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Biomedical subjects

N Motoyama

Publications and source records attributed to N Motoyama.

At least 55 records · Page 3Linked to original sources
PubMed1992

Paroxysmal nocturnal hemoglobinuria due to hereditary nucleotide deletion in the HRF20 (CD59) gene.

HRF20 (CD59) is a membrane glycoprotein which protects cells from the membrane attack reaction of homologous complement. A patient who is completely deficient in HRF20 expression and is suffering from paroxysmal nocturnal hemoglobinuria (PNH) was studied. His parents are cousins and both have decreased HRF20 expression, suggesting that the deficiency is genetic. We established a cultured cell line (NCU1) which is HRF20 deficient from the patient's lymphocytes by Epstein-Barr-virus (EBV) infection. Northern blot analysis revealed HRF20 mRNA signals, indicating that HRF20 mRNA were transcribed. HRF20 cDNA was amplified by the polymerase chain reaction (PCR) method. Sequencing of the cDNA from the NCU1 showed two single-base deletions at amino acid 16 and 96 from the N terminus of the mature protein. Deletion in the genomic DNA of peripheral blood lymphocytes was confirmed by the DNA sequence of an HRF20 open reading frame containing amino acid 16. Furthermore, the patient's parents and sister possessed both intact and deleted genomic HRF20 DNA while his brother's DNA was intact. These findings demonstrate that the HRF20 deficiency was genomic in origin, and that complete deletion was brought about by a homozygous abnormality in the HRF20 gene. The base deletion caused a codon frame shift resulting in failure to produce intact HRF20 protein in the patient.

Antigens, CD↗
PubMed1992

Kinetics of inter-heavy chain disulfide bond formation of liganded and unliganded human immunoglobulin G by radioimmunoassay.

Monoclonal antibodies (mAbs) to the hinge region of human IgG1 immunoglobulins were prepared by immunization with a proteolytic fragment of hinge segment coupled to keyhole limpet hemocyanin. A mAb, 4G3, was obtained capable of binding to intact IgG but not to partially reduced IgG. Using this mAb, inter-heavy (H) chain disulfide bond formation from partially reduced anti-tetanus antibodies (Abs) in the presence or absence of antigens (Ags) was studied by solid phase radioimmunoassay. When the Abs were partially reduced in solution and then coated to plastic plates, only 10% regeneration of inter-H chain disulfide bonds occurred after reoxidation, although 100% formation occurred in solution [Kishida et al., J. Biochem. (Tokyo) 79, 91-105 (1976)]. This difference in the extent of disulfide bond formation can be explained by the fact that there are two convertible isomers in solution, Conformer I and II, one of which (Conformer I) can form disulfide bonds but is present as a minor component. Since the motion of IgG molecules on plates is restricted by hydrophobic interactions, the two conformers are not convertible as in solution. Therefore only Conformer I which existed before coating formed inter-H chain disulfide bonds. Similar kinetic measurements were performed using plates coated with tetanus toxoid. Abs, partially reduced in solution and then allowed to react with Ags on the plate were able to completely regenerate their inter-H chain disulfide bonds, although the rate of reaction was slow. These results can also be explained by the fact that the two isomers are convertible since Ag-Ab complexes are dissociable. Ag-binding therefore did not significantly perturb conformation of the hinge segments. In addition, no difference between liganded and unliganded Abs was observed in the binding of anti-hinge mAbs. These results imply little or no contribution of the hinge region to transmission of the signal produced by Ag-binding to Fc.

Amino Acid Sequence↗
PubMed1992

Effect of synergists on the oral and topical toxicity of azamethiphos to organophosphate-resistant houseflies (Diptera: Muscidae).

