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Biomedical subjects

A Murakami

Publications and source records attributed to A Murakami.

At least 19 recordsLinked to original sources
PubMed1992

Isolation of human retinal genes: recoverin cDNA and gene.

A human retina cDNA library enriched for retina-specific clones was prepared by subtraction with a non-retina population of cDNA in combination with polymerase chain reaction (PCR) amplifications. A highly retina-specific cDNA clone (1190 bp) was obtained through this library encoding a 200 amino acid protein with three calcium binding sites and 87% homology to the bovine photoreceptor protein, recoverin, which has been shown to mediate the recovery of the dark current after photoactivation, and 58% homology to the calcium-binding chick cone protein, visinin. Analysis of the gene indicated a 9-10 kb single-copy gene with at least three exons and two introns. The three exons contained the entire coding sequence, and all of the calcium-binding EF-hand regions were in putative exon 1. The recoverin gene was mapped to human chromosome 17 by hybridization to a panel of human-rodent hybrid DNAs.

Amino Acid Sequence↗
PubMed1992

A deletion in the ornithine aminotransferase gene in gyrate atrophy.

Gyrate atrophy (GA) is an autosomal recessive chorioretinal degenerative disease of the eye caused by an inborn defect of the nuclear encoded mitochondrial enzyme ornithine aminotransferase (OAT). We have described previously a GA patient with a 5.0-kilobase pair truncated EcoRI OAT gene fragment and the absence of OAT mRNA on Northern blot analysis. Cloning and sequencing analysis of the truncated gene fragment revealed a 1,072-base pair (bp) deletion including the entire exon 6, starting in intron 5, 172 bp upstream of exon 6 and ending in intron 6, 772 bp downstream of exon 6. A short direct repeat sequence (AGGAGC), resembling the sequence shown to cause DNA polymerase alpha to pause, and sequences capable of forming hairpin loops were both present at the 5' and 3' break-points of the deletion. Reverse transcription-polymerase chain reaction amplification of the patient's RNA with OAT primers yielded DNA fragments of two different sizes, consistent with a low level expression of OAT mRNA. Direct sequencing of the smaller fragment demonstrated the complete absence of exon 6 sequence in the mRNA predicted from the deletion, causing a reading frame shift which results in a premature termination codon at position 192. The mutation in the other allele has been demonstrated by polymerase chain reaction, denaturing gradient gel electrophoresis, and direct sequencing also to be a premature termination codon in exon 6. The absence of detectable OAT mRNA in this patient is consistent with these premature termination mutations because they have been shown to decrease the level of mRNA, especially if present early in the coding sequence.

Amino Acid Sequence↗
PubMed1992

Purification of serine hydroxymethyltransferase from Bacillus stearothermophilus with ion-exchange high-performance liquid chromatography.

The gene of serine hydroxymethyltransferase (SHMT) of a thermophilic bacterium Bacillus stearothermophilus was expressed in Escherichia coli, and SHMT was successfully purified from the crude extract of E. coli in two steps while maintaining the enzymatic activity. The purification steps involved ammonium sulphate precipitation followed by high-performance liquid chromatographic separation using the anion-exchange column Fractogel EMD DEAE-650(S). In addition to the DEAE column, three other types of anion- and cation-exchange columns were also studied for their ability to separate SHMT, and the performance of the four columns were compared.

Ammonium Sulfate↗
PubMed1992

Hydroxyl radical formation by UV-irradiated epidermal cells.

To elucidate the mechanism of sunlight-induced skin damage, guinea pigs were exposed to UV light (280-320 nm, UV B, 4 J/cm2) and a homogenate of the epidermis was examined by means of the thiobarbituric acid (TBA) test. Three hours after the exposure, TBA-malondialdehyde adducts had increased while glutathione reductase activity had decreased, indicating lipid peroxidation. To detect the initial species, spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to a suspension of illuminated epidermal cells (0.5 J/cm2). An ESR signal obtained only with irradiation comprised a 1:2:2:1 quartet [a(N)= a(beta H) = 1.49 mT] attributable to a spin adduct of hydroxyl radicals. These results suggest that sunlight exposure of skin may lead to hydroxyl radical generation and simultaneous lipid peroxidation.

