[Single extract of Forsythia Suspense versus the prepared drug in pieces: comparison of their anti-inflammatory, antitumor and antibacterial effects in zebrafish].

OBJECTIVE: To compare the anti-inflammatory, antitumor and anti-bacterial effects of the single extract (in granules) and the prepared drug in pieces of Forsythia Suspense (Lianqiao, a traditional Chinese herbal medicine). METHODS: In zebrafish embryo models of CuSO4 exposure, tail transection and LPS microinjection-induced inflammation, the anti-inflammatory effects of 10 µg/mL DEX, single extract of Forsythia Suspense, and the water extract of the prepared drug (400, 600, and 800 µg/mL) were evaluated by observing neutrophil counts, RT- qPCR, HE staining and survival analysis. Zebrafish embryo models bearing different human tumor cell xenografts were used to assess the anti-tumor effect of the drugs in different dosage forms by fluorescence staining and HE staining. The microbroth dilution method was used to evaluate the antibacterial efficacy of the drugs. RESULTS: In the zebrafish embryo models of inflammation, both of the two dosage forms of Forsythia Suspense significantly inhibited neutrophil aggregation, reduced the mRNA expressions of TNF-α, IL-6, P38, Jnk, Erk and P65, and increased the survival rate of zebrafish. They both showed obvious inhibitory effects against xenografts of different human cancer cells including colon cancer cells (HCT116), pancreas adenocarcinoma cells (PANC-1), lung cancer cells (A549), liver cancer cells (Hep3B) and cervical carcinoma cells (Hela) in zebrafish embryos, and exhibited strong anti-bacterial effects at the concentration of 15.63 mg/mL. CONCLUSION: The two dosage forms of Forsythia Suspense have similar anti-inflammatory, antitumor and antibacterial effects, but their effects for inhibiting IL-6, P65, and Jnk mRNA expressions and HCT116 cell proliferation differ significantly at low doses in zebrafish.

[Isodopharicin C inhibits NLRP3 inflammasome activation and alleviates septic shock in mice].

OBJECTIVE: To investigate the effect of Isodopharicin C (Iso C), a traditional Chinese herbal medicine extract, on NLRP3 inflammasome activation and lipopolysaccharide (LPS)-induced septic shock in mice. METHODS: Murine bone marrow-derived macrophages (BMDM) and human monocytic THP-1 cells were stimulated with LPS before treatment with different NLRP3 inflammasome agonists to activate canonical NLRP3 inflammasomes. The non-canonical NLRP3 inflammasomes were activated by intracellular LPS transfection, and AIM2 inflammasomes were activated with poly A: T. The cleavage of caspase-1 induced by NLRP3 activation was measured using Western blotting. The levels of NLRP3-dependent and -independent pro-inflammatory cytokines in the cell culture supernatant were detected using ELISA, and the intracellular potassium ion concentration was measured using ICP-OES. In the animal experiment, C57BL/6J mouse models of septic shock (induced by intraperitoneal LPS injection) were treated with Iso C, and the levels of IL-1ß, TNF-α and IL-6 in the serum and peritoneal lavage fluid were detected using ELISA. The survival time of the mice was observed within 48 h after LPS injection and a survival curve was plotted. RESULTS: In BMDM cells, Iso C dose-dependently inhibited the activation of canonical NLRP3 inflammasomes and non-canonical NLRP3 inflammasomes (P<0.05) without obviously affecting the secretion levels of TNF-α and IL-6 (P>0.05), the activation of AIM2 inflammasomes (P>0.05), or K + efflux, the upstream signaling of NLRP3 activation (P>0.05). Iso C inhibited the activation of canonical NLRP3 inflammasomes in human THP-1 cells. In septic C57BL/6J mice, Iso C treatment significantly reduced IL-1ß levels in the serum and peritoneal lavage fluid, and prolonged the survival time of the mice (P<0.05). CONCLUSION: Iso C specifically inhibits NLRP3 inflammasome activation and alleviates septic shock in mice, and can serve as a potential small molecule compound for treatment of inflammatory diseases.

Garlic skin induces shifts in the rumen microbiome and metabolome of fattening lambs.

