Hepatoprotective effect of resveratrol against ethanol-induced oxidative stress through induction of superoxide dismutase in vivo and in vitro.

The present study aimed to investigate the hepatoprotective effect of resveratrol (RSV) against ethanol-induced oxidative stress in vivo, and investigate the underlying mechanisms by which RSV exerts its anti-oxidative effects on hepatic cells. C57BL/6J mice were divided into four groups: Untreated control, ethanol-treated, RSV-treated, and ethanol + RSV-treated. The plasma lipid profile, hepatic lipid accumulation and antioxidative enzyme activities were analyzed. HepG2 cells were used as a cellular model to analyze the effects of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and peroxisome proliferator-activated receptors (PPARs) in the RSV-mediated protection of ethanol-induced oxidative stress. In C57BL/6J mice, ethanol caused a significant increase in plasma triglyceride levels and hepatic lipid accumulation (P<0.05), whereas RSV notably increased SOD activity. In HepG2 cells, SOD activity was enhanced in the RSV-treated HepG2 cells, whereas the activity of CAT and GPx was not affected. Western blot and quantitative polymerase chain reaction analyses demonstrated that RSV significantly increased SOD protein and mRNA expression levels (P<0.05). Using a transient transfection assay, PPARγ was observed to participate in the regulation of SOD gene expression in RSV-administered HepG2 cells. To conclude, the results from the present study suggest that RSV may contribute towards the protection of hepatic cells from ethanol-induced oxidative stress via the induction of SOD activity and gene expression.

Interaction of acupuncture and electroacupuncture on the pharmacokinetics of aspirin and the effect of brain blood flow in rats.

Acupuncture and electroacupuncture have been used to improve the brain and motor functions of poststroke patients, and aspirin is used for the prevention of stroke recurrence. Our hypothesis is that acupuncture and electroacupuncture treatments may interact with aspirin in terms of pharmacokinetics via affecting the brain blood flow. The aim of this study is to investigate the potential interactions of acupuncture and electroacupuncture on the pharmacokinetics of aspirin. The effects of acupuncture treatments on brain blood flow were measured by the laser Doppler blood flow imager. The parallel pharmacokinetic study design included three groups: control, acupuncture, and electroacupuncture groups. Two acupoints, namely, Quchi (LI 11) and Zusanli (ST 36), were needled and stimulated electronically in anaesthetized rats. The concentrations of aspirin and its metabolite, salicylic acid were determined by microdialysis and HPLC analysis after aspirin administration (30 mg/kg, i.v.). The brain blood flow responded to electroacupuncture treatments, but the pharmacokinetic parameters of aspirin and salicylic acid in blood and brain were not significantly changed by acupuncture and electroacupuncture treatments. This study may, in part, offer some evidence to support the contention that there is no significant interaction for the combination of aspirin with acupuncture or electroacupuncture.

Identification of multiple ingredients for a Traditional Chinese Medicine preparation (bu-yang-huan-wu-tang) by liquid chromatography coupled with tandem mass spectrometry.

Bu-yang-huan-wu-tang (BYHWT) is a popular Traditional Chinese Medicine formula consisting of seven herbal medicines (Astragalus membranaceus, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, Carthamus tinctorius, Amygdalus persica and Pheretima aspergillum), that has been used in China for centuries to overcome stroke-induced disability. To ensure the consistency of quality, a reliable analytical method is required, therefore, we developed a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the major constituents in BYHWT. The herbal ingredients consisting of the cycloartane-type triterpene glycosides of astragaloside I, astragaloside II and astragaloside IV; isoflavones of formononetin, ononin calycosin, calycosin-7-O-ß-d-glucoside; ligustilide and paeoniflorin were separated on a C18 column with gradient elution of methanol/10 mM ammonium acetate buffer-formic acid (100:0.1, v/v). This study was performed by a mass spectrometer using electrospray ionization (ESI) with positive ionization ions monitored in the multiple reaction-monitoring (MRM) mode. The linearity, accuracy, precision, limit of detection (LOD) and lower limit of quantification (LLOQ) were validated for this quantification method, and the sensitivity, reliability and reproducibility were all confirmed. The experiments provided a good method for analyzing BYHWT extracts. This study also quantitated the active components in various brands of commercially available products. The results indicated that the pharmaceutical industrial products of BYHWT exhibited considerable variation in their contents of the herbal compounds.