Animals↗
PubMed1992

Effects of calphobindin II (annexin VI) on procoagulant and anticoagulant activities of cultured endothelial cells.

Effects of human placental calphobindin II (CPB-II) on the protein C activation and prothrombin activation on the cell surface of cultured calf pulmonary arterial endothelial cells have been investigated. CPB-II inhibited thrombin generation by factor Xa bound to the surface of the cultured endothelial cells in a dose-dependent manner. The amount (IC50) of CPB-II causing the inhibition at 50% was estimated to be approximately 10 nM. CPB-II was found to be ineffective, however, in the protein C activation by thrombin-thrombomodulin (TM) complex on the cell surface. Assay using purified TM revealed that CPB-II was able to exhibit the inhibitory potency for the protein C activation exclusively in the reconstituted system with negatively charged phospholipids. These results suggest that the neutral phospholipids participate in the protein C activation through the thrombin-TM system on the endothelial cell surface. The ability of CPB-II to inhibit procoagulant activity without affecting anticoagulant activity on the cultured endothelial cells is probably related to its potential physiological function, while it is able to exert various degrees of influence upon these activities in blood coagulation by interacting with negatively charged phospholipids in vitro.

Amino Acid Sequence↗
PubMed1992

Chalcone tetramers, lophirachalcone and alatachalcone, from Lophira alata as possible anti-tumor promoters.

Two chalcone tetramers were isolated as inhibitors of Epstein-Barr virus (EBV)-activation induced by a tumor promoter, teleocidin B-4, from a medicinal plant in tropical west Africa, Lophira alata (Ochnaceae). One of them was identified as lophirachalcone. The other, named alatachalcone, was new, and the structure was determined by spectral properties. Both compounds also showed potent inhibitory activities against teleocidin B-4-induced inflammation on mouse ear. In an initiation-promotion experiment on mouse skin, alatachalcone (16 nmol) significantly inhibited tumor promotion caused by 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.6 nmol).

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Essential oil phenyl propanoids. Useful as .OH scavengers?

In order to search for radical scavengers which could be used as raw materials for cosmetics, phenyl propanoids (eugenol, isoeugenol, dehydrodieugenol, dehydrodieugenol B and coniferyl aldehyde) were examined for their hydroxyl radical (.OH) scavenging ability. A Fenton system was used to produce .OH. In order to see scavenging by these phenyl propanoids, competition reactions between a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and these phenyl propanoids for .OH were studied. The relative yield of the spin adduct of .OH (DMPO-OH) was measured by electron spin resonance spectroscopy. The approximate rate constants of the reactions between these phenyl propanoids and .OH estimated by measuring the reduced height of the ESR signals of DMPO-OH were found to be at least in the order of 10(9) M-1 s-1 (diffusion-controlled). Also, using the TBA tests, the reactions between .OH and several compounds reactive with .OH were investigated in the presence of the phenyl propanoids and it was found that the phenyl propanoids compete with such reactive compounds for .OH. These results indicate that these phenyl propanoids can be used as antioxidants for skin damage perhaps caused by .OH generated by UV-light.

Animals↗
PubMed1992

Interaction of antisense DNA with nucleic acids/proteins.

In order to study interaction of various types of labeled antisense DNAs were prepared. Fluorescein and 2,2,6,6-tetramethypiperidine-N-oxyl were the label molecules, which were introduced to 5'-end of oligonucleotides and their analogs. Interactions of labeled antisense DNAs with nucleic acids or proteins such as HSA, HIG and TF, were studied by UV, fluorescence depolarization spectroscopy, and ESR spectroscopy. Hybrid formation of antisense DNAs with oligonucleotides in solution could be monitored by the increase in fluorescence anisotropy (r) and by intensity change in ESR spectra. When phosphorothioate type antisense molecules anchoring fluorescein (F-OPT) were mixed with proteins, r drastically increased, whereas ODN slightly increased. These results suggest that OPTs have much more affinity for proteins than ODNs.

