Recombinant sea urchin flagellar adenylate kinase

Adenylate kinase (AK) is localized in sea urchin sperm flagella and embryonic cilia. To investigate sea urchin Strongylocentrotus purpuratus AK (SpAK) enzymatic characteristics, the full-length recombinant protein of 130 kDa (SpAKr) and each of its three catalytic domains were expressed in Escherichia coli. Although the full-length SpAK had high enzymatic activity, each of the three catalytic domains had no activity. The Km for ATP synthesis from ADP was 0.23 mM and the Vmax was 4.51 mumol ATP formed per minute per milligram of protein. The specific AK inhibitor, Ap5A, blocks SpAKr enzymatic activity with an IC50 of 0.53 microM. The pH optimum for SpAKr is 8.1, as compared to 7.7 for the natural SpAK. Calcium inhibits SpAKr activity in a dose-dependent manner. Although SpAKr has three cAMP-dependent protein kinase phosphorylation sites, and can be phosphorylated in vitro, the enzymatic kinetics after phosphorylation are not significantly altered. SpAK and Chlamydomonas flagellar AKs are the only AKs with three catalytic sites. Further study of the SpAKr will aid in understanding the active site of this interesting and important ATP synthase.     

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.     

Possible molecular evolution of biomembranes: from single-chain to double-chain lipids

We have studied a possible evolution process permitting a 'primitive' membrane to evolve towards a membrane structure with an outer wall, similar to that of bacteria. We have investigated whether a polysaccharide bearing hydrophobic phytyl or cholesteryl chains coats giant vesicles made of single- or double-chain lipids. Phytyl-pullulan 5b was found to bind to the surface of vesicles made of either single- or double-chain lipids. In contrast, cholesteryl-pullulan 5a only coated the surface of vesicles made of double-chain lipids. These results indicate that there must be a close match between the size and shape of membrane constituents and the hydrophobic molecules to be inserted. This process could, thus, provide a selection mechanism of lipid-membrane constituents during the course of biomembrane evolution. The presence of the above 'hydrophobized' polysaccharides on the surface of different giant vesicles was identified by lectin binding. Both concanavalin A and annexin V were shown by fluorescence microscopy to bind spontaneously to vesicles made of double-chain lipids. Our experiments exemplify that self-organization of amphiphiles into closed vesicles in aqueous solution automatically leads to the coating of vesicles by 'hydrophobized' polysaccharides, which then permit lectin binding. This is a possible mechanism for the evolution of primitive membranes towards 'proto-cells'.     

The genetic structure of endangered indigenous populations of the amago salmon, <Emphasis Type="Italic">Oncorhynchus</Emphasis><Emphasis Type="Italic">masou</Emphasis><Emphasis Type="Italic">ishikawae</Emphasis>, in Japan

The amago salmon, Oncorhynchus masou ishikawae, is an endemic subspecies of O. masou in Japan. Owing to the extensive stocking of hatchery fish throughout Japan, indigenous populations of O. m. ishikawae are now on the verge of extinction. We examined the genetic effects of stocking hatchery fish on wild populations in the River Koza, Japan, using microsatellite and mitochondrial DNA (mtDNA) markers. For mtDNA, haplotype mt1, which is common in wild populations, was present exclusively in isolated wild populations assumed to be unaffected by previous stocking, while it was never observed in hatchery fish. Genetic diversity was much higher in wild populations in the stocked area, which shared many mtDNA haplotypes with hatchery fish, than in isolated wild populations with haplotype mt1. Pairwise F _ST estimates based on microsatellites showed significant differentiation among the isolated populations with many microsatellite loci monomorphic. Significant deviation from Hardy–Weinberg equilibrium was observed in wild populations in the area subject to stocking, where a Bayesian-based assignment test showed a high level of introgression with hatchery fish. These results suggest that wild populations with haplotype mt1, which became isolated through anthropogenic environmental change in the 1950–1960s, represent indigenous populations of O. m. ishikawae in the River Koza. They have low genetic diversity, most likely caused by genetic bottlenecks following damming and environmental deterioration, while stocking of hatchery fish over the past 30 years apparently had a large impact on the genetic structure of wild populations in the main channel of the River Koza.     

