Electron transport inhibitor in Cyperus javanicus.

The natural quinone, hydroxydietrichequinone (3-heptadec-8-enyl-2-hydroxy-5-methoxy-[1,4]benzoquinone) is a secondary metabolite of Cyperus javanicus. We found that this quinone inhibited both mitochondrial respiration and photosynthesis in their electron transportation systems. The quinone was found to have a mode of action against the ubiquinone reductase site from the results of different electron donor experiments on intact mitochondria from rat liver. The electron transport system, photosystem-II (PS-II), in chroloplast from spinach leaves was inhibited by the quinone in a similar way to that of the triazin sires herbicide, atrazin, with its mode of action against PS II. This natural quinone has a long aliphatic chain (C17) including an unsaturated bond at its midpoint. We recognized 8-9 unsaturated bonds in the aliphatic chain from an MS analysis of the methylthio-addact, and spectral data presumed a configuration of cis. form.

Utility of Etest in choosing appropriate agents to treat fungal keratitis.

PURPOSE: To evaluate the utility of Etest in choosing the appropriate treatment of fungal keratitis. METHODS: Etest was used to determine the drug sensitivities of isolates from the eyes of three patients with fungal keratitis, and the clinical outcomes of treatment with selected drugs were evaluated. RESULTS: In all cases, drug sensitivity demonstrated by Etest accorded with clinical efficacy of the drugs. CONCLUSION: The results in these cases suggest that evaluating drug sensitivities with Etest is an efficient means of selecting optimal pharmacotherapy for fungal keratitis.

Preparation of a monoclonal antibody to N(epsilon)-(Hexanonyl)lysine: application to the evaluation of protective effects of flavonoid supplementation against exercise-induced oxidative stress in rat skeletal muscle.

The monoclonal antibody to N(epsilon)-(hexanonyl)lysine (HEL), a novel adduct formed by the reaction of linoleic acid hydroperoxide and lysine, has been prepared and characterized. The obtained antibody specifically recognized the HEL moiety. Using the monoclonal antibody, we evaluated the protective effects of feeding eriocitrin, which is one of flavonoids in lemon fruit, on oxidative modification induced by exercise in rats. The supplementation of eriocitrin significantly suppressed the increase in HEL in the skeletal muscle by exercise. The result suggests that the determination of HEL may be a good method for evaluation of the protective effect of beneficial food factors against oxidative stress.

Suppression of glycogen consumption during acute exercise by dietary branched-chain amino acids in rats.

The effects of a diet supplemented with branched-chain amino acids (BCAA; 4.8% or 6.2%) on BCAA catabolism and glycogen metabolism in rats were examined. Rats were fed a BCAA diet or control diet for 4 wk and part of the rats were subjected to exercise training during the experimental period. Feeding the BCAA diet increased serum BCAA concentrations and activity of the hepatic branched-chain alpha-keto acid dehydrogenase complex, the rate-limiting enzyme in the catabolism of BCAA, suggesting that dietary BCAA promotes BCAA catabolism. Although the serum glucose concentration and glycogen contents in the liver and gastrocnemius muscle of rested rats were not significantly affected by feeding of the BCAA diet, those in rats exhausted by acute exercise were 2-4-fold higher in rats fed the BCAA diet than in rats fed the control diet. The activity of pyruvate dehydrogenase complex in the liver and gastrocnemius muscle after acute exercise showed reverse trends; the complex activities (especially in liver) tended to be less in the BCAA diet group than in the control diet group. These results suggest that dietary BCAA spares glycogen stores in liver and skeletal muscle during exercise and that the decrease in pyruvate dehydrogenase complex activity in these tissues by dietary BCAA is involved in the mechanisms.

BTEB2, a Krüppel-like transcription factor, regulates expression of the SMemb/Nonmuscle myosin heavy chain B (SMemb/NMHC-B) gene.

