Inhibition of acyl-CoA synthetase by triacsins.

Triacsin A, 1-hydroxy-3-(E,E-2',4'-undecadienylidine) triazene and triacsin C, 1-hydroxy-3-(E,E,E-2',4',7'-undecatrienylidine) triazene are potent inhibitors of acyl-CoA synthetase (EC 6.2.1.3). The concentrations of triacsin A required for 50% inhibition of acyl-CoA synthetase from Pseudomonas aeruginosa and from rat liver are 17 and 18 microM, and those of triacsin C are 3.6 and 8.7 microM, respectively. Kinetic analysis indicates that inhibition of triacsin A is non-competitive with respect to the two substrates ATP and coenzyme A, but is competitive with respect to long-chain fatty acids. The apparent Ki value is 8.97 microM when oleic acid is used as substrate. Acid hydrolysis of triacsins results in corresponding polyenic aldehydes with no activity. This suggests that the N-hydroxytriazene moiety is essential for inhibitory activity against acyl-CoA synthetase.

F-244 specifically inhibits 3-hydroxy-3-methylglutaryl coenzyme A synthase.

A beta-lactone isolated from Scopulariopsis sp. shows a potent inhibition of cholesterogenesis. The structure of this beta-lactone, termed F-244, is 3,5,7-trimethyl-12-hydroxy-13-hydroxymethyl-2,4-tetradecadiendioic acid 12,14-lactone. The inhibition site of F-244 in cholesterol synthesis was studied. The growth of Vero cells was inhibited at 6.25-12.5 micrograms/ml of F-244. The inhibition of growth was overcome by the addition of mevalonate to the culture medium, but not by the addition of acetate. In a rat liver enzyme system, the incorporations of [14C]acetate and [14C]acetyl-CoA into digitonin-precipitable sterol were 50% inhibited by 0.58 microgram/ml of F-244. The incorporation of [14C]mevalonate was not affected. Studies on the effects of F-244 on the three enzymes involved in mevalonate biosynthesis demonstrated that the drug specifically inhibits HMG-CoA synthase with IC50 value of 0.065 microgram/ml. The effect of analogs of F-244 on HMG-CoA synthase was also investigated.

Central vs peripheral nerve conduction. Before and after treatment of subacute combined degeneration.

Central and peripheral nerve conduction was studied in two patients with subacute combined degeneration by using the short-latency somatosensory evoked potentials and the peripheral nerve conduction study during treatment with cyanocobalamin. Before the treatment, somatosensory evoked potentials with median nerve stimulation were normal, but those with peroneal nerve stimulation revealed prolonged central conduction indicating dysfunction within the posterior column. Peripheral sensory and motor nerve action potentials were reduced with normal or slightly reduced conduction velocity. After treatment, marked shortening of the central conduction time (by 24% and 31%, respectively) was observed with mild or no recovery of peripheral nerve action potentials. These physiologic findings suggest that the main pathologic changes in the central nervous system may be demyelination in the posterior column in addition to axonal degeneration in the peripheral nerve. The former was responsive to treatment but the latter was poorly responsive to treatment. Sensory symptom in subacute combined degeneration appears to be, at least partially, attributed to the spinal cord lesion.

Voice tremor: dysregulation of voluntary expiratory muscles.

We studied three patients: two with voice and hand tremor, and one with voice tremor. Voice tremor was associated with synchronous rhythmic contraction of cricothyroid and rectus abdominis muscles, but not always vocalis muscle. Voice tremor was manifested only in voluntary phonation or expiration, not in involuntary phonation, voluntary inspiration, or involuntary expiration and inspiration (breathing at rest). Impaired regulation of the CNS programs innervating the voluntary expiratory muscles probably causes voice tremor. Clonazepam and propranolol were helpful in blinded studies.

Effect of oxymetholone on left ventricular dimensions in heart failure secondary to idiopathic dilated cardiomyopathy or to mitral or aortic regurgitation.

