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Biomedical subjects

T Aoyagi

Publications and source records attributed to T Aoyagi.

At least 397 records · Page 22Linked to original sources
PubMed1981

Epidermal cyclic GMP is increased in psoriasis lesions.

Cyclic GMP levels in epidermis of normal subjects and of psoriatic patients were measured with a highly sensitive radioimmunoassay method. Technical improvements for the assay are 2-fold: (1) skin samples were frozen in vivo before biopsy and local injection of any anesthetic was avoided to overcome ischemia effect which could lower cyclic GMP artificially; (2) epidermis was microdissected to avoid contamination of dermis and keratin layers. The results show that on a per mg tissue dry weight basis the cyclic GMP levels are about 200 fmol in the involved lesional epidermis and 70 fmol in the uninvolved or normal epidermis. Similarly increases in the cyclic GMP levels in the lesional epidermis are observed when the data are expressed either on a DNA or protein basis. The cyclic GMP level in normal epidermis from nonpsoriatic subjects is the same as that in the uninvolved epidermis of psoriasis patients.

Cyclic GMP↗
PubMed1981

The effect of histamine on epidermal outgrowth: its possible dual role as an inhibitor and stimulator.

We have investigated the effect of histamine on pig epidermal cell outgrowths in vitro. Histamine inhibited the epidermal cell outgrowths (and also mitosis). This inhibition was partially counteracted by a specific H2 antagonist, cimetidine. Inhibition was maximal at a histamine concentration of 10(-4) M and was less at 10(-3) M. These histamine concentrations respectively coincide with the optimal concentrations for accumulating intracellular cyclic AMP (via H2 receptors) and cyclic GMP (via H1 receptors) in the same pig epidermal slice system. 4-Methyl-histamine, a pure H2 agonist, which only increased the intracellular cyclic AMP level but not the cyclic GMP level, caused a maximal outgrowth inhibition at 10(-3) M. Attempts to counteract the histamine effects due to cyclic GMP accumulation by various H1 antagonists (so that 10(-3) M histamine would have caused maximal outgrowth inhibition) were unsuccessful, since the addition of each H1 antagonist alone strongly inhibited the outgrowth. These data strongly suggest a dual role of histamine through the cyclic nucleotide system; i.e., histamine inhibits epidermal cell growth by elevating the intracellular cyclic AMP level via an H2 receptor, while histamine at high concentrations (10(-3) M) partially counteracts the inhibition by increasing cyclic GMP via an H1 receptor.

Animals↗
PubMed1981

Inhibition of epidermal adenyl cyclase by lithium carbonate.

An in vitro floating system was used to investigate the effect of lithium carbonate (Li2CO3) on the activity of adenyl cyclase in normal pig epidermis. Li2CO3 decreased the responsiveness of adenyl cyclase to stimulation by histamine, adenosine monophosphate (AMP) and epinephrine. This abnormality is similar but not identical to the previously described impaired responsiveness of adenyl cyclase to epinephrine and PGE2 in psoriatic plaques compared to normal adjacent skin. Involved and uninvolved skin from a psoriatic on lithium therapy demonstrated decreased responsiveness to in vitro stimulation by epinephrine, histamine and adenosine when compared to skin from psoriatics who were not on lithium therapy. These results are consistent with the observation that lithium therapy worsens psoriatic lesions.

Adenylyl Cyclase Inhibitors↗
PubMed1981

Effect of hydrocortisone on the adenylate cyclase system of the skin--in vitro explant study.

We have previously reported the effect of hydrocortisone (HC) on the adenylate cyclase system of pig epidermis. HC had no effect on the basal level of cyclic AMP but there was an increased adrenaline-induced cyclic AMP accumulation when epidermal slices were incubated for more than 6 h with HC. This incubation did not alter the responsiveness to histamine. We are interested in the effects on epidermal cells of HC, which may act through the adenylate cyclase system. It is well documented that adrenaline, histamine and adenosine stimulate adenylate cyclase and cause an accumulation of cyclic AMP in pig epidermis. An increased cyclic AMP results in the inhibition of mitosis and epidermal outgrowth. Employing the rate of epidermal outgrowth and mitotic index as indicators of cyclic AMP effect, we studied the effect of HC on the adenylate cyclase system. The pretreatment with HC (100 microM) magnified the inhibitory effect of adrenaline on epidermal outgrowth and mitosis, whereas the inhibitory effect of histamine or adenosine was not affected by HC. It is suggested that HC induced an increased responsiveness of adenylate cyclase to adrenaline, resulting in a magnified inhibitory effect of adrenaline on epidermal outgrowth and mitosis.

Adenosine↗
PubMed1981

Various enzyme activities in muscle and other organs of dystrophic mice.

