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Biomedical subjects

K Adachi

Publications and source records attributed to K Adachi.

At least 685 records · Page 38Linked to original sources
PubMed1977

Purification and properties of glutamate synthase from Thiobacillus thioparus.

Glutamate synthase was purified about 250-fold from Thiobacillus thioparus and was characterized. The molecular weight was estimated as 280,000 g/mol. The enzyme showed absorption maxima at 280, 380, and 450 nm and was inhibited by Atebrin, suggesting that T. thioparus glutamate synthase is a flavoprotein. The enzyme activity was also inhibited by iron chelators and thiolbinding agents. The enzyme was specific for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and alpha-ketoglutarate, but L-glutamine was partially replaced by ammonia as the amino donor. The Km values of glutamate synthase for NADPH, alpha-ketoglutarate, and glutamine were 3.0 muM, 50 muM, and 1.1 mM, respectively. The enzyme had a pH optimum between 7.3 and 7.8. Glutamate synthase from T. thioparus was relatively insensitive to feedback inhibition by single amino acids but was sensitive to the combined effects of several amino acids. Enzymes involved in glutamate synthesis in T. thioparus were studied. Glutamine synthetase and glutamate synthase, as well as two glutamate dehydrogenases (NADH and NADPH dependent), were present in this organism. This levels of glutamate synthase and glutamate dehydrogenase were similar in T. thioparus grown on 0.7 or 7.0 mM ammonium sulfate. The sum of the activities of both glutamate dehydrogenases was only 1/25 of that of glutamate synthase under the assay conditions. It was concluded that the glutamine pathway is important for ammonia assimilation in this autotrophic bacterium.

Adenosine Triphosphate↗
PubMed1977

A study on the reaction mechanism of adenosine 5'-phosphosulfate reductase from Thiobacillus thioparus, an iron-sulfur flavoprotein.

The reaction mechanism of adenosine 5'-phosphosulfate (APS) reductase (EC 1.8.99.2) from Thiobacillus thioparus was studied using difference spectrum and stopped-flow techniques. The enzyme-bound FAD was rapidly reduced by sulfite with a first order rate constant of 97.1 s-1. The addition of AMP induced further spectral changes in the reduced enzyme which were consistent with the oxidation of FADH2 to the red (anionic) semiquinone FADH-) and the concomitant reduction of nonheme iron to the ferrous state. Superoxide dismutase (EC 1.15.1.1) or anaerobiosis inhibited the reduction of cytochrome c by the enzyme only to the extent of 25-35%, indicating the existence of a direct reduction of cytochrome c by the enzyme without involving O2-. the activity of enzyme with cytochrome c was inhibited by increasing the potassium phosphate concentration, the inhibition being more pronounced with horse heart cytochrome c than with Candida krusei cytochrome c.

Adenosine Monophosphate↗
PubMed1977

Denatured hemoglobin in sickle erythrocytes.

To study the nature of numerous inclusion bodies seen in red cells from patients with sickle cell disease (Hb SS), we have prepared red cell ghosts free of oxyhemoglobin and analyzed them by spectrophotometric and heme extraction methods. The absorption spectrum in the visible region of the ghost suspensions was typical of hemichromes. The spectrum was similar to that of denatured hemoglobin repared by treatment of oxyhemoglobin S with mechanical shaking or heat. Similar treatment of cells containing only normal hemoglobin (Hb AA) showed a very small amount of denatured hemoglobin, approximately one-fifth of the amount in Hb SS cells. The amount of denatured hemoglobin determined after solution of membrane with 2.5% sodium dodecyl sulfate was 0.158+/-0.070% (1 SD) of the total cellular heme in Hb SS patients. In controls, the amount was 0.030+/-0.016%. Persons with Hb AA and reticulocytosis did not have an elevated amount of membrane-associated heme. In patients with hereditary spherocytosis and autoimmune hemolytic anemia, denatured stromal hemoglobin was normal or slightly elevated before and after splenectomy. The increased amount of denatured hemoglobin in Hb SS red cells may be related to the instability of sickle oxyhemoglobin.

Adolescent↗
PubMed1977

Phosphodiesterase inhibitors: their comparative effectiveness in vitro in various organs.

