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Biomedical subjects

K Akama

Publications and source records attributed to K Akama.

At least 37 records · Page 2Linked to original sources
PubMed1996

Plant nuclear tRNA(Met) genes are ubiquitously interrupted by introns.

We have isolated three independent clones for nuclear elongator tRNA(Met) genes from an Arabidopsis DNA library using a tRNA(Met)-specific probe generated by PCR. Each of the coding sequences for tRNAMet in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA(Met) is dispersed at at least eight loci in the genome. The unspliced precursor tRNA(Met) intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA(Met) genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5'-distal end of each intron.

Arabidopsis↗
PubMed1996

Interaction of nucleosome core DNA with transition proteins 1 and 3 from boar late spermatid nuclei.

The DNA binding properties of boar transition protein 1 and 3 (TP1 and TP3) were studied by means of physicochemical techniques. The ultraviolet difference absorption spectra upon TP1 and TP3 binding to rat liver nucleosome core DNA (double-stranded DNA) showed TP1- and TP3-induced hyperchromicity at 260 nm, which is suggestive of local melting of DNA. CD measurements of TP1-DNA and TP3-DNA complexes indicated that the binding of TP1 and TP3 induced different conformational changes in DNA, probably including local melting of DNA. Thermal melting studies on the binding of TP1 and TP3 to DNA showed that although at 1 mM NaCl TP1 and TP3 caused slight stabilization of the DNA against thermal melting, destabilization of the DNA was observed at 50 mM NaCl. From the results of quenching of the tyrosine fluorescence of TP1 and the tryptophan fluorescence of TP3 upon their binding to double-stranded and single-stranded boar liver nucleosome core DNA at 50 mM NaCl, the apparent association constants for the binding of TP1 to double- and single-stranded DNA were calculated to be 8.0 x 10(4) and 1.3 x 10(5) M-1, respectively, and those for the binding of TP3 to double- and single-stranded DNA to be 7.1 x 10(4) and 1.8 x 10(5) M-1, respectively. These results suggest that TP1 and TP3, having higher affinity for single-stranded DNA, induce local destabilization of DNA, probably through the stacking of Tyr32 and Trp18 with nucleic acid bases, respectively.

Amino Acid Sequence↗
PubMed1996

Inhibition of endothelium-dependent relaxation by hemoglobin in rabbit aortic strips: comparison between acellular hemoglobin derivatives and cellular hemoglobins.

Hemoglobin (Hb)-based artificial oxygen carriers are supposed to induce vasoconstriction through the inactivation of endothelium-derived relaxing factor (EDRF). We examined the vasoconstrictive activity of acellular Hb and cellular Hb solutions in rabbit aortic strips. Unmodified Hb, pyridoxalated Hb, bovine unmodified Hb, haptoglobin-Hb complex (Hp-Hb), and polyoxyethylene glycol-conjugated Hb (PEG-Hb) were used as acellular Hbs having different molecular masses. Cellular Hbs included liposome-encapsulated Hb and red blood cells (RBC). In the first experiment, Hb (10 ng/ml to 1 mg/ml) was cumulatively added to the tissues in which steady-state relaxation was evoked by acetylcholine (ACh) after precontraction induced by phenylephrine. Although all Hb solutions induced a dose-dependent reversal of ACh-induced relaxation, the most potent vasoconstrictive effect was noted with acellular Hbs, and their contractile activities were almost the same independent of molecular mass. On the other hand, liposome-Hb and RBC showed reduced potencies in this order. These results indicate the importance of cellularity as the major factor determining Hb-related EDRF inactivation. In another experiment, the tissues were exposed to Hb at 0.01, 0.1, or 1 mg/ml for 30 min and ACh-induced relaxation was recorded after the complete removal of Hb in an organ bath chamber. Exposure to unmodified Hb at > 0.1-mg/ml concentrations significantly reduced the ACh-induced relaxation, whereas the relaxation was not affected by PEG-Hb, Hp-Hb, liposome-Hb, or RBC. These results suggest that unmodified Hb might be persistently associated with tissues and thereby inhibit ACh-induced relaxation. From these findings, we propose two attributes of Hb-related inhibition of endothelium-dependent relaxation: Acellular Hbs inhibit EDRF more efficiently in the luminal space than cellular Hbs, and unmodified Hb can also inhibit it adluminally and/or adventitially.

Acetylcholine↗
PubMed1995

The amino acid sequence and interaction with the nucleosome core DNA of transition protein 4 from boar late spermatid nuclei.