To elucidate the metabolic abnormality of musclar dystrophy, 27 kinds of enzyme activity in various organs of control and dystrophic mice were examined. The organs examined included muscle, bone, heart, testis, uterus, spleen, thymus, submaxillary gland, stomach, pancreas, liver, kidney, brain, and lung. The activities of 14 different aminopeptidases, 5 endopeptidases, 4 glycosidases, phosphatase, esterase, and ribonuclease were measured. Most of the enzyme activities were significantly elevated in muscles and bones of dystrophic mice. These organs were similar in their patterns of enzyme abnormality. Among the 14 kinds of aminopeptidase activity studied, the degree of increased activity was greater for the aminopeptidases (AP):Ala-AP, Leu-AP, Met-AP, Phe-AP, Trp-AP, Gly-Pro-Leu-AP. In addition to aminopeptidases, there were significant increases in activities of chymotrypsinlike enzyme, cathepsin C, cathepsin D, several glycosidases and neutral ribonuclease in the muscles of dystrophic mice. Similarly increased enzyme activity was also observed in organs other than muscle and bone. Furthermore, protein content in most organs was higher in dystrophic mice than in those of control mice. These abnormalities were seen in both males and females. The present results suggest that there are extensive abnormalities in the protein metabolism in dystrophic mice. It seems therefore that the therapeutic approach to muscular dystrophy should be studies not only from the well-known abnormality of intramuscular endopeptidases, but from other aspects as well.

Animals↗
PubMed1981

Intramuscular enzyme abnormalities of dystrophic chickens compared to those of dystrophic mice.

The present study was performed to investigate the enzymatic changes in dystrophic chickens compared to those of dystrophic mice. The activities of 14 kinds of aminopeptidases, 5 kinds of endopeptidase, 4 kinds of glycosidases, phosphatase, esterase, and ribonuclease were measured in muscles of control and dystrophic chickens. When the enzyme activities were expressed as specific activity per unit weight of organs, only some of them were found to be significantly elevated in dystrophic chickens; e.g., alanine aminopeptidase (Ala-AP), Gly-AP and cathepsin D. On the contrary, the activities of alpha-D-glycosidase, alpha-D-galactosidase and alpha-D-mannosidase were significantly decreased. Muscular protein contents of dystrophic chickens also tended to be lower than those of controls. These observations offer a striking contrast with the one obtained in the study on dystrophic mice. However, when expressed as specific activity per mg protein, many enzyme activities were found to be significantly elevated suggesting an extensive abnormality of metabolism in dystrophic chickens. Among 14 kinds of aminopeptidase activities, highly significant elevations were seen especially in AP-A, AP-B, Gly-AP, Ala-AP, Ser-AP, Pro-AP, Leu-AP, Met-AP and Trp-AP. Interestingly enough, a statistical approach suggested a significant correlation between the aminopeptidase changes of dystrophic chickens with those of dystrophic mice. In addition to aminopeptidases, there were highly significant increases in the activities of cathepsin D, alpha-D-glucosidase, beta-D-galactosidase, alpha-D-mannosidase, esterase and RNase. These results indicate that the intramuscular metabolic abnormality of dystrophic chickens are generally different from but partly resembled with those of dystrophic mice.

Aminopeptidases↗
PubMed1981

Epinephrine-induced cyclic AMP accumulation in the psoriatic epidermis.

Although there are many reports concerning different beta-adrenergic responsiveness in involved and uninvolved skin of psoriasis, previous experiments have been done mainly by using keratome-sliced skin, which contains unknown amounts of stratum corneum, dermis, skin appendages, etc. In order to determine the effect of epinephrine on the cyclic AMP level of 'pure' epidermis in psoriasis, a microdissection technique was employed. Basal levels of cyclic AMP in the involved epidermis were slightly higher than in the uninvolved epidermis (involved 1.9 +/- 0.3 pmoles/mg dry weight; uninvolved 1.3 +/- 0.3 pmoles/mg d.w.). This difference was not statistically significant (p greater than 0.1). The response to epinephrine by the involved epidermis (8.4 +/- 1.0 pmoles/mg d.w.) was much lower than that in the uninvolved epidermis (23.3 +/- 4.3 pmoles/mg d.w.). The difference was statistically highly significant (p less than 0.005). Our data show that psoriatic involved epidermis per se had a reduced beta-adrenergic responsiveness, which might be significantly involved in the pathophysiology of psoriasis.

Adolescent↗
PubMed1981

[The experimental study of epidermal growth factor -effects of epidermal growth factor on the cyclic nucleotide system and protein phosphorylation in pig epidermis- (author's transl)].

Incubation of pig skin slices with epidermal growth factor (EGF) caused an increase in intracellular cyclic GMP concentration of pig epidermis. A significant increase was found after 1 hr of incubation and reached a peak by 6 hrs. EGF caused no change in the cyclic AMP level, nor did it affect epinephrine-induced cyclic AMP responses. EGF also stimulated protein phosphorylation in pig epidermis. The stimulation was both time and dose dependent, and was not inhibited by the simultaneous addition of cycloheximide. The addition of cyclic GMP did not mimic the EGF treatment and EGF stimulated protein phosphorylation in 20 minutes. Phosphoproteins stimulated by EGF were identified by SDS-PAGE and autoradiographic analysis. The identified phosphoproteins were fibrous keratin proteins (MW. 65,000, 60,000, 56,000 and 51,000) and soluble protein (MW. 30,000). Calcium ion potentiated the phosphorylation of keratin proteins by EGF. Ionophore A23187 plus calcium ion also enhanced the phosphorylation of keratin proteins.