Garlic (Allium sativum L.) and its constituents have been shown to modify rumen fermentation and improve growth performance. Garlic skin, a by-product of garlic processing, contains similar bioactive components as garlic bulb. This study aimed to investigate the effects of garlic skin supplementation on growth performance, ruminal microbes, and metabolites in ruminants. Twelve Hu lambs were randomly assigned to receive a basal diet (CON) or a basal diet supplemented with 80 g/kg DM of garlic skin (GAS). The experiment lasted for 10 weeks, with the first 2 weeks serving as the adaptation period. The results revealed that the average daily gain and volatile fatty acid concentration were higher (P < 0.05) in lambs fed GAS than those in the CON group. Garlic skin supplementation did not significantly (P > 0.10) affect the α-diversity indices, including the Chao1 index, the abundance-based coverage estimator value, and the Shannon and Simpson indices. At the genus level, garlic skin supplementation altered the ruminal bacterial composition by increasing (P < 0.05) the relative abundances of Prevotella, Bulleidia, Howardella, and Methanosphaera and decreasing (P < 0.05) the abundance of Fretibacterium. Concentrations of 139 metabolites significantly differed (P < 0.05) between the GAS and the CON groups. Among them, substrates for rumen microbial protein synthesis were enriched in the GAS group. The pathways of pyrimidine metabolism, purine metabolism, and vitamin B6 metabolism were influenced (P < 0.05) by garlic skin supplementation. Integrated correlation analysis also provided a link between the significantly altered rumen microbiota and metabolites. Thus, supplementation of garlic skin improved the growth performance of lambs by modifying rumen fermentation through shifts in the rumen microbiome and metabolome.

Berberine binds RXRα to suppress ß-catenin signaling in colon cancer cells.

Berberine, an isoquinoline alkaloid, is a traditional oriental medicine used to treat diarrhea and gastroenteritis. Recently, we reported that it could inhibit the growth of intestinal polyp in animals and in patients with the familial adenomatous polyposis by downregulating ß-catenin signaling. However, the intracellular target mediating the effects of berberine remains elusive. Here, we provide evidence that berberine inhibits ß-catenin function via directly binding to a unique region comprising residues Gln275, Arg316 and Arg371 in nuclear receptor retinoid X receptor alpha (RXRα), where berberine concomitantly binding to and synergistically activating RXRα with 9-cis-retinoic acid (9-cis-RA), a natural ligand binding to the classical ligand-binding pocket of RXRα. Berberine binding promotes RXRα interaction with nuclear ß-catenin, leading to c-Cbl mediated degradation of ß-catenin, and consequently inhibits the proliferation of colon cancer cells. Furthermore, berberine suppresses the growth of human colon carcinoma xenograft in nude mice in an RXRα-dependent manner. Together, our study not only identifies RXRα as a direct protein target for berberine but also dissects their binding mode and validates that berberine indeed suppresses ß-catenin signaling and cell growth in colon cancer via binding RXRα, which provide new strategies for the design of new RXRα-based antitumor agents and drug combinations.

Iron intake, serum iron indices and risk of colorectal adenomas: a meta-analysis of observational studies.

To provide a quantitative assessment of the association between iron intake, serum iron indices and the risk of colorectal adenoma (CRA), we summarised the evidence from observational studies. Relevant studies were identified in MEDLINE and EMBASE until 31 March 2015. Summary relative risks (SRRs) with 95% confidence intervals (CIs) were pooled with a random-effects model. A total of 10 articles, involving 3318 subjects with CRA, were used in this meta-analysis. The SRR of CRA was 0.83 (95% CI: 0.71-0.98; Pheterogeneity  = 0.694, I2  = 0) for the highest versus the lowest level of dietary iron intake. The SRR was 0.93 (95% CI: 0.62-1.42) for total (dietary and supplemental) iron intake, 1.23 (95% CI: 1.03-1.48) for haem iron intake and 0.73 (95% CI: 0.54-0.97) for supplemental iron intake. Serum iron indices were not associated with CRA risk (serum ferritin: SRR = 1.16, 95% CI: 0.81-1.66; serum iron: SRR = 1.02, 95% CI: 0.75-1.38; serum transferrin saturation: SRR = 1.01; 95% CI: 0.82-1.50). Increased intake of haem iron is associated with significantly increased risk of CRA, whereas intake of non-haem or supplemental iron is inversely associated with risk of CRA. Limited data indicate null associations between serum iron indices and CRA risk.

Genetic variability and diversity of the main resources of lily assessed via phenotypic characters, pollen morphology, and ISSR markers.

