The substrate deacylation mechanisms of serine-β-lactamases (classes A, C and D) were investigated by theoretical calculations. The deacylation of class A proceeds via four elementary reactions. The rate-determining process is the tetrahedral intermediate (TI) formation and the activation energy is 24.6 kcal/mol at the DFT level. The deacylation does not proceed only by Glu166, which acts as a general base, but Lys73 also participates in the reaction. The C3-carboxyl group of the substrate reduces the barrier height at the TI formation (substrate-assisted catalysis). In the case of class C, the deacylation consists of two elementary processes. The activation energy of the TI formation has been estimated to be 30.5 kcal/mol. Tyr150O_η is stabilized in the deprotonated state in the acyl-enzyme complex and works as a general base. This situation can exist due to the interaction with two positively charged side chains of lysine (Lys67 and Lys315). The deacylation of class D also consists of two elementary reaction processes. The activation energy of the TI formation is ca. 30 kcal/mol. It is thought that the side chain of Lys70 is deprotonated and acts as a general base. When Lys70 is carbamylated, the activation energy is reduced to less than 20 kcal/mol. This suggests that the high hydrolysis activity of class D with carbamylated Lys70 is due to the reduction of activation energy for deacylation. From these results, it is concluded that the contribution of the lysine residue adjacent to the serine residue is indispensable for the enzymatic reactions by serine-β-lactamases.

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We described the main pathways of bacitracin (Bc) decomposition, chromatographically set the position of its major degradation products and evaluated microbiological activity of isolated components of Bc and its degradation products. All processes of Bc decomposition under stress and accelerated test conditions were monitored with HPLC, performed mainly on a new type reversed-phase (RP-18e) monolithic silica column (Chromolith^®) enabling fast separation times and some of them also on conventional HPLC columns. Diode array detection, preparative HPLC and FAB mass spectrometry were used for identification of individual Bc components. We found that the major decomposition mechanism in water solutions of Bc is oxidation, and in alkaline solutions, deamidation. In oxidation process the components B1, B2 and B3 and A are oxidized into their corresponding oxidative products H1, H2, H3 and F respectively by the same mechanism. A detailed study of oxidative degradation products revealed that HPLC separation with an acid mobile phase caused splitting of peaks of components H2, H3 and F into two peaks but the peak of component H1 did not split due to its special structural properties. For the component A we confirmed gradual formation of desamido product through an intermediate. We found oxidative degradation products of Bc to be relatively stable, and desamido degradation products to be rather unstable. The estimation of kinetics of Bc decomposition was presented with a semi-quantitative model. Microbiological activity of individual isolated active components of Bc was established and the negligible antimicrobial activity of the degradation products was confirmed.

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We previously reported the cloning of a calcium-activated chloride channel (CLCA) from rat brain (Biochem. Biophys. Res. Commun., 334, 569—576 (2005)), which we designated rbCLCA1. We further showed that rbCLCA1 is expressed in the central nervous system and peripheral organs, and may be functionally expressed in mammalian HEK293 cells. In the present study, we report the successful cloning of a second CLCA from rat cerebrum (designated rbCLCA2), using reverse transcription-PCR (RT-PCR) with primers specific for rbCLCA1. We have begun to clone this cDNA based on the rbCLCA1-like sequence. The full-length rbCLCA2 cDNA, obtained via 5′ and 3′ rapid amplification of cDNA ends (RACE), is 2900 bp long and encodes a putative polypeptide of 905 amino acids having at least two major transmembrane domains and showing 85.2% identity to rbCLCA1. RT-PCR analysis revealed that, similar to rbCLCA1, rbCLCA2 was predominantly expressed in the rat cerebrum, cerebellum, kidney, stomach, spinal cord, lung and small intestine, but not in the heart, large intestine, liver, orand spleen. Whole-cell patch clamp studies in HEK293 cells transiently co-transfected with expression vectors encoding rbCLCA2 and EGFP allowed us to identify the presence of niflumic acid (a CLCA channel blocker)-sensitive and voltage-dependent chloride currents in cells expressing rbCLCA2 but not EGFP alone. Treatment of these cells with ionomycin, a Ca²⁺ ionophore, significantly increased the novel currents in cells expressing rbCLCA2 and EGFP, but not those expressing EGFP alone, indicating that activation of the rbCLCA2 current is Ca²⁺-dependent. In sum, we herein report the cloning of a second member of the rbCLCA family from rat brain and its functional expression in vitro, thus adding to our knowledge of anion channels and facilitating future exploration of brain and other organ physiology.

