Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.

Time-dependent, irreversible inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by the antibiotic citrinin.

The inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase by citrinin, an antibiotic, has been studied. The inhibition was the mixed type with respect to 3-hydroxy-3-methylglutaryl-CoA and non-competitive with respect to NADPH. When the enzyme was preincubated with citrinin prior to enzyme assay, however, it caused a time-dependent, irreversible inhibition, possibly by binding to a site distinct from the active center on the enzyme protein.

Effects of ML-236B on cholesterol metabolism in mice and rats: lack of hypocholesterolemic activity in normal animals.

ML-263B (compactin), a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, is very effective in lowering serum cholesterol levels in animal species such as hens, dogs, monkeys and man. In the present studies, the effect of this drug on cholesterol metabolism in several strains of mice and rats was studied. The results indicate that, when administered for a longer period, the drug showed no hypocholesterolemic activity in these species under either normo- or hypercholesterolemic conditions, except for rats treated with the detergent Triton WR-1339. The administration of ML-236B caused a significant decrease in fecal excretion of bile acids and in the hepatic levels of cholesterol 7 alpha-hydroxylase, and produced a marked increase in hepatic levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity, resulting in no inhibition of hepatic sterol synthesis, even in the presence of the drug in the active form(s). It is concluded that the lack of hypocholesterolemic activity of ML-236B in mice and rats could, at least partly, be explained by these unexpected results.

Trehazolin, a slow, tight-binding inhibitor of silkworm trehalase.

Mechanisms of enzyme inhibition by trehazolin, a new inhibitor of trehalase (Ando et al. (1991) J. Antibiot. 44, 1165), were investigated using purified soluble silkworm trehalase and other glycosidases. Trehazolin inhibited trehalase with an IC50 value of 27 nM, whereas some other exo-alpha-glucosidases were inhibited only weakly, with IC50 values ranging from 7 to 370 microM. Other glycosidases tested were not inhibited by 500 microM trehazolin. The inhibition of trehalase by trehazolin was competitive with respect to trehalose. A notable feature of the inhibition was a slow progression of the association and dissociation of the enzyme-inhibitor complex. Preincubation of the enzyme and the inhibitor at 37 degrees C potentiated the inhibition by 10-times in a time-dependent manner up to 6 h. Dialysis of the inactivated enzyme recovered the enzymatic activity very slowly, and the rate constant for the dissociation at 37 degrees C was (7.3).10(-2) h-1. Trehalamine, a deglucosylated form of trehazolin, inhibited both silkworm trehalase and exo-alpha-glucosidases only weakly. The inhibition of trehalase by trehalamine was reversible. Rat isomaltase inhibition by trehazolin and sucrase inhibition by trehalamine were also reversible. Taken together, trehazolin is a specific slow, tight-binding inhibitor of trehalase, and the glucose moiety of the inhibitor is essential to the tight binding.

CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species.

CS-514 is a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in cholesterol synthesis. For the microsomal enzyme from rat liver, the mode of inhibition is competitive with respect to hydroxymethylglutaryl-CoA, and the Ki value is 2.3 X 10(-9) M. CS-514 also strongly inhibited the sterol synthesis from [14C]acetate in cell-free enzyme systems from rat liver and in freshly isolated rat hepatocytes; the concentrations required for 50% inhibition were 0.8 ng/ml and 2.2 ng/ml, respectively. On the other hand, the inhibition by CS-514 was much less in the cells from nonhepatic tissues such as freshly isolated rat spleen cells, and cultured mouse L cells and human skin fibroblasts. In addition, the cellular uptake of 14C-labeled CS-514 by isolated rat spleen cells or mouse L cells was less than one-tenth of that by isolated hepatocytes. These differences between hepatic and nonhepatic cells were further confirmed by the fact that CS-514 orally administered to rats inhibited sterol synthesis selectively in liver and intestine, the major sites of cholesterogenesis. CS-514 markedly reduced serum cholesterol levels in dogs, monkeys and rabbits, including Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia in man, but did not reduce those in rats and mice. In the former case, preferential lowering of atherogenic lipoproteins was observed in all of the animals tested. The biliary neutral sterols significantly decreased, whereas the amount of biliary bile acids was not affected by administration of the drug to dogs.

Endothelin converting enzyme-1 gene expression in the kidney of spontaneously hypertensive rats.