Lily (Lilium spp), which belongs to Lilium, is one kind of monocotyledon. As a perennial ornamental plant with extremely high esthetic, edible, and medicinal value, lily has gained much favor due to its mostly showy flowers of various colors and elegant shape. In this research, we studied experimental materials in a sample of 49 individuals including 40 cultivars, nine species of wild lily, and their variants. The collection of 40 cultivars covered all six hybrids in the genus, i.e., Asiatic hybrids, Oriental hybrids, Longiflorum hybrids, LA hybrids, LO hybrids, and OT hybrids. Genetic diversity and inter-relationships were assessed through analysis of phenotypic characteristics, pollen morphology, and ISSR molecular markers. Quantitative characters were selected to analyze phenotypic variation, with results indicating greater variability in petiole length as compared to other characters. Pollen morphological observations suggested that the largest variation coefficient between all hybrids and wild species was the lumina. ISSR makers demonstrated that both cultivars and wild species possess a high level of genetic diversity. Specifically, the genetic diversity of wild lily was higher than cultivars.

[Changes of brain pain center and default mode network an electro acupuncture in Weizhong and Dachangshu acupoints: a task-fMRI study].

OBJECTIVE: To investigate the functional brain pain center and default mode network response to electro acupuncture stimulate in weizhong acupoints(BL40) and dachangshu acupoints(BL25). METHODS: During January to February 2015, volunteers were enrolled in this study from the staff and student interns of Gansu Province Traditional Chinese Medicine Hospital. A total of 20 healthy, right-handed subjects, male 9, female 11, age (23±3) years, participated in this study. Block design task functional magnetic resonance imaging(fMRI) 3.0 T was performed in all subjects by electro acupuncture stimulating at BL40 and BL25 from the same experienced acupuncturist.The needle connected electric acupuncture apparatus through tow long coaxial-cable. A block design with five 120 s blocks of rest time (OFF block, electric acupuncture turn off ) interspersed between five 60 s blocks of stimulation (ON block, electric acupuncture turn on) fMRI scan. Magnetic resonance data of brain function was collected and FSL(fMRI Software Library) software was used to analyze the data. RESULTS: All subjects' data were analyzed except 2 cases whose head movement were more than 2 mm. Activated brain function regions by electro acupuncture stimulate included temporal lobe lateral sulcus, lobus insularis, thalamus, supramarginal gyrus, prefrontal medial frontal gyrus. Negative activated brain regions included middle frontal gyrus, parahippocampal gyrus, cingulate cortex abdominal segment, parietal cortex.The functional pain central and default mode network were changed when electro acupuncture stimulate in(BL40) and(BL25). CONCLUSION: There are several brain activation regions and negative activated brain regions when administering electro acupuncture stimulation at BL40 and BL25.

In vivo effects of high dietary copper levels on hepatocellular mitochondrial respiration and electron transport chain enzymes in broilers.

The diet of broiler chickens supplemented with increasing concentrations of copper (Cu) was assessed for the effect of Cu on liver mitochondrial function. A total of 160, 1-d-old Cobb 500 broilers (Gallus domesticus) were randomly assigned in equal numbers into 4 groups, which differed in the concentration of copper supplements in the diet; 11 (control), 110, 220 and 330 mg of Cu/kg dry matter. Liver mitochondrial function was recorded at 12, 24, 36, 48 and 60 d of age. Supplementation with 110 mg Cu/kg dry matter enhanced mitochondrial function and activities of complexes I-V, and this was significant at 36 d of age compared with the other diets (P < 0.05). Supplementation with 220 mg Cu/kg dry matter and 330 mg Cu/kg dry matter enhanced mitochondrial function and activities of complexes I-V at 12, 24 and 36 d of age, but displayed reduced function (P < 0.05) at 48 and 60 d of age except in complex IV (P > 0.05). Mitochondrial hydrogen peroxide (H2O2) production was also increased (P < 0.05) with an increase of copper supplementation in the diet. The results indicate that appropriate dietary copper supplements are sufficient for improving mitochondrial function and activities of the respiratory complexes. Higher concentrations of copper, on the other hand, lead to copper toxicity by affecting certain respiratory complexes.

Otx1 promotes basal dendritic growth and regulates intrinsic electrophysiological and synaptic properties of layer V pyramidal neurons in mouse motor cortex.