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Lipid droplets (LDs) are intracellular storage sites of neutral lipids, which accumulate in fatty liver disease. Here, we investigated the effects of fatty acids and glucose on LD formation in a cultured human hepatocyte, HuH7, by adding them to culture media. Fatty acids with carbohydrate chains C12—C18 efficiently induced LDs, but those of C8 and C10 were ineffective. Glucose did not induce LD formation even in the presence of insulin. Oleic acid induced significant increases in cellular neutral lipids, and cell fractionation revealed that most of the newly synthesized neutral lipids were concentrated in LDs together with LD proteins. The LD formation was not abrogated by removal of medium glucose but was significantly inhibited by an ACSL inhibitor, triacsin C. These results demonstrate that long-chain fatty acids contribute to LD formation to a greater extent than glucose, possibly by being taken up into the cells, activated by ACSL, reconstituted into neutral lipids and then stored in LDs. Pregnenolone and lithium did not suppress oleic acid-dependent LD formation, despite previous reports of their ability to inhibit LD formation in macrophages and adipocytes suggesting differences among LD formations in these cells.

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Oxidatively modified low-density lipoprotein (OxLDL) is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis. In the present study, in order to clarify the structure-binding activity relationship of Asp-hemolysin-related peptides to OxLDL, we investigated the interaction between Asp-hemolysin-related peptides consisting of 4 to 29 amino acid residues and OxLDL. The incubation of OxLDL with each Asp-hemolysin-related peptide resulted in the formation of an Asp-hemolysin/OxLDL complex. In particular, the tetrapeptide, YKDG (P-4), bound to OxLDL and inhibited the OxLDL-induced macrophage proliferation in a dose-dependent manner. Furthermore, we demonstrated that lysophosphatidylcholine (LysoPC) extracted from OxLDL inhibited the binding of P-21 to OxLDL in a dose-dependent manner and synthetic [¹⁴C]LysoPC bound to P-21. We propose here that the YKDG region is one of the important sites for the binding of these peptides to OxLDL, and LysoPC as a typical lipid moiety of OxLDL is attributed to the binding of OxLDL to these peptides.

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Testis-specific genes are essential for the spermatogenesis in mammalian male reproduction. We have identified a novel gene, Tsc24, from the results of the Affymetrix Genechip analysis in the developmental stage of days 4, 9, 18, 35, 54 and 6 months of postnatal Balb/C mouse testis. The scaling signal intensities of Tsc24 in the six stages of mouse testis showed that the expression of Tsc24 was not detected on day 4, 9 or 18 but on day 35, 54 and 6 months. The full cDNA length of mouse Tsc24 was 899 bp, with a 624 bp open reading frame encoding a 207 amino acid protein with a predicted molecular weight of 23.997 kDa. The results of semi-quantitative RT-PCR showed that the expression of mouse Tsc24 can only be detected after the mouse was 35 d old and the expressing level increased gradually from day 35 to 6 months. Of the eight tissues (liver, spleen, heart, lung, brain, kidney, epididymis, and testis) examined in mice, and of the 12 tissues (liver, kidney, muscle, brain, spleen, adipose, lung, heart, epididymis, testis, ovary and uterus) examined in human, Tsc24 was exclusively expressed in the testis, but in none of the other studied tissues. The result of subcellular localization of GFP-Tsc24 fusion protein in Cos-7 cells supports that Tsc24 protein is expressed in nuclear. Our study should be a basis for function characterization of the Tsc24 gene, leading to the elucidation of the molecular events underlying mammalian male reproduction.

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The WRNIP1 protein interacts with WRN, the product of the causative gene for Werner syndrome. Mutation of the Saccharomyces cerevisiae gene MGS1, the yeast counterpart of WRNIP1, confers synthetic lethality with mutation of RAD18. To examine the functional relationship between WRNIP1 and Rad18 in higher eukaryotic cells, we generated WRNIP1^−/−/−/RAD18^−/− lines from chicken DT40 cells and compared them with single mutant cell lines. Unlike the corresponding yeast mutant, WRNIP1^−/−/−/RAD18^−/− cells are viable but grow more slowly than single mutants and wild type cells, and they show an additive or synergistic elevation in the frequency of sister chromatid exchanges. As reported, WRNIP1^−/−/− cells and RAD18^−/− cells are moderately and severely sensitive to camptothecin (CPT), respectively. Unexpectedly, the severe CPT sensitivity of RAD18^−/− cells is slightly suppressed by disruption of the WRNIP1 gene.

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Citral, trans-cinnamaldehyde, (−)-perillaldehyde, (−)-citronellal, eugenol and carvacrol were tested for their influence on microbial count in air by vaporizing with an air washer. The highest antibacterial activity was observed when (−)-perillaldehyde was sprayed. The average reduce of germ count was 53%. On the other hand, the antimicrobial activity of eugenol was the lowest of these six compounds. The average reduction of germ count was 13%. When water without volatile compounds was sprayed, the colony forming units increased. These results suggest the utility of selected aroma-compounds for the control of bacteria in the room.

