Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting ...at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc(1) complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q(i) site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q(o) site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A(3) in a fashion similar to the Q(o) site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q(i) and the Q(o) sites of the fungal cytochrome bc(1) complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc(1) complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q(o) and Q(i) sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc(1) complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q(i) and Q(o) sites. Moreover, crystal structure of chicken cytochrome bc(1) complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q(i) and Q(o) sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc(1) inhibitor that acts at both of the active sites of this enzyme.
We have previously found that a pyrazole derivative 1 possesses antibacterial activity and inhibitory activity against DNA gyrase and topoisomerase IV. Here, we synthesized new pyrazole derivatives ...and found that 5-(E)-2-(5-chloroindol-3-yl)vinylpyrazole 16 possesses potent antibacterial activity and selective inhibitory activity against bacterial topoisomerases. Many of the synthesized pyrazole derivatives were potent against clinically isolated quinolone- or coumarin-resistant Gram-positive strains and had minimal inhibitory concentration values against these strains equivalent to those against susceptible strains.
Citrus depressa Hayata (commonly known as shiikuwasa) is cultivated in the northern areas of Okinawa, Japan, and used as a juice. In this study, we examined the anti-obesity effects and mechanism of ...action of shiikuwasa peel extract (SE) using high-fat diet (HFD)-induced obese mice. Mice were fed a low-fat diet (LFD), HFD or HFD containing 1% or 1.5% (w/w) SE (HFD
+
1 SE and HFD
+
1.5 SE, respectively) for 5 weeks. The body weight gain and white adipose tissue weight were significantly decreased in the HFD
+
1.5 SE group compared with the HFD group. The plasma triglyceride and leptin levels were also significantly reduced in the HFD
+
1.5 SE group compared with the HFD group. Histological examinations showed that the sizes of the adipocytes were significantly smaller in the HFD
+
1.5 SE group than in the HFD group. The HFD
+
1.5 SE group also showed significantly lower mRNA levels of lipogenesis-related genes, such as activating protein 2, stearoyl-CoA desaturase 1, acetyl-CoA-carboxylase 1, fatty acid transport protein and diacylglycerol acyltransferase 1, than the HFD group. These results suggest that the anti-obesity effects of SE may be elicited by regulating the expressions of lipogenesis-related genes in white adipose tissue.
A weak hydrolyzing activity against bis (2-ethylhexyl) phthalate (DEHP) was discovered in a commercial crude lipase (EC 3.1.1.3) preparation from porcine pancreas. DEHP was hydrolyzed to mono ...(2-ethylhexyl) phthalate (MEHP) not by a pancreatic lipase but by a cholesterol esterase (CEase, EC 3.1.1.13), a trace contaminant in the crude lipase preparation. Enzymatic hydrolysis of phthalic acid esters (PAEs), suspected to be endocrine-disrupting chemicals, was investigated using CEases from two species of mammals and a microorganism. Eight structurally diverse PAEs, namely diethyl phthalate (DEP), di-
n-propyl phthalate (DPrP), di-
n-butyl phthalate (DBP), di-
n-pentyl phthalate (DPeP), di-
n-hexyl phthalate (DHP), DEHP,
n-butyl benzyl phthalate (BBP), and dicyclohexyl phthalate (DCHP), were hydrolyzed to their corresponding monoesters by both porcine and bovine pancreatic CEases, while a microbial CEase from
Pseudomonas sp. had no hydrolyzing activity against these PAEs. The hydrolysis experiments with bovine pancreatic CEase (50 U) indicated complete hydrolysis of every PAE (5 μmole) except for BBP and DCHP within 15
min; BBP and DCHP were hydrolyzed within 30
min and 6
h, respectively. The rates of PAE hydrolysis could be affected by the bulkiness of alkyl side chains in the PAEs. This study provides important evidence that mammalian pancreatic CEases, such as those from porcine and bovine sources, are potential enzymes for nonspecific degradation of structurally diverse PAEs.
LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS ...stimulated osteoclast formation and PGE(2) production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE(2) is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE(2) production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE(2) production.