This study suggests that the short-term administration of a small dosage of anabolic steroids may have a beneficial effect on the deteriorated myocardium, although long-standing exposure to a larger dosage of anabolic steroids may induce myocardial and systemic complications.

Comparison of protective effects of preinfarction angina pectoris in acute myocardial infarction treated by thrombolysis versus by primary coronary angioplasty with stenting.

The protective effects of preinfarction angina were evaluated in acute myocardial infarction (AMI) treated by primary percutaneous transluminal coronary angioplasty (PTCA) and stenting. We studied 613 patients with AMI. Group 1 (n = 306) was treated by conventional medical therapies and coronary thrombolysis and group 2 (n = 307) was treated by primary PTCA supported by stenting. Each group was subdivided into those with and without preinfarction angina within 24 hours before the onset of AMI. There was no significant difference in clinical characteristics between the subgroups of groups 1 and 2. In group 1, there were differences between patients with preinfarction angina (n = 84) and those without (n = 222) in in-hospital mortality (11% vs 18%), pump failure (Killip classes 3 and 4) (11% vs 21%, p <0.05), left ventricular ejection fraction at discharge (52 +/- 13% vs 48 +/- 14%, p <0.05), and peak creatine kinase (2,106 +/- 1,637 vs 2,764 +/- 2,154 U/L, p <0.02). In group 2, however, there was no significant difference between those with preinfarction angina (n = 82) and those without (n = 225) in mortality (6% vs 6%), pump failure (12% vs 12%), left ventricular ejection fraction (50 +/- 13% vs 50 +/- 13%) and peak creatine kinase (3,285 +/- 2,306 vs 3,291 +/- 2,262 U/L). Multivariate analysis indicated that preinfarction angina was an independent determinant of in-hospital death and pump failure in group 1, but not in group 2. We conclude that the protective effects of preinfarction angina in AMI are not evident in those treated by primary PTCA and stenting, possibly because of the overwhelming protective effects of complete coronary revascularization provided by primary PTCA and stenting.

Instability of coronary lesions in unstable angina assessed by C-reactive protein values following coronary interventions.

The instability of coronary lesions was evaluated in 30 patients with stable angina and 60 patients with unstable angina. The grade of instability of coronary lesions as predicted by the increases in C-reactive protein induced by PTCA and/or stenting was closely correlated with Braunwald's classification of unstable angina.

Possible prevention of postangioplasty restenosis by ascorbic acid.

In this preliminary study to assess the possibility of using ascorbic acid to prevent post-percutaneous transluminal coronary angiography (PTCA) restenosis, the incidence of restenosis was significantly less in 50 patients receiving 500 mg/day of oral ascorbic acid than in 51 control patients. Thus, ascorbic acid, a potent natural antioxidant, appeared to be possibly effective in attenuating post-PTCA restenosis.

Evaluation of intracardiac thrombus with computed tomography.

Left atrial (LA) and left ventricular (LV) thrombus was evaluated by computed tomography in 56 patients. The patients were divided into 2 groups: Group I, 28 patients with mitral valve disease, and Group II, 28 patients with myocardial infarction. Computed tomography and 2-dimensional echocardiography were performed in all the patients studied. Cineangiocardiography was performed in all Group I and in 13 Group II patients. Open heart surgery or autopsy was performed in all Group I and 4 Group II patients. The sensitivity in detecting LA thrombus was 100% with computed tomography, 70% with angiocardiography, and 60% with 2-dimensional echocardiography. The specificity in detecting LA thrombus was 91% with computed tomography, 86% with 2-dimensional echocardiography, and 88% with angiocardiography. Thrombi located at the LA appendage were associated with great difficulties in detection by other methods, but were well delineated with computed tomography. LV thrombus was also visualized by computed tomography with similar or greater accuracy than other diagnostic methods, although the sensitivity and specificity were not ascertained because surgery or autopsy was performed in only a minority of Group II patients. Therefore, as far as the detection of intracardiac thrombus is concerned, computed tomography has the advantage of offering uniform slices of the heart in an attempt to detect thrombi in unknown areas of cardiac chambers, including the LA appendage or LV apex, without being disturbed by the surrounding cardiac and noncardiac structures. Thus, computed tomography has excellent accuracy in the detection of intracardiac thrombus.