Dermal and oral toxicities of azamethiphos were determined in two organophosphate-resistant and one susceptible strain of houseflies, Musca domestica L. The 594vb strain was 1,967-fold more resistant to azamethiphos when compared with the susceptible Chemical Specialties Manufacturers Association (CSMA) strain by dermal application. When the compound was administered orally to the 594vb strain, we observed only a 15-fold resistance. In contrast, the Yachiyo strain, which show 1,500-fold resistance to diazinon and which has known multiple mechanisms for organophosphate resistance, showed only 6-fold resistance to azamethiphos by topical application and 4-fold resistance by oral administration. Azamethiphos administered dermally and orally was equally toxic to the CSMA and Yachiyo strains. However, when azamethiphos was administered to the 594vb strain, the insecticide was 71 times more toxic orally than by the dermal route. This result indicated involvement of a cuticular penetration factor in the resistance mechanism. The effect on azamethiphos toxicity of piperonyl butoxide (PB), an inhibitor of the monooxygenases, and tributylphosphorotrithioate (DEF), an esterase inhibitor, was investigated in the three strains. Pretreatment of the flies with PB, DEF, or PB+DEF before topical application of azamethiphos resulted in a significant decrease in LD50s in all the strains. The degree of synergism, however, varied depending upon the strains and the synergists. Similar results were obtained when azamethiphos was administered orally following pretreatment of the flies with PB+DEF. We attribute the high level of azamethiphos resistance in the 594vb strain partly to increased degradation by oxidative and hydrolytic enzymes. The hydrolytic enzymes are more important, but other factors including reduced cuticular penetration and insensitive acetylcholinesterase may be involved.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Somatic mutation in constant regions of mouse lambda 1 light chains.

To study the distribution of somatic mutation, we determined nucleotide sequences of rearranged lambda 1-chain genomic DNA from four hybridomas obtained from C57BL/6 mice that had been immunized with (4-hydroxy-3-nitrophenyl)acetyl-conjugated chicken gamma globulin. In total, 114 nucleotide substitutions were observed, with neither insertion nor deletion. Sixty-one mutations occurred in the variable-joining region genes (V lambda 1-J lambda 1) and 49 in joining-constant (J lambda 1-C lambda 1) introns. Although frequency decreased with distance from the V lambda 1-J lambda 1 coding region, somatic mutations occurred in the entire J lambda 1-C lambda 1 intron and even in the C lambda 1 region. We found four nucleotide substitutions in C lambda 1 genes, all of which were replacement mutations. Therefore, the mechanism responsible for somatic mutation is operative into the C lambda 1 exons. Nucleotide sequences of rearranged but inactive lambda 2-chain genes from two hybridomas were also examined and compared with those of lambda 1-chain genes. The clustering of replacement mutations in complementarity-determining regions in the inactive lambda 2-chain genes similar to the active lambda 1-chain genes suggested a mechanism that induces somatic mutation preferentially in this region even in the absence of antigenic selection.

Animals↗
PubMed1991

Studies on the resistance to various insecticides of a house fly strain (Diptera: Muscidae) selected with azamethiphos.

A colony of azamethiphos-resistant house flies, Musca domestica (L.), was obtained from Denmark and further selected in the laboratory with azamethiphos for four generations. LD50s for various insecticides were determined and compared with those of a susceptible house fly strain. The selected flies showed cross-resistance to all insecticides evaluated. The flies were highly resistant to most organophosphorus, carbamate, and chlorinated hydrocarbon insecticides except prothiophos, p,p'-DDT, and the pyrethroids. We conclude that the main mechanisms responsible for resistance are presumed to be factors other than acetylcholinesterase sensitivity and nerve sensitivity due to knockdown resistance.

Animals↗
PubMed1989

Comparison between in vivo and in vitro cutaneous penetration of fenvalerate in tobacco budworm (Lepidoptera: Noctuidae).

In vivo and in vitro penetration of fenvalerate were compared in the 3-d-old fifth-instar tobacco budworm, Heliothis virescens (F). In the in vitro studies, a piece of cuticle was excised and mounted on a diffusion cell, and the amount of radioactivity present in the cuticle and medium was determined at various time intervals. At 24 h, approximately 7.4% of the dose was located in the cuticle, whereas only 1.6% actually penetrated. In the in vivo studies, approximately 7.4% of the radioactivity was recovered from the body after cuticular wash.

