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Biomedical subjects

K Yagi

Publications and source records attributed to K Yagi.

At least 523 records · Page 29Linked to original sources
PubMed1987

[Bronchioloalveolar carcinoma diagnosed by bronchoalveolar lavage].

A case of bronchioloalveolar carcinoma of the lung is reported. A 63-year-old man identified during a chest mass-survey program, came to our hospital for a precise examination. He had no subjective complaints though his plain chest X-ray film showed a diffuse alveolar pattern in the bilateral hilar regions mimicking a butterfly shadow. Further, cytological examinations of both the sputa and specimens from brushings obtained on bronchoscopy failed to demonstrate malignant cells, and only a cytological examination of the fluid obtained by bronchoalveolar lavage (BAL) revealed the presence of a malignancy. Thus, BAL would seem to be a simple and reasonable method for the detection of suspected malignant cells in bronchioloalveolar carcinoma.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1987

[Basic studies on chemotherapy of brain tumors by means of liposomes: affinity of sulfatide-inserted liposomes to human glioma cells].

In order to utilize liposomes for the treatment of brain tumors, we examined the interaction between the cells and the liposomes prepared from phosphatidylcholine, cholesterol, and sulfatide (molar ratio, 7:2:1), which were able to deliver the encapsulated materials into the brain through the blood-brain barrier. With a variety of human cell lines, the incorporation of the liposomes and the release of the liposomal contents into cells were studied by spectrofluorometry and flow cytometry by use of encapsulated 6-carboxy-fluorescein. It was found that the amounts of liposomes incorporated into cells were dependent on the dose of liposomes and type of cells. At the same concentration of liposomes, the highest incorporation was found for glioma cells, which was further confirmed by electron microscopy with ferritin-containing liposomes. These results indicate that the liposomes composed of sulfatide, phosphatidylcholine and cholesterol have a high affinity for human glioma cells and should be useful for the chemotherapy of glioma when antitumor drugs are encapsulated into them.

Antineoplastic Agents↗
PubMed1987

In vitro synthesis of L-gulono-gamma-lactone oxidase by rabbit reticulocyte lysate.

In vitro synthesis of L-gulono-gamma-lactone oxidase [L-gulono-gamma-lactone:oxygen 2-oxidoreductase, EC 1.1.3.8], one of the microsomal flavin enzymes, was performed with poly(A)+ RNA of rat liver using the rabbit reticulocyte lysate system in order to study the biosynthesis of the enzyme. The apparent molecular weight of the synthesized enzyme protein was almost the same as that of the purified L-gulono-gamma-lactone oxidase from rat liver. It was demonstrated that the enzyme protein was not detectable when guinea pig poly(A)+ RNA was used for the translation, indicating that the mRNA for the enzyme is absent in the guinea pig or, if it exists, is not translatable.

Animals↗
PubMed1987

A simple fluorometric method to discriminate adriamycin-resistant subline from adriamycin-sensitive parental P388 murine leukemia cell line.

A fluorometric method to estimate adriamycin accumulation in cells was used to discriminate adriamycin- and vincristine-resistant sublines from sensitive parental P388 murine leukemia cells. It was easily checked that adriamycin accumulation was lower in the adriamycin- and vincristine-resistant cells than in the sensitive ones. With this method we found that tamoxifen as well as diltiazem increased adriamycin accumulation in the resistant cells to a level similar to that in the sensitive ones. The effect of progesterone was much less.

Animals↗
PubMed1987

Purification and properties of a phospholipase C that has high activity toward sphingomyelin from Aspergillus saitoi.