We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15-bp sequence, designated SE1, located at -105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this cis-regulatory element. We screened a lambdagt11 cDNA library prepared from C2/2 cells, a rabbit-derived SMC line, by using a radiolabeled concatenated oligonucleotide containing SE1 as a probe. Sequence analysis revealed that one of the cDNA clones corresponds to the rabbit homologue of basic transcriptional element binding protein-2 (BTEB2), which has previously been identified as one of the Krüppel-like transcription factor. Gel mobility shift assays and antibody supershift analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factor:SE1 complexes. Furthermore, a glutathione S-transferase-BTEB2 fusion protein binds to the SE1 in a sequence-specific manner. In support of the functionality of BTEB2 binding, basal promoter activity and BTEB2-induced transcriptional activation were markedly attenuated by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was most highly expressed in intestine, urinary bladder, and uterus. BTEB2 mRNA levels were downregulated in rabbit aorta during normal development. Moreover, immunohistochemical analysis indicated a marked induction of BTEB2 protein in the neointimal SMC after balloon injury in rat aorta. These results suggest that BTEB2 mediates the transcriptional regulation of the SMemb/NMHC-B gene and possibly plays a role in regulating gene expression during phenotypic modulation of vascular SMC.

Dietary maltitol decreases the incidence of 1,2-dimethylhydrazine-induced cecum and proximal colon tumors in rats.

Maltitol is fermented in the colon due to only partial hydrolysis in the small intestine. In the present study, we examined effects of dietary maltitol on dimethylhydrazine-induced intestinal tumor in rats. In experiment 1, rats were fed a fiber-free diet or diets supplemented with 1 or 5 g/100 g maltitol for 27 wk. Each group of rats was injected with dimethylhydrazine or vehicle alone for the first 14 wk of the experimental period. Maltitol supplementation at 1 g/100 g of the diet significantly reduced tumor incidence in the cecum and the 5% supplement reduced tumor incidence in both the cecum and proximal colon in dimethylhydrazine-treated rats. In experiment 2, we investigated the effect of the 1 g/100 g maltitol diet on the short chain fatty acid concentrations in cecal contents of placebo and dimethylhydrazine-treated rats. Intake of the 1 g/100 g maltitol diet doubled (P < 0.05) the concentration of butyrate but did not affect acetate or propionate in the cecal contents. These results suggest that dietary maltitol has a protective effect against dimethylhydrazine-induced tumors in rat cecum and proximal colon and that butyrate produced by bacterial fermentation of maltitol in the cecum may be involved in the protection.

Sympathetic activity is lower in rats fed a beef tallow diet than in rats fed a safflower oil diet.

Effects of dietary fats consisting of different fatty acids on sympathetic activity and body fat accumulation were studied in rats. Rats were meal-fed an isoenergetic diet based on either beef tallow or safflower oil for 8 weeks. Carcass fat content was greater (P < .05) in rats fed the beef tallow diet than in rats fed the safflower oil diet. Norepinephrine (NE) turnover rate was significantly lower (P < .05) in interscapular brown adipose tissue (IBAT) and pancreas in rats fed the beef tallow diet than in rats fed the safflower oil diet, resulting in a decreased (P < .05) diet-induced thermogenesis (DIT) and an increased (P < .05) serum insulin concentration in the former. To confirm the effects of dietary fats on sympathetic activity in relation to body fat accumulation, rats were chemically sympathectomized. Sympathectomy abolished the differences in body fat accumulation, DIT, and serum insulin concentration between the two dietary groups. These results suggest that the beef tallow diet promotes body fat accumulation by reducing sympathetic activity as compared with intake of the safflower oil diet.

Diet-induced thermogenesis is lower in rats fed a lard diet than in those fed a high oleic acid safflower oil diet, a safflower oil diet or a linseed oil diet.

