Mg2+-, Ca2+-dependent unwinding of DNA by poly-L-glutamic acid

In order to examine a possibility that the high acidic amino acid region in the nonhistone protein HMG(1+2) is concerned with the Mg2+-, or Ca2+-dependent unwinding of DNA by the HMG(1+2) (1,2), poly-L-glutamic acid was employed as an acidic model peptide for thermal melting temperature analysis. The poly-L-glutamic acid bound to DNA either in the presence or absence of Mg2+. The poly-L-glutamic acid unwound DNA double-helix to a similar extent to HMG(1+2) in the presence of Mg2+ or Ca2+, but not in the absence of them. These results may suggest that the high acidic amino acid region in HMG(1+2) participates in Mg2+-, Ca2+-dependent unwinding of DNA double-helix.     

Determination of genotypes of human aldehyde dehydrogenase ALDH2 locus

Virtually all Caucasians have two major aldehyde dehydrogenase isozymes, ALDH1 and ALDH2, in their livers, while approximately 50% of Japanese and other Orientals are "atypical" in that they have only ALDH1 and are missing ALDH2. We previously demonstrated the existence of an enzymatically inactive but immunologically cross-reactive material (CRM) in atypical Japanese livers. Among 10 Japanese livers examined, five had ALDH1 but not ALDH2 isozyme. These are considered to be homozygous atypical at the ALDH2 locus. Four had both ALDH1 and ALDH2 components detected by starch gel electrophoresis, that is, they are apparently usual. However, biochemical and immunological studies revealed that three of these four livers contained CRM. These three livers should be heterozygous atypical in the ALDH2 locus, that is, genotype ALDH2(1)/ALDH2(2). A Japanese liver, as well as control Caucasian livers, had no CRM, and they must be homozygous usual ALDH2(1)/ALDH2(1). Although the number of liver specimens examined is limited, the frequencies of three genotypes determined in this study are compatible with the values calculated based on the genetic model that two common alleles ALDH2(1) and ALDH2(2) for the same locus are codominantly expressed in Orientals. The remaining liver had only ALDH2 isozyme and was missing ALDH1. This type was not previously found in Caucasians and Orientals. The two-dimensional crossed immunoelectrophoresis revealed the existence of a CRM corresponding to ALDH1 in this liver. The abnormality can be considered to be due to structural mutation at the ALDH1 locus producing a defective ALDH1 molecule, although other possibilities such as post-translational modifications are not ruled out.     

Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function

Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis.     

Physiological Effects of a and {alpha} Sexual Agglutination Substances on Mating Reaction in the Yeast Saccharomyces cerevisiae

The sex-specific glycoprotein agglutination substance, responsiblefor sexual agglutination, solubilized from the surface of haploidcells of a or a mating type by the autoclave method had thefollowing effects on mating reaction in Saccharomyces cerevisiae.Sexual agglutination was inhibited by the agglutination substanceof the opposite mating type in living cells as well as in heat-killedcells. Formation of zygotes was completely inhibited, when botha and a cells were treated with the agglutination substanceof the opposite mating type. The a and a agglutination substanceswere inactivated by cells of the opposite mating type, withthe degree of inactivation being greater for the former. Theenzyme responsible for the inactivation of a agglutination substanceseems to be carboxypeptidase Y. ¹ This paper is dedicated to the late Professor J. Ashida, KyotoUniversity. ² Present address: Department of Plant Pathology, Universityof California, Davis, CA. 95616, U.S.A. (Received November 1, 1982; Accepted January 19, 1983)     

Effect of triazinone on root growth and gravireaction

The effects of a synthetic growth promoter, 4-ethoxy-l-( p -tolyl)-S-triazine-2,6 (1H, 3H)-dione [TA], on growth and gravireaction of Zea mays L. (cv. LG 11) roots were investigated. In horizontal, intact roots, pretreatment with TA at 4 × 10⁻⁴ M inhibited the gravireaction. If the pretreated roots were rinsed with a buffer solution before incubation, the TA effect was reduced, indicating that a continuous presence of TA was necessary for its maximal activity. On the other hand, the TA pretreatment (1×10⁻⁵, 1×10⁻⁴ and 4 × 10⁻⁴ M ) promoted the elongation of these roots. The TA effect was stronger for illuminated roots than for those kept in darkness. TA also decreased the lateral curvature of half-decapitated roots maintained vertically in light. This indicates that the action of TA could be associated with some growth inhibiting substances produced or released in cap cells.     

Neolignans from Ocotea catharinensis

The trunk bark of Ocotea catharinensis yielded, besides the known bicyclo(3.2.1)octanoid neolignans canellin-C and 5′-methoxycanellin-C, two epimers rel-(1R,4S and 4R,5S,6R,7S,8R)-1-allyl-4,8-dihydroxy-3,5-dimethoxy-7-methyl-6-piperonyl-bicyclo(3.2.1)oct-2-enes and rel-(1R,5S,6R,7S,8R)-1-allyl-3,8-dihydroxy-5-methoxy-7-methyl-6-piperonyl-4-oxobicyclo(3.2.1)oct-2-ene. The hydrobenzofuranoid neolignans are represented by the equally novel (2S,3S,5R)-5-allyl-5,7-dimethoxy-3-methyl-2-piperonyl-2,3,5,6-tetrahydro-6-oxobenzofuran and (2R,3S,3aS)-3a-allyl-5,7-dimethoxy-3-methyl-2-piperonyl-2,3,3a,6-tetrahydro-6-oxobenzofuran.     

Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycol mediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromosome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.     

Restriction map of CELO virus DNA

A restriction map of chicken embryo lethal orphan (CELO) virus DNA was reported with ten restriction endonucleases (XbaI, XhoI, SalI, HindIII, EcoRI, BglI, KpnI, BamHI, PstI and SstI). CELO virus DNA was estimated by comparing CELO virus DNA fragments with marker DNA fragments to have a molecular weight of 29.3·10⁶.     

Amino acid sequence of the active site of human pseudocholinesterase

The usualE ₁ ^u and atypicalE ₁ ^a human pseudocholinesterases (acylocholine acylhydrolase, EC 3.1.1.8) were purified to homogeneity. The active-site serine residue was conjugated with diisopropyl fluorophosphate and digested with trypsin. The tryptic peptide containing the active site was isolated by gel filtration followed by two-dimensional paper chromatography and electrophoresis. The amino acid sequence of the active site peptide obtained from the usualE ₁ ^u enzyme was found to be Gly-Glu-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu. A remarkable structural homology exists between the human and the horse enzymes in their active sites. From the difference in electrophoretic mobility of the active-site peptides obtained from the usual and atypical enzymes, the probable structure of the atypical human enzyme was deduced as Gly-His-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu.