Phosphorylation of five histone H1 subtypes of L5178Y cells at the exponential growth and mitotic phases

Histone H1 of cells of L5178Y, a mouse lympholeukemic cell line, consists of five molecular species designated as H1-I, II, III, IV, and V. The phosphorylation of these H1 subtypes was examined at the exponential growth phase and during mitosis, by BioRex 70 column chromatography and two-dimensional polyacrylamide gel electrophoresis. In exponentially growing cells, the degree of phosphorylation was different for each subtype. H1-II was the most highly phosphorylated, 1.8 phosphate residues per molecule, followed by H1-IV/V, 1.4, I, 0.8, and III, 0.5. In the mitotic phase, H1-II was also the most highly phosphorylated 6.0 phosphate residues per molecule, H1-IV/V, 3.5, I, 2.7, and III, 1.2. The phosphorylation started simultaneously among the subtypes after colcemid addition, and phosphorylated H1 subtypes accumulated linearly. The rate of incorporation of 32P into each H1 subtype was almost constant during colcemid treatment. During 4 h after colcemid addition, the phosphate residues incorporated into H1 did not dephosphorylated. The H1 kinase activities increased to six times higher during the colcemid treatment.     

Effects of homocysteine on the binding of extracellular-superoxide dismutase to the endothelial cell surface

Homocysteine is known to be a risk factor for several vascular diseases. Previously, we found a significant association between plasma homocysteine and plasma extracellular-superoxide dismutase (EC-SOD) levels. The binding of EC-SOD to human and bovine aortic endothelial cell cultures showed significant decreases after incubation with 10 microM homocysteine, whereas the expression of EC-SOD in fibroblast cell cultures was inhibited with a high concentration (1 mM) of homocysteine. Furthermore, binding of EC-SOD to heparin immobilized on plates was decreased with homocysteine. These observations suggested that homocysteine decreases the binding of EC-SOD to vascular endothelial cell surfaces by degradation of endothelial heparan sulfate proteoglycan, which results in a loss of the ability to protect endothelial cell surfaces from oxidative stress.     

Overview of the transcriptome profiles identified in hagfish, shark, and bichir: current issues arising from some nonmodel vertebrate taxa

Because of their crucial phylogenetic positions, hagfishes, sharks, and bichirs are recognized as key taxa in our understanding of vertebrate evolution. The expression patterns of the regulatory genes involved in developmental patterning have been analyzed in the context of evolutionary developmental studies. However, in a survey of public sequence databases, we found that the large-scale sequence data for these taxa are still limited. To address this deficit, we used conventional Sanger DNA sequencing and a next-generation sequencing technology based on 454 GS FLX sequencing to obtain expressed sequence tags (ESTs) of the Japanese inshore hagfish (Eptatretus burgeri; 161,482 ESTs), cloudy catshark (Scyliorhinus torazame; 165,819 ESTs), and gray bichir (Polypterus senegalus; 34,336 ESTs). We deposited the ESTs in a newly constructed database, designated the "Vertebrate TimeCapsule." The ESTs include sequences from genes that can be effectively used in evolutionary developmental studies; for instance, several encode cartilaginous extracellular matrix proteins, which are central to an understanding of the ways in which evolutionary processes affected the skeletal elements, whereas others encode regulatory genes involved in craniofacial development and early embryogenesis. Here, we discuss how hagfishes, sharks, and bichirs contribute to our understanding of vertebrate evolution, we review the current status of the publicly available sequence data for these three taxa, and we introduce our EST projects and newly developed database.     

Common variants in CDKN2B-AS1 associated with optic-nerve vulnerability of glaucoma identified by genome-wide association studies in Japanese

Background

To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated.

Methods and Principal Findings

We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10⁻¹⁰). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients.

Conclusions and Significance

In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.     

On the similar spatial arrangement of active site residues in PAPS-dependent and phenolic sulfate-utilizing sulfotransferases

Mammalian sulfotransferases (STs) utilize exclusively the sulfuryl group donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to catalyze the sulfurylation reactions based on a sequential transfer mechanism. In contrast, the commensal intestinal bacterial arylsulfate sulfotransferases (ASSTs) do not use PAPS as the sulfuryl group donor, but instead catalyze sulfuryl transfer from phenolic sulfate to a phenol via a Ping-Pong mechanism. Interestingly, structural comparison revealed a similar spatial arrangement of the active site residues as well as the cognate substrates in mouse ST (mSULT1D1) and Escherichia coli CFT073 ASST, despite that their overall structures bear no discernible relationship. These observations suggest that the active sites of PAPS-dependent SULT1D1 and phenolic sulfate-utilizing ASST represent an example of convergent evolution.     

Spectroscopic evidence that salicylic acid converts a temporally inactivated form of horseradish peroxidase (compound III) to the irreversibly inactivated verdohemoprotein (P-670)

We obtained spectroscopic evidence in support of salicylate-dependent inactivation of horseradish peroxidase-C. Addition of salicylate to the enzyme arrested at a temporal inactive state (Compound III) in the presence of H2O2, resulted in rapid and irreversible inactivation of the enzyme yielding verdohemoproteins (P-670). Multiple roles for salicylate in peroxidase-catalyzed reactions are discussed.     

Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii.

Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and gamma-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

Production of transgenic lily plants by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation

A system for the production of transgenic plants was developed for the Oriental hybrid lily, Lilium cv. Acapulco, by Agrobacterium-mediated genetic transformation. Filament-derived calli were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm, which harbored a binary vector carrying the neomycin phosphotransferase II, hygromycin phosphotransferase, and intron-containing -glucuronidase genes in the T-DNA region. Six hygromycin-resistant (Hyg^r) culture lines were obtained from 200 calli by scratching them with sandpaper prior to inoculation and using NH₄NO₃-free medium for co-cultivation and a hygromycin-containing regeneration medium for selection. Hyg^r culture lines regenerated shoots, which developed into plantlets following transfer to a plant growth regulator-free medium. All of these plantlets were verified to be transgenic by GUS histochemical assay and inverse PCR analysis.Abbreviations AS Acetosyringone (3,5-dimethoxy-4-hydroxy-acetophenone) - BA Benzyladenine - CaMV Cauliflower mosaic virus - GUS -Glucuronidase - HPT Hygromycin phosphotransferase - Hyg^r Hygromycin-resistant - NOS Nopaline synthase - NPTII Neomycin phosphotransferase II - PGR Plant growth regulator - PIC Picloram (4-amino-3,5,6-trichloropicolinic acid)Communicated by H. Ebinuma     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.