The objectives of the present study were to examine the effects of dietary fats differing in fatty acid composition on diet-induced thermogenesis, sympathetic activity in brown adipose tissue and body fat accumulation in rats. Rats were meal-fed for 12 wk an isoenergetic diet based on lard, high oleic acid safflower oil, safflower oil or linseed oil, and norepinephrine turnover rates in brown adipose tissue were then estimated. Whole-body oxygen consumption after the meal indicated that diet-induced thermogenesis was significantly lower in rats fed the lard diet than in those fed the other diets. The norepinephrine turnover rate in the interscapular brown adipose tissue was also significantly lower in the lard diet group than in the other diet groups. The carcass fat content was significantly higher in the lard diet group than in the other diet groups, whereas the abdominal adipose tissue weights were the same in all diet groups. These results suggest that the intake of animal fats rich in saturated fatty acids, compared with the intake of vegetable oils rich in monounsaturated or polyunsaturated fatty acids, decreases diet-induced thermogenesis by a decline of sympathetic activity in brown adipose tissue, resulting in the promotion of body fat accumulation.

Herpetic epithelial keratitis caused by acyclovir-resistant strain.

Dendritic keratitis occurred during oral acyclovir (ACV) therapy in a 60-year-old man. Corneal stromal edema and iritis were found at his first visit. Herpetic keratouveitis was suspected, based on clinical findings and previous history. Treatment with steroid eyedrops and ACV ointment was initiated. However, ACV [corrected] ointment was changed to oral ACV, since conjunctival ulcer occurred as an adverse effect of the ointment. Subsequently, he received long-term oral ACV medication and steroid eyedrops for recurrent keratitis. The fifth recurrence was also treated with oral ACV and steroid eyedrops. At this time, although the stromal keratitis had improved, there was an outbreak of dendritic keratitis. The lesion healed spontaneously after only a reduction in the steroid. The 50% effective dose (ED)50 of the isolated virus to ACV was 4.4 +/- 0.15 micrograms/ml (mean +/- SD), a level considered ACV-resistant in vitro. The clinical course of this case emphasizes that it is important to consider the route and duration of ACV administration and the use of steroids in the treatment of stromal keratitis or keratouveitis.

Altered monoamine metabolism in the hypothalamus of the genetically obese yellow (Ay/a) mouse.

Changes in hypothalamic monoamine metabolism were investigated in the genetically obese yellow (Ay/a) mouse. At the age of 6 weeks when there was no difference in body weight between black (a/a) and yellow (Ay/a) mice, the contents of norepinephrine (NE), dopamine (DA) and their main metabolites (MHPG, DOPAC) were already significantly reduced in yellow (Ay/a) mice. Reduction of 5-hydroxytryptamine (5-HT) level and an increasing 5-HIAA/5-HT ratio has been observed. When a significant increase in body weight in the yellow (Ay/a) mouse at the age of 12 weeks was present, both NE and DA contents have been increased in the hypothalamus of the obese mouse. MHPG level was lower than in the lean mouse, resulting in an increase of MHPG/NE ratio. The present study suggests that the observed reduction in hypothalamic NE and DA metabolism might be involved in the development of overweight gain in the yellow (Ay/a) mouse.

Sucrose feeding at weaning alters the preference for sucrose in adolescence.

The present studies were undertaken to examine the hypothesis that sucrose feeding at weaning may alter the preference for sucrose in the adolescence. Chronological changes of hypothalamic dopamine (DA), its metabolite, 3,4-dihydroxy-phenylacetic acid (DOPAC) and norepinephrine (NE) contents were also measured by HPLC. In 21 day-aged rats, 10% sucrose or water was given as drinking water for 3 weeks. From 9 weeks, all animals were maintained under a free choice between 30% sucrose solution and water. Sucrose-ingested rats more preferred to 30% sucrose solution than control rats and body weight gain of sucrose group was significantly greater than that of controls. Hypothalamic DA content was significantly decreased at 6 and 13 weeks and the DOPAC/DA ratio increased at 3, 6, and 13 weeks later. In contrast, hypothalamic NE concentration was not changed at all. The data obtained herein suggest that sucrose feeding at weaning alters the preference for sucrose, resulting in an overweight gain, and that the observed increase of hypothalamic DA metabolism may be involved in the altered preference for sucrose.

