro studies demonstrate for the first time that Hsp70.1 and Hsp70.3 have an essential role in maintaining genomic stability under stress conditions.     

Partial purification of chlorophyll degrading enzymes from cavendish banana (Musa Cavendishi)

Cavendish banana (Musa Cavendishi, subgroup AAA) remains green upon ripening at tropical temperature (25-30 degrees C), due to incomplete degradation of chlorophyll (Chl). Earlier, evidence for the existence of two distinct degradative pathways--chlorophyllase and chlorophyll oxidase pathways in these bananas was provided. Here, an attempt has been made to understand further the mechanism of inhibition of Chl degradation at different stages of ripening and detecting various enzyme activities by partial purification. Soluble and Triton-solubilized protein fractions obtained from peel acetone powder from green-unripe, green-ripe and yellow-ripe bananas efficiently degraded Chl a. About 2-fold increase in Chl hydrolyzing/oxidizing and magnesium-dechelatase activities was observed in ripe, as compared to green-unripe bananas. The electrophoretic pattern of the soluble and detergent-solubilized proteins from the three stages of ripening revealed that the latter fraction contained only three slow moving proteins, which were found to be glycoproteins, as revealed in PAS staining. The soluble enzyme fraction contained all other bands along with the above three bands, as observed in the Native-PAGE of DEAE-Sepharose purified fractions. Only soluble fraction from 'green-ripe' bananas, catalyzed formation of an unknown intermediate (retention time 8.6 min), which was formed by the action of Triton-solubilized enzyme fractions, obtained from 'green-unripe' and 'yellow-ripe' bananas. The enzyme responsible for the formation of this intermediate might be involved in the stay-green character and could be a component of Chl oxidase pathway. Partial purification of soluble protein fraction by DEAE-Sepharose showed the presence of chlorophyllase, magnesium-dechelatase, pheophorbide a oxygenase, red fluorescent catabolite reductase and Chl oxidase. Native PAGE of pooled fractions showed separation of proteins in different bands. Pooled fractions IV and VI showed the presence of a single major band, resulting in almost a homogenous preparation in a single step. Fraction IV catalyzed dechelation of Mg by Mg-dechelatase, while fraction VI catalyzed the formation of 132-OH-Chl a by chlorophyll oxidase. Chlorophyll oxidase activity was stimulated by linolenic acid, indicating involvement of lipoxygenase in oxidative Chl degradation, thereby resulting in the formation of 13(2)-OH-Chl a as product. The results show the presence of various enzymes of chlorophyllase and chlorophyll oxidase pathways in soluble enzyme fraction.     

Computational method for discovery of estrogen responsive genes

Estrogen has a profound impact on human physiology and affects numerous genes. The classical estrogen reaction is mediated by its receptors (ERs), which bind to the estrogen response elements (EREs) in target gene's promoter region. Due to tedious and expensive experiments, a limited number of human genes are functionally well characterized. It is still unclear how many and which human genes respond to estrogen treatment. We propose a simple, economic, yet effective computational method to predict a subclass of estrogen responsive genes. Our method relies on the similarity of ERE frames across different promoters in the human genome. Matching ERE frames of a test set of 60 known estrogen responsive genes to the collection of over 18,000 human promoters, we obtained 604 candidate genes. Evaluating our result by comparison with the published microarray data and literature, we found that more than half (53.6%, 324/604) of predicted candidate genes are responsive to estrogen. We believe this method can significantly reduce the number of testing potential estrogen target genes and provide functional clues for annotating part of genes that lack functional information.     

The biology of triploid fish

This review deals with major areas of triploidy research in fish. It includes not only methods for induction and detection of triploidy but also the impact of triploidy on morphology, anatomy, growth, haematology, energetics, behaviour, endocrinology and gonads in various species of fish, studied so far. The future prospects of research on triploid fish are discussed inviting researchers with diverse areas of interest in fish biology.     

A novel interaction between calreticulin and ubiquitin-like nuclear protein in rice

Calreticulin (CRT), a major Ca²⁺-sequestering protein, has beenimplicated in a variety of cellular functions such as Ca²⁺ storage,signaling and chaperone activity within the cytoplasm and endoplasmicreticulum. To investigate the biological role of CRT in rice,21 partial cDNAs, encoding proteins that interacted with riceCRT in a yeast two-hybrid interaction-cloning system, were characterizedand the nucleotide sequences were found to be identical to eachother. A full-length cDNA of 3.5 kb, obtained from ricegenomic sequence data and 5' RACE, codes for a novel proteinof 966 amino acid residues and was designated as CRTintP (CRTinteracting protein). Primary sequence analysis of CRTintP showedno sequence homology with the known functional proteins; however,a potential ubiquitin-like domain at the N-terminal togetherwith a putative leucine zipper, a nuclear localization signaland several sites for serine/threonine kinases were evident.Cellular localization of CRTintP demonstrated its role in directinggreen fluorescent protein to the nucleus in onion epidermalcells. Northern and immunoblot analysis showed increased expressionof CRT and CRTintP in response to cold stress. Co-immunoprecipitationusing anti-CRT antibodies confirmed the existence of the CRT-CRTintPcomplex in vivo in the stressed leaf tissue, suggesting theirpotential role in regulating stress response. ⁴ Corresponding author: E-mail, skomatsu{at}affrc.go.jp; Fax, +81-298-38-7464.