Cupric ion-dependent inhibition of lysosomal acid cholesteryl ester hydrolase in the presence of hydroxylamine

In the presence of hydroxylamine or ascorbic acid, the inhibitory effects of Cu²⁺ on lysosomal acid cholesteryl ester hydrolase (acid CEH) partially purified from rat liver were studied. Hydroxylamine stimulated the inhibition of acid CEH activity by Cu²⁺ but not that by Zn²⁺, Fe²⁺, Co²⁺, Mn²⁺, Ca²⁺, Mg²⁺ and Hg²⁺. This Cu²⁺-dependent inhibition of acid cholesterol ester hydrolase (CEH) activity was completely prevented by ethylenediamine tetraacetic acid (EDTA), EGTA and o-phenanthroline, a chelator with a stability constant for Cu²⁺, and also by sulfhydryl agents and cytoplasmic reducing agents such as cysteine, glutathione and mercaptoethanol. In addition, the stimulative effects of hydroxylamine on Cu²⁺-dependent inhibition were maintained even after preincubation of Cu²⁺ with hydroxylamine. On the other hand, ascorbic acid was found to replace the stimulation by hydroxylamine of the Cu²⁺-dependent inhibition of acid CEH activity but the effects of ascorbic acid progressively became smaller with prolongation of the preincubation time. Moreover, addition of chemical radical scavengers to the reaction mixture did not prevent the Cu²⁺-dependent inhibition of acid CEH activity in the presence of ascorbic acid. These results suggest that Cu²⁺ causes inhibition of lysosomal acid CEH activity through the formation of Cu¹⁺ in a reductive medium.     

Electron-transfer function of NAD+-immobilized alginic acid

Polymerized NAD+ (Alg-NAD+) was prepared and its electrochemical properties were investigated. NAD+ has been covalently immobilized at the carboxyl group of alginic acid using water soluble carbodiimide (EDC) and then Alg-NAD+s of various NAD+ density were obtainable depending on NAD+ concentration in the reaction mixture. Absorbance of 260 nm of Alg-NAD+s showed that 3.4 to 17.6% of carboxyl groups of alginic acid were coupled with NAD+. The coenzyme activity of immobilized NAD+ has reached 80 to 90% on each Alg-NAD+. A cathodic peak in the cyclic voltammogram of Alg-NAD+ appeared at -1.2 V (vs. SCE) corresponding to the reduction wave of free NAD+. The anodic wave of NAD dimer was not observed in the presence of 2.0 mM methyl viologen and 5 units of diaphorase and NAD+ immobilized on the composite electrode could be reduced to the normal NADH. The ratio of apparent diffusion coefficient (Dapp.) of Alg-NAD+ and free NAD+ was evaluated from the variation of ipc with the square root of sweep rate (v 1/2). Despite the high molecular weight of Alg-NAD+, Dapp. Alg-NAD+/Dapp. free NAD+ are larger than that expected. These results indicate that electron transfer occurred effectively between each NAD+ molecule immobilized onto the polymer chain. It is also confirmed by a conjugated redox enzyme reaction with Alg-NAD+.     

Effect of cupric ions on serum and liver cholesterol metabolism

Cupric ions were administered subcutaneously to male Sprague-Dawley rats at a single dose of 200 μmol/kg. At 24 hr after administration, a remarkable increase of total and free cholesterol was seen in the rat serum. Also, when lecithin-cholesterol acyltransferase (LCAT) (E.C. 2.3.1.43) activity was expressed as the percentage of the total serum that free cholesterol esterified, the acyltransferase activity in rats treated with cupric ions showed a slight decrease while the triglyceride content in rat serum and liver decreased by 54% and 61%, respectively. However, the content of hepatic cholesterol in rats treated with cupric ions did not show such a marked change. On the other hand, acid cholesteryl ester hydrolase activity (Acid CEH) (E.C. 3.1.1.14) in liver lysosomes of rats treated with cupric ions showed a marked decrease with increasing cupric ion concentration both in vivo and in vitro. Furthermore, cupric ions caused a marked release of the lysosomal enzymes cathepsin D and β-glucuronidase into the cytosolic fraction. The changes in acid cholesteryl ester hydrolase activity induced by cupric ions appear to be a direct effect of cupric ions on the enzyme. These results suggest that excessive cupric ion concentrations could cause various disorders in lipid metabolism.     

Radiation-induced grafting of N,N-dimethylacrylamide onto polytetrafluoroethylene

Grafting of N,N-dimethylacrylamide (DMAA) onto polytetrafluoroethylene (PTFE) has been carried out by using gamma rays from a ⁶⁰Co source. Degree of grafting was affected by grafting conditions. Especially, solvent for the grafting was found to be a very significant parameter to obtain a higher grafting yield. When ethylacetate or acetone was used as the solvent, grafting yield greater than 5% was obtainable while the graftcopolymerization scarcely occurred in the presence of other kinds of solvent such as ethanol or water. Apparent activation energy of the grafting in ethylacetate was calculated to be 7.90 Kcal/mol, and initial grafting rate of it was proportional to 0.55 power of dose rate.     

