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Antipain Y, a new antipain analog that inhibits neurotransmitter release from rat dorsal root ganglion neurons

We investigated the age-dependency of serum levels of 9 kinds of proteases. The results showed that the enzymatic activities corresponding to urokinase, plasmin, and thrombin, all involved in blood clotting and fibrinolysis, were inversely correlated with age. This suggests that there is some similarity between the normal process of aging and the pathologic process of Alzheimer's disease. Compared with our previous data on Alzheimer patients, the present results indicate that some derangement in the aging process is involved in the pathogenetic mechanisms of Alzheimer's disease.

Previously, we reported protease changes in cerebral tissues, as well as in serum, of patients with Alzheimer-type dementia. In the present study, we investigated enzymatic activities in their cerebrospinal fluid. In the Alzheimer patients the activities of dipeptidyl peptidase III (DPP-III), DPP-IV, and cathepsin B were significantly increased when compared with those of the control subjects. In vascular dementia patients the activities of Leu-AP and cathepsin B were increased but the activity of DPP-III was decreased. These results, as well as those of a multivariate study on these enzymatic changes, suggest the independency of the cerebral changes in the Alzheimer patients from those in vascular dementia. The results may also be of use for clinical diagnoses of these pathologic states.

Osteoblasts are the cells responsible for bone formation during embryonic development and adult life. Small compounds that could induce osteoblast differentiation might be promising sources of therapies for bone diseases such as osteoporosis. During screening for inducers of osteoblast differentiation of mouse pluripotent mesenchymal C3H10T1/2 cells, we isolated a small compound from the fermentation broth of Penicillium verruculosum CR37010. This compound, named decalpenic acid, bears a decalin moiety with a tetraenoic acid side chain. Treatment of C3H10T1/2 cells with decalpenic acid alone induced the expression of early osteoblast markers, such as alkaline phosphatase activity and osteopontin mRNA, but did not induce the late osteoblast marker osteocalcin mRNA or adipocyte markers under our experimental conditions.

Cyclooctatin has been isolated from Streptomyces melanosporofaciens MI614-43F2 as part of a program designed to find microorganism-produced inhibitors of lysophospholipase. It was purified by chromatography on silica gel, Capcell Pak C18 (HPLC) and Sephadex LH-20 followed by solvent extraction and then isolated as a colorless powder. Cyclooctatin has the molecular formula of C20H34O3. It is competitive with the substrate, and the inhibition constant (Ki) was 4.8 x 10(-6) M.

Benastatins have been isolated as part of a program designed to find microorganism-produced inhibitors of glutathione S-transferase from Streptomyces sp. MI384-DF12. They were purified by chromatography of reversed-phase silica gel, silica gel and Capcell Pak C18 (HPLC) followed by solvent extraction and then isolated as yellow powders. Benastatins A and B have the molecular formulae, C30H28O7 and C30H30O7, respectively. They were competitive with 3,4-dichloronitrobenzene as the substrate, and the inhibition constants (Ki) of benastatins A and B were 5.0 x 10(-6) and 3.7 x 10(-6), respectively.

WHT/Ht mice are known to have GM2 deficiency especially in the liver. In order to know the relationship of this abnormality to the general metabolism in organs or cells, we compared 17 hydrolytic enzyme activities in the liver, brain, spleen, and kidney between the WHT/Ht mice and control BALB/c mice or without administration of GM2 and GM3. Regardless of the GM2 or GM3 treatment, the galactosidase activity was markedly low in all of the tested organs in the WHT/Ht mice when compared with that in the control animals. This consistency stands in contrast with the known organ-dependent heterogeneity of ganglioside expression and thus indicates independency of the GM2 deficiency from the galactosidase deficiency in this model animal.

Poststatin, a new inhibitor of prolyl endopeptidase (PEP) was discovered in the fermentation broth of Streptomyces viridochromogenes MH534-30F3. It was purified by Diaion HP-20, Sephadex LH-20 and YMC-gel (ODS-A) column chromatography and then isolated as a colorless powder. Poststatin has the molecular formula C26H47N5O7. The IC50 value of poststatin against the PEP of partially purified porcine kidney was 0.03 microgram/ml. It has low acute toxicity. No deaths occured after iv injection of 250 mg/kg of this agent to mice.

Our previous report suggested that increased serum angiotensin-converting enzyme (ACE) activity plays a central part in the pathogenesis of essential hypertension in man. In the present study, we investigated whether such findings can be seen in spontaneously hypertensive rats (SHR). The results suggested that serum carboxypeptidase N and kallikrein, rather than ACE, plays a central part in this experimental model. The ACE activity was decreased rather than increased in spontaneously hypertensive rat when compared with that in Wistar-Kyoto rats.