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In order to determine whether Pseudomonas aeruginosa alkaline protease AprA is involved in facilitating siderophore-mediated iron-acquisition from human transferrins, we measured bacterial growth, the production of siderophore and AprA, iron-acquisition from transferrins, and the proteolytic cleavage of transferrins in an alkaline minimal medium (pH 8.3) containing human transferrins as an iron source and compared these on a time scale. The growth of P. aeruginosa was found to be stimulated in proportion to the iron-saturation levels of transferrins. AprA production and the proteolytic cleavage of transferrins began concomitantly with siderophore production from the early growth phase when P. aeruginosa was actively growing and consuming most iron for growth. However, the AprA-free, but siderophore-containing, culture ultrafiltrates could also remove iron from transferrin. These results indicate that alkaline protease AprA can facilitate the siderophore-mediated iron-uptake of P. aeruginosa via the proteolytic cleavage of transferrins. However, the proteolytic cleavage by AprA is not essentially required for iron-acquisition from transferrins.

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Since phenoxazine is an essential structure of actinomycin D, which exerts a strong anticancer effect, we examined the anticancer effect of 2-aminophenoxazine-3-one (Phx-3) on mouse malignant melanoma B16 cells in vitro and in vivo. Phx-3 inhibited proliferation of the B16 cells in a dose-dependent manner in vitro. We furthermore studied the in vivo effects of Phx-3 on mouse malignant melanoma B16 cells transplanted in female C57BL/6Cr Slc mice. Treatment with Phx-3 (0.5 mg/kg) completely suppressed the growth of mouse malignant melanoma B16 cells transplanted in mice as compared with the control group. Phx-3 was found to exert few adverse effects, in terms of bodyweight loss, changes in serum levels of blood biochemical parameters such as aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN) and creatinine, dysfunction of the liver and the kidney examined by pathological methods, piloerection and wasting, when mice were treated with a dose of 0.5 mg/kg. These results suggest that Phx-3 may be used to treat patients affected by malignant melanoma in future.

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The potential role of the methanolic extract of Heliotropium zeylanicum (BURM.F) LAMK (MEHZ) in the treatment of diabetes along with its antioxidant and antihyperlipidemic effects was studied in streptozotocin-induced diabetic rats. Oral administration of (MEHZ) 150 and 300 mg/kg/d for 14 d significantly decreased the blood glucose level and considerably increased the body weight, food intake, and liquid intake of diabetic-induced rats. MEHZ significantly decreased thiobarbituric acid reactive substances and significantly increased reduced glutathione, superoxide dismutase and catalase in streptozotocin-induced diabetic rats at the end of 14 d of treatment. The study also investigated the antihyperlipidemic potential of MEHZ. The results show that the active fraction of MEHZ is promising for development of a standardized phytomedicine for the treatment of diabetes mellitus.

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Oral administration of tea catechin dose-dependently prevented absolute ethanol-induced (50, 100, 200 mg/kg) or restraint plus water immersion stress-induced acute gastric mucosal injury (300, 400 mg/kg) in rats. When the effect of test compound was evaluated on the 15th day after acetic acid injection to rats, repeated oral administration of tea catechin (25, 50, 100 mg/kg twice daily) dose-dependently accelerated the healing of acetic acid-induced chronic gastric ulcers. Tea catechin (10⁻⁵—10⁻¹ g/100 ml) concentration-dependently scavenged superoxide anions in vitro. Tea catechin (100, 200 mg/kg orally) markedly inhibited the increase in thiobarbituric acid-reactive substances in the injured mucosa of rats treated with 50% ethanol. Tea catechin (50, 100 mg/kg twice orally, daily) markedly inhibited the increase in content of thiobarbituric acid-reactive substances in the ulcerated region of acetic acid-induced gastric ulcers on the 7th and 15th days. In addition, at 50, 100 and 200 mg/kg orally, it dose-dependently prevented the decrease in gastric mucosal hexosamine content induced by absolute ethanol, although it failed to inhibit the basal gastric acid secretion. These results suggest that tea catechin may primarily protect gastric mucosa from acute gastric mucosal injury and promote the healing of chronic gastric ulcers by its antioxidant activity and gastric mucus-increasing actions.

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Prostaglandin E₂ (PGE₂) works as a common final mediator of the febrile. Guizhi-Tang, one of the most famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions, was previously reported to reduce the production of PGE₂ in rats. 2-Methoxycinnamaldehyde is a principle compound isolated from Guizhi-Tang. The aim of the present study was to investigate the effects of 2-methoxycinnamaldehyde on PGE₂ production of rat cerebral endothelial cells (CECs). 2-Methoxycinnamaldehyde dose-dependently inhibited interleukin (IL)-1β-induced PGE₂ production in CECs with IC₅₀ values of 174 μM. IL-1β stimulation increased the protein, activity and mRNA expression of cyclooxygenase (COX)-2 but not COX-1. 2-Methoxycinnamaldehyde reduced IL-1β-induced protein and activity of COX-2, but did not influence the COX-2 mRNA expression. Our results show that prostaglandin production in CECs during stimulated conditions is sensitive to inhibition by 2-methoxycinnamaldehyde and suggest that 2-methoxycinnamaldehyde may reduce COX-2 protein level and activity but not COX-2 mRNA.