An enzyme hydrolyzing sphingomyelin was purified from extracts of solid cultures of Aspergillus saitoi 7041 by fractionation with isopropanol followed by successive column chromatographies on DEAE-Sepharose CL-6B, butyl-Toyopearl 650 M, and phenyl-Sepharose CL-4B. The preparation of purified enzyme was homogeneous and had an activity increased 81-fold over that of the isopropanol fraction. The yield was about 65%. The molecular weight was estimated to be 54,000 by sodium dodecyl sulfate-gel electrophoresis. The enzyme solution had a violet color and contained iron atoms. The enzyme catalyzed the hydrolysis of sphingomyelin to N-acylsphingosine and phosphorylcholine. The optimum pH for hydrolytic activity was around 3.5. The Km values for sphingomyelin and 2-hexadecanoylamino-4-nitrophenylphosphorylcholine were 0.11 and 0.33 mM, respectively. The enzyme also catalyzed the hydrolysis of other phospholipids; the order of its hydrolytic activity at a substrate concentration of 2.5 mM was phosphatidylcholine greater than or equal to sphingomyelin = phosphatidylethanolamine = lysophosphatidylethanolamine greater than phosphatidyl DL-glycerol = phosphatidyl L-serine greater than phosphatidylinositol. From these results, this enzyme appears to be a new type of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3).

Aspergillus↗
PubMed1986

6-Azido- and 6-aminoflavins as active-site probes of flavin enzymes.

6-Azidoflavins have been bound to the apoproteins of five representative flavoproteins and their properties, before and after light irradiation, compared with those of the same proteins containing the appropriate 6-aminoflavin. In the dark the 6-azidoflavoproteins are quite stable, except for L-lactate oxidase, where spontaneous conversion to the 6-amino-FMN enzyme occurs slowly at pH 7. 6-Azido-FMN Old Yellow Enzyme is converted to the 6-amino-FMN enzyme by aerobic turnover with NADPH, and 6-azido-FAD D-amino acid oxidase is converted to the 6-amino-FAD enzyme by treatment with D-alanine. Light irradiation of 6-azidoriboflavin bound to riboflavin-binding protein does not result in any covalent fixation of the flavin to the protein. Light irradiation of 6-azido-FMN flavodoxin gives only a small amount of covalent linkage. In contrast, 6-azido-FMN Old Yellow Enzyme undergoes a very facile light-induced change, in which approximately 50% of the flavin is attached in a stable covalent linkage to the protein. The resulting flavoprotein form has lost the ability to bind phenols, a distinctive characteristic of the native enzyme; it does, however, bind NADPH, but the latter cannot reduce the covalently bound flavin. 6-Azido-FAD D-amino acid oxidase also undergoes a facile light modification, in which almost quantitative fixation of the flavin to the protein takes place. The resulting flavoprotein cannot bind benzoate, an active-site ligand for the native enzyme, nor is it reduced anaerobically by D-alanine. The covalent linkage is fairly labile and is destroyed on denaturation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Azides↗
PubMed1986

6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins.

6-Thiocyanatoflavins have been found to be susceptible to nucleophilic displacement reactions with sulfite and thiols, yielding respectively the 6-S-SO3--flavin and 6-mercaptoflavin, with rate constants at pH 7.0, 20 degrees C, of 55 M-1 min-1 for sulfite and 1000 M-1 min-1 for dithiothreitol. The 6-SCN-flavin binds tightly to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-lactate oxidase, and apo-Old Yellow Enzyme as the FMN derivative, and to apo-D-amino acid oxidase as the FAD derivative. The riboflavin-binding protein derivative is inaccessible to dithiothreitol attack, and the lactate oxidase and D-amino acid oxidase derivatives show only limited accessibility. However, the flavodoxin and Old Yellow Enzyme derivatives react readily with dithiothreitol, indicating that the flavin 6-position is exposed to solvent in these proteins. The lactate oxidase and D-amino acid oxidase derivatives convert slowly but spontaneously to the 6-mercaptoflavin enzyme forms in the absence of any added thiol, indicating the presence of a thiol residue in the flavin binding site of these proteins. The reaction rates have been investigated of 6-mercaptoflavins with iodoacetamide, N-ethylmaleimide, methyl methanethiosulfonate, H2O2, and m-chloroperbenzoate, in both the free and protein-bound state. The results confirm the conclusions drawn from the studies with 6-SCN-flavins described above and from 6-N3-flavins [Massey, V., Ghisla, S., & Yagi, K. (1986) Biochemistry (preceding paper in this issue)]. The spectral properties of the protein-bound 6-mercaptoflavin vary widely among the five proteins studied and show stabilization of the neutral flavin with flavodoxin and riboflavin-binding protein and of the anionic species by Old Yellow Enzyme, lactate oxidase, and D-amino acid oxidase. In the case of the latter two enzymes, the stabilization appears to be due to interaction of the negatively charged flavin with a positively charged protein residue located near the flavin pyrimidine ring. This positively charged residue appears to be responsible also for the strong stabilization of the two-electron oxidation state of the mercaptoflavin as the 6-S-oxide. With the other flavoproteins studied this oxidation level is stabilized as the 6-sulfenic acid or 6-sulfenate.