Less body fat accumulation in rats fed a safflower oil diet than in rats fed a beef tallow diet.

The effects on body fat accumulation of long-term feeding of high fat diets of differing fatty acid composition were studied in rats. The rats were meal-fed isoenergetic diets based on safflower oil or beef tallow for 4 mo. Each diet was freshly prepared every day throughout the experimental period. Oxygen consumption and carbon dioxide production for 6 h after meals were measured between the 50th and 54th d of the experimental period. Oxygen consumption for 3 h after meals was significantly greater in the safflower oil diet group than in the beef tallow diet group, indicating greater diet-induced thermogenesis in the former group. From the assessment of respiratory quotient, the fat oxidation rate was also higher in the former. After the experimental period (4 mo), body fat accumulation was significantly less in the rats fed safflower oil. This difference was, at least in part, ascribed to increased diet-induced thermogenesis and fat oxidation. Serum triacylglycerol level was markedly lower in the rats fed safflower oil than in those fed beef tallow. The lipoprotein lipase activities in heart and soleus muscle after meals appeared to be higher in the former than in the latter. These results suggest that the consumption of the safflower oil diet increased lipoprotein lipase activity in heart and skeletal muscle, resulting in the elevation of fat oxidation rate and the depression of serum triacylglycerol level.

Effects of interleukin-1 and anti-inflammatory drugs on the degradation of human articular cartilage.

It has been suggested that metalloproteases produced by chondrocytes play an important role in cartilage breakdown in joint diseases. The aim of this study was to investigate changes in enzyme activities in human rheumatoid and osteoarthritic articular cartilage. Cartilage fragments were incubated with various drugs for 48 hours. The concentrated culture media were used as enzyme solutions. Collagenase was assayed using FITC-collagen as the substrate. Proteoglycanase (PGase) was measured either by the release of 35S-labelled proteoglycans from cartilage into the medium, or by enzyme assay using proteoglycan monomer bound to fluorescein-conjugated hyaluronic acid as the substrate. Collagenase and proteoglycanase were found only in trace amounts in the concentrated media of healthy cartilage. Interleukin-1 (IL-1) enhanced the enzyme activities significantly. Marked increases of enzyme activities were observed in the concentrated media of rheumatoid (RA) and osteoarthritic (OA) cartilage. The sensitivity to interleukin-1 was also higher in OA and RA cartilage compared with healthy cartilage. Dexamethasone (10(-6) mol/L) markedly depressed enzyme activity. Tiaprofenic acid (4 x 10(-5) mol/L) also decreased enzyme activity, whereas indomethacin (4 x 10(-6) mol/L) and naproxen (3 x 10(-4) mol/L) had no effect.

Simulation of the initial stage of endochondral ossification: in vitro sequential culture of growth cartilage cells and bone marrow cells.

Growth cartilage cells were isolated from the ribs of young rats and cultured at high cell density in Ham's F-12 medium supplemented with 10% fetal calf serum. During 7 days, glycosaminoglycans and proteoglycans were actively synthesized and secreted, forming a metachromatic matrix. When cultured together with growth cartilage cells precultured and biosynthetically prelabeled with 35SO4(2-) in their glycosaminoglycans, bone marrow cells caused release of 35S-labeled material into the culture medium. Glycosaminoglycan was also released by addition of conditioned medium obtained from cultures of bone marrow cells or peritoneal macrophages to the growth cartilage cell cultures. Electron microscopic studies of the extracellular matrix of growth cartilage cells cocultured with bone marrow cells showed that needles of apatite mineral were deposited within and in close apposition to the surfaces of matrix vesicles. These findings suggest that enzymes released from bone marrow cells or macrophages removed glycosaminoglycan or proteoglycans, which may be inhibitors of mineral growth, and consequently mineralization was initiated. From these findings, sequential culture of growth cartilage cells and bone marrow cells is promising as an experimental system for investigating the mechanism of the initial stage of endochondral ossification.