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Biomedical subjects

M Shin

Publications and source records attributed to M Shin.

At least 127 records · Page 7Linked to original sources
PubMed1985

Photo CIDNP study on the complex formation of lambda cro protein with OR3.

A photo CIDNP spectrum of lambda cro repressor protein showed that one of the three tyrosines and His 35 are quite accessible to the photosensitive dye. For the remaining two tyrosine residues one is slightly accessible, but the other is inaccessible. In comparison with the result of differential nitration at tyrosine side chains followed by the peptide analysis, it can be concluded that Tyr 26 is mostly exposed and Tyr 51 is slightly exposed on the surface of the cro dimer. On the addition of OR3 17mer, His 35 and Tyr 26 are no longer accessible to the dye, which indicates that they are involved in interaction. However, a similar phenomenon was observed by adding CAP binding site 22mer. The interaction mechanism will be discussed.

Bacteriophage lambda↗
PubMed1985

Effects of short-term treatment with calcium on the parathyroid gland of the rat, under particular consideration of the alteration of storage granules.

Short-term effects of CaCl2-treatment on parathyroid cells of the rat, especially on their storage granules, were studied at the ultrastructural level. After an injection of 4% CaCl2, serum calcium levels (SCL) rapidly increased from 9.1 mg/dl (controls) to a maximum of 14.9 mg/dl at 20 min. At 5 min after the injection, the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged, in spite of elevated SCL (12.4 mg/dl). As soon as SCL rose to 13.2 mg/dl at 7.5 min, NSG-I gradually decreased to a minimum at 30 min; in contrast, NSG-II gradually increased to a maximum at 30 min. Vacuolar bodies also increased together with the augmentation of type-II storage granules. The average diameter of the core of the storage granules decreased significantly after the injection. Protein A-gold method for immunocytochemistry showed that the cores of these granules contain parathormone. Acid-phosphatase activity was occasionally found in storage granules of both types, especially in those of type II. It is concluded that type-I storage granules may be transformed into vacuolar bodies via type-II granules as a result of hydrolysis, and that these processes may be accelerated during hypercalcemia.

Acid Phosphatase↗
PubMed1985

Effect of leucine on intestinal absorption of tryptophan in rats.

1. To elucidate the causal relation between leucine and the lowering of hepatic NAD content of rats fed on a leucine-excessive diet (Yamada et al. 1979), the effect of leucine on intestinal absorption of tryptophan was investigated. 2. Co-administration of [3H]tryptophan and leucine, with leucine at ten times the level of tryptophan, delayed absorption of L-[side chain 2,3-3H]tryptophan from the digestive tract and incorporation of [3H]tryptophan into portal blood, the liver and a protein fraction of the liver. After 120 min, more than 95% of tryptophan was absorbed whether [3H]tryptophan was administered with or without leucine. 3. Co-administration of a mixture of ten essential amino acids, in proportions simulating casein, with [3H]tryptophan markedly delayed absorption of tryptophan from the digestive tract. The addition of supplementary leucine to the amino acid mixture, however, caused no further delay. 4. In rats prefed a leucine-excessive diet for 1 week [3H]tryptophan was absorbed at the same rate as in rats fed on a control diet. 5. The results indicate that competition between tryptophan and leucine for intestinal absorption did not cause lowering of hepatic NAD.

Amino Acids, Essential↗
PubMed1985

Effects of pilocarpine treatment and of electrical stimulation of the vagus nerve on the rat parathyroid gland, with special reference to the alteration of storage granules.

Effects of pilocarpine treatment and of electrical vagal stimulation on the rat parathyroid were studied ultrastructurally. The number of type I storage granules with a narrow halo (NSG-I) and that of type II storage granules having a wide halo (NSG-II) were calculated. After pilocarpine treatment, NSG-I gradually decreased and reached a minimum at 30 min; in contrast, NSG-II gradually increased and reached a maximum at 20 min, but thereafter it slightly decreased and instead vacuolar bodies increased. Excluding these alterations, the ultrastructure of parenchymal cells showed no remarkable changes. Electrical vagal stimulation furthermore confirmed these results. Acid phosphatase activity was occasionally found in storage granules of both types in control and experimental rats. It was concluded that storage granules normally may be transformed from type I into type II and finally into vacuolar bodies as a result of hydrolysis, and that these processes may be accelerated by parasympathetic stimulation.