The primary structure of transition protein 4 (TP4) from boar late spermatid nuclei was determined by automated Edman degradation of S-pyridylethylated protein and of peptides generated by cleavage with Staphylococcus aureus V8 protease, lysyl endopeptidase and CNBr. Boar TP4 is a basic protein consisting of a highly basic amino-terminal half (residues 1-73) and a less basic carboxy-terminal half (residues 74-138). The latter half includes a highly hydrophobic segment, a four-times tandemly repeated sequence, N(G)QNKR(K)X, and a carboxy-terminal segment containing Trp126. Ultraviolet absorption and CD spectra of TP4-rat-liver-nucleosome-core-DNA (double-stranded DNA) complexes suggest a TP4-induced local melting of DNA. Although at 1 mM NaCl TP4 brought about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From the results of quenching of tryptophan (Trp126) fluorescence of TP4 upon its binding to double-stranded and single-stranded boar liver nucleosome-core DNA at 50 mM NaCl, the apparent association constants for the binding of TP4 to double-stranded and single-stranded DNA were calculated to be 7.3 x 10(3) M-1 and 4.1 x 10(3) M-1, respectively. These results suggest that TP4, having different domain structures from TP1-3 and a higher affinity for double-stranded DNA, induces a local destabilization of DNA probably through the stacking of Trp126 with nucleic acid bases.

Amino Acid Sequence↗
PubMed1995

Touch-inducible genes for calmodulin and a calmodulin-related protein are located in tandem on a chromosome of Arabidopsis thaliana.

Genes for calmodulin and calmodulin-related proteins in Arabidopsis are up-regulated by a variety of physical stimuli, which include rain, wind and touch [Braam and Davis (1990) Cell 60: 357]. We have isolated five genes for calmodulin (AtCAL1, 2, 3, 5, 6) and one gene for a calmodulin-related protein (AtCAL4) from an Arabidopsis genomic library. Touch stimulus of Arabidopsis plants induces the accumulation of mRNA transcribed from AtCAL4 and AtCAL5, but not from the other isolated genes. The two touch-inducible genes are arrayed in tandem with a short intergenic region of 700 bp but they show different organ-specific patterns of expression.

Arabidopsis↗
PubMed1994

The amino acid sequence of a boar transition protein 3.

The boar transition protein 3 (TP3) was extracted with acid from the isolated late spermatid nuclei or directly from the testes, fractionated with trichloroacetic acid, and reduced and carboxymethylated (RCM-). RCM-TP3 from the nuclei was purified by HPLCs on Nucleosil 300 7C18 and Hitachi #3057, and that from the testes, by ion-exchange chromatography on CM-Sephadex C-25 and HPLCs on Nucleosil 300 7C18 and Chemcosorb 7C8. The two TP3 preparations were identical in acid-urea- and SDS-gel electrophoretic mobilities and amino acid composition. The primary structure of TP3 was determined by manual Edman degradation of the peptides obtained by lysyl endopeptidase-digestion or by alpha-chymotrypsin-digestion of RCM-TP3 from the testes, and by automated Edman degradation of it. Boar TP3 is a basic protein of 76 residues: H-AKVTEKSWQPQTTSTKRWKKRKTPSQPRSRGKVRKIYKKVKRPLHVCSRKKYSPKVITTSRRQKRAR RANKFETIP-OH, and it shows 27% homology with boar TP1. TP3 is composed of an N-terminal region (1-19) having two characteristic tryptophan residues (8 and 18) which is absent in the known TP1 group, and a C-terminal region (20-76) having a close resemblance to boar TP1.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of a novel acrosin-like enzyme from boar cauda epididymal sperm.

A trypsin-like protease was extracted with 1% cetyltrimethylammonium bromide (CTAB) at pH 7.0 from boar cauda epididymal sperm nuclei whose acrosin had previously been removed by acid extraction. The CTAB-extracted sperm protease (CSP) was purified by ion-exchange chromatography on CM-23, gel filtration on Sephadex G-100, affinity chromatography on benzamidine-CH-Sepharose 4B, and HPLC on CM-5PW. CSP is a two chain protein composed of M(r) 2.6K and M(r) 37K chains, which are covalently cross-linked by disulfide bonds. CSP exhibited a pH optimum between pH 8.0 and 9.0, and was inhibited by diisopropyl phosphorofluoridate, antipain, leupeptin, and 1-chloro-3-tosylamide-7-amino-L-2-heptanone. The activity of CSP was enhanced about 1.2-fold with 50 mM CaCl2, with which acrosin is enhanced 2.0-fold. The catalytic efficiency (kcat/Km) of CSP toward Bz-L-Arg-OEt, Tos-L-Arg-OMe, and Tos-L-Lys-OMe in the presence of 50 mM CaCl2 differed from that of acrosin by factors of 0.53, 1.2, and 0.80, respectively. Amino acid sequencing of V8-digested peptides of CSP, and its L- and H-chains showed that the amino acid sequence of CSP was closely related to, but different from, that of acrosin. These results suggest that CSP is a novel acrosin-like enzyme that differs from acrosin in its location in the sperm head, the effect of calcium ions on its activity, and its substrate specificity.

Acrosin↗
PubMed1994

Acellular and cellular hemoglobin solutions as vasoconstrictive factor.