Binding Sites↗
PubMed1986

In vitro synthesis of pig kidney general acyl CoA dehydrogenase.

In vitro synthesis of general acyl CoA dehydrogenase [EC 1.3.99.3], one of the mitochondrial flavoenzymes, was carried out to elucidate its biosynthetic mechanism. Poly(A)+ RNA isolated from pig kidney was translated in vitro using wheat germ lysate system and the synthesized enzyme was immunoprecipitated by the antibody against purified pig kidney general acyl CoA dehydrogenase. The apparent molecular weight of the synthesized protein was estimated to be approximately 1,000 daltons larger than that of the mature enzyme, indicating that general acyl CoA dehydrogenase in pig kidney is synthesized as a precursor with a larger molecular weight.

Acyl-CoA Dehydrogenase↗
PubMed1986

Preparation and some properties of 6-substituted flavins as active site probes for flavin enzymes.

6-Azidoflavins, 6-thiocyanatoflavins, and 6-mercaptoflavins at the lumiflavin, riboflavin, FMN, and FAD level were prepared from the corresponding 6-aminoflavins and some of their properties investigated. They are bound tightly by apoflavin enzymes which bind either riboflavin, FMN, or FAD. 6-Azidoflavins undergo facile photolysis. One major product was identified as 6-aminoflavin. A further product, which was formed also during acid decomposition of the azide, results from opening of the flavin benzene ring and is proposed to have a lumazine structure. 6-Thiocyanatoflavins are easily converted by dithiothreitol to 6-mercaptoflavins. The latter are stabilized against dimerization in the presence of reducing thiols. 6-Mercaptoflavins have a pK of 5.9, which corresponds to ionization of the 6-SH function. The neutral form is yellow, while the anion is green, due to a long-wavelength band (lambda max approximately 600 nm) extending beyond 700 nm. These properties suggest the use of these 6-substituted flavins for probing the active site of flavin enzymes. Because their reactive substituents are in close proximity to the flavin N(5)-position, these 6-substituted derivatives should also serve as useful probes of the environment around the flavin N(5), a position known to be involved in all flavin-mediated redox processes.

Binding Sites↗
PubMed1986

Isolation of hydrogen-oxidation gene from Alcaligenes hydrogenophilus and its expression in Pseudomonas oxalaticus.

A gene bank of a megaplasmid encoding the hydrogen-oxidizing enzyme system (Hox) in Alcaligenes hydrogenophilus was constructed using a broad host range cosmid vector pVK102, and established in Escherichia coli. Hybrid cosmids containing hox genes were identified by transferring the bank into Pseudomonas oxalaticus OX1 and screening colonies for the ability of H2-dependent autotrophic growth. About 800 colonies were formed under autotrophic conditions. One of the Hox+ transconjugants was isolated and its hydrogenases activities were measured. Although soluble hydrogenase was not detected, the Hox+ transconjugant had four times the membrane-bound hydrogenase activity of A. hydrogenophilus.