Acid Phosphatase↗
PubMed1985

Ultrastructural changes in development and aging of the interrenal cell of the salamander, Hynobius nebulosus.

The fine structure of interrenal cells in the salamander, Hynobius nebulosus, from prometamorphic larvae to its adult stage, was observed with electron and light microscopes. In all the animals examined the interrenal cell clusters were located at the medial edge of the ventral surface along the total length of each kidney, suggestive of a primitive nature. In larval salamanders, the interrenal cells contained small to moderate numbers of lipid droplets, and their cytoplasm was filled with a tubular network of smooth endoplasmic reticulum (SER) and numerous mitochondria with tubulo-vesicular cristae. In the adult and young adult salamanders, however, most interrenal cells were filled with lipid droplets, so that both tubular SER and mitochondria were decreased in amount in inverse proportion to the increased lipid droplets. These mitochondria frequently contained a crystalloid structure composed of closely packed tubules which were continuous with the cristae. These findings suggest that the interrenal cells in the larvae are more active than those in the adult or young adult salamanders. In addition, the interrenal cells at the end of metamorphosis contained a greater number of enlarged mitochondria with loosely distributed tubular cristae and with a less dense matrix and more numerous membrane-bounded dense bodies, 0.1-0.3 micron in diameter, than those at the prometamorphic stage. These findings suggest that the cells are their most active at this stage. In the prometamorphic larvae, bundles of filaments frequently occurred in the cytoplasm, especially around the nucleus. These filaments gradually decreased in number with the advance of age, and in young adult salamanders they appeared only occasionally.

Adrenal Glands↗
PubMed1985

Specificity of the interaction between lambda cro repressor protein and operator DNA fragments.

Cro repressor protein is known to interact with specific sites in the operator DNA. The cro protein of lambda phage was isolated and the mode of its interaction with three different DNA fragment, lambda-OR3 17mer, phi 80-OR2 19mer and CAP binding site 22-mer, were examined by the use of proton NMR. Some of the imino proton resonances of lambda-OR3 shifted and were broadened remarkably on addition of lambda-cro protein, which indicated the induction of conformational change with complexation. In the spectrum of phi 80-OR2 which has a six base pair sequence common to lambda-OR3 the signals of the common base pairs revealed slight shifts on addition of lambda-cro protein. The imino proton signals of the CAP site DNA, however, did not show any change at all on mixing with lambda-cro. Combining the data of photo CIDNP of lambda-cro, we could postulate the mode of interaction between lambda-cro repressor and operator DNA.

Bacteriophage lambda↗
PubMed1984

Butyl-Toyopearl 650 as a new hydrophobic adsorbent for water-soluble enzyme proteins.

Butyl-Toyopearl 650, a butyl derivative of Toyopearl HW-65, was synthesized for use in hydrophobic chromatography. Water-soluble enzyme proteins were adsorbed on butyl-Toyopearl 650 in the presence of ammonium sulfate and eluted easily in the absence of the salt. Cytochrome c, myoglobin, and chymotrypsinogen A were successfully separated on a butyl-Toyopearl 650 column in order of their individual hydrophobicity by decreasing the concentration of ammonium sulfate contained in the buffer eluant. Based on these results, the use of butyl-Toyopearl 650 is demonstrated for the hydrophobic separation of water-soluble enzyme proteins.

Adsorption↗
PubMed1984

Artificial electron transport mediated by trinitrophenyl groups introduced into protein molecules by means of chemical modification.

Spinach ferredoxin was found to lose its iron-sulfur center during trinitrophenylation. The loss of the iron-sulfur center resulted in disappearance of its activities as electron mediators in the NADP+ photoreduction and NADPH-cytochrome c reduction systems, whereas it did not affect the activity in the cytochrome c photoreduction system. Based on the experimental evidence that ovalbumin, originally inactive in electron transport, became active in the cytochrome c photoreduction system after trinitrophenylation, the retained activity of the modified ferredoxin was concluded to be due not to the original function of the iron-sulfur center, but to artificial electron transport mediated by TNP groups introduced into the protein molecule. Trinitrophenylated cytochrome c was also active as a mediator, but lost its ability as a terminal acceptor in the system.

Animals↗
PubMed1984

Effects of epinephrine treatment on the rat parathyroid gland, with special reference to the frequency of storage granules.