Diversity of Ca2+-induced morphology revealed by morphological phenotyping of Ca2+-sensitive mutants of Saccharomyces cerevisiae

Yeast cell morphology can be treated as a quantitative trait using the image processing software CalMorph. In the present study, we investigated Ca(2+)-induced morphological changes in Ca(2+)-sensitive (cls) mutants of Saccharomyces cerevisiae, based on the discovery that the characteristic Ca(2+)-induced morphological changes in the Ca(2+)-sensitive mutant zds1 reflect changes in the Ca(2+) signaling-mediated cell cycle control pathway. By applying hierarchical cluster analysis to the quantitative morphological data of 58 cls mutants, 31 of these mutants were classified into seven classes based on morphological similarities. The patterns of morphological change induced by Ca(2+) in one class differed from those of another class. Based on the results obtained using versatile methods for phenotypic analysis, we conclude that a high concentration of Ca(2+) exerts a wide variety of effects on yeast and that there are multiple Ca(2+)-regulatory pathways that are distinct from the Zds1p-related pathway.     

Norlichexanthone produced by cultured endolichenic fungus induced from Pertusaria laeviganda and its antioxidant activity

Endolichenic fungi, nonobligate microfungi that live in lichen, are promising as new bioresources of pharmacological compounds. We found that norlichexanthone isolated from the endolichenic fungus in Pertusaria laeviganda exhibited high antioxidant activity. Norlichexanthone produced by endolichenic fungus had the antioxidant activity with same level of ascorbic acid. This is the first report of high antioxidant activity of norlichexanthone.

Abbreviations: AAPH: 2,2?-azobis (2-methylpropionamidine) dihydrochloride; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FL: fluorescein sodium salt; HPLC-PDA: high-performance liquid chromatography with photodiode array; LC-ESI-MS: liquid chromatography with electrospray ionization mass spectrometry; ORAC: oxygen radical absorbance capacity; PB: phosphate buffer; ROS: reactive oxygen species; TLC: thin-layer chromatography     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.     

Severely reduced production of klotho in human chronic renal failure kidney

We recently identified a novel gene, termed klotho (kl) that is involved in the development of a syndrome in mice resembling human aging. A defect of the kl gene expression in mice leads to multiple disorders including arteriosclerosis, osteoporosis, ectopic calcification, and skin atrophy together with short life-span and infertility. Patients with chronic renal failure (CRF), develop multiple complications that are reminiscent of phenotypes observed in kl mutant mice. Furthermore, the kl gene is mainly expressed in kidney and brain. These evidences above suggest the possible involvement of Klotho function in the complications arising in CRF patients. To investigate the above possibility, we examined the kidneys of 10 clinically or histologically diagnosed CRF cases. The level of kl gene expression was measured by utilizing RNase protection assay. The expression of Klotho protein was assayed by utilizing Western blot analysis and by immunohistochemistry. The levels of kl mRNA expression were greatly reduced in all CRF kidneys. Moreover, the production of Klotho protein was also severely reduced in all CRF kidneys. These results suggest that the decrease in kl gene expression in CRF patients may underlie the deteriorating process of multiple complications in the CRF patients.     

Variation of hepatic methotrexate 7-hydroxylase activity in animals and humans

This study deals with individual and species variations in the converting activity of methotrexate (MTX) to 7-hydroxymethotrexate in animals and humans. When MTX 7-hydroxylase was assayed in six human liver cytosols, a 48-fold range of intersubject variation of the activity was observed. The variations were correlated to the concentrations of aldehyde oxidase activity in human subjects assayed with benzaldehyde as a substrate. Species differences of liver MTX 7-hydroxylase activity were also observed. The activity was highest in rabbits, followed by rats, hamsters, and monkeys but was undetectable in dogs. Strain differences of MTX 7-hydroxylase activity based on aldehyde oxidase activity were also observed in rats and mice. The results suggest that aldehyde oxidase functions as MTX 7-hydroxylase in livers of animals and humans, and the observed differences of MTX 7-hydroxylase activity are due to variations in the amount of aldehyde oxidase present.