Surface morphology of myocardium, Purkinje fibers, and transitional cells.
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Animals↗
Biomedical subjects
T Noguchi
Publications and source records attributed to T Noguchi.
[The correlation of host-immunocompetence and host-humoral states to the take incidence and proliferative activity of implantation C-6 glioma cells].
Recently it has become evident that "second growth factor" of growth hormone (GH), such as somatomedins, has an effect on the proliferation and growth of tumor cells derived from nervous tissue. Effects of host-immunocompetence and the host-humoral states on the take incidence and proliferative activity of brain tumor cells were studied using two animal models: nude mouse and pituitary Snell dwarf mouse. Nude mouse is known to be immunodeficient. Pituitary Snell dwarf mouse is characterized by lack of circulating GH, TSH, prolactin, in addition to immunodeficiency. Cell line used in this experiment was C-6 cell of rat glioma cell. After intracranial implantation of C-6 glioma cells in the animals, the take incidence and growth rate of C-6 glioma cells were followed up and measured over a period of 2 months. Tissues of implants were studied immunohistochemically and biochemically. Regardless of cell line, successful take incidence in the different animal species was found to be greater in the descending order of nude mouse, dwarf mouse. This confirmed the role of immune status for the successful take of iso-, or heterologous tumor cells after implantation. We are now investigating the effect of exogenous GH on the growth rate of cells implanted in the dwarf mouse. This may clarify the effect of growth factors on proliferative activity of implanted tumor cells.
Animals↗
Human M2-type pyruvate kinase: cDNA cloning, chromosomal assignment and expression in hepatoma.
Two overlapping clones, covering the entire coding sequence of human M2-type pyruvate kinase (PK) cDNA, were isolated and sequenced. Nucleotide sequencing results showed that they contained the 109-bp 5'-untranslated region, the 1593-bp coding region and the 585-bp 3'-untranslated region. Nucleotide sequence homology was 90% and 69% with rat M2-type and L-type PK cDNA, respectively. In situ hybridization using the human M2-type PK cDNA probe disclosed that the gene for M2-type PK is located at band q22 on chromosome 15. Northern blot analysis with RNA from human hepatoma demonstrated that M2-type PK was predominantly expressed in hepatoma cells, whereas L-type PK was preferentially expressed in the non-tumor portion of the liver.
Amino Acid Sequence↗
Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry.
A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.
Animals↗
Four species of cDNAs for cytochrome P450 isozymes immunorelated to P450C-M/F encode for members of P450IID subfamily, increasing the number of members within the subfamily.
A chicken antibody mono-specific to cytochrome P450C-M/F, which exists in untreated male and female rat liver and catalyses the 2- and 16 alpha-hydroxylation of estrogens (1), was used to screen a cDNA library of male Sprague-Dawley rat liver. Four cDNA clones which encoded P450 isozymes, CMF1a, CMF1b, CMF2 and CMF3, were isolated. CMF1a and CMF2 deduced consisted of 504 and 500 amino acid residues, respectively, while C-terminal 487 and 324 residues for CMF1b and CMF3, respectively, were deduced from the 5'-truncated cDNAs. The isozymes were more than 72% similar in amino acid sequences to each other and to rat P450db1, P450db2 (2), and to a mouse male specific C-P45016 alpha (3), suggesting that they belonged to a new P450 subfamily, P450IID. CMF1a and db1, and CMF2 and db2, respectively, were 99.2% and 99.0% similar in amino acid sequences, suggesting that they were virtually identical. CMF1a and CMF1b were different but 96.1% similar, and CMF3 was between 76% and 78% similar to other members of the rat P450IID family.
Amino Acid Sequence↗
pmcf.2-11, a single copy clone from chromosomal region 12q12-q13.1 [D12S32].
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Chromosomes, Human, Pair 12↗
A single amino acid substitution converts cytochrome P450(14DM) to an inactive form, cytochrome P450SG1: complete primary structures deduced from cloned DNAS.
Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.
Amino Acid Sequence↗
Synergistic in vitro and in vivo anti-tumor effect of daunomycin-anti-96-kDa melanoma-associated antigen monoclonal antibody CL 207 conjugate and recombinant IFN-gamma.