The transcription factor Otx1 is specifically expressed in layer V pyramidal cells (L5PCs) in the cerebral cortex. Otx1 null mutant mice have a defect in the developmental axon pruning of L5PCs and show epileptic seizures. However, the role of Otx1 in electrophysiology, morphology and synaptology of the cortical neurons has not been fully investigated. This study examines the influences of Otx1 on neuronal properties of L5PCs by loss- and gain-of-function approaches. Mice with an Otx1-null mutation had decreased structural measurements of basal dendrites in L5PCs. In contrast, the size of basal dendrites was increased in the Otx1-over-expressed pyramidal cells (PCs) in L2/3 where the gene normally does not express. PCs showed burst and non-burst firing patterns of action potentials. The proportion of burst firing neurons was reduced in the Otx1 mutant but increased in the neurons over-expressing Otx1. Although the burst firing population decreased, the proportion of those bursting neurons with a low threshold increased in the Otx1 mutant mice. Moreover, excitatory facilitating synaptic connections formed between L5PCs were predominant in the Otx1 mutant mice, which greatly contrasted with the predominant depressing synaptic connections in the controls. Taken together, it suggests an enhanced activity of neuronal network in the cortex of Otx1 mutant mice. These data indicate that the Otx1 expression is essential for the normal development of dendritic morphology, intrinsic electrophysiology and synaptic dynamics of L5PCs. This study provides new insights into molecular mechanisms underlying the spatial and temporal regulation of neuronal and synaptic properties of L5PCs, and improves our understanding on the generation of epileptic seizures.

Characterization of 236 novel alleles at the HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 loci from China Marrow Donor Program.

Two hundred and thirty-six novel human leukocyte antigen (HLA) alleles are described from volunteer donors of the China Marrow Donor Program: 71 HLA-A alleles, 79 HLA-B alleles, 43 HLA-C, 16 HLA-DRB1 alleles, 26 HLA-DQB1 and 1 HLA-DPB1. Two hundred and thirteen (90.3%) of the 236 novel alleles are single nucleotide substitution variants when compared with their most homologous allele. Seventy-eight of these single nucleotide variants are silent substitutions. The remaining novel alleles differ from their most similar allele by two to four nucleotide substitutions. Some of the novel alleles encode amino acid changes at positions not previously reported to be polymorphic, such as codons 57, 62, 67, 41 and 52 in HLA-A alleles; codons 133, 156, 201 and 215 in HLA-B alleles; codons 74, 208 and 225 in HLA-C; codons 25, 32 and 72 in HLA-DRB1; codons 20, 39 and 77 in HLA-DQB1.

Influence of organic zinc supplementation on the antioxidant status and immune responses of broilers challenged with Eimeria tenella.

An experiment was conducted to investigate the influence of Zn supplementation on the performance, antioxidant status, and immune responses of broilers challenged with Eimeria tenella. A total of 384 male broilers (1 d old) were assigned to 8 treatments consisting of 8 replicates of 6 chicks each. A basal corn-soybean meal diet (29.6 mg of Zn/kg) was supplemented with methionine hydroxyl analog-Zn chelate at 0, 20, 40, and 60 mg/kg of diet. At 21 d of age, birds were orally gavaged with 1.5 × 10(4) sporulated E. tenella oocysts. Dietary Zn supplementation had no effect on growth performance of either the challenged or nonchallenged birds. Activities of Cu-Zn superoxide dismutase and glutathione peroxidase were increased (P < 0.001) with increasing Zn levels in both the challenged and nonchallenged groups. Lipid peroxidation tended to be reduced (P = 0.08) at Zn inclusion of 20 and 40 mg/kg. In vitro lymphocyte proliferation responses to mitogen concanavalin A and LPS were not influenced by dietary Zn or challenge. The main effects of Zn level and challenge were significant for secretory IgA on d 28 (P < 0.01) and 35 (P < 0.001). During both periods, secretory IgA of birds receiving dietary Zn supplementation was higher (P < 0.05) than that of those receiving no Zn supplementation. Birds fed Zn supplementation excreted fewer oocysts in the excreta than those receiving no Zn supplement (P < 0.001). Results indicated that organic Zn supplementation reduced oxidative stress and improved some immune responses irrespective of whether birds were healthy or challenged with E. tenella.

Effect of different rumen-inert fatty acids supplemented with a dietary antioxidant on performance and antioxidative status of early-lactation cows.