Effects of epinephrine treatment on the rat parathyroid gland were studied morphologically. The mean number of storage granules per cell section (NSG) was rapidly decreased as early as 5 min after an injection of epinephrine and seemed to reach a minimum between 5 and 30 min. During this period, serum calcium levels (SCL) gradually rose and reached a maximum at 30 min. The ultrastructure of chief cells in these epinephrine-injected rats showed no marked difference as compared with that in control rats. In slightly hypocalcemic rats, induced previously by 2% EDTA-treatment, NSG was more rapidly decreased. It was suggested that storage granules may be released promptly by epinephrine treatments in spite of high SCL and that they are more promptly released under hypocalcemia.

Animals↗
PubMed1984

High resolution NMR study of CAP binding site 22mer in H2O solution.

High resolution proton NMR were measured for the deoxyoligonucleotide 22mer duplex corresponding to the CAP (catabolite gene activator protein) binding site of lac promotor. The spectra in the lower field region than the water resonance were taken with the time-shared Redfield pulse method by using a JEOL 500 MHz NMR spectrometer. In the imino proton region 18 peaks were separately observed, but the area intensity at 10 degrees C corresponds to 20 protons. By selective irradiation at each peak position NOEs (nuclear Overhauser effects) were observed between the imino and adenine C2H protons and between imino proton themselves. By tracing sequential NOE train carefully, 17 imino proton signals could be unambiguously assigned to each base pair except five AT base pairs at terminals. With the elevation of temperature the peaks showed gradual broadening and disappeared, which indicates the stepwise base pair opening of the duplex. Referring to the above peak assignments it can be concluded that GC20 and AT4 pairs close to terminals relax first and the base pair opening proceeds toward central GC13 and 14.

Base Sequence↗
PubMed1983

Lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by acid-activated serum.

Erythrocytes from paroxysmal nocturnal hemoglobinuria patients (PNH-E) are much more susceptible to lysis by acid-activated human serum than normal human erythrocytes. Acidification of normal human serum to pH 6.4 in the absence of erythrocytes generates this lytic activity independently of the alternative pathway of complement activation. A shift of pH of a mixture of purified human C5 and C6 to 6.4 at 0 degrees C generates a similar activity C(56)a that lyses PNH-E together with C7-C9 much more efficiently than normal erythrocytes. Since acid-activation of normal human serum occurs in the absence of C3, the acid-activated C56 appears to be the lytic principle in acidified human serum.

Animals↗
PubMed1983

Protein-primed initiation of phage phi 29 DNA replication.

We recently reported the development of an in vitro replication system for bacteriophage phi 29 DNA. We have used this system for the isolation of replication activity associated with gene 3 protein (terminal protein) from phi 29-infected Bacillus subtilis cells. We utilized two assay systems: (i) DNA replication dependent on phi 29 DNA with the 5' end covalently linked to terminal protein (DNA-protein) and (ii) the formation of complex between the terminal protein and dAMP. The DNA-replication and the complex-forming activities were purified together through all steps. The complex of terminal protein and dAMP formed in the purified fraction was shown to serve as an effective primer for successive chain elongation in the presence of dNTPs by a pulse-chase experiment. The protein fraction purified from cells infected with a temperature-sensitive phi 29 mutant in gene 3 was thermolabile compared to the wild-type activity in the assay system for complex formation. This shows that the purified fraction having replication activity includes the gene 3 product of phi 29. Both the DNA replication and the complex formation activities are highly specific for phi 29 DNA-protein as template. The product analysis of elongated DNA revealed that the replication starts at both termini of the phi 29 genome. These results are consistent with the basic elements of the protein-priming model for the initiation of linear DNA synthesis.

Bacillus subtilis↗
PubMed1983

Toyopearl HW-65C: ammonium sulfate as a new column chromatographic adsorbent for enzyme purification.

We found that Toyopearl HW-65C gel matrix adsorbed ferredoxin and ferredoxin-NADP+ reductase in the presence of concentrated ammonium sulfate. Ferredoxin was strongly adsorbed on the gel in 80% saturated ammonium sulfate, and ferredoxin-NADP+ reductase was adsorbed in 40% saturated ammonium sulfate. The phenomenon was utilized for purification of ferredoxin and the reductase on a Toyopearl HW-65C: ammonium sulfate column. The technique greatly simplified the early stage of purification of ferredoxin and the reductase. The improved purification methods further involved column treatments with DEAE-Toyopearl 650M and Matrex Red A. The effectiveness of the columns is reported. Since a number of other proteins such as cytochrome c, myoglobin, chymotrypsinogen A, ovalbumin, and glucose oxidase were also adsorbed well in an appropriately concentrated ammonium sulfate solution, the method may be of general use in enzyme purification.