Alcaligenes↗
PubMed1986

Synergistic activation of rat supraoptic neurosecretory neurons by noxious and hypovolemic stimuli.

The effects of saphenous nerve stimulation on discharge activity of supraoptic neurosecretory (NS) cells were studied in anesthetized rats. Of 112 supraoptic neurosecretory cells, 62 exhibited a 'phasic' discharge pattern. The nerve stimulation transiently excited 46 of these 62 'phasic' units, as well as 35 of the 50 remaining 'non-phasic' units. No appreciable blood pressure change was noted using PSTHs with 1-ms resolution. Though the nerve stimulation also evoked a flexor reflex of the ipsilateral hind limb, blockage of the hind limb movement with gallamine did not alter the amplitude of the supraoptic cell excitation. The threshold of the nerve stimulation was higher for the excitation than for the flexor reflex. Effects of hypovolemic and hyperosmotic stimuli on discharge activity of 'phasic' cells during saphenous nerve stimulation were studied to find a possible interaction between these stimuli. Hemorrhage potentiated the transient excitation evoked by the nerve stimulation in all of the 8 'phasic' cells tested, while no such effect was seen after an injection of hypertonic sodium chloride solution in the 7 'phasic' cells tested. These electrophysiological data suggest that hypovolemic and noxious stimuli potentiate VP secretion in a synergistic manner but that hyperosmotic and noxious stimuli do not.

Animals↗
PubMed1986

Epidemiological studies of hemorrhagic fever with renal syndrome (HFRS) related virus infection among urban rats in Hokkaido, Japan.

Seroepidemiological studies of hemorrhagic fever with renal syndrome (HFRS) virus infection were carried out among urban rats (Rattus norvegicus and Rattus rattus) and small field rodents in Hokkaido, Japan. An urban rat colony that was seropositive to SR-11 strain of HFRS virus (laboratory rat origin) was demonstrated in February 1983 at a dumping ground area of Kami-iso Town near Hakodate port. An HFRS-related virus, named KI-262 strain, was isolated from the lung tissue of a seropositive rat using Vero-E6 cell culture. Antigenicity of the isolate was closely related to Hantaan 76-118 and SR-11 strains by the indirect immunofluorescent antibody (IFA) test. No seropositive rat was found among the 861 rats captured in 38 other regions. It is unclear whether or not the infected rats in the positive area were introduced from abroad, though the area is located near Hakodate International Port. Furthermore, higher positive rates of urban rats in the Kami-iso area were observed in the spring and winter than in the summer and fall. Significantly high proportion of positive cases was observed among adult rats (six months or older) than among younger animals. The seasonal and age distribution of positive cases suggested that the virus was not readily transmitted from one infected rat to another. One seropositive case of a small field mouse (Clethrionomys rufocanus bedfordiae) was detected around the Kami-iso area.

Age Factors↗
PubMed1986

Modulation by phospholipids of the activity of monoamine oxidase purified from pig liver.

Monoamine oxidase was purified from pig liver mitochondria to homogeneity. The enzyme sample contained a large amount of phospholipids. Depletion of lipids from the enzyme sample resulted in a decrease in its activity, while activity was restored by the binding of the lipid-depleted enzyme to phosphatidylcholine, phosphatidylethanolamine, or mitochondrial lipids. Upon binding the lipid-depleted enzyme to the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), the enzymatic activity toward serotonin was elevated over that of the purified enzyme, but not toward benzylamine, suggesting a change in substrate specificity. Upon lipid depletion, inhibition by deprenyl became weaker, while that by clorgyline became stronger. This alteration was reversed by the binding to lipids. By the binding of the lipid-depleted enzyme to some lipids such as the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), inhibition by clorgyline became even weaker than for the original enzyme sample.

Animals↗
PubMed1986

Studies in a model system on the effect of hydrogen bonding at hetero atoms of oxidized flavin on its electron acceptability.