When administered to mice, enzyme inhibitors exert extensive effects that cannot be expected from their direct actions. On the basis of our previous observations [1], we tested how glycosidase inhibitors influence the intracerebral activities of various hydrolytic enzymes including glycosidases. The results showed that, when administered intraperitoneally, mannostatin and nagstatin stimulated, while siastatin and pyridindolol suppressed, intracerebral activities of glycosidases. The effects showed dose dependency. Although the above inhibitors influenced various other enzymatic activities as well, no particular significance could be attached to such effects because of the absence of dose dependency.

Muscular dystrophy is known to occur mainly in hindlimb muscle, rather than in forelimb muscle, of dystrophic mice. In order to explain this phenomenon, we measured the activities of 12 hydrolytic enzymes, including exopeptidases, endopeptidases, and glycosidases, in muscle of forelimb and hindlimb. In spite of similar enzymatic changes in both limbs, multivariate analysis enabled us to extract two important factors that may explain the difference in the metabolism between forelimb muscle and hindlimb muscle of dystrophic mice. Of the two factors, one was closely correlated to mannosidase, DPP-IV, PEP, and cathepsin B, while the other was correlated to DPP-III, kallikrein, and AP-A. Of these enzymes, DPP-III was particular in that its activity was decreased in forelimb, but not in the hindlimb, muscle of dystrophic mice. The lack of a significant decrease in this enzymatic activity may prompt the muscular breakdown in the hindlimb of dystrophic mice.

We compared the activities in serum of 12 kinds of proteases including renin, angiotensin-converting enzyme, aminopeptidases, kallikrein, and other endopeptidases, between control subjects and patients with essential hypertension. In the patients, the activity of angiotensin-converting enzyme was increased by more than two times, although the activities of most of other proteases were significantly decreased, when compared with the age-matched controls. A multivariate study using discriminant function analysis suggested that angiotensin-converting enzyme plays an important role in the protease changes in the patients. The results altogether indicate that the pathogenesis of essential hypertension is closely related to an imbalance between vasoconstrictive and vasodilatative peptides such as angiotensins and kinins.

New inhibitors of dipeptidyl peptidaseIII (EC 3.4.14.4) from human placenta, designated as fluostatins A and B, were discovered in the fermentation broth of a strain isolated in our institute. The strain has been identified as Streptomyces sp. TA-3391 on the basis of taxonomic studies. Fluostatins A and B were purified by Diaion HP-20 chromatography, ethyl acetate extraction, silica gel chromatography and reverse phase preparative HPLC. With the synthetic substrate, arginyl-arginine-2-naphthylamide, the IC50 values of fluostatins A and B were 0.44 and 24.0 micrograms/ml, respectively. Fluostatins A and B were slightly inhibitory against other dipeptidyl peptidases. Fluostatin A showed mixed-type (competitive and noncompeptitive) inhibition with human leucine-enkephalin as a substrate, and the inhibition constant (Ki) was 14.2 microM.

A new inhibitor of dipeptidyl peptidase II (DPP-II, EC 3.4.14.2), designated as epostatin, was discovered in the fermentation broth of a strain isolated in our institute. The strain has been identified as Streptomyces sp. MJ995-OF5 on the basis of taxonomic studies. Epostatin was obtained as a yellow powder after seqential purification by chromatography on Diaion HP-20, n-butanol extraction, Sephadex LH-20 column chromatography and centrifugal partition chromatography (CPC). Epostatin inhibited DPP-II competitively in a dose dependent manner. The compound was slightly inhibitory against other dipeptidyl peptidases.

Benastatins have been isolated as part of a program designed to find microorganism-produced inhibitors of glutathione S-transferase from Streptomyces sp. MI384-DF12. They were purified by chromatography of reversed-phase silica gel, silica gel and Capcell Pak C18 (HPLC) followed by solvent extraction and then isolated as yellow powders. Benastatins A and B have the molecular formulae, C30H28O7 and C30H30O7, respectively. They were competitive with 3, 4-dichloronitrobenzene as the substrate, and the inhibition constants (Ki) of benastatins A and B were 5.0 × 10-6 and 3.7 × 10-6, respectively.

New benzo[a]naphthacenequinone metabolites, designated bequinostatins A and B, have been isolated from the culture broth of the benastatin-producing strain Streptomyces sp. MI384-DF12. The structures of bequinostatins A and B were determined by spectral analyses to be 5,6,8,13-tetrahydro-1,6,7,9,11-pentahydroxy-8,13-dioxo-3- pentylbenzo[a]naphthacene-2-carboxylic acid and 2-decarboxybequinostatin A, respectively. Bequinostatin A showed considerable inhibitory activity against human pi class glutathione S-transferase (GST pi).