HPLC-MS/MS analysis of a traditional Chinese medical formulation of Bu-Yang-Huan-Wu-Tang and its pharmacokinetics after oral administration to rats.

Bu-yang-huan-wu-tang (BYHWT) is one of the most popular formulated traditional Chinese medicine prescriptions, and is widely for prevention of ischemic cardio-cerebral vascular diseases and stroke-induced disability. A specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been developed and validated for simultaneous quantification of the nine main bioactive components, i.e., astragaloside I, astragaloside II, astragaloside IV, formononetin, ononin, calycosin, calycosin-7-O-ß-d-glucoside, ligustilide and paeoniflorin in rat plasma after oral administration of BYHWT extract. This method was applied to investigate the pharmacokinetics in conscious and freely moving rats. No significant matrix effects were observed. The overall analytical procedure was rapid and reproducible, which makes it suitable for quantitative analysis of a large number of samples. Among them, three astragalosides and four isoflavones in A. membranaceus, ligustilide in Radix Angelicae Sinensis and Rhizoma Ligustici Chuanxiong and paeoniflorin in Radix Paeoniae Rubra were identified. This developed method was then successfully applied to pharmacokinetic studies of the nine bioactive constituents after oral administration of BYHWT extracts in rats. The pharmacokinetic data demonstrated that astragaloside I, astragaloside II, astragaloside IV and ligustilide presented the phenomenon of double peaks. The other herbal ingredients of formononetin, ononin, calycosin, calycosin-7-O-ß-d-glucoside and paeoniflorin appeared together in a single and plateau absorption phase. These phenomenona suggest that these components may have multiple absorption sites, regulation of enterohepatic circulation or the gastric emptying rate, or there is ingredient-ingredient interaction. These pharmacokinetic results provide a constructive contribution to better understand the absorption mechanism of BYHWT and to support additional clinical evaluation.

Simultaneous determination and pharmacokinetics of protein unbound aspirin and salicylic acid in rat blood and brain by microdialysis: an application to herbal-drug interaction.

Aspirin is commonly used for the prevention of myocardial infarction and ischemic stroke; whereas the Chinese people employ the bu-yang-huan-wu-tang (BYHWT) as a routine herbal formulation for the treatment and prevention of transient ischemic stroke. The current study develops a microdialysis technique coupled to a validated liquid chromatography system to measure free-form aspirin and salicylic acid for herbal-drug interaction in rat blood and brain. The intra- and inter-day precisions in biological dialysates were within 0.1-9.4% in the concentration ranges of 0.1-50 µg/mL and the accuracies ranged from -4.7 to 6.1%. The pharmacokinetic data demonstrate that the area under the concentration time curve (AUC) of the aspirin was 2031 ± 266 min µg/mL after aspirin administration (100mg/kg, i.v.). The AUC of salicylic acid was 12660 ± 1799 min µg/mL, which suggests that aspirin is quickly hydrolyzed to salicylic acid in blood and the metabolite can also be detected within 15 min in brain dialysate. The herbal-drug pharmacokinetic interaction showed no significant effect in blood and brain. The results of pharmacodynamics for the bleeding time suggested that there were no significant differences between the aspirin alone group and the BYHWT pretreated group. However, the bleeding time has been prolonged when compared aspirin alone or the group pretreated with BYHWT to the blank control. The conclusion provides practical information for clinical practice for the herbal formulation BYHWT and aspirin used concurrently.

Therapeutic potential of human elafin.