The inhibitory effects of acellular and cellular hemoglobin (Hb) solutions on endothelium-dependent vasorelaxation were investigated in rabbit thoracic aortic strips. As acellular Hb solutions, 2,3-diphosphoglycerate (DPG)-depleted Hb and pyridoxylated Hb were examined. Cellular Hb solutions included washed human fresh red cells and liposome Hb encapsulated with pyridoxal-5'-phosphate (PLP). The tissues were precontracted with phenylephrine (PE), after which acetylcholine (ACh) was added to elicit a steady-state relaxation. Acellular Hb solutions cumulatively reversed ACh-induced relaxation, and these inhibitory effects reached a plateau at 10 micrograms/ml. Increasing oxygen affinity by pyridoxylation had little effect on this. In contrast, both red cells and liposome Hb solution showed moderate inhibitory effects, and they reached a plateau at 1 mg/ml. These findings indicate that acellular Hb solutions are more potent inhibitors than cellular Hb solutions by a factor of about 100, and that the encapsulation of Hb is a preferable method to mimic the red cell.

2,3-Diphosphoglycerate↗
PubMed1994

Oxygen transport and in vivo parameters of artificial red cells (ARC).

Artificial red cells (ARC) are prepared by encapsulating Hb with a polymerizable phospholipid. Their physical stability is very high and long-term preservation is possible in the frozen state. We examined the effect of blood parameters on the hematological and biochemical findings in transfused rats. The oxygen transport capacity of ARC in vivo were also tested by exchange transfusion in beagles. The oxygen binding parameters were almost the same as those of red blood cells (i.e., P50, Hill's coefficient, and oxygen transport efficiency (OTE) were 30 mmHg, 2.5, and 30%, respectively). The blood parameters after transfusion showed no significant changes when compared with the control. The oxygen transport capacity was of the same efficiency as red blood cells.

Animals↗
PubMed1994

Isolation of intact transition protein 4 from boar late spermatid nuclei.

Boar transition protein 4 was extracted with acid from the late spermatid nuclei, and separated from the transition protein-degrading proteases by ion-exchange chromatography on Fractogel EMD SO3- 650 (M). The transition protein was further purified by HPLCs on Nucleosil 300 7C18 and Diol-200. The circular dichroic spectra of the protein with and without dithiothreitol showed that the protein had beta-form predominantly. Although sodium dodecyl sulfate affected the tertially structure of the protein, the beta-form was well retained. These indicate that the protein has a structure-forming potential for the beta-structure.

Animals↗
PubMed1994

The amino acid sequence and phosphorylation sites of a boar transition protein 1.

Boar transition protein 1 was extracted with acid from the testes, purified by chromatographies on CM-Sephadex C-25 and Sephadex G-50, and reduced and carboxymethylated. The modified protein was purified by HPLC on Nucleosil 300 7C18. The primary structure of the protein was determined by automated Edman degradation of the C-terminal peptide of the BrCN-cleaved protein and of the whole protein, and by carboxypeptidase digestion of it. The study of phosphorylation sites showed that Ser36 and Ser39 in the very conserved sequence 29-42 were partly phosphorylated, suggesting the involvement of this region in the interaction with DNA.

Amino Acid Sequence↗
PubMed1994

Isolation of intact transition protein 1 and 3 from boar late spermatid nuclei.

Boar transition protein 1 and 3 were extracted with acid from the late spermatid nuclei, separated from the TP-degrading proteases by ion-exchange chromatography on Fractogel EMD SO3- 650 (M), and further purified by HPLCs on Diol-120 and on Hitachi #3057, respectively. The circular dichroic spectra of the transition proteins with and without dithiothreitol showed that they have beta-form predominantly. Although sodium dodecyl sulfate partially induced helical structure, the beta-form was considerably retained. These indicate that the transition proteins have a structure-forming potential for the beta-structure.

Animals↗
PubMed1992

Stability and blood compatibility of polylipid/Hb.

The Polylipid/Hb vesicle is a new artificial red cell (ARC) based on liposome-encapsulated Hb. Advantages are derived from the stabilized liposomal bilayer membranes, obtained by polymerization of 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phospho choline (DODPC). Furthermore, blood compatibility in vitro are good.

Blood Coagulation↗
PubMed1990

Boar transition protein 2 and 4 isolated from late spermatid nuclei by high-performance liquid chromatography.

The boar late spermatid nuclei retaining transition proteins (TPs) could be obtained from the testis by the use of antipain to inhibit TP-degrading proteinases of the nuclei. The enzymes detected in acid extract including the basic proteins were inactivated by reduction and carboxymethylation of the proteins. The reduced and carboxymethylated basic proteins were fractionated by differential precipitation between 3% trichloroacetic acid (TCA) and 3-20% TCA. From the 3% TCA-precipitate, boar TP2 and TP4 were isolated by high-performance liquid chromatography (HPLC) on Nucleosil 300 7C18. The two TPs were characterized by acid urea- and SDS-polyacrylamide gel electrophoreses and amino acid analysis. Boar TP2 closely resembled rat and mouse TP2s, and ram protein 3 in its high content of serine and basic amino acids, the presence of cysteine and molecular weight. Boar TP4 was similar to ram protein P1 in its high content of basic amino acids, the presence of cysteine and molecular weight. But the TP2 and TP4 differed in electrophoretic mobility on acid urea-gel and solubility in 3% TCA from those of the other species. The HPLC used here also enabled us to efficiently separate boar TP1, TP2, TP3 and TP4, and to estimate that the amount of the TP2, TP3 and TP4 was about 1/8, 1/4 and 1/4 that of the TP1, respectively.

Amino Acids↗