The objective of the study was to evaluate the effect of diets supplemented with fatty acids of different degrees of saturation, in the absence or presence of an antioxidant (AOX; Agrado Plus, Novus International Inc., St. Charles, MO), on dairy cow lactation performance. Calcium salts of long-chain fatty acids were supplemented as a source of lower saturation fatty acid, and a palm acid product was supplemented as the higher saturation fatty acid source. Sixty early-lactation Chinese Holstein cows (100+/-23 d in milk) were randomly allocated to 4 dietary treatments in a 2 x 2 factorial design: (1) lower saturation fatty acid (LS), (2) LS and AOX, (3) higher saturation fatty acid (HS), and (4) HS and AOX. The Ca salts of long-chain fatty acids and palm acid product were supplied at 1.8 and 1.5% on a dry matter basis, respectively, to form isoenergetic diets. The AOX was added at 0.025% in the ration. The experiment lasted 9 wk, including 1 wk for adaptation. Lactation performance was recorded and milk was sampled and analyzed weekly. Blood samples were taken from the coccygeal vein to determine metabolism parameters on d 16, 36, and 56 during the experiment. Neither fatty acid type nor AOX supplementation showed a significant effect on dry matter intake during the study. Milk yield was lower in the LS-fed cows compared with the cows fed HS. Milk fat and milk protein concentrations were not affected by fatty acid type or AOX supplementation. Adding AOX increased the yield of milk in the LS-fed cows, but did not affect those fed HS. Activity of plasma superoxide dismutase was significantly lower, plasma glucose tended to be lower, and plasma malondialdehyde was higher in the LS-fed animals compared with those fed HS. Addition of AOX decreased both plasma nonesterified fatty acids and hydrogen peroxide contents and increased total antioxidant capacity across the fatty acid types. Plasma beta-hydroxybutyrate was not affected by fatty acid type or AOX treatment. Cows fed LS had higher cis-9C(18:1) and trans-10, cis-12C(18:2) in milk at the expense of C(18:0), whereas AOX addition increased milk cis-9C(18:1) at the expense of milk C(12:0), C(16:0), and trans-10, cis-12C(18:2). It is inferred that feeding LS resulted in inferior lactation performance, whereas addition of antioxidant partially alleviated these negative effects.

Pharmacokinetic studies of a Chinese triple herbal drug formula.

Yin Chen Hao Tang preparation (YCHTP) is a classic traditional Chinese medicine formula, which is commonly used for clinical treatment of hepatological diseases. In this study, a rapid and validated high-performance liquid chromatography (HPLC) method was developed to simultaneously identify 6,7-dimethylesculetin and geniposide in rat plasma. This assay was performed on a Dikma Diamonsil RP(18) column (200 mm×4.6 mm, 5 µm) with acetonitrile-methanol-water (0.1% formic acid) as the mobile phase, showing acceptable linearity, intra- and inter-day precision and accuracy (R.S.D.=5%), and absolute recovery for two analytes (74%); the limits of quantitation were 0.4 and 1.12 µg/ml, and the limits of detection were 0.06 and 0.09 µg/ml for two analytes. The developed method was successfully applied to study the effect of formula compatibility on the pharmacokinetics of 6,7-dimethylesculetin and geniposide in YCHTP when orally administrating an effective human daily dose of YCHTP to rats. We surmise that formula compatibility can significantly influence the pharmacokinetics of YCHTP, and we have elucidated and validated the compatible administration of YCHTP.

Evidence for suppression of electroacupuncture on spinal glial activation and behavioral hypersensitivity in a rat model of monoarthritis.

Our previous study demonstrated that single intrathecal (i.t.) application of fluorocitrate, a glial metabolic inhibitor, synergized electroacupuncture (EA) antagonizing behavioral hypersensitivity in complete Freund's adjuvant (CFA)-induced monoarthritic rat. To further investigate the relationship between spinal glial activation and EA analgesia, the present study examined the effects of multiple EA on spinal glial activation evoked by monoarthritis (MA). The results showed that (1) unilateral intra-articular injection of CFA produced a robust glial activation on the spinal cord, which was associated with the development and maintenance of behavioral hypersensitivity; (2) multiple EA stimulation of ipsilateral "Huantiao" (GB30) and "Yanglingquan" (GB34) acupoints or i.t. injection of fluorocitrate (1 nmol) significantly suppressed spinal glial activation; (3) inhibitory effects of EA on spinal glial activation and behavioral hypersensitivity were significantly enhanced when EA combined with fluorocitrate, indicating that disruption of glial function may potentiate EA analgesia in inflammatory pain states. These data suggested that analgesic effects of EA might be associated with its counter-regulation to spinal glial activation, and thereby provide a potential strategy for the treatment of arthritis.

Acaricidal activities of extracts of Kochia scoparia against Tetranychus urticae, Tetranychus cinnabarinus, and Tetranychus viennensis (Acari: Tetranychidae).

Extracts