Elafin is an endogenous human protein composed of an N-terminal transglutaminase substrate motif and a C-terminal WAP (whey acidic protein)-domain with antiproteolytic properties. Elafin is expressed predominantly in epithelial tissue and potently inhibits the neutrophil-derived serine proteases elastase and proteinase-3 by a competitive tight-binding mechanism. Furthermore, it inhibits EVE (endogenous vascular elastase). Studies on several animal models show that antiprotease augmentation with human elafin is an effective strategy in the treatment of inflammatory vascular, systemic and pulmonary diseases and of inflammation triggered by reperfusion injury. This raises the possibility that elafin might be effective in the treatment of a variety of human inflammatory diseases. In a Phase I clinical trial, elafin was well tolerated. Phase II trials are underway to investigate the therapeutic effects of elafin on post-operative inflammation and the clinical consequences of major surgery. Of particular interest is the reduction of post-operative morbidity after oesophagus cancer surgery, coronary artery bypass surgery and kidney transplantation.

Reduction of CMP-N-acetylneuraminic acid hydroxylase activity in engineered Chinese hamster ovary cells using an antisense-RNA strategy.

Rodent cells, widely used for the industrial production of recombinant human glycoproteins, possess CMP-N-acetylneuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase; EC 1.14.13.45) which is the key enzyme in the formation of the sialic acid, N-glycolylneuraminic acid (Neu5Gc). This enzyme is not expressed in an active form in man and evidence suggests that the presence of Neu5Gc in recombinant therapeutic glycoproteins may elicit an immune response. The aim of this work was, therefore, to reduce CMP-Neu5Ac hydroxylase activity in a Chinese Hamster Ovary (CHO) cell line, and thus the Neu5Gc content of the resulting glycoconjugates, using a rational antisense RNA approach. For this purpose, the cDNA of the hamster hydroxylase was partially cloned and sequenced. Based on the sequence of the mouse and hamster cDNAs, optimal antisense RNA fragments were selected from preliminary in vitro translation tests. Compared to the parental cell line, the new strain (CHO-AsUH2), which was transfected with a 199-bp antisense fragment derived from the mouse CMP-Neu5Ac hydroxylase cDNA, showed an 80% reduction in hydroxylase activity. An analysis of the sialic acids present in the cells' own glycoconjugates revealed a decrease in the percentage of Neu5Gc residues from 4% in the parental cells to less than 1% in the CHO-AsUH2 cell line.

Isolation and characterization of cytidine-5'-monophosphate-N-acetylneuraminate hydroxylase from the starfish Asterias rubens.

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is formed by cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase (EC 1.14.13.45). The enzyme from mammals exhibits several unusual characteristics, raising questions about its evolution. Since echinoderms are the most primitive organisms possessing glycoconjugate-bound Neu5Gc, studies on the hydroxylase from members of this phylum may yield insights into the origin and development of the hydroxylase. Investigations on crude CMP-Neu5Ac hydroxylase in gonads from the starfish Asterias rubens revealed that it shares many properties with its mammalian counterpart. However, the echinoderm hydroxylase also exhibits fundamental differences, particularly its association with a membrane and a requirement for high ionic strength for optimal activity. Here, we describe the isolation of the CMP-Neu5Ac hydroxylase from A. rubens gonads using anion exchange chromatography and chromatography on immobilized cytochrome b(5). The enzyme was enriched 137-fold with a yield of 13%. The preparation exhibited a main polypeptide of 76 kDa, consistent with a cDNA sequence published earlier, and a minor protein of 64 kDa. A kinetic characterization showed that salt activation of this enzyme results from an increase in affinity for CMP-Neu5Ac. Evidence for the formation of a ternary complex of hydroxylase, CMP-Neu5Ac and cytochrome b(5) is also presented. The mechanistic and physiological significance of these results is discussed.

Regulation of N-glycolylneuraminic acid biosynthesis in developing pig small intestine.