Abnormal renal handling of water and sodium is implicated in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). Alteration of renal endothelin-1 synthesis is also reported in SHR. Endothelin-1, a potent vasoconstrictor and regulator of sodium reabsorption in the nephron, has a pathophysiological potential in the development of hypertension. Because synthesis of bioactive endothelin-1 requires endothelin converting enzyme-1 (ECE-1), we investigated whether renal ECE-1 gene expression is altered in the kidney of SHR. Kidneys from both 4- and 12-week-old SHR and age-matched Wistar-Kyoto rats (WKY) were studied. ECE-1 mRNA in microdissected nephron segments was assessed by reverse transcription-competitive polymerase chain reaction, and ECE-1 protein level by Western blot. In 4-week-old SHR, ECE-1 mRNA was significantly increased in the proximal straight tubule, medullary thick ascending limb, cortical thick ascending limb, and inner medullary collecting duct. ECE-1 protein level was increased in both the outer and inner medulla. In 12-week-old SHR, ECE-1 gene expression was significantly increased in the proximal straight tubule, medullary thick ascending limb, and also in the glomeruli. Glomerular preproendothelin-1 mRNA expression was not different between the two strains at both 4 and 12 weeks. We conclude that high ECE-1 gene expression in the nephron, via increase of endothelin-1 synthesis, may promote sodium retention that contributes to the development and/or maintenance of hypertension in SHR.

Identification and characterization of two isoforms of an endothelin-converting enzyme-1.

We report the cloning and sequencing of 5'-terminal region of a beta form of rat ECE-1 cDNA which is different only in its N-terminal amino-acid sequence to the cDNA we have cloned previously (alpha form [K. Shimada et al. (1994) J. Biol. Chem. 269, 18275-18278]). No significant difference was found in the specific activity and substrate specificity between the two isoforms. The expression level of ECE-1 alpha mRNA was higher than that of ECE-1 beta in various rat cells and tissues, suggesting that the physiologically important isoform is ECE-1 alpha. The present findings verified the presence of two forms of ECE-1 over many species, which are created probably through alternative splicing.

Inhibition of ADAMTS4 (aggrecanase-1) by tissue inhibitors of metalloproteinases (TIMP-1, 2, 3 and 4).

ADAMTS4 (aggrecanase-1) is considered to play a key role in the degradation of aggrecan in arthritides. The inhibitory activity of tissue inhibitors of metalloproteinases (TIMPs) to ADAMTS4 was examined in an assay using aggrecan substrate. Among the four TIMPs, TIMP-3 inhibited the activity most efficiently with an IC(50) value of 7.9 nM, which was at least 44-fold lower than that of TIMP-1 (350 nM) and TIMP-2 (420 nM) and >250-fold less than that of TIMP-4 (2 microM for 35% inhibition). These results suggest that TIMP-3 is a potent inhibitor against the aggrecanase activity of ADAMTS4 in vivo.

Growth-promoting activity of tissue inhibitor of metalloproteinases-1 (TIMP-1) for a wide range of cells. A possible new growth factor in serum.

Human tissue inhibitor of metalloproteinases-1 (TIMP-1), but not TIMP-2, has potent growth-promoting activity for a wide range of human and bovine cells. TIMP-1 seems to be a new cell-growth factor in serum and to stimulate the cells independently of its inhibitory activity.

Adenophostin-medicated quantal Ca2+ release in the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1.

Kinetics of Ca2+ release by adenophostin, a novel agonist of inositol 1,4,5-trisphosphate (IP3) receptor, in the purified and reconstituted IP3 receptor type 1 (IP3R1) was investigated using the fluorescent Ca2+ indicator fluo-3. Submaximal concentrations of adenophostin caused quantal Ca2+ release from the purified IP3R1 as IP3 did. Adenophostin-induced Ca2+ release by the purified IP3R1 exhibited a high positive cooperativity (nH = 3.9 +/- 0.2, EC50 = 11 nM), whereas the IP3-induced Ca2+ release exhibited a moderate one (nH = 1.8 +/- 0.1, EC50 = 100 nM). Inhibition of [3H]IP3 binding to the purified IP3R1 by adenophostin exhibited a positive cooperativity (nH = 1.9, Ki = 10 nM), whereas IP3 did not (nH = 1.1, Ki = 41 nM).

Sequencing, expression and biochemical characterization of the Porphyromonas gingivalis pepO gene encoding a protein homologous to human endothelin-converting enzyme.

We have determined the nucleotide sequence of the clone pAL2 obtained from Porphyromonas gingivalis 381 in the previous study [Ansai et al. (1995) Microbiology 141, 2047-20521. The DNA sequence analysis of this fragment revealed one complete ORF and one incomplete ORF. The ORF encoded a protein (PgPepO) of 690 amino acids with a calculated molecular weight of 78796. The deduced amino acid sequence exhibited a significant homology with human endothelin-converting enzyme (ECE)-1. Recombinant PgPepO was purified to homogeneity and characterized. The purified enzyme was strongly inhibited by phosphoramidon, and converted big endothelin-1 to endothelin-1. Furthermore, the purified PgPepO strongly cross-reacted with a monoclonal antibody against rat ECE-1. These results indicate that PgPepO has striking similarity to mammalian ECE in structure and function.