The effect of hydrogen bonding at hetero atoms of oxidized flavin on its electron acceptability was studied by the ab initio molecular orbital method. The calculations were carried out for all possible lumiflavin-H2O complexes and for some lumiflavin-formamide complexes. Calculated data showed that the magnitudes of hydrogen bonding energy at the hetero atoms are in the order of N(3)H greater than N(5) greater than O(12) greater than N(1) greater than O(14). It was found that the atomic orbital coefficient of the lowest unoccupied molecular orbital is the largest at N(5) and that hydrogen bonding at N(1), N(5), O(12), and O(14) increases the electron acceptability of the oxidized flavin at N(5), while hydrogen bonding at N(3)H decreases it.

Chemical Phenomena↗
PubMed1986

Distribution of 8 alpha-hydroxyriboflavin in rat organs.

From a survey of flavin compounds using a high performance liquid chromatograph equipped with a fluorescence detector, 8 alpha-hydroxyriboflavin, its derivative, riboflavin, FMN, and FAD were found in rat organs. The derivative of 8 alpha-hydroxyriboflavin was purified from the kidney sequentially by DEAE column chromatography, paper chromatography, and high performance liquid chromatography, and was determined to be 8 alpha-hydroxyriboflavin 5'-phosphate. The amount of 8 alpha-hydroxyriboflavin in the brain, heart, liver, and kidney of the rat was calculated to be 0.010, 0.026, 0.172, and 0.103 nmol per g of wet tissue, respectively. The amount of 8 alpha-hydroxyriboflavin 5'-phosphate in the brain, heart, and liver was less than 0.002 nmol, while that in the kidney was 0.330 nmol, per g of wet tissue. On the other hand, we could not detect 7 alpha-hydroxyriboflavin in the organ extracts, even though it was excreted into human urine along with 8 alpha-hydroxyriboflavin. This is the first reported occurrence of 8 alpha-hydroxyriboflavin and 8 alpha-hydroxyriboflavin 5'-phosphate in higher animal organs.

Animals↗
PubMed1986

Purification and characterization of a phosphoprotein phosphatase that dephosphorylates myosin and the isolated light chain from chicken gizzard smooth muscle.

A phosphatase that dephosphorylates myosin and the isolated light chain has been purified to near homogeneity from chicken gizzard smooth muscle. The molecular weight of the enzyme was estimated to be 100,000 and 35,000 under native and denatured conditions, respectively. It requires Mg2+ or Mn2+. The activity was measured quantitatively with a coupled enzyme system with the aid of myosin light chain kinase. The Vm and Km were determined to be 23.4 mumol/mg/min and 4.2 microM, respectively, with the isolated light chain as substrate under the optimal conditions (5 mM Mg2+ at pH 8.45). The specific activity with myosin as substrate at a concentration of 0.9 microM was found to be 1.25 mumol/mg/min, which was about one-fifth of the activity for the isolated light chain under the same conditions. The phosphatase seems to be specific to gizzard myosin. It may play an important role in the regulation of the myosin-actin interaction in smooth muscle.

Animals↗
PubMed1986

Monoclonal antibodies distinguish between the head portions of two myosin isozymes from chicken breast muscle.

Monoclonal antibodies against chicken breast myosin and its subfragment-1(S-1) were produced. One antibody, 2G41, reacted with S-1 containing a light chain 3 (LC3), but not with another S-1 containing a light chain 1 (LC1) or a mixture of the light chains. A structural difference can be assumed to exist between the head portions of the two myosin isozymes. Antigenicity of S-1 toward 2G41 could not be detected after tryptic digestion into three fragments of 50K, 27K, and 20K daltons. Another monoclonal antibody, M68, was obtained from mice immunized with myosin. M68 preferably recognized the heavy chain from S-1 containing LC3 rather than that from that containing LC1 or S-1. M68 reacted with the 27K fragment among the three.

Animals↗