Potential advantages in therapeutic index of OK-432 ointment to abdominal skin over systemic applied in a mouse model.

A mixture of OK-432 powder and an ointment base, lanolin, was applied to mouse abdominal skin. After application of the mixture of 5 KE OK-432 and 0.2g lanolin, the leucocyte count and IL-b content in the abdominal cavity increases to up to 3.6 x 10(6) cells and 1.4 ng, respectively. INF-gamma content in the cavity after application of a mixture of 10 KE OK-432 and 0.2 g lanolin also increases, although not after application of the 5 KE OH-432 mixture of dorsum subcutaneous injection of a saline solution of 5 KE OK-432. These results indicate that an application of OK-432 mixed in lanolin to the mouse abdomen is able to exert an influence on the immunomodulatory effect in the abdominal cavity. The concentrations of IL-6 in serum after applying the ointment mixture, and subcutaneous of intraperitoneal injection of the saline solution of 5 KE OK-432, were <20, 370, and 430 pg/ml, respectively. After application of the ointment mixture of 5 KE OK-432, serum IL-6 did not increase. IL-6 levels in the abdominal cavity after 5 KE OK-432 ointment of subcutaneous injection increases to up to 1.4 and 2.1 ng/cavity. From these results, the immunomodulatory systemic response of OK-432 brought about by applying OK-432 ointment to the abdominal skin is lower than that brought about by injecting OK-432 subcutaneously. Therefore, it is thought that application of OK-432 ointment might be most suitable for potentiating local immune response.

Nm23 expression in human gastric cancers - possible correlation of nm23 with lymph-node metastasis.

In order to elucidate the relationship between nm23 and metastasis in human gastric cancers, we analyzed gene and protein expression of nm23-H1 using Northern blot and immunohistochemical techniques. nm23-H1 gene expression was identified in 17 out of 19 gastric cancer tissues. The signals in the tumor tissues presenting regional lymph node metastasis seem to be lower than those in the tumor tissues without regional lymph node metastasis, suggesting a role of nm23-H1 in the regional lymph node metastasis in the gastric cancers. However, the protein expression detected immunohistochemically was not correlated to the gene expression, partly because of difficulty in quantifying the amount of protein. Expression of the nm23-H1 gene as well as the nm23-H1 protein in the tumor tissues was higher than those in the corresponding normal mucosae. This suggests a linkage of nm23-H1 in the process of the gastric cancer progression. We also analyzed the sequence abnormalities of the nm23-H1 gene in the gastric cancer tissues using a direct sequencing technique, no mutations were observed.

Dissociation between indices of pump performance and contractility in patients with coronary artery disease and acute myocardial infarction.

An analysis of ventricular performance comparing pump function and muscle function indices was performed in 13 patients with acute myocardial infarction, ten patients with normal coronary arteries, and 15 patients with coronary artery disease. Pump function was described by plotting left ventricular stroke work index as a function of left ventricular end diastolic pressure. This description provided a clear separation between normal patients, and surviving and nonsurviving patients with acute myocardial infarction. Values of contractile element velocity (VCE5 as an estimate of Vmax) did not separate between normals and surviving or nonsurviving patients with acute myocardial infarction. In 15 patients with acute coronary artery disease there was no correlation between values of VCE5 and the ventricular function curve. Changes in performance following the stress of ventriculography, angiotensin infusion, or isometric hand grip exercise also did not show any correlation between pump function and muscle function indices. It is concluded that pump function indices are a better indicator of ventricular performance in patients with acute myocardial infarction and coronary artery disease.