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Chronic hypertension shifts cerebral blood flow (CBF) autoregulation towards higher blood pressure. We examined whether or not benidipine, a long-lasting dihydropyridine calcium channel blocker (CCB), improves the CBF autoregulation in spontaneously hypertensive rats (SHRs). CBF was analyzed by laser-Doppler flowmetry during stepwise hypotension by controlled bleeding. The lower limit of CBF autoregulation was calculated as the mean arterial blood pressure at which CBF decreased by 10% of the baseline. Mean arterial blood pressure and cerebral vascular resistance in SHRs were higher than those in normotensive Wistar rats. Oral administration of benidipine (3 mg/kg) for 8 d lowered the mean arterial blood pressure and cerebral vascular resistance, which were equivalent to the effects of amlodipine (3 mg/kg), another CCB, or candesartan (1 mg/kg), an Angiotensin II type-1 receptor blocker. The lower limit of CBF autoregulation in SHRs (142±4 mmHg) was significantly shifted to a higher-pressure level compared with Wistar rats (59±2 mmHg). The lower limit of CBF autoregulation was significantly lower in the benidipine-treated group (91±4 mmHg) than that in the control SHRs, and similar to that of the amlodipine group (97±6 mmHg). Benidipine reduced the lower limit of CBF autoregulation more effectively than candesartan (109±4 mmHg). In conclusion, benidipine shifted the limit of CBF autoregulation towards lower blood pressure in SHRs under hypotensive conditions by hemorrhage. These results suggest that benidipine may be useful for the treatment of hypertensive patients with the elderly or cerebrovascular disorders, in whom autoregulation of CBF is impaired.

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The relationship between the structure and antibacterial activity of 22 polyphenols was analyzed by using minimum inhibitory concentration (MIC) as a criterion against 26 species of bacteria which can grow in Mueller–Hinton medium. There was no clear correlation between Gram-staining and bacterial susceptibility to polyphenols, and the extent of the susceptibility was approximately dependent on the species of bacteria. In the same Gram-negative bacteria, the antibacterial activity of the polyphenols against Aeromonas hydrophila, Vibrio parahaemolyticus and Vibrio vulnificus was comparatively strong. On the other hand, the activity against 11 species of the Enterobacteriaceae was comparatively weak, and the activity against six species of aerobic bacteria causing plant disease was moderate. Polyphenols having pyrogallol groups showed strong antibacterial activity, and those with catechol and resorcinol rings showed lower activity. The structure–activity relationship was extended to 26 polyphenol-rich plant extracts which could have potent antibacterial activity suitable for commercial use.

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Phenolic compounds are numerous and ubiquitous in the plant kingdom, being particularly present in health-promoting foods. Epidemiological evidences suggest that the consumption of polyphenol-rich foods reduces the incidence of cancer, coronary heart disease and inflammation. Chlorogenic acid (CGA) is one of the most abundant polyphenol compounds in human diet. Data obtained from in vivo and in vitro experiments show that CGA mostly presents antioxidant and anti-carcinogenic activities. However, the effects of CGA on the inflammatory reaction and on the related pain and fever processes have been explored less so far. Therefore, this study was designed to evaluate the anti-inflammatory, antinociceptive and antipyretic activities of CGA in rats. In comparison to control, CGA at doses 50 and 100 mg/kg inhibited carrageenin-induced paw edema beginning at the 2nd hour of the experimental procedure. Furthermore, at doses 50 and 100 mg/kg CGA also inhibited the number of flinches in the late phase of formalin-induced pain test. Such activities may be derived from the inhibitory action of CGA in the peripheral synthesis/release of inflammatory mediators involved in these responses. On the other hand, even at the highest tested dose (200 mg/kg), CGA did not inhibit the febrile response induced by lipopolysaccharide (LPS) in rats. Additional experiments are necessary in order to clarify the true target for the anti-inflammatory and analgesic effects of CGA.

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The present study evaluated the effect of the crude extract of the leaves of Nectandra falcifolia (NEES) Castiglioni and its fractions in different experimental models of inflammation (paw edema, pleurisy, and ear edema). Carrageenan-induced edema of the paw and pleurisy were evaluated in Wistar rats (180—220 g), which were treated with different doses of the total extract (250, 500 mg·kg⁻¹). Edema of the ear, induced by croton oil, and determination of myeloperoxidase activity were evaluated in Swiss mice (25—35 g). In this experiment, the crude extract of Nectandra falcifolia (Nf) (1.25, 2.5, 5.0, 7.5 mg) and the hexane, chloroform, ethyl-acetate and hydromethanol fractions (5.0 mg) were applied topically, immediately after application of the oil. The crude extract of Nf (500 mg·kg⁻¹) significantly reduced edema of the paw compared to the control group. Similarly, at doses of 250 and 500 mg·kg⁻¹ it significantly reduced the volume of pleural inflammatory exudate compared to the control animals. However, it did not change the number of migrated cells. At doses of 2.5, 5.0 and 7.5 mg, the crude extract significantly inhibited edema of the ear and the influx of neutrophils. The fractions from Nectandra falcifolia (hexane, chloroform, ethyl acetate and hydromethanol) also inhibited edema of the ear. Taken together, the results demonstrated that the crude extract and its fractions administered to animals orally or topically showed an anti-inflammatory effect.