Benastatin C, a new member of the benastatins, has been isolated from the culture broth of Streptomyces sp. MI384-DF12. The structure of benastatin C was elucidated as 2-decarboxy-benastatin A by NMR studies. Benastatin D, 2-decarboxybenastatin B, was derived from benastatin B. Benastatins C and D showed inhibitory activities against human pi class glutathione S-transferase (GST pi) and excellent stimulatory activities on the murine lymphocyte blastogenesis in vitro.

Nagstatin, a new inhibitor of N-acetyl-beta-D-glucosaminidase (NAG-ase) was discovered in the fermentation broth of Streptomyces amakusaensis MG846-fF3. It was purified by chromatography on Dowex 50W, Avicel and Sephadex LH-20 followed by the treatment of active carbon and then isolated as colorless powder. Nagstatin has the molecular formula of C12H17N3O6. It is competitive with the substrate, and the inhibition constant (Ki) was 1.7 x 10(-8) M.

Our previous studies suggested that the administration of exogenous gangliosides to the body modulates enzymatic networks in the brain. In the present study, we tested whether that is the case with another organ, spleen. By testing the dose response relationship, we found that there is a optimum dose for the effect of enzymatic modulation of GM1 (monosialoganglioside 1) administration. Although the optimum level varied depending on each of the examined hydrolytic enzymes, it usually fell in the range around 50 micrograms/kg body weight. The findings led us to conclude that the enzyme-modulating actions of gangliosides come not merely from the bizarre actions in vivo of high molecular exogenous substances.

We found benarthin, a new inhibitor of pyroglutamyl peptidase, in the fermentation broth of Streptomyces xanthophaeus MJ244-SF1. It was purified by column chromatography and centrifugal partition chromatography (CPC) and then was isolated as a colorless powder. The binding of benarthin was competitive with substrate and its inhibition constant (Ki) was 1.2 x 10(-6) M.

In order to search for more proximal factors in the pathogenesis of Alzheimer's disease, we studied the activities of various enzyme in the brains of patients, as well as control cases, by postmortem autopsy. In addition to the findings already known, such as the increase in prolyl endopeptidase (post-proline cleaving enzyme, PPCE) activity and the decrease in kallikrein activity, we found, anew, an increase in aminobutyrate aminotransferase (GABA-T) activity in the Alzheimer brain. This may be an important impetus for the reduction of gamma-aminobutyric acid (GABA) in the brain, one of the neurotransmitters. It has to be determined whether the former two abnormalities offer a background for such an abnormality of the neurotransmitter.

Experimental allergic encephalomyelitis (EAE) is one of the experimental models of human demyelinating diseases and recently is regarded also as a useful model for studying cell-mediated autoimmune diseases. Because of the possibility of induction of systemic changes in this model, we investigated enzymatic changes in serum and main organs of the diseases animals, including brain, spinal cord, limb muscle, heart muscle, spleen, liver and kidney. The enzymes measured consisted of 7 aminopeptidases, 5 endopeptidases, 3 glycosidases, creatine kinase, phosphatase and esterase. Significant changes of many enzymatic activities occurred in all the organs tested in 1 to 2 weeks after the administration of EAE antigen, myelin basic protein (MBP). Interesting correlations of the pattern of enzymatic changes were seen among most of the organs tested. Those patterns changed in the course of the 2 weeks and there remained marked changes characteristic for each organ. This model may represent some type of systemic autoimmune diseases.

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.

We tested the effect of bestatin [(2S, 3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine] on the serum levels of various hydrolytic enzymes in a patient with progressive muscular dystrophy. Long-term administration of bestatin significantly suppressed the serum levels of aspartate aminopeptidase, arginine aminopeptidase, carboxypeptidase, alkaline phosphatase, and esterase. The results of multivariate study performed on the enzyme networks are compatible with the notion that this drug has a synchronizing effect on the movements of enzyme networks. Although in this middle-aged patient with an established disease the levels of enzymes of muscular origin were not significantly suppressed by this treatment, the present findings may warrant a therapeutic trial with this agent on young patients with initial symptoms of the disease.