Cerulenin resistance in a cerulenin-producing fungus. II. Characterization of fatty acid synthetase from Cephalosporium caerulens.

Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium caerulens, is a potent inhibitor of fatty acid synthetase systems of various microorganisms and animal tissues. This antibiotic specifically blocks the activity of beta-ketoacyl thioester synthetase (condensing enzyme) by binding to the functional cysteine-SH in the active center of the condensing enzyme domain (the peripheral SH-group). However, fatty acid synthetase from C. caerulens is much less sensitive to cerulenin than fatty acid synthetases from other sources. The properties of C. caerulens synthetase were investigated and compared to those of Saccharomyces cerevisiae synthetase, which is sensitive to the antibiotic. The molecular weight of the enzymically active form of C. caerulens synthetase was 2.53 X 10(6). The enzyme consisted of two multifunctional proteins, alpha and beta, which are arranged in a complex, alpha 6 beta 6. The synthetase was inactivated by iodoacetamide. At 0 degrees C and pH 7.15, the second-order rate constant of k = 15.6 M-1 X s-1 was obtained for the inactivation by iodoacetamide. This value was about 15 times greater than that for S. cerevisiae synthetase. Treatment of C. caerulens synthetase with iodoacetamide, while impairing the synthetase activity, induced malonyl-CoA decarboxylase activity. When S. cerevisiae synthetase was preincubated with cerulenin, malonyl-CoA decarboxylase activity could not be detected even after treatment of the enzyme with iodoacetamide (Kawaguchi, A., Tomoda, H., Nozoe, S., Omura, S., & Okuda, S. (1982) J. Biochem. 92, 7-12). In the case of C. caerulens synthetase, on the other hand, malonyl-CoA decarboxylase activity was induced by iodoacetamide even after the preincubation of the enzyme with cerulenin.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of action of cerulenin on fatty acid synthetase. Effect of cerulenin on iodoacetamide-induced malonyl-CoA decarboxylase activity.

Cerulenin, an antibiotic with the structure of (2R)(3S)-2,3-epoxy-4-oxo-7,10-dodecadienoylamide, irreversibly inactivates yeast fatty acid synthetase. Of all catalytic activities of the synthetase, only the condensation reaction is inhibited by cerulenin. At 0 degrees C and pH 6.5, the second-order rate constant of k = 88 M-1 . S-1 was obtained for the inactivation by cerulenin. This value was about 90-times greater than the rate constant for the inactivation of the enzyme by iodoacetamide. The enzyme was protected against the action of cerulenin by prior treatment with acetyl-CoA but not malonyl-CoA. Treatment of the enzyme with iodoacetamide, while impairing the synthetase activity, induced malonyl-CoA decarboxylase activity [Kresze, G.-B., Steber, L., Oesterhelt, D., and Lynen, F. (1977) Eur. J. Biochem. 79, 191-199]. Cerulenin had no effect on the malonyl-CoA decarboxylase activity of the iodoacetamide-treated enzyme. N-Ethylmaleimide, in contrast, inhibited the iodoacetamide-induced malonyl-CoA decarboxylase activity. When the enzyme was preincubated with cerulenin, malonyl-CoA decarboxylase activity could not be detected even after treatment of the enzyme with iodoacetamide. These results indicated that the reaction of cerulenin with the peripheral SH-groups of the synthetase is responsible for the inactivation.

Complete inhibition of mouse macrophage-derived foam cell formation by triacsin C.