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Production of free radical species in cells and body tissues is known to cause many pathological disorders. Therefore, free radical scavengers play an important role in the prevention of various human diseases. Bamboo grass, Sasa senanensis, is a native Japanese plant. Sasa has been used for medicine in Japan for many centuries. In this study, we investigated the antioxidative activity of Absolutely Hemicellulose Senanensis (AHSS), a novel extract from Sasa. In the first part of this study, we found that AHSS has antioxidant activities by the assay using superoxide anion-2-methyl-6-methoxyphenylethynylimidazopyrazynone (MPEC) reaction kit. We then confirmed its antioxidative activity using a rat ischemia and subsequent reperfusion (I/R) injury model. Breakdown of the intestinal wall caused by intestinal I/R was attenuated by pretreatment with AHSS. Moreover, AHSS inhibited the production of lipid peroxide by intestinal I/R. AHSS could be an important source of ingredients for use in functional foods and other applications.

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The aim of this study was to investigate the effects of sarsasapogenin from Anemarrhena asphodeloides BUNGE (Liliaceae) on the forced swimming test, and the central noradrenergic, dopaminergic and serotonergic activities in mice. Our results showed that sarsasapogenin treatment at 12.5, 25 and 50 mg/kg (p.o.) for 14 d significantly reduced the duration of immobility in the forced swimming test. These doses that affected the immobile response did not affect locomotor activity. In addition, the neurochemical assays showed that sarsasapogenin produced a marked increase of noradrenaline and serotonin levels at 50 mg/kg in both the hypothalamus and the hippocampus. Moreover, sarsasapogenin showed a monoamine oxidase inhibitory activity in the mouse brain. These findings suggest that the antidepressant activity of sarsasapogenin may involve the central monoaminergic neurotransmitter systems.

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Biological activities of 2α-substituted 1α,25-dihydroxyvitamin D₃ analogues were evaluated in vitro. Their binding affinity was examined with calf thymus cytosolic vitamin D receptor (VDR) and rat plasma vitamin D-binding protein (DBP). In addition, the transcriptional activity of the analogues was measured using a rat 25-hydroxyvitamin D₃-24-hydroxylase gene promoter, a human osteocalcin gene promoter, and VDR-GAL4 system. This study investigated the biological activities of 2α-substituted analogues in comparison with 2β-substitued analogues at the molecular level, with regard to the structural differences of alkyl, hydroxyalkyl, hydroxyalkoxy substituents at the 2-position of 1α,25-dihydroxyvitamin D₃.

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The Korean genuine medicine “Gigukjiwhangwhangami (GJWGM)” has long been used for various cerebrovascular diseases. However, the exact mechanism that accounts for the anti-inflammatory effect of GJWGM is not completely understood. The aim of the present study is to elucidate how GJWGM modulates the inflammatory reaction in lipopolysaccaride (LPS)-stimulated peripheral mononuclear cells from patients with cerebral infarction. Production of cytokine was measured by the ELISA and RT-PCR method. The level of nuclear factor-kappaB (NF-κB)/Rel A protein and NF-κB DNA binding activity were determined by the Western blot analysis and TF-EIA method. We showed that GJWGM inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-6, and IL-8 induced by LPS in dose dependent manner (p<0.05). Maximal inhibition rate of TNF-α, IL-1β, IL-6 and IL-8 production by GJWGM was about 54.34%, 41.37%, 44.04%, and 54.46%, respectively. GJWGM inhibited the TNF-α and IL-8 mRNA expression. In addition, we showed that the inhibitory mechanism of GJWGM is through the suppression of NF-κB pathway. Our study suggests that an important molecular mechanism by GJWGM reduce inflammation, which may explain its beneficial effect in the regulation of inflammatory reactions.

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Ethanol extract from Arrabidaea triplinervia leaves showed in vitro activity (ED₁₀₀ 5.0 mg/ml) against trypomastigotes of Trypanosoma cruzi, the etiologic agent of Chagas` disease. Bioactivity-directed fractionation of this extract led to the isolation of ursolic and oleanolic acids as trypanocidal compounds besides pomolic acid (not tested) and alpinetine (inactive). A series of natural and synthetic derivatives of ursolic and oleanolic acids was simultaneously assayed for structure activity relationships (SAR) studies. Ursolic acid (ED₁₀₀ 0.4 mg/ml) was four times more active than oleanolic acid (ED₁₀₀ 1.6 mg/ml). The presence of free hydroxy and/or carboxy groups is necessary for the trypanocidal activity as could be deduced from the effect of the acetates, methyl ester, and aldehyde derivatives.