The mAb CL 207 recognizes a 96-kDa melanoma-associated Ag. The latter is not modulated by antibody but is highly susceptible to induction by immune IFN-gamma on human carcinoma and melanoma cells. The mAb CL 207 does not mediate C- and cell-dependent lysis of human carcinoma and melanoma cells. Conjugation of daunomycin with mAb CL 207 causes a slight reduction of its immunoreactivity and affinity but does not affect its serologic specificity or the toxicity of daunomycin. In combination with IFN-gamma, the daunomycin-mAb CL 207 conjugate displays a selective in vitro and in vivo toxic effect on tumor cells that express the 96-kDa MAA. The synergistic effect of IFN-gamma and daunomycin-mAb CL 207 conjugate is specific. The in vivo toxicity is influenced by the interval between injection of human tumor cells into nude mice and that of IFN-gamma and daunomycin-mAb CL 207 conjugate. The present results suggest that the 96-kDa melanoma-associated Ag may be a useful model to investigate the combined use of IFN-gamma and mAb for the selective destruction of tumor cells.
Animals↗
Lactate dehydrogenase isoenzymes are present in matrix vesicles.
Matrix vesicles were isolated from epiphyseal growth plates of young rabbits. Lactate dehydrogenase activity was detected in the isolated matrix vesicles only in the presence of detergents, suggesting that NADH, the cofactor for the assay, does not penetrate the membrane of matrix vesicles. In contrast, the activity of alkaline phosphatase, a marker enzyme of the outer surface of matrix vesicles, was detected in the matrix vesicles using p-nitrophenyl phosphate as the substrate both in the presence and absence of detergents. Lactate dehydrogenase activity was detected only in the cytosol of chondrocytes of the epiphyseal growth plates but not in other subcellular fractions, showing that lactate dehydrogenase is not from the plasma membrane and membranes of intracellular organelles of chondrocytes. The isolated matrix vesicles contained all five lactate dehydrogenase isoenzymes but did not possess other cytosolic enzymes. These results show that lactate dehydrogenase is located in the matrix vesicles and suggest the presence of a mechanism for the specific uptake of cytosolic lactate dehydrogenase and the possibility of enzymatic quantification of the matrix vesicles at various calcification sites.
Animals↗
Transcriptional and posttranscriptional regulation of malic enzyme synthesis by insulin and triiodothyronine.
To investigate the transcriptional and posttranscriptional regulation of malate dehydrogenase (EC 1.1.1.40) induction by insulin, the transcriptional rate, mRNA concentration and enzyme induction of malic enzyme were compared in livers of normal and diabetic rats fed a high-carbohydrate diet. When rats were fed the diet for 4 days, the enzyme induction and mRNA concentration in livers of diabetic rats were only about 10% and 39%, respectively, of the values of normal rats, and the transcriptional rate was about 64%. Insulin treatment restored the transcriptional rate and mRNA concentration in 8 h and the enzyme induction in 4 days. Thus, it is suggested that insulin is involved in malic enzyme transcription of the gene and also possibly in the translation of the cytoplasm. On the other hand, by giving triiodothyronine treatment, the transcriptional rate and mRNA concentration were increased about twice and the enzyme induction, about 10-times in the diabetic animals. Triiodothyronine appears to stimulate malic enzyme transcription and possibly post-transcriptional steps even at a very low insulin level.
Animals↗
Detection of a low frequency of activated ras genes in human melanomas using a tumorigenicity assay.
We have used an assay combining DNA-mediated gene transfer and tumorigenicity in Swiss athymic mice to look for activated ras genes in solid human sporadic melanomas. This assay can detect ras oncogenes mutated at codons 12, 13, or 61. We examined a panel of 13 independent surgical specimens of primary tumors and metastases. No H- or K-ras oncogenes were detected; an N-ras oncogene, mutated at codon 61, was identified in one of the 13 samples. No N-ras genes mutated at codon 13 were detected. Thus, the tumorigenicity assay detects a low frequency of ras gene activation in melanomas.
Amino Acid Sequence↗
Cytochrome P-450C-M/F, a new constitutive form of microsomal cytochrome P-450 in male and female rat liver with estrogen 2- and 16 alpha-hydroxylase activity.