We found that penicillin-induced glutamate production by Corynebacterium glutamicum is inhibited when a de novo protein synthesis inhibitor, chloramphenicol, is added simultaneously with penicillin. ...When chloramphenicol was added 4 h after penicillin addition, glutamate production was essentially unaffected. ³H-Leucine incorporation experiments revealed that protein synthesis continued for 1 h after penicillin addition and then gradually decreased. These results suggest that de novo protein synthesis within 4 h of penicillin treatment is required for the induction of glutamate production. To identify the protein(s) necessary for penicillin-induced glutamate production, proteome analysis of penicillin-treated C. glutamicum cells was performed with two-dimensional gel electrophoresis. Of more than 500 proteins detected, the amount of 13 proteins, including OdhI (an inhibitory protein for 2-oxoglutarate dehydrogenase complex), significantly increased upon penicillin treatment. Artificial overexpression of the odhI gene resulted in the decreased specific activity of the 2-oxoglutarate dehydrogenase complex and increased glutamate production without any triggers. These results suggest that the de novo synthesis of OdhI is the necessary factor for penicillin-induced glutamate overproduction by C. glutamicum. Moreover, continuous glutamate production was achieved by overexpression of odhI without any triggers. Thus, the odhI-overexpressing strain of C. glutamicum can be useful for efficient glutamate production.
Lignin is one of the major water insoluble substances that support the physical properties of plants and can be solubilized by sulfite or alkaline treatment in accordance with pulpification. The ...lignin derivatives produced by both the sulfite and the kraft processes are called lignosulfonate (LS) and kraft lignin (KL), respectively, and both derivatives show a broad spectrum of optical absorbance from ultraviolet to visible light. When the spore suspension of an isolated Penicillium sp., Penicillium sp. A, was inoculated to a medium containing 0.1% commercial LS, absorbance at 480 nm (A480) almost completely disappeared after 5 days of cultivation. Maximum decolorization of the culture broth was observed when the microbe was cultured in the 0.8% LS medium reaching 88%, and the amount of LS removed was calculated to be 7 g/L. In a similar assay with the dark-liquid containing KL, 80% of the A480 of a 20% (v/v) dark-liquid medium disappeared after 5 days of culturing and the amount of KL removed was calculated to be 2.9 g/L. These values significantly exceeded the previously reported amounts with respect to substrate concentration and decolorization. Furthermore, since similar results were obtained in the cases of both LS and KL, it is expected that the present strain is able to non-specifically adsorb a wide range of lignin derivatives, because most of the colored substances were recovered in the culture sediments. Thus, the strain can be used to clean up waste fluids containing water soluble lignin derivatives, adsorb lignin derivatives in waste fluids before dehydration.
Although quercetin has suppressed bone resorption in several animal studies, its target cells and the mechanism of its action related to bone resorption has not been fully elucidated. We investigated ...the effect of quercetin on the differentiation and activation of osteoclasts. We used cocultures of mouse spleen cells and ST2 cells, and cultures of osteoclast progenitor cells {M-CSF-dependent (MD) cells from mouse bone marrow and murine monocytic RAW 264 (RAW) cells}. Quercetin dose-dependently inhibited osteoclast-like (OCL) cell formation at 2—5 μM concentration in both the coculture and MD cell culture. Quercetin inhibited the increase of tartrate-resistant acid phosphatase (TRAP) activity of mononuclear preosteoclasts (pOCs) induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) in both MD and RAW cell cultures. Quercetin reversely induced the disruption of actin rings in OCLs. Quercetin also suppressed both pit formation induced by osteoclasts on dentine slices and PTH-stimulated 45Ca release in mouse long bone cultures. These results suggest that osteoclast progenitors as well as mature osteoclasts, are quercetin's target cells in relation to bone resorption, and that quercetin's suppressive effect on bone resorption results from both its inhibitory effect on the differentiation of osteoclast progenitor cells into pOCs and from its disruptive effect on actin rings in mature osteoclasts.
Platelet-derived growth factor (PDGF)-BB is one of the most potent factors in the development and progression of various vascular disorders such as restenosis and atherosclerosis. Chrysoeriol is a ...flavonoid with antioxidant and anti-inflammatory activities. In this study, we investigated the effect of chrysoeriol on the proliferation of human aortic smooth muscle cells (HASMC). Chrysoeriol significantly inhibited PDGF (20 ng/mL)-induced migration and 3H-thymidine incorporation into DNA at concentrations of 5 and 10 μM without any cytotoxicity. Chrysoeriol also blocked PDGF-stimulated dissociation of actin filament and inhibited PDGF beta-receptor (Rβ) phosphorylation in a concentration-dependent manner. As a result, the downstream signal transduction pathways of PDGF-Rβ, including ERK1/2, p38, and Akt phosphorylation, were also inhibited by chrysoeriol in the same pattern. These findings suggest that in addition to its antioxidant and anti-inflammatory activities, chrysoeriol may be used for the prevention and treatment of vascular diseases and during restenosis after coronary angioplasty.
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