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The H₂O, H₂O/MeOH (1 : 1) extracts from the wood of Taxus yunnanensis showed a remarkable inhibitory effect on induced histamine release from the human basophilic cell line, KU812. The eleven constituents purified from the wood extracts of Taxus yunnanensis were tested by an in vitro histamine release inhibition assay. Among them, secoisolarciresinol and taxiresinol were found to show inhibitory activities. A new neolignan, 2-[2-hydroxy-5-(3-hydroxypropyl)-3-methoxyphenyl]-1-(4-hydroxy-3-methoxyphenyl)propane-1,3-diol, was isolated from the wood of Taxus yunnanensis.

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Correct genotype identification of medicinal plant material remains important for botanical drug industry. Limitations of chemical and morphological approaches for authentication have generated need for newer methods in quality control of botanicals. The present study was carried out to develop DNA based marker for identification of Phyllanthus emblica LINN. A putative marker (1.1 kb) specific for P. emblica was identified by Random Amplified Polymorphic DNA (RAPD) technique. Sequence Characterized Amplified Region (SCAR) marker was developed from the RAPD amplicon. The SCAR marker was found useful for identification of P. emblica in its commercial samples and Triphalachurna, a multi-component Ayurvedic formulation.

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Ethanol extract of the aerial portion of Chelidonium majus L. inhibited acetylcholinesterase (AChE) activity without a significant inhibition of butyrylcholinesterase (BuChE). Using mass spectrometry and NMR studies, three active constituents were isolated and identified: 8-hydroxydihydrochelerythrine (1), 8-hydroxydihydrosanguinarine (2), and berberine (3). Compounds 1—3 showed potent inhibitory activity against AChE, with IC₅₀ (μM) values of 0.61—1.85. Compound 1 exhibited competitive and selective inhibition for AChE.

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Dykellic acid, a novel factor initially identified from the culture broth of Westerdykella multispora F50733, has been shown to inhibit matrix metalloprotease 9 activity, caspase-3 activity, B cell proliferation and LPS-induced IgM production, suggesting that this factor may have anti-cancer effects. In an effort to further address the possible anti-tumoral effects of dykellic acid, we used wound healing, invasion and RhoA-GTP assays to examine the effects of dykellic acid on cell migration, invasion and angiogenesis. Our results revealed that dykellic acid dose-dependently inhibits B16 cell migration and motility, and inhibits HUVEC tube formation. Western blot analysis of the active form of RhoA (RhoA-GTP) showed that dykellic acid treatment decreased the levels of RhoA-GTP. These findings collectively suggest that dykellic acid may have both anti-metastatic and anti-angiogenic acitivites, and provides the first evidence for the involvement of RhoA in dykellic acid-induced effects.

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The combination of cisplatin (CDDP) and 5-fluorouracil (5-FU) has been reported to show marked therapeutic effects on experimental tumors and human malignancies, such as head and neck cancer. In these clinical studies, CDDP was administered on day 1 and followed by a 5-d continuous infusion of 5-FU. However, it was repeatedly shown that the sequence of 5-FU followed by CDDP is more active and less toxic in tumor-bearing animals. Thus, the optimal administration schedule of CDDP and 5-FU against L1210 murine leukemia was examined and compared with that of the combination of 5-FU and carboplatin (JM-8). The combinations of 5-FU (days 1 to 5) and CDDP, given either on day 1 or on day 5, showed a similar level of antitumor activity and toxicity. On the other hand, the combinations of 5-FU (days 1 to 5) and JM-8 given on day 5 showed significantly higher antitumor activity and rather less toxicity, as compared with the combinations on the reverse sequence. Thus, the treatment sequence of platinum compounds followed by a 5-d continuous infusion of 5-FU in many clinical studies appears to be extremely favorable to CDDP than JM-8. In addition, pathological examinations of died mice showed that accumulation of ascites and pleural effusion was inhibited most effectively by JM-8, given alone or in combination with 5-FU. These results strongly suggest that the combination of 5-FU followed by JM-8 will be expected to show more excellent antitumor activity against human malignancies, and may be especially useful in patients who are unable to tolerate CDDP-related toxicity.

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Objective: The goal of this study was to evaluate the influence of congestive heart failure (CHF) on the clearance of mexiletine. Methods: The mexiletine clearance/bioavailability (CL/F) ratio was estimated in 584 inpatients receiving mexiletine therapy. The study population consisted of 210 patients with CHF [CHF group; 116 inpatients with New York Heart Association (NYHA) class I—II (group NYHA I—II) CHF and 94 inpatients with NYHA class III—IV (group NYHA III—IV) CHF] and 374 inpatients without CHF (Non-CHF group). Serum levels of mexiletine were determined by high performance liquid chromatography (HPLC). Results: Mexiletine clearance was significantly lower in the CHF group when compared with the Non-CHF group (0.264±0.093 vs. 0.393±0.082 l/h/kg, mean±S.D., p<0.05). Further, the CL/F ratio was 50% lower in group NYHA III—IV when compared with the Non-CHF group, and the CL/F ratio tended to change in inverse proportion to NYHA class. Conclusion: CHF status significantly affects mexiletine clearance. Therefore, dose adjustments and careful monitoring are likely required in CHF patients receiving mexiletine.