A new cytochrome P-450 isozyme, P-450C-M/F, has been purified from untreated rat liver microsomes. The purified preparation was electrophoretically homogeneous and contained 12-15 nmol of P450/mg of protein and had a minimum molecular weight of 48,500. The NH2-terminal amino acid sequence of P-450C-M/F was different from that of other P-450's. Immunoblot analysis of microsomes demonstrated that P-450C-M/F was present in the liver of untreated male as well as female rats. Treatment of rats with phenobarbital, 3-methylcholanthrene, or beta-naphthoflavone did not induce P-450C-M/F. Cytochrome P-450C-M/F exhibited little activities of 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation or hydroxylation of arylhydrocarbon, testosterone, androstenedione, and progesterone. In contrast, it was highly active in N-demethylation of ethylmorphine and benzphetamine and in 2- and 16 alpha-hydroxylation of estrogens, particularly that of estradiol. These studies establish that cytochrome P-450C-M/F is constitutively present in both male and female rats and suggest that it may be involved in the oxidative metabolism of estradiol, particularly in the formation of estriol, the uterotropic metabolite of estradiol.
Amino Acid Sequence↗
Identification of mammalian aminotransferases utilizing glyoxylate or pyruvate as amino acceptor. Peroxisomal and mitochondrial asparagine aminotransferase.
The subcellular distribution of asparagine:oxo-acid aminotransferase (EC 2.6.1.14) in rat liver was examined by centrifugation in a sucrose density gradient. About 30% of the homogenate activity after the removal of the nuclear fraction was recovered in the peroxisomes, about 56% in the mitochondria, and the remainder in the soluble fraction from broken peroxisomes. The mitochondrial asparagine aminotransferase had identical immunological properties with the peroxisomal one. Glucagon injection to rats resulted in the increase of its activity in the mitochondria but not in the peroxisomes. Immunological evidence was obtained that the enzyme was identical with alanine:glyoxylate aminotransferase 1 (EC 2.6.1.44) which had been reported to be identical with serine:pyruvate aminotransferase (EC 2.6.1.51) (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The same results as described above were obtained with mouse liver. All of alanine:glyoxylate aminotransferase 1 in livers of mammals other than rodents, which cross-react with the antibody against rat liver alanine:glyoxylate aminotransferase 1, had no asparagine aminotransferase activity.
Animals↗
A monoclonal antibody to the carbohydrate chain on human hepatocellular carcinoma-associated antigen which suppressed tumor growth in nude mice.
There have been few reports stating that monoclonal antibody alone inhibits human solid tumor growth in vivo. The present study demonstrated that monoclonal antibody S1 (IgG2a), which recognized the antigenic determinant of the carbohydrate moiety, showed antibody-dependent cell (or macrophage)-mediated cytotoxicity (ADCC or ADMC) in conjunction with murine splenocytes of both BALB/c and athymic mice. In vivo experiments demonstrated that the antibody S1 clearly prolonged the survival of athymic mice which had been inoculated with a human liver carcinoma cell line. In addition, the antibody S1 significantly suppressed the human hepatoma line transplanted s.c. into nude mice. 125I-Labeled monoclonal antibody S1 revealed that the antibody accumulated significantly in the tumor mass. Many mononuclear cells were observed surrounding tumor cells when the antibody was given. This model system might be useful for analyzing the ADCC (or ADMC) mechanism in vivo.
Animals↗
Restoration of microcephalic cerebrum with hypomyelination in the growth hormone-deficient mouse (lit): stimulatory effects of GH restricted to the first 20 days of postnatal life.
We administered bovine growth hormone to the Little (lit), a promising model of isolated growth hormone deficiency, during the first and second 20 days after birth. Positive results were obtained only when bovine growth hormone was given during the first 20 days of postnatal life. We observed a distinct increase in cerebral weight, DNA content, and 2',3'-cyclic nucleotide 3'-phosphohydrolase activity. The latter administration of bovine growth hormone was ineffective. These data prove that growth hormone has an independent action on cerebral development, apart from the complementary or synergistic action of thyroid hormones, and that the administration of exogenous growth hormone led to increased myelinogenesis through its stimulatory effects on glial proliferation, as evidenced by the increase in cerebral DNA content.