KK mice are used as model animals for non-insulin-dependent diabetes. In order to better understand the biochemical features of these animals, we compared the activities of various hydrolytic enzymes, including aminopeptidases, endopeptidases, carboxypeptidase, esterase, and so forth in the pancreas and liver of these animals with the same enzymes in ICR mice as controls. The enzymatic changes in the model animals were extensive not only in pancreas but also in liver. Multivariate analysis showed that the metabolic changes in liver rather than in pancreas are closely related to the abnormal glucose metabolism in KK mice. This may be somehow related to the insulin resistance in peripheral tissues in this model.

We performed longitudinal studies on patients with two different types of rheumatic diseases, systemic erythematodes (SLE)-like and rheumatoid arthritis (RA)-like, to elucidate the relationship between hydrolytic enzymes and immunological disturbances. The plasma levels of 9 hydrolytic enzymes and subpopulations of T-cells, OKT4 and OKT8 cells, in peripheral blood were checked periodically for 6 months. Analysis of the obtained data by a method of spectral analysis utilizing Akaike's autoregressive approach clearly demonstrated the existence of a dynamic network between the hydrolytic enzymes and the immune system. In testing the behavior of this network, we obtained data suggesting that the response of OKT4 cells when an impulse is given to OKT8, is abnormal in the SLE-like syndrome, and that this abnormality may be related to the malfunction of suppresser T-cells in this pathological condition. Principal component analysis suggested that the behaviors of kallikrein and carboxypeptidase B are quite different between the two types of immunological disturbances. The network relationships between the hydrolytic enzymes and the immune system seem to play important roles in the pathophysiology of rheumatic diseases.

In order to confirm the pathophysiological isgnificance of the dissociation between the plasma levels of aminopeptidases A and B in spontaneously hypertensive rats, we tested the effects of several enzyme inhibitors including inhibitors of aminopeptidase B on the course of hypertension in these rats. In addition to an ihibitor of angiotensin-converting enzyme, inhibitors of aminopeptidase B (arphamenine B and bestatin) significantly suppressed the development of hypertension in these animals. This finding may represent an important clue to the pathogenesis of essential hypertension.

The present study was undertaken to compare the peculiarity of enzymatic changes in dystrophic hamsters and mice. Various enzymatic activities in muscle, bone, heart, spleen, liver and kidney were measured. The enzymes tested include 7 aminopeptidases, 5 endopeptidases, 3 glycosidases, creatine kinase, phosphatase and esterase. In dystrophic mice, the enzymatic changes were chiefly confined to muscle and bone. In dystrophic hamsters, on the other hand, extensive and pronounced changes in enzymatic activities were seen not only in skeletal muscle but also in bone, heart muscle, spleen, liver and kidney. Furthermore, resemblance of pattern of enzymatic changes was seen among several organs including skeletal muscle, heart muscle, bone and spleen in the hamster model. Comparing the enzymatic changes in these two models, dystrophic mouse may be regarded as more specific a model for musculoskeletal diseases. Dystrophic hamster may be related more to multiorgan diseases possibly associated with immunological or other systemic diseases. These models may represent two different disease categories, respectively.

We compared the therapeutic effects of various low-molecular-weight enzyme inhibitors on dystrophic mice. Leupeptin, bestatin, forphenicinol and forphenicine significantly affected the enzymatic activities in the dystrophic muscles. The pattern of enzymatic changes in the muscles of forelimb and hindlimb caused by these inhibitors were similar in spite of the variety of their inhibitory spectra in vitro. However, comparing the pattern of enzymatic changes in spleen, forphenicinol differed from the other inhibitors tested. This may be related to the peculiar effects of this inhibitor on immunologically responsive cells.

Leupeptins, leupeptin Pr and leupeptin Ac, strongly inhibit proteolysis by plasmin, trypsin and papain, but do not inhibit proteolysis by α-chymotrypsin. The inhibition is competitive with substrates. The inhibitory effect on esterolysis by plasmin and trypsin is weaker than on proteolysis. The results with derivatives of leupeptins which contain carboxyl or alcohol instead of aldehyde and of di-n-butyl acetals of leupeptins indicate that the free aldehyde group plays a role in the activities. Leupeptins are absorbed orally and at least about 25 % is excreted in urine. Oral administration of leupeptins exhibited an anti-inflammatory effect on edema. Leupeptins inhibited thrombokinase reaction and coagulation of blood of human and rabbit. Type of inhibition was different from heparin. Coagulation of blood of rats and dogs are not inhibited. The effects of leupeptins on thrombokinase, thrombin, plasmin, trypsin, papain, kallikrein and α-chymotrypsin were compared with those of e-aminocaproic acid, trans-4-aminomethylcyclohexanecarboxylic acid, soybean trypsin inhibitor and trasylol.