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To study the effect of hydration on skin absorption, we investigated penetration across human skin of twelve model chemicals having steroidal structure but different molecular weight and compared the steady-state penetration rate (J) and lag-time (t) across hydration intact skin (J_h and t_h) with that across dehydrated intact skin (J_d and t_d). Stratum corneum (SC) thickness of hydrated (52 μm) is 3.3 times that of dehydrated skin (16 μm). Transepidermal water loss (TEWL) of hydrated (7.6±2.1 g/m²/h) is twice that of dehydrated skin (3.4±1.6 g/m²/h, p<0.05) which are similar to in vivo values, suggesting the SC barrier function was recovered. The ratio of J_h/J_d ranged between 0.7 and 3.6 (average of 1.9). On the other hand, the ratio of t_h/t_d was almost constant (average of 0.8). Ratios of J_h/J_d and t_h/t_d were independent of MW and K_o/w. In percutaneous absorption experiments in vitro, skin was preserved in culture medium until use and SC might swell during that time. Therefore, we consider the possibility that J and t varied between hydrated and dehydrated skin. We confirmed the difference of J and t between hydrated and dehydrated skin in vitro and now need to define these results under in vivo condition.

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Grapefruit juice (GJ) contains components that may increase the bioavailability of drugs; however, approaches to the removal of these components have been little investigated. It is known that furanocoumarin derivatives (FCs), such as bergamottin (BG) and 6′,7′-dihydroxybergamottin (DHB) in GJ, induce such drug interactions. In the present study, it was found that the heat treatment of grapefruit juice decreases concentrations of BG and DHB as well as their interactions both in vitro and in vivo. We incubated GJ for 10, 20, 30, 40, 50, and 60 min at 37, 62, 72, and 95 °C; FCs in each sample were then measured, using high-performance liquid chromatography (HPLC). The concentrations of BG and DHB were decreased in a time- and temperature-dependent manner, by 82.5 and 97.9% respectively, after incubation for 1 h at 95 °C. In contrast, the concentration of bergaptrol (BT) increased in a time- and temperature-dependent manner (27.7% after 60 min at 95 °C). In addition, the effect of each GJ sample on testosterone 6β-oxidation in human liver microsomes was observed. The inhibitory effects of GJ heated to 95 °C were decreased in a time-dependent manner, as in the case of BG and DHB concentrations. Furthermore, 2 ml of GJ treated for 60 min at 95 °C was administered into the rat duodenum. After 30 min, nifedipine (NFP) was administered intraduodenally at a dose of 3 mg/kg body weight. The concentrations of NFP in the plasma samples were determined by HPLC. No significant increase in the AUC of NFP was observed in the rats given heat-treated GJ. These results suggest that the heat treatment of GJ reduces the concentrations of FCs, thus eliminating the potential for drug interactions.

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We previously observed that rhinacanthins-C, -N and -Q, three main naphthoquinone esters isolated from the roots of Thai medicinal plant; Rhinacanthus nasutus KURZ. (Acanthaceae) induced apoptosis of human cervical carcinoma HeLaS3 cells. Since these rhinacanthins showed limited solubility in aqueous medium, we attempted to entrap them into liposomal membrane: Liposomalization enabled injection of the drugs and the drugs were expected to transfer to lipoproteins in the bloodstream. Liposomal formulations of rhinacanthins-C, -N and -Q showed strong antiproliferative activity against HeLaS3 cells with the IC₅₀ values of 32, 17, 70 μM; 19, 17, 52 μM and 2.7, 2.0 and 5.0 μM for the exposure time of 24, 48, and 72 h, respectively. These liposomes suppressed the tumor growth in Meth-A sarcoma-bearing BALB/c mice at the dose of 5.0 mg/kg/d for 10 d. Among rhinacanthins, liposomal rhinacanthin-N significantly suppressed solid tumor growth. Based on these results, our findings demonstrated that rhinacanthin-N suppressed tumor growth in vivo, and suggested that liposomes are useful for preparing injectable formulation of hydrophobic drugs.

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We compared particulate and microbial contamination in residual solutions of peripheral intravenous admixtures after the termination of drip infusion between intravenous fluids admixed with glass ampoule drugs and those admixed with pre-filled syringe drugs. The mean number of particles ≥1.3 μm in diameter per 1 ml of residual solution was 758.4 for fluids (n=60) admixed with potassium chloride in a glass ampoule (20 ml volume), 158.6 for fluids (n=63) admixed with potassium chloride in a pre-filled syringe (20 ml volume), 736.5 for fluids (n=66) admixed with sodium chloride in a glass ampoule (20 ml volume), 179.2 for fluids (n=15) admixed with sodium chloride in a pre-filled syringe (20 ml volume), 1884.5 in fluids (n=30) admixed with dobutamine hydrochloride in 3 glass ampoules (5 ml volume), and 178.9 (n=10) in diluted dobutamine hydrochloride in pre-filled syringes (50 ml volume: For these samples alone, particulate and microbial contamination were evaluated in sealed products.) Thus, for potassium chloride or sodium chloride for injection, the number of particles ≥1.3 μm in diameter in the residual intravenous solution was significantly higher for fluids admixed with glass ampoule drugs than for those admixed with pre-filled syringe drugs (p<0.0001). For dobutamine hydrochloride for injection, the number of particles ≥1.3 μm in diameter in the residual intravenous solution was estimated to be higher for fluids admixed with its glass ampoule drug than for those admixed with its pre-filled syringe drug. Observation of the residual solutions of fluids admixed with potassium chloride, sodium chloride, or dobutamine hydrochloride in glass ampoules using an electron microscope with an X-ray analyzer showed glass fragments in each residual solution. Therefore, for the prevention of glass particle contamination in peripheral intravenous admixtures, the use of pre-filled syringe drugs may a useful method. No microbial contamination was observed in any of the residual solutions of 5 types of admixture.