The inhibitor constants of several inhibitors for cyclic AMP- and cyclic GMP-phosphodiesterase from various organs are compared. The inhibitors were classical theophyline, papaverine, and some of newly developed inhibitors: an imidazolidinone compound, RO20-1724, and two phthalazinol compounds, EG 467 and EG 626. Among the inhibitors tested, papaverine and EG 626 were found to be the most potent. Both compounds were extremely inhibitory to platelet and arterial phosphodiesterases. EG 626 was much more inhibitory to cyclic AMP phosphodiesterase than to cyclic GMP phosphodiesterase in platelet- and brain-extract and RO20-1724 was inhibitory to cyclic AMP- but not cyclic GMP-phosphodiesterase in brain-extract. When the skin adenyl cyclase was activated by AMP, the addition of theophylline blocked this activation, but EG 626 or EG 467 further potentiated the activation. These in vitro studies may serve as basic screening tests for the effectiveness of the specific phosphodiesterase inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

Ovum aging and pH imbalance as a cause of chromosomal anomalies in the hamster.

Using female hamsters mated in estrus, we have produced triploid embryos as manifestations of pregnancy wastage by combining delays of 3 to 4 hours in ferilization with exposure of the animals to hypoxia. Among the triploid embryos only XXX and XXY karotypes occurred, indicating derivation from XX-containing ova. These findings have relevance to human reproduction.

Animals↗
PubMed1976

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase.

Adenosine, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phosphodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell surface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides had no effect on the accumulation of cyclic AMP. Among other adenine nucleotides we tested, adenosine 5'-monophosphoramidate, but not adenosine 5'-monosulfate significantly increased cyclic AMP especially with the addition of papaverine. Neither 2'- nor 3'-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

Interaction of serum albumin with normal and sickle hemoglobins.

The stability of oxyhemoglobin S during mechanical shaking was enhanced by the addition of human serum albumin. The stabilizing effect was maximum when the concentration of serum albumin approached that of oxyhemoglobin, suggesting a molecular level interaction between them. The effects of serum albumin on oxyhemoglobin A were essentially similar to those on oxyhemoglobin S. Deoxy- and methemoglobins were also stabilized by serum albumin. The addition of human serum albumin to a solution containing sickle cell oxyhemoglobin slowly formed a compound which had an absorbance peak at 620 nm. After purification by Sephadex G-200 column chromatography, this compound was identified as methemalbumin. Comparison of the rates of formation of methemalbumin from hemoglobin with various ligand states and human serum albumin showed that the rate of formation from hemichrome was much faster than from met-, oxy- and deoxyhemoglobin. About 60% of the heme was transferred from hemichrome to albumin when the mixture was kept standing at room temperature for 5 min, in contrast to only 5% from methemoglobin. This result suggests that hemichrome, rather than methemoglobin, is the intermediate in the formation of methemalbumin from oxyhemoglobin and human serum albumin. This hypothesis is supported by the finding that the rate of formation of methemalbumin was faster at alkaline pH values than at acid pH values. Serum albumin from various animal sources showed different stabilizing effects. The formation of methemalbumin from these animal albumins was far less than that from human albumin.

Apoproteins↗
PubMed1976

Multiple forms of cyclic nucleotide phosphodiesterase in pig epidermis.

Pig epidermal cyclic nucleotide phosphodiesterases (EC 3.1.4.16) have been partially purified by DEAE-cellulose column chromatography. At least three different forms of the epidermal phosphodiesterases were identified. They were cyclic GMP-specific, cyclic GMP- and cyclic AMP-hydrolyzing and apparently a cyclic AMP-specific enzyme: the first two forms were soluble and the last was the particulate enzyme. The cyclic GMP-specific soluble fraction had a relatively low Km, the cyclic GMP- and cyclic AMP-hydrolyzing fraction had a high Km for the respective substrates and the third particulate enzyme had both high and low Km values for cyclic AMP. The cyclic GMP-hydrolyzing enzyme was localized almost entirely in the soluble fraction, whereas cyclic AMP-hydrolyzing enzyme was distributed to both soluble and particulate fractions. Thus, our studies show that the multiple forms of pig epidermal enzyme differ distinctly in their substrate affinity, specificity and subcellular distribution.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1976

High resolution shadowing for electron microscopy by sputter deposition.

The sputtering process by an ion beam well collimated and highly accelerated provides a valuable means of high resolution shadowing, replication of a fine object by a combination of pre-shadowing and deposition as well as a preparation of supporting films. High resolution shadowing and films with grains smaller than 1 nm can be obtained by argon ion-sputtering targets of tungsten and tungsten/tantalum alloy. The resolution of carbon replicas pre-shadowed with tungsten/tantalum is determined from the radius of curvature of replicated magnesium oxide crystal corners.