Primary mouse peritoneal macrophages effectively take up and metabolize phosphatidylcholine/cholesterol liposomes containing a small amount of phosphatidylserine, which results in the massive accumulation in the cytoplasm of oil red O positive lipid droplets consisting of cholesteryl ester (CE) and triacylglycerol (TG) [Nishikawa et al. (1990) J. Biol. Chem. 265, 5226-5231]. A number of inhibitors of CE synthesis have been reported, but their effects on the lipid droplet formation have not been fully examined. Furthermore, the contribution of TG synthesis to lipid droplet formation has been poorly investigated. We have investigated the relationship between CE and TG syntheses and cytosolic lipid droplet formation in macrophages cultured in the presence of inhibitors with different modes of action. When macrophages were cultured with liposomes and [14C]oleic acid in the presence of triacsin C, a potent inhibitor of long chain acyl-CoA synthetase, both [14C]CE and [14C]TG syntheses were inhibited to similar extents with IC50 values of 0.19 and 0.10 microM, respectively. On the other hand, pregnenolone, a well-known inhibitor of cellular cholesterol transport, and CL-283,546, a potent inhibitor of acyl-CoA:cholesterol acyltransferase, inhibited [14C]CE synthesis specifically with IC50 values of 5.0 and 0.038 microM, respectively. Microscopic observation revealed that the inhibitors of cholesterol metabolism produce only a partial decrease in cytosolic lipid droplets even at the highest doses which cause almost complete inhibition of [14C]CE synthesis. However, the triacsin C-dose dependent inhibition of lipid droplet formation was almost complete at 0.59 microM, a concentration that inhibits [14C]CE and [14C]TG syntheses by about 90%. These results show that inhibiton of acyl-CoA synthetase by triacsin C causes a decrease in the cellular levels of acyl-CoA, the common substrate for CE and TG syntheses, leading to an inhibiton of neutral lipid synthesis and eventually to the complete disappearance of cytosolic lipid droplets from macrophages. These findings suggest that TG synthesis, as well as CE synthesis, is responsible for macrophage-derived foam cell formation, and is therefore a potential target for new antiatherosclerotic agents.

Binding site of cerulenin in fatty acid synthetase.

An antibiotic cerulenin, (2R, 3S)-2,3-epoxy-4-oxo-7,10-trans,trans- dodecadienamide, irreversibly inhibits fatty acid synthetase from Saccharomyces cerevisiae. Three moles of cerulenin were bound to 1 mol of the enzyme with concomitant loss of its activity. Pretreatment of the enzyme with iodoacetamide reduced the amount of cerulenin bound to the enzyme. Since iodoacetamide is known to specifically bind to the cysteine residue on the condensing reaction domain, cerulenin is considered to bind to the same domain. Tryptic digestion of the [3H] cerulenin-treated enzyme gave a radioactive peptide; its amino acid composition was Asx 1, Thr 1, Ser 1, Glx 2, Pro 1, Gly 1, Ala 1, Val 1, Ile 1, and Leu 2. This composition included all the amino acids of the condensing reaction site (Thr-Pro-Val-Gly-Ala-Cys) previously reported by Kresze et al. (Eur. J. Biochem., 79, 181 [1977] except for Cys. When the enzyme was treated with [3H]cerulenin and digested successively with trypsin and carboxypeptidase P, a [3H] cerulenin-cysteine adduct was isolated as the sole product. This was identified with the adduct chemically synthesized from non-labeled cerulenin and cysteine, and its structure was elucidated by 1H-, 13C-NMR, and fast atom bombardment mass spectrometry. These results indicate that cerulenin, forming a hydroxylactam ring, reacts at its epoxide carbon (C-2 position) with the SH-group of the cysteine residue in the condensing reaction domain of yeast fatty acid synthetase.

Cerulenin resistance in a cerulenin-producing fungus. III. Studies on active-site peptides of fatty acid synthetase from Cephalosporium caerulens.