N -Glycolylneuraminic acid (Neu5Gc), an abundant sialic acid in animal glycoconjugates, is formed by the enzyme CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase. The amount of Neu5Gc relative to other sialic acids is highly dependent on the species, tissue and developmental stage. Although the activity of the hydroxylase is a key factor in controlling Neu5Gc incorporation in adult animals, little is known about the regulation of hydroxylase expression and the role of this enzyme in determining changes in Neu5Gc during development. Using pig small intestine as a model system, the appearance of total sialic acid and the regulation of Neu5Gc biosynthesis during development were studied in various regions of this tissue. The amount of total sialic acid and Neu5Gc declined markedly in 2 weeks after birth. Although in subsequent developmental phases there were no positional differences in total sialic acid, a significant proximal-to-distal increase in Neu5Gc was detected. In all cases, a good correlation between the amount of Neu5Gc, the activity of the hydroxylase and the level of hydroxylase mRNA was observed. However, Western-blot analysis revealed considerable accumulation of less active enzyme in the post partum period, which persisted until adulthood. No evidence for cytosolic factors influencing the hydroxylase activity or for the formation of truncated enzyme was found, raising the possibility that other regulatory mechanisms are involved. The relevance of these results in the formation of Neu5Gc as a receptor for certain pig enteric pathogens is also discussed.

Calcium channel blockade limits transcriptional, translational and functional up-regulation of the cardiac calpain system after myocardial infarction.

Abnormal Ca(2+) inward current through cardiac Ca(2+) channels during ischemia has been shown to be an initial signal for activation of myocardial Ca(2+)-dependent enzymes. This study investigated the contribution of cardiac L- and T-type Ca(2+) channels in the calpain-mediated myocardial damage following myocardial infarction. Myocardial infarction was induced by permanent ligation of the left coronary artery. Infarcted rats were orally treated with placebo, amlodipine (L-channel blockade; 4 mg/kg/day) or mibefradil (L-/T-channel blockade; 10 mg/kg/day) beginning 7 days before induction of myocardial infarction. Gene expression, protein levels and enzyme activity of calpains I and II were measured 1, 3, 7 and 14 days postcoronary occlusion in the noninfarcted and infarcted myocardium. Infarct size, left ventricular dilation and interstitial collagen volume fraction were determined in picrosirius red-stained hearts. Myocardial infarction induced an up-regulation of calpain I mRNA, protein and activity in the noninfarcted myocardium (maximum 14 days postinfarction), whereas mRNA, protein and activity of calpain II were maximally increased in the infarcted myocardium 3 days postinfarction. Fourteen days postinfarction, infarct size was 49%, the left ventricle was dilated and interstitial collagen volume fraction was increased. Amlodipine-inhibited mRNA, protein and activity up-regulation of calpain I decreased interstitial collagen volume fraction and infarct size. Mibefradil-attenuated mRNA, protein and activity up-regulation of calpain II at all four time points measured and of calpain I at 7 and 14 days postinfarction reduced infarct size and prevented left ventricular dilation. Infarction-induced cardiac hypertrophy was accompanied by an up-regulation of calpain I, whereas calpain II was up-regulated in the infarcted myocardium. Cardiac L- and T-type Ca(2+) channel blockade differentially reduced postinfarction remodeling associated with selective inhibition of cardiac calpains I and II, respectively.

Activity profile of calpains I and II in chronically infarcted rat myocardium--influence of the calpain inhibitor CAL 9961.

1. The calpains have been proposed to be activated following cardiac ischaemia and to contribute to myocyte damage after myocardial infarction (MI). In this study, the activity of calpains I and II in the infarcted and non-infarcted rat myocardium and the action of the selective calpain inhibitor, CAL 9961, has been investigated. 2. MI was induced by permanent ligation of the left coronary artery. One, 3, 7 and 14 days post MI, the enzymes calpain I and II were separated from homogenates of the interventricular septum (IS) and left ventricular free wall (LVFW) by chromatography on DEAE-Sepharose. The activity of the calpains was measured in sham-operated and MI animals chronically treated with placebo or CAL 9961 (15 mg kg(-1) d(-1) s.c.) in a synthetic substrate assay. Treatment was started 3 days before MI induction. 3. Calpain I activity reached highest values in IS 14 days post MI, whereas maximum activity of calpain II was measured in LVFW 3 days post MI. In experiments in vitro, CAL 9961 completely inhibited both calpains. In vivo, chronic treatment of MI animals with CAL 9961 partially prevented the increase in calpain I activity in IS and reduced calpain II activity in LVFW to sham levels. 4. Our findings demonstrate that calpains I and II are activated after MI, however, both enzymes differ in their regional and temporal activation within the infarcted myocardium. Chronic inhibition of these enzymes with CAL 9961 might limit the calpain-induced myocardial damage and preserve cardiac structural integrity post MI.