Alloys↗
PubMed1976

Carcinogen-induced cutaneous neoplasms in nonhuman primates.

Dimethylbenzanthracene (DMBA) combined with UV light, dodecylbenzene (DDB), or both was oncogenic when applied to the skin of nonhuman primates. Ten years after the cutaneous application of DMBA and a cocarcinogen, 2 groups of rhesus monkeys (Macaca mulatta) displayed dermal melanosis, papillomas, basal cell tumors, and mesodermal sarcomas (accompanied in one case by hepatic metastasis). One group had been painted for 15 months; the other, for 6 1/2 years. Eight years after the initiation of a 4-year regimen of biweekly applications of DMBA plus a cocarcinogen, papillomas, basal cell tumors, and basosquamous cell epidermal tumors with satellite lipomas were observed in galagos (Galago crossicaudatus). The same carcinogen applied to the skin of pottos (Perodicticus potto) caused death in 6 of the 7 animals within 9 weeks. Neoplasms occurred in 17 of the 19 primates that lived longer than 9 weeks after the cutaneous application of DMBA combined with either UV light or DDB.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1976

Effects of short chain alcohols and hydrocarbon compounds on the adenylate cyclase of the skin.

The cyclic AMP content of epidermal slices is increased by incubation with ethanol, the effect of which is dose-dependent from I to 5% concentration in the incubation media. n-Propanol and acetone are also effective at a concentration equimolar to 5% ethanol. Experimental results suggest that this effect of ethanol is due to activation of adenylate cyclase, rather than to the inactivation of cyclic AMP-phosphodiesterase.

1-Propanol↗
PubMed1975

Mechanical precipitation of hemoglobin köln.

Hb Köln (beta 98 Val leads to Met) was found to precipitate rapidly during mechanical shaking. The rate of precipitation of Hb Köln is 5-6 times faster than that of Hb S. The kinetics of precipitation of the patient's hemolysate, which is a mixture of Hb Köln and Hb A, showed a biphasic curve indicating that Hb Köln precipitates independently from Hb A. The instability of Hb Köln may be attributed to the conformational change in the vicinity of heme. The mechanical shaking may be used as a new method for detection and quantitation of hemoglobin Köln and other unstable hemoglobins.

Chemical Precipitation↗
PubMed1975

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.

Adenosylmethionine Decarboxylase↗
PubMed1975

Cyclic AMP and psoriasis.

Evidence that an adenyl cyclase system is present in all mammalian epidermis is reviewed. This adenyl cyclase is stimulated by at least two separate types of chemicals: catecholamines, which act at a beta-adrenergic receptor site, and prostaglandins of the E series, which act at a separate site. In the psoriatic lesion, the response to these stimulators, especially to the catecholamines, is reduced. Despite this lack of response to external agents which elevate cyclic AMP, the concentration of cyclic AMP within the epidermis of the psoriatic lesion is no lower than in noninvolved skin. How cyclic nucleotides act to control cell proliferation and cell differentiation remains unclear.

Adenosine Monophosphate↗
PubMed1975

Micro-determination of cyclic AMP levels in human epidermis, dermis and haif follicles.

In order to study the biological and possible pathological roles of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in the skin, it is mandatory to measure cyclic AMP in 50-100 mug of microdissected epidermis, dermis or appendages. In the present study, we offer a method of extracting cyclic AMP from less than 100 mug of tissue, removing contaminating nucleotides and scaling down Gilman's method to fit the analysis of small amounts of tissue. Cyclic AMP levels in the dermis, epidermis, and hair follicles (bulbs) were approximately 1, 2 and 3-5 pmols/mug dry weight tissue respectively. This procedure is applicable to the measurement of cyclic AMP levels in limited foci of healthy or diseased skin.

Adenine Nucleotides↗
PubMed1975

Cyclic AMP in skin: effects of acute ischaemia.

The cyclic adenosine 3',5'-monophosphate (cyclic AMP) content of pig skin was measured several seconds to minutes after removal of the skin from the body. It increased very rapidly, reached a maximum by 2 min after removal (4 times higher than the initial level), then decreased very slowly. Propranolol injected into the animal before or added after the removal of the skin did not suppress this phenomenon. The practical significance of this finding (increase of cyclic AMP level in skin after ischaemia) is obvious-in order to measure cyclic AMP level in vivo, the sample must be frozen immediately to avoid an 'artificial' increase in cyclic AMP.

Animals↗