2',3'-Cyclic-Nucleotide Phosphodiesterases↗
Structure and distribution of lymphatic capillaries and fenestrated blood capillaries in the conduction system of the rabbit heart.
The distribution and structure of lymphatic and blood capillaries in the rabbit heart conduction system were investigated by transmission electron microscopy. The sinuatrial node, atrioventricular node, and atrioventricular bundle possessed a rich network of lymphatic capillaries, which were situated not only at the periphery but also in the interior of the conduction system. The fine structure of these lymphatic capillaries was essentially similar to those within the atria and the ventricles. Although blood capillaries within working myocardium were nonfenestrated, the heart conduction system was often supplied by fenestrated blood capillaries. In the atrioventricular node and bundle especially, fenestrated blood capillaries and lymphatic capillaries were topographically associated, forming an extensive microcirculatory system. The presence of fenestrated capillaries suggests that a fast transcapillary passage of metabolites occurs in these regions, while the lymphatic capillaries may play an important role in the removal of macromolecules and excess intercellular fluid.
Animals↗
Protective effect of alpha-human atrial natriuretic polypeptide (alpha-hANP) on chemical-induced pulmonary edema.
It has been established that alpha-hANP, the newly discovered peptide extracted from human cardiac atria, has potent natriuretic and hypotensive actions. Our present investigation is the first to demonstrate that alpha-hANP is capable of protecting against pulmonary edema caused by various chemicals, using isolated perfused guinea pig lung system. Lungs were perfused via pulmonary artery with Krebs-Ringer bicarbonate buffer at 5.0 ml/min, and wet weight of lungs and perfusion pressure of pulmonary artery (Pa) were monitored. Bolus injection of Triton-X or CHAPS into cannulated pulmonary artery produced edema as indicated by a massive increase in wet weight and a slight increase in Pa. Constant infusion of alpha-hANP through pulmonary artery at 200 ng/ml was effective in causing decrease in wet weight of lung. Perfusion of lung with paraquat or PGF2 alpha, and repeated bolus injection of arachidonic acid or PGE2 caused elevation in both wet weight of lung and Pa. The treatment with alpha-hANP similar to that described above also protected against edema caused by paraquat or arachidonic acid. Bolus administration of epinephrine induced a slight increase in wet weight and Pa, and alpha-hANP was effective in decreasing the elevated lung wet weight and Pa of lungs. Infusion or bolus administration of alpha-hANP into control lungs increased cGMP level in outflow perfusate as well as in lung tissue significantly. In lungs with edema which were induced by Triton-X or paraquat, there was a slight increase in cGMP level in Triton-X treated and no increase in paraquat treated lung tissues. In either cases, was there any increase in cGMP level in perfusate. The specific binding study of [125I]alpha-hANP revealed that the lack of increase in cGMP was not due to a loss of receptor in Triton-X or paraquat treated lungs. Thus our study demonstrated that alpha-hANP had a direct anti-edematic action(s) in lung which was not secondary to the systemic natriuretic and/or hypotensive action(s).
Animals↗
Properties of two toxins isolated from the blue-green alga Microcystis aeruginosa.
Attempts were made to characterize the two toxins (P-1 and P-2) isolated from the blue-green alga Microcystis aeruginosa, by amino acid analysis, mass spectrometry, 1H- and 13C-NMR. P-2, the major toxin, had a molecular weight of 1044, and consisted of one molecule each of beta-methylaspartic acid, D-Glu, D-Ala, L-Arg, L-Tyr, N-methyldehydroalanine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda). P-1, with a molecular weight of 994, appeared to have almost the same composition except that it contained L-Leu instead of L-Tyr in P-2. Mass spectrometric studies, along with a negative ninhydrin reaction, indicated that each toxin was a cyclic peptide. P-2 showed an LD99 of 70 micrograms/kg mice when injected i.p. and [alpha]D24 of -72.42 degrees (c = 0.095 in methanol), and was decomposed at around 218 degrees C.
Amino Acids↗