Blood Proteins↗
PubMed1992

Syntheses and properties of fluorescent labeled oligonucleotides containing deoxyethenoadenosine at 5' end.

The fluorescent labeled oligodeoxyribonucleotides which contain deoxyethenoadenosione (d epsilon A) at their 5' end were prepared by treating CPG bound oligonucleotides with 5'-DMTr-deoxyethenoadenosine-3'-H-phosphonate. The hybrid formation of d epsilon A-oligonucleotide with its complementary DNA was studied by fluorescence spectroscopy. The fluorescence of d epsilon A in a single strand was largely quenched by stacking interaction with the base at 3' position. When d epsilon A-oligonucleotides hybridized with their complementary strands, relative fluorescence quantum yields (Qrel) against d epsilon A changed in specific manners. These results suggest that d epsilon A-oligonucleotides are applicable to study the local structure of DNA in solution.

Base Sequence↗
PubMed1992

Separation and characterization of diastereoisomeric oligonucleotide.

For developing the antisense method, two types of oligonucleotides suitable for antisense molecules were explored for their essential properties. One is oligonucleoside phosphorothioate, and all their possible R/S diastereoisomers were separated and purified by reversed phase liquid chromatography. Isolated diastereoisomers have been investigated for the correlation between their configuration and hybridization manner. Separation, R/S characterization and spectroscopical properties of these oligonucleotides will be discussed.

Chromatography, High Pressure Liquid↗
PubMed1992

Effects of base damages on DNA replication--mechanism of preferential purine nucleotide insertion opposite abasic site in template DNA.

DNA polymerase preferentially inserts purine nucleotides opposite non-instructive lesions such as abasic sites during DNA replication. In order to elucidate the mechanism of the preferential insertion, a DNA template containing a model abasic site and primers containing 4 different nucleotides (A,G,C,T) at primer terminus were synthesized. The stability of the primer terminus nucleotide placed opposite the abasic site was evaluated on the basis of its sensitivity to 3'-5' exonuclease associated with DNA polymerase.

DNA↗
PubMed1992

NMR studies on solution structure of single-stranded oligonucleotides causing line broadening.

Unusual line broadening of 1H-NMR lines attributable to the proton (8H) of guanine residues was observed for all tetradeoxyoligonucleotides tested here which have a specific base sequence of dGXXG (X = A or T). For the same samples, line broadening was also obtained in the 31P-NMR spectra. These broadened signals did not become sharp up to 60 degrees C. This unusual spectral phenomenon has been attributed by 2D-NMR and differential NOE to the compact solution structure of the oligonucleotides.

Magnetic Resonance Spectroscopy↗
PubMed1992

Sequence-specific DNA recognition by basic peptides.

A chiral template with C2 symmetry has been used for modeling a dimeric interface of DNA binding protein. An oligopeptide derived from the basic region of MyoD, a recently described "helix-loop-helix" class of DNA binding protein, has been tethered to the template. Among the four models which differ in chirality and polarity with respect to the arrangement of two subunits, only one dimer model with right-handed and C-terminus to C-terminus arrangement of the peptide subunits binds DNA containing native MyoD binding sequence.

DNA↗
PubMed1992

[Magnetic resonance imaging: evaluation of the Blalock-Taussig shunts and anatomy of the pulmonary artery].