Animals↗
PubMed1981

The relationships between enzyme inhibitors and function of mammalian cells.

We have been searching for enzyme inhibitors in culture filtrates of microbes and have found leupeptin, antipain, chymostatin, elastatinal, pepstatin, hydroxypepstatin, pepstanone and phosphoramidon as specific inhibitors of serine, thiol, carboxyl and metallo proteases. We found significant activities of aminopeptidases, phosphatase and esterase on surface membranes of various mammalian cells. We discovered bestatin, amastatin, forphenicine, esterastin and ebelactones A and B as specific inhibitors against these enzymes. These inhibitors were proved to bind to cells and modify immune responses. The usefulness of bestatin in cancer treatment has been suggested by clinical studies. It has been shown by several investigators that some endopeptidases such as Ca2+-activated neutral proteases and some other serine proteases may play important roles in muscular dystrophy. In addition to these endopeptidases, we found an abnormal increase in various enzyme activities in dystrophic mice and chickens. Especially, aminopeptidase activities are markedly increased. Moreover, its inhibitor bestatin became interesting on the aspects of its binding to cell surfaces. Bestatin and leupeptin which inhibit Ca2+-dependent protease showed some therapeutic effects against mouse dystrophy. Investigating enzyme activities in synovial fluid of patients with rheumatoid arthritis and osteoarthritis, we found increased activities of aminopeptidases, chymotrypsin-like enzyme, and phosphatase in rheumatoid arthritis but not in osteoarthritis. In chronic hemodialysis patients, RNase activity in serum is markedly elevated. Thus, enzyme inhibitors are increasing their potential usefulness in treatment of various diseases.

Animals↗
PubMed1980

Purification and properties of N-formylmethionine aminopeptidase from rat liver.

A specific enzyme for the liberation of N-terminal N-formylmethionine from N-formylmethionyl peptides was purified 4750-fold from rat liver by successive applications of (NH4)SO4 precipitation, DEAE-cellulose, N-formylbestatin-AH-Sepharose 4B and AH-Sepharose 4B chromatography followed by Sepharose CL-6B gel filtration. The molecular weight was determined by gel filtration on Sepharose CL-6B as 290 000 +/- 5000. This was suggested to be a tetramer consisting of a subunit which was shown by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis to have a 72 000 +/- 2000 molecular weight. The optimum pH of the enzyme was 7.8. Cd2+ and Hg2+ were highly toxic to the enzyme. Michaelis constants of N-formylmethionyl leucine and N-formylmethionine beta-naphthylamide were 0.03 and 0.2 mM, respectively.

Aminopeptidases↗
PubMed1980

Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney.

1. Amastatin, a specific inhibitor of aminopeptidase A (L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7), was linked to an agarose matrix and by this affinity chromatography aminopeptidase A of pig kidneys was purified as a single protein shown by acrylamide gel electrophoresis. 2. Aminopeptidase A which was purified 710-fold, hydrolyzed only acidic amino acid beta-naphthylamide. The optimum pH and the optimum temperature was 7.5 and 45-50 degrees C, respectively. 3. The molecular weight was approx. 300 000 as determined by Sephadex G-200 gel filtration. 4. The activity of aminopeptidase A was not affected by sulfhydryl agents, S-S dissociating agents and serine proteinase inhibitor, but was inhibited strongly by metal chelating agents, and enhanced by alkaline earth metals. 5. Amastatin inhibited aminopeptidase A in a competitive manner with L-glutamic acid beta-naphthylamide, and the Ki value was calculated to be 2.5 x 10(-7) M. The inhibitory effect of amastatin on aminopeptidase A was not reversed by addition of Ca2+.

Aminopeptidases↗
PubMed1980

Stimulation of protein phosphorylation by epidermal growth factor in pig skin (epidermis).

Incorporation of 32P-orthophosphoric acid into pig epidermal proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incorporation was observed in 2 major and 2 minor peaks with approximate molecular weights of 52,000 and 27,000, 17,000, and 12,000 respectively. Treatment with epidermal growth factor (EGF) consistently increased 32P incorporation into all 4 peaks. The stimulation was both time- and dose-dependent, and was not inhibited by the simultaneous addition of cycloheximide. In our previous study (submitted to J Invest Dermatol), we observed that EGF increased the cyclic GMP level in the same pig skin (epidermal) system. The possibility that the increased cyclic GMP leads to the protein phosphorylation observed in the present study was considered but ruled out because (1) the addition of cyclic GMP does not mimic the EGF treatment, (2) simultaneous addition of EGF and cyclic GMP stimulate phosphorylation to the same degree as the addition of EGF alone and (3) EGF stimulates phosphorylation in 20 min whereas the EGF induced increase in the cyclic GMP level takes 60 min.

Animals↗