Moving mask deep X-ray lithography system with multi stage for 3-D microfabrication

 Based on a moving mask deep X-ray lithography concept, a new deep X-ray exposure system with multi stage has been built up, which can fabricate 3 dimensional microstructures with controllable free shaped wall such as inclined, curved and vertical wall. The system has 6 stages, an X-stage and a Y-stage for substrate scanning, a substrate tilt stage and a substrate rotation stage for controlling an incident X-ray angle to a substrate, an X–Y stage for mask movement and X–Y stage for substrate and mask alignment. The system performance has been confirmed by fabricating microstructures such as gratings, micro grid and micro prism. Received: 10 August 2001/Accepted: 24 September 2001     

Effect of additives on gelation and tissue adhesion of gelatin-poly(L-glutamic acid) mixture

Gelation and tissue adhesion of mixtures of gelatin and poly (L-glutamic acid) (PLGA) aqueous solution were investigated in the presence of additives following the addition of a water-soluble carbodiimide (WSC) that induced chemical cross linking between gelatin and PLGA. To prevent spontaneous gelation of the mixed solution through physical cross linking between gelatin molecules at room temperature, additives were added to the mixed solution. Among the additives studied, starch and urea were effective in preventing the spontaneous physical gelation. The mixed gelatin and PLGA solution set to a cross-linked hydrogel within scores of second by WSC addition, irrespective of the presence of urea, whereas the viscosity of the solution with added starch was too high to measure the gelation time. The cross-linked gelatin-PLGA hydrogels with and without urea showed higher bonding strength to soft tissues than fibrin glue. This was in marked contrast to gelatin-PLGA hydrogels with soluble starch. Irrespective of the presence of urea, the gelatin-PLGA hydrogels gradually biodegraded in the back subcutis of mice over 3 months and no severe inflammatory response to the hydrogels was observed. These findings indicate that urea is promising as an additive to prevent spontaneous physical gelation of the mixed gelatin and PLGA aqueous solution without changing the characteristics of WSC-induced cross linking and tissue adhesion of the formed hydrogel.     

Changes in insulin-stimulated glucose transport and GLUT-4 protein in rat skeletal muscle after training

After running training, which increased GLUT-4 protein content in rat skeletal muscle by <40% compared with control rats, the training effect on insulin-stimulated maximal glucose transport (insulin responsiveness) in skeletal muscle was short lived (24 h). A recent study reported that GLUT-4 protein content in rat epitrochlearis muscle increased dramatically ( approximately 2-fold) after swimming training (J.-M. Ren, C. F. Semenkovich, E. A. Gulve, J. Gao, and J. O. Holloszy. J. Biol. Chem. 269, 14396-14401, 1994). Because GLUT-4 protein content is known to be closely related to skeletal muscle insulin responsiveness, we thought it possible that the training effect on insulin responsiveness may remain for >24 h after swimming training if GLUT-4 protein content decreases gradually from the relatively high level and still remains higher than control level for >24 h after swimming training. Therefore, we examined this possibility. Male Sprague-Dawley rats swam 2 h a day for 5 days with a weight equal to 2% of body mass. Approximately 18, 42, and 90 h after cessation of training, GLUT-4 protein concentration and 2-[1,2-3H]deoxy-D-glucose transport in the presence of a maximally stimulating concentration of insulin (2 mU/ml) were examined by using incubated epitrochlearis muscle preparation. Swimming training increased GLUT-4 protein concentration and insulin responsiveness by 87 and 85%, respectively, relative to age-matched controls when examined 18 h after training. Forty-two hours after training, GLUT-4 protein concentration and insulin responsiveness were still higher by 52 and 51%, respectively, in muscle from trained rats compared with control. GLUT-4 protein concentration and insulin responsiveness in trained muscle returned to sedentary control level within 90 h after training. We conclude that 1) the change in insulin responsiveness during detraining is directly related to muscle GLUT-4 protein content, and 2) consequently, the greater the increase in GLUT-4 protein content that is induced by training, the longer an effect on insulin responsiveness persists after the training.     

Direct intracerebral invasion from skull metastasis of large cell lung cancer

A 56-year-old Japanese woman was referred to us for the treatment of lung cancer. On admission, the patient showed multiple bone metastases, including the skull, without brain metastasis. During chemoradiotherapy for the primary tumor and bone metastasis involving the thoracic spine, she suffered a fatal intracerebral hemorrhage. Since the patient had no risk factors for intracerebral hemorrhage, the skull bone metastasis was thought to be responsible for this event. At autopsy, penetration of the metastatic tumor from the skull bone into the dura, with direct invasion of the brain tissue, was confirmed histologically. A hematoma also was identified at the same site adjacent to the skull bone metastasis. To our knowledge, direct tumor invasion to the brain from a skull metastasis of non-small cell lung cancer has not been previously reported.