Adsorption↗
PubMed1983

Trinitrophenylation of spinach ferredoxin and its effect on the functions.

Spinach ferredoxin was trinitrophenylated by reaction with 2,4,6-trinitrobenzenesulfonate. Four amino groups in the ferredoxin could be modified of the total of five amino groups. The trinitrophenylated ferredoxin formed a complex with ferredoxin-NADP+ reductase just as native ferredoxin did. The modified ferredoxin also retained the activity of electron transport in the cytochrome c photoreduction system of chloroplasts, but could neither donate electrons to ferredoxin-NADP+ reductase in the NADP+ photoreduction system, nor accept electrons from the reductase in the NADPH-cytochrome c reduction system in vitro. Furthermore, it lost the inhibitory effect against the NADPH-diaphorase activity of the reductase. These results suggest that the complex formation of ferredoxin with ferredoxin-NADP+ reductase is a phenomenon essentially independent of the function of electron transport between the two proteins.

Chloroplasts↗
PubMed1983

Origin of slow conformer conversion of triostin A and interaction ability with nucleic acid bases.

The n.m.r. pattern of triostin A in weakly polar solvents was explained by the presence of two symmetrical conformers. Its S-benzyl derivative still shows the n.m.r. pattern of two conformers, while des-N-tetramethyltriostin A, which lacks the N-methyl groups, gives one conformer. Therefore the slow interconversion of two conformers arises from the cis-trans isomerization of the N-methyl peptide bonds, but not from the reversal of the chirality around the S-S bond. Only one of the two conformers of triostin A interacts with adenosine and guanosine derivatives. Des-N-tetramethyltriostin A can also interact with the purine nucleosides, but more preferentially with the adenosine derivative.

Adenosine↗
PubMed1983

Effect of nicotinamide administration to rats on the liver microsomal drug metabolizing enzymes.

A single injection or 3 successive injections of nicotinamide (500 mg/kg body wt) increased NADPH-cytochrome c reductase and aniline hydroxylase activities of rat liver microsomes without changing cytochrome P-450 content. Oral administration of nicotinamide for 2 weeks resulted in significant increase in cytochrome P-450, indicating that nicotinamide was an inducer of cytochrome P-450 though its potency was weak. A kinetic study indicated that microsomes isolated from control rats contained only high affinity (low Km) form of aniline hydroxylase while microsomes isolated from nicotinamide-treated rats contained more high affinity form and a newly appeared-low affinity (high Km) form. These results suggest that there exist at least 2 different cytochrome P-450s participating in aniline hydroxylation in rat liver microsomes and nicotinamide induces the high Km form. Ethanol or nicotinamide consumption for 2 weeks resulted in enhancement of high affinity form and appearance of low affinity form but with slightly different Km values.

Aminopyrine N-Demethylase↗
PubMed1983

Effect of leucine and alpha-ketoisocaproic acid on NAD biosynthesis from tryptophan or nicotinic acid in the isolated rat liver cells.

The effects of leucine and alpha-ketoisocaproic acid on the NAD biosynthesis from (carboxyl-14C) nicotinic acid or (benzene ring-U-14C) tryptophan were investigated in the isolated rat liver cells. The quantities of NAD formed from tryptophan and nicotinic acid increased linearly during 60 min-incubation. Leucine, which was essentially not metabolized by the isolated liver cells, had no inhibitory effect on the NAD synthesis. On the other hand, alpha-ketoisocaproic acid, alpha-keto acid analogue of leucine, which was rapidly metabolized to ketone bodies, diminished a quantity and a specific radioactivity of NAD synthesized from both tryptophan and nicotinic acid to a similar extent. Palmitic acid, which was oxidized to ketone bodies by the isolated rat liver cells, also diminished an amount and a specific radioactivity of NAD synthesized from nicotinic acid. Ketone bodies exogenously added to the incubation medium exerted a similar inhibitory effect on NAD synthesis. Results demonstrate that substances which can be oxidized to ketone bodies are inhibitory to NAD biosynthesis from tryptophan and nicotinic acid in the isolated rat liver cells.

Animals↗