Hypolipidemic effects in dogs of ML-236B, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

ML-236B, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, significantly reduced both serum cholesterol and phospholipid levels in dogs, when used at a dosage higher than 10 mg/kg per day. Triglyceride levels were not consistently changed, but beta- and pre-beta-lipoproteins were preferentially reduced. Serum cholesterol levels were reduced by 44--45% at the higher dosage of 100--400 mg/kg per day (for 5 weeks) but ML-236B caused no significant changes in the cholesterol content of the liver and aorta and in the activities of serum GOT, GPT, CPK and lecithin : cholesterol acyltransferase. Fecal excretion of neutral sterols was unaffected but that of bile acids was markedly elevated by the drug. Under these conditions, hepatic cholesterol 7alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, showed no detectable changes.

Hypolipidemic effects of CS-500 (ML-236B) in WHHL-rabbit, a heritable animal model for hyperlipidemia.

Oral administration of CS-500, a competitive inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, lowered the serum cholesterol level of both normal and WHHL-rabbits (the first example of heritable hyperlipidemic animals) at doses higher than 5 mg/kg/day. Phospholipids decreased concomitantly, whereas triglycerides did not in either normal or WHHL-rabbits.

Identification of aggrecanase activity in medium of cartilage culture.

Erosion of cartilage is a major feature of joint diseases, i.e., osteoarthritis and rheumatoid arthritis, which leads with time to a loss of joint function. Proteolytic cleavage of the aggrecan core protein is a key event in the progress of these joint diseases. Aggrecan degradation has been believed to be mediated by a putative proteinase, aggrecanase. We identified aggrecanase activity in conditioned medium from explant culture of bovine nasal cartilage stimulated by retinoic acid. The activity was partially purified more than 10,000-fold. The enzyme cleaves at the aggrecanase site (Glu(373)-Ala(374)) but not at the MMP site (Asn(341)-Phe(342)) in the interglobular domain of the aggrecan. It also cleaves at Glu(1971)-Leu(1972), which is located in the gap region in the chondroitin sulfate attachment region prior to the aggrecanase site. The enzyme is a typical Ca(2+)-dependent metalloproteinase with a unique salt-dependency and is inhibited by several hydroxamate-based inhibitors for matrix metalloproteinases. Heparin and chondroitin sulfate inhibited the enzyme in a dose-dependent manner, suggesting that the large carbohydorate in aggrecan is important for substrate recognition by aggrecanase.

Matlystatins, new inhibitors of typeIV collagenases from Actinomadura atramentaria. II. Biological activities.

Matlystatin A, the main component of matlystatins, inhibits 92 kDa and 72 kDa typeIV collagenases with IC50 values of 0.3 microM and 0.56 microM, respectively, while 7- to 11-fold greater concentrations are required to inhibit thermolysin and aminopeptidase M. The inhibition is reversible and competitive with respect to gelatin. It inhibits the invasion of basement membrane Matrigel by human fibrosarcoma HT1080 dose-dependently with an IC50 value of 21.6 microM.

Matlystatins, new inhibitors of type IV collagenases from Actinomadura atramentaria. IV. Synthesis and structure-activity relationships of matlystatin B and its stereoisomers.

The first total synthesis of matlystatin B (1a), a low molecular weight inhibitor of type IV collagenases, was accomplished, and its absolute configuration was unambiguously determined. Furthermore, ten stereoisomers of 1a were synthesized, and the inhibition of the 92 kDa type IV collagenase and of other metalloproteinases by each stereoisomer was investigated.

Leualacin, a novel calcium blocker from Hapsidospora irregularis. I. Taxonomy, fermentation, isolation, physico-chemical and biological properties.

A new calcium blocker, designated leualacin, has been isolated from Hapsidospora irregularis. The compound inhibits the binding of 3H-nitrendipine, a well known synthetic calcium blocker, to cardiac Ca channel in a competitive manner, although its structure is completely different from dihydropyridines.

A-72363 A-1, A-2, and C, novel heparanase inhibitors from Streptomyces nobilis SANK 60192, II. Biological activities.

Inhibitory activities of A-72363 A-1, A-2 and C, the diastereomers of a neuraminidase inhibitor siastatin B, against various glycosidases were tested in comparison to siastatin B. Despite these compounds differing only in their configuration, each compound showed strikingly different specificities towards the various glycosidases tested. A-72363 C inhibited bovine liver beta-glucuronidase and tumor cell heparanase with IC50 values of 1.6 microM and 12 microM, respectively.

Folipastatin, a new depsidone compound from Aspergillus unguis as an inhibitor of phospholipase A2. Taxonomy, fermentation, isolation, structure determination and biological properties.

A new inhibitor of phospholipase A2 was isolated from the fermentation broth of Aspergillus unguis. The structure, with a depsidone carbon skeleton, was assigned by spectroscopic experiments.

Matlystatins, new inhibitors of typeIV collagenases from Actinomadura atramentaria. I. Taxonomy, fermentation, isolation, and physico-chemical properties of matlystatin-group compounds.

During the course of a screening for inhibitors of typeIV collagenases, new metabolites, designated matlystatins, have been isolated from an actinomycete strain, which was identified as a strain of Actinomadura atramentaria. Matlystatins were composed of five congeners, which were separated and purified by n-butanol extraction and chromatography.