Reversal of the adult IgE high responder phenotype in mice by maternally transferred allergen-specific monoclonal IgG antibodies during a sensitive period in early ontogeny.

IgE is an important trigger in allergy and asthma, diseases whose development is suggested to depend on an initial sensitization in early life. While induction of murine IgE responses requires both a genetically based IgE high responder phenotype and defined experimental conditions, maternally transferred IgG can override these prerequisites and suppress IgE formation in an allergen-specific manner. Here, we show that maternally transferred monoclonal IgG, irrespective of their subclass and recognized epitopes, induce IgE unresponsiveness, which is effective for parenteral immunization with bee venom phospholipase A2 as well as for airway-immunization with nebulized ovomucoid-containing ovalbumin. This IgE suppression is detectable in the offspring during the first 4 months of life, but not thereafter and not in the dams. However, when the initial immunization at an age of 3 or 4 months was followed by further application of both allergens via their respective routes, IgE suppression persisted up to an age of more than one year. If applicable to man, these findings may allow the development of a new strategy for the prevention of allergy and asthma by maternally transferred or neonatally injected allergen-specific mAb in combination with natural or prophylactic exposure to the respective allergens during early childhood.

Alternative splicing of fructose 1,6-bisphosphate aldolase transcripts in Drosophila melanogaster predicts three isozymes.

The genes that encode fructose 1,6-bisphosphate aldolase of Drosophila melanogaster have been isolated and characterized. These genes exist in a single copy 8-kilobase pair locus in the Drosophila genome which is located at cytogenetic position 97A-B. The nucleotide sequence and transcript mapping suggest that three overlapping protein isozyme genes may be encoded at this locus. These isozyme genes all share a single promoter, a 5'-untranslated first exon, and two other protein coding exons. The isozyme-specific carboxyl-terminal amino acids are encoded by one of three alternatively utilized fourth exons: 4A, 4B, or 4C by alternative splicing. The transcript containing exon 4C, whose sequence has been reported previously, is abundant throughout development and has a developmental profile similar to other glycolytic gene transcripts; however, it shows developmental specificity in the alternative use of two polyadenylation signals which result in a 2.4-kilobase and a 1.9-kilobase transcript. The transcript containing exon 4B is 1.6 kilobases in size and is most abundant during the larval stages and during the time of eclosion. The transcript containing exon 4A is in low abundance and found only during the adult stage. Sequence comparisons of the alternative fourth exons indicate that the duplication leading to the multiple exons is quite old and preceded the origin of the genus Drosophila.

Structure and expression of the triose phosphate isomerase (Tpi) gene of Drosophila melanogaster.

We report the isolation of the genomic sequence that encodes the enzyme triose phosphate isomerase of Drosophila melanogaster. There is a single copy of the Tpi sequence in the genome of Drosophila, as judged by Southern blots and in situ hybridization to salivary gland chromosomes. The sequence of 3414 nucleotides from the Tpi region was determined. The gene has an intron in the 5' untranslated region of the transcript and a second intron in the coding region at an evolutionarily conserved position. Transcripts initiate at a single site which does not have a TATA box in the usual position. Northern blot analysis of RNA prepared from different developmental stages revealed that Tpi mRNA is present in substantial amounts in oocytes, declines in abundance in early embryos, and begins to increase during mid-embryogenesis. Transcript abundance follows a pattern typical of enzymes involved in intermediate metabolism. A peak is found during third instar followed by a decline during pupal stages and then a second rise near the time of eclosion.