Animals↗
PubMed1989

Inhibition of growth and development of tobacco hornworm (Lepidoptera: Sphingidae) larvae by cyromazine.

When fed to larvae of tobacco hornworm, Manduca sexta (L.), cyromazine strongly inhibited normal growth and development. Depending on the dose or period of feeding, symptoms were cuticular melanization, swellings in intersegmental regions, cuticular lesions, rupture of the body wall, and death. At greater than 10 ppm, cyromazine in the diet initiated symptoms in fifth instars, whereas greater than 20 ppm induced symptoms which led to death during the instar or at molting. The ratio of chitin to larval body weight in both cyromazine-exposed and control fifth instars increased slightly from ecdysis to 42 h. However, this ratio did not differ between the two groups, indicating that cyromazine had no immediate or direct effect on chitin production. When cyromazine was ingested by fifth instars, it was excreted rapidly, and a small amount (less than 5%) was metabolized to melamine. The amount of cyromazine found in the hemolymph remained relatively constant during the feeding period, whereas the amount present in the body wall increased with time and was localized in the KOH-soluble fraction. Cyromazine may inhibit growth or expansion of the body wall (or both) sufficiently to prevent normal internal growth, producing the observed symptoms and leading to abnormal development.

Animals↗
PubMed1988

Cell cycle-dependent inhibition of DNA synthesis by prostaglandin I2 in cultured rabbit aortic smooth muscle cells.

The role of prostaglandin I2 (PGI2) in the control of DNA synthesis during the cell cycle was investigated in cultured rabbit aortic smooth muscle cells (SMC). SMC at confluency in the G0 state reached the S phase about 16 h after stimulation with serum, as judged by measurement of [3H]thymidine incorporation into DNA (DNA synthesis). Cyclooxygenase inhibitors such as indomethacin and aspirin enhanced DNA synthesis, suggesting that endogenously synthesized prostaglandins inhibit DNA synthesis. Added PGE1 or PGE2 had little effect on DNA synthesis. PGI2 inhibited DNA synthesis only when added from 10 to 16 h after stimulation of SMC in the G0 state with serum. Addition of CS-570, a stable PGI2 analogue, inhibited DNA synthesis at any time after serum stimulation. The endogenous syntheses of PGI2 and DNA were negatively correlated. These results suggest that PGI2 inhibits DNA synthesis by acting on the progression stage of the G1 state.

Animals↗
PubMed1987

Purification and properties of inositol-1,4-bisphosphate 4-phosphohydrolase from rat brain.

Inositol-1,4-bisphosphate 4-phosphohydrolase (inositol-1,4-bisphosphatase) was highly purified from a soluble fraction of rat brain. On SDS-polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band and its molecular weight was estimated to be 42000. The isoelectric point of the enzyme was 4.3. The enzyme specifically hydrolyzed the 4-phosphomonoester linkage of inositol 1,4-bisphosphate. The Km value for inositol 1,4-bisphosphate was 30 microM, and it required Mg2+ for activity. Ca2+ was a competitive inhibitor with a Ki value of 60 microM as regards the Mg2+ binding. Li+, which is known to be a strong inhibitor of inositol 1-phosphatase (EC 3.1.3.25), inhibited the enzyme activity and caused 50% inhibition at a concentration of 1 mM (IC50 = 1 mM). Li+ was an uncompetitive inhibitor of substrate binding with a Ki value of 0.6 mM. These inhibitory parameters of Li+ were quite similar to those for inositol 1-phosphatase (IC50 = 1 mM, Ki = 0.3 mM). Thus, the effect of Li+ on decreasing the free inositol level with a subsequent decrease in agonist-sensitive phosphoinositides, is caused by its inhibition of multiple enzymes involved in conversion of inositol 1,4-bisphosphate to inositol.

Animals↗
PubMed1987

Phosphotyrosine phosphatase: a novel phosphatase specific for phosphotyrosine, 2'-AMP and p-nitrophenylphosphate in rat brain.