The morphology and circulation of the pulmonary arteries and shunt vessels were evaluated by magnetic resonance imaging (MRI) in 8 patients with cyanotic heart disease after a Blalock-Taussig shunt operation. Their ages ranged from one month to 17 years. MRI permitted assessment of the size and patency of the Blalock-Taussig shunts, as well as the size and morphology of the pulmonary arteries in all patients. Measurements of the vessel diameters on MRI correlated well with the angiographic measurements (main pulmonary artery, r = 0.98; right pulmonary artery, r = 0.98; left pulmonary artery, r = 0.98; and Blalock-Taussig shunt, r = 0.97). MRI successfully imaged 3 of 4 shunt obstructions and 3 of 4 pulmonary stenoses with high resolution. In assessing peripheral pulmonary stenosis or obstruction, MRI was superior to echocardiography, the latter being unable to image peripheral pulmonary arteries satisfactorily. We concluded that MRI is an excellent noninvasive method for serially evaluating the anatomy and function of Blalock-Taussig shunts and pulmonary arteries, which is particularly useful for children with cyanotic congenital heart disease.

Adolescent↗
PubMed1992

Monomer-dimer equilibrium of nitroso-aromatic spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid, sodium salt (DBNBS), in aqueous solution.

3,5-Dibromo-4-nitrosobenzenesulfonic acid, sodium salt (DBNBS), which is widely used as a spin trap in biological systems, was allowed to form the monomer-dimer equilibrium in aqueous solution, and its equilibrium constant (Kc) was determined by NMR measurement and visible spectrum analysis. The Kc values obtained from the two different methods were virtually identical and evaluated as 1.3 x 10(-3) mol dm-3 at 25 degrees C. Further, the molar extinction coefficient of DBNBS monomer (epsilon 760) was determined to be 34 dm3mol-1cm-1. The present results indicate that only a minor portion (ca. 20%) of dissolved DBNBS (10 mmol dm-3) is practically capable of spin trapping under the conditions most widely used in biological studies.

Benzenesulfonates↗
PubMed1992

Comparison of 2,5,5-trimethyl-1-pyrroline-N-oxide (M3PO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin traps.

2,5,5-Trimethyl-1-pyrroline-N-oxide (M3PO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO) were examined for their potential as spin traps used in biological samples, and the results obtained for M3PO and M4PO were compared with those of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) reported previously. The rate constants for the reactions of M3PO and M4PO with hydroxyl radicals were in the same order of that constant of DMPO. The hyperfine splitting constants of M4PO spin adducts were as informative as those of DMPO adducts. In the presence of ferric ions, M3PO and M4PO formed 2-hydroxy-2,5,5-dimethyl-1-pyrrolidinyloxy (M3PO-OH) and 2-hydroxy-3,3,5,5-tetramethyl-1-pyrrolidinyloxyl (M4PO-OH), respectively, by the nucleophilic addition of a water molecule, as we have already reported for DMPO. M4PO-OH was subjected to oxidation by ferric ions with a rate much slower than that for DMPO-OH, while M3PO-OH was not. The results indicate that M4PO is a spin trap suitable for biological applications and that nitrones having a methyl group at C2 represent improved spin traps.

Cyclic N-Oxides↗
PubMed1992

[A successful operative case of congenital mitral stenosis associated with double outlet right ventricle, patent ductus arteriosus, persistent left superior vena cava and severe pulmonary hypertension in adult].

A 29-year-old woman, who was diagnosed as congenital mitral stenosis associated with DORV, PDA, PLSVC and severe pulmonary hypertension, was reported. The mitral valve, consisting of leaflet dysplasia, shortened and thickened chordae tendineae, obliteration of interchordal spaces, under-development of papillary muscle, and stenotic supravalvular membrane, was replaced with 25 SJM prosthesis. Simultaneously, patch closure of VSD and intrapulmonary direct closure of PDA was performed. Her postoperative course was uneventful and systolic pulmonary arterial pressure fell down from 95 mmHg to 56 mmHg. She is doing well after correction. To our knowledge, this is very rare corrective case with congenital mitral stenosis in adult to be reported in the literature in Japan.

Adult↗