Active-site peptides of acetyl transferase, condensing enzyme and acyl carrier protein in the neighborhood of the prosthetic group, 4'-phosphopantetheine, of Cephalosporium caerulens fatty acid synthetase were investigated. The enzyme was reacted with [14C]acetyl-CoA or [14C]iodoacetamide. 14C-Labeled enzyme was digested with pepsin, trypsin or both. 14C-Labeled peptides were isolated by several purification procedures. The amino acid sequence of the active site of condensing enzyme was determined to be Tyr-Gln-Val-Glu-Ser-Cys-Pro-Ile-Leu-Glu-Gly-Lys and that of acetyl transferase was Phe-Ser-Gly-Ala-Thr-Gly-His-Ser-Gln-Gly. The amino acid composition around the 4'-phosphopantetheine-carrying serine was determined to be Asx2, Thr, Ser, Glx3, Gly2, Ala, Ile, Leu3, and Lys. When these active-site peptides were compared with those of Saccharomyces cerevisiae synthetase, a high degree of homology was observed in the active-site peptides of the acetyl transferase and acyl carrier protein domains. However, that of the condensing enzyme domain gave lower homology. These findings may support the assumption that the low reactivity of cerulenin with C. caerulens synthetase is a consequence of the structure of the condensing enzyme domain.

Surgical resection of pulmonary metastases from colorectal adenocarcinoma. Special reference to repeated pulmonary resections.

Pulmonary resection of metastatic lesions from colorectal adenocarcinoma was performed in 35 patients. The cumulative 5-year survival was 38%. The primary site of cancer was the colon in about half of the patients. Patients with a solitary metastasis or tumors smaller than 3 cm in diameter survived longer than did patients with multiple metastases or tumors larger than 3 cm but the differences were not significant. Other factors, including age, sex, histologic grade of tumor, location and stage of primary carcinoma, location of pulmonary metastases, disease-free interval, and type of pulmonary resection, had no apparent influence on survival time. The lung was the major site of recurrence following pulmonary resection. Seven patients underwent two or more pulmonary resections for metastasis from a colorectal carcinoma. At the time of last follow-up, four patients were alive and free of recurrent disease at 5, 34, 39, and 58 months after the second pulmonary resection. These data suggest that some patients will survive for a long time following pulmonary resection of colorectal metastases, and for highly selected patients, repeated pulmonary resection may further extend survival.

Advantages and safety of local treatment with MMC/Beriplast P for cancer tumors.

To target the treatment of small points of cancer, Beriplast P, already used clinically as a physiological tissue adherent drug carrier, was mixed with the anticancer drug, mitomycin C (MMC). In this in vitro study, MMC did not release quickly from the clot of MMC/Beriplast P. The antitumor effect of this mixture was examined for its effect on cancer growth. In one series of experiments, tumor tissues were inoculated with MMC/100 microliters Beriplast P and in another series, MMC/100 microliters Beriplast P was injected into tumors at a weight of 300 mg. In the first series of experiments, tumor tissue treated with 0.3 mg MMC/100 microliters Beriplast P was replaced with plasma cells and lymphocytes, and no viable cancer cells could be found. In the second series, MMC/100 microliters Beriplast P delayed tumor growth, and the survival of Balb/c mice injected with 0.08 mg MMC/100 microliters Beriplast P was significantly longer than that of mice injected with 0.08 mg MMC/100 microliters saline solution (P = 0.026). In addition, the abdominal aorta, vena cava, and intestine around the area of treatment with 1.6 mg MMC/100 microliters Beriplast P were not damaged. These results indicate that the mixture of Beriplast P and MMC is more effective than MMC solution alone in the local treatment of residual cancer.

Volitional movement is not preceded by cortical slow negativity in cerebellar dentate lesion in man.

Slow negative potential preceding voluntary self-paced middle finger extension, as recorded from scalp electrodes by backward averaging technique, was absent in two patients with dyssynergia cerebellaris myoclonica (Ramsay Hunt syndrome); but present in two patients with cerebellar cortical degeneration. As the main pathological lesion in Ramsay Hunt syndrome is in the dentate nucleus and its efferent pathway, the present results are in conformity with the experimental finding that the premotor and motor cortices receive strong inputs from the cerebellar efferent system.