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The model penetrants oxaprozin, nimesulide, gliclazide, and ribavirin, because of their different lipophilicities, were selected to assess the enhancing activity of pre-treatment solutions consisting of isopropyl palmitate (IP) in ethanol (5%, 10%, 15%and 20%, w/w, respectively) across excised rat skin using Franz diffusion cells and HPLC detection. All pre-treatment solutions produced a significant increase in the flux and permeation of all four penetrants (p<0.001) and a relationship between penetrant lipophilicity and enhancement effect was observed. The general order of IP effectiveness at concentration was 20%>15%>10%>5% (w/w). The lag-time of drugs did not significantly change except for ribavirin.

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Glucagon-like peptide 2 (GLP-2) is a potent intestinal epithelium-specific growth factor that has been shown to reduce the severity of inflammatory disorders of the intestine in rodent models. We examined whether a relationship exists between plasma level of GLP-2 and the degree of intestinal injury induced by chemotherapeutic agents in the rat. Methotrexate (MTX) was administrated orally for 6 consecutive days at doses of 1.25, 2.5, and 5.0 mg/kg body weight per day. Mucosal samples of rat duodenum, jejunum, and ileum were used for assessment of mucosal weight, DNA and protein content. Plasma GLP-2 levels were measured on day 8. MTX significantly reduced body weight. The values of all indices tended to decrease in all segments with increases in MTX dose. Plasma GLP-2 levels were significantly higher in the MTX 2.5 mg/kg/d group (p<0.05) and the MTX 5.0 mg/kg/d group (p<0.01) than in the control group. Correlations were found between plasma GLP-2 levels and mucosal weight, DNA and protein content. We concluded that plasma GLP-2 levels reflect the degree of intestinal injury following MTX administration in this preclinical model.

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Thalidomide has been used for the treatment of refractory multiple myeloma, the dosage in Japan is lower than in other countries; however, there is little information on the pharmacokinetics and their relationship with the drug response. The aim of this study was to characterize the pharmacokinetics of low-dose thalidomide in Japanese patients with refractory multiple myeloma, and to examine the relationship between pharmacokinetics and adverse events. On the first and second days, a 100 mg capsule was administered to 8 Japanese patients after breakfast and blood samples were obtained. The plasma concentrations were measured using HPLC and analyzed based on a one-compartment model. If intolerable adverse events were not observed for 14 d, the dose was increased to 200 mg. The average apparent volume of distribution (Vd/F), apparent total clearance (CL/F) and area under the plasma concentration–time curve from 0 to infinity (AUC_0—∞), which were 45.3 l, 5.5 l/h and 21.7 μg·h/ml, respectively, with smaller Vd/F and CL/F and larger AUC_0—∞ than in Caucasian populations. This pharmacokinetic difference may explain the dose difference between Japan and other countries. Adverse events were associated with AUC_0—∞, which was best correlated with plasma concentration at 12 h after administration. The 12-h time point was suggested to be a capable indicator for “safety-oriented” therapeutic drug monitoring of thalidomide.

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We examined the protective ability of tea melanin against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity in C57BL6J mice. Reduced tea melanin (RTM) and non-reduced tea melanin (NRTM) were incorporated to distinguish anti-oxidant activity from alternative pathways. The mice were given a single oral dose of TCDD (100 μg/kg body weight) and then they were administered daily with NRTM or RTM (40 mg/kg, p.o.) for next 14 d. RTM protected the animals against TCDD-induced lipid peroxidation, inhibition of glutathione peroxidase, alteration in reduced and oxidized glutathione concentrations, loss of body weight, and increased relative liver weight. NRTM was less effective as compared to RTM because of its inferior antioxidant activity, but it still displayed a strong protective effect against TCDD toxicity owing to its similar suppression of the activity of the aryl hydrocarbon receptor. Both NRTM and RTM suppressed the expression of CYP1A1 gene and prevented the activation of cytochrome P450 isozyme in the livers of animals exposed to TCDD. These results suggest that tea melanin might be a potential agent offering dual protection against the development of TCDD-induced oxidative stress.

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