A unique phosphatase that selectively hydrolyzed phosphotyrosine and 2'-AMP at alkaline pH and p-nitrophenylphosphate at neutral pH was isolated from a cytosolic fraction of rat brain. The purified enzyme appeared homogenous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 42,000. The molecular weight of the native enzyme was 45,000 as determined by molecular sieve chromatography. These findings indicate that the native enzyme is a monomer protein. At pH 8.6, the enzyme hydrolyzed L-phosphotyrosine, D-phosphotyrosine, 2'-AMP, p-nitrophenylphosphate, 3'-AMP, 2'-GMP, and 3'-GMP; the ratio of its activities with these substrates was 100:96:115:68:39:25:16. Its Km values for L-phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate were 0.8 X 10(-4) M, 1.4 X 10(-4) M, and 1.7 X 10(-4) M, respectively. At pH 7.4, the enzyme hydrolyzed p-nitrophenylphosphate, L-phosphotyrosine, and D-phosphotyrosine; the ratio of its activities with these compounds was 100:17:17, and its Km values for L-phosphotyrosine and p-nitrophenylphosphate were 1.8 X 10(-4) M and 2.0 X 10(-4) M, respectively. The enzyme activity was dependent on Mn2+ or Mg2+, and was strongly inhibited by 5'-nucleotides, pyrophosphate, and Zn2+. The enzyme was not sensitive to inhibitors of some well-characterized phosphatases such as NaF, molybdate, L(+)tartrate, tetramisole, vanadate, and lithium salt. The physiological role of the enzyme is discussed with respect to its activities toward phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate.

Adenosine Monophosphate↗
PubMed1984

Biochemical mechanisms of resistance to insecticides.

The principal biochemical mechanisms of resistance to insecticides involve either modified, less sensitive cholinesterase, esterase action, glutathione S-transferase action or cytochrome P-450-dependent monooxygenation. Both quantitative and qualitative differences in cytochrome P-450 isozymes are under genetic control and both are related to resistance. Recent characterization studies involving ligand binding and multiplicity of isozymes in Musca domestica (the housefly) are discussed in relation to resistance. The recent demonstration that multiple isozymes of glutathione S-transferase exist in susceptible and resistant insects is of interest, and some re-examination of their role in the mechanism of resistance is required. Esterases are a heterogeneous group of enzymes whose role in resistance has often been suggested but seldom rigorously defined. Purification studies in the green rice leafhopper, Nephotettix cincticeps, have involved an enzyme with carboxylesterase, phosphotriesterase and pyrethroid esterase activities. A similar enzyme, but without pyrethroid esterase activity, is also found in the housefly. In resistance such enzymes may serve either to catalyse hydrolysis or as binding proteins. It has been suggested, from time to time, that regulator genes, enzyme induction and gene magnification all play a part in controlling biochemical mechanisms of resistance, although clearly defined evidence has not always been brought forward. These hypotheses are re-examined.

Acetylcholinesterase↗
PubMed1979

Comparative studies on the molecular weight of glutathione S-transferases from mammalian livers and an insect.

1. A comparative study was conducted on the molecular weights of glutathione S-transferases in the housefly and liver of the mouse and rat using Sephadex G-100 gel chromatography. 2. The values varied depending upon the buffers used in gel filtration. Molecular weights of 44,600, 53,600 and 43,000 daltons respectively were obtained with 0.01 M potassium phosphate buffer, pH 6.7; 0.05 M Tris-HCl buffer, pH 8.0; and 0.05 M Tris-HCl buffer containing 0.1 M KCl, pH 8.0, respectively. 3. There was no difference in the molecular weights of the enzymes obtained from the insect and from the mammalian livers. Purified enzymes eluted in the same fractions as those from the crude extracts, suggesting little modification in the molecular size of the enzymes during purification. 4. The presence of a large volume of stabilizer(s) in the enzyme solutions applied to the column delayed the elution of the activity peaks and resulted in erroneous values. Therefore, different literature values of molecular weights for glutathione S-transferases may be the result of different buffers and stabilizers used in gel filtration and probably do not represent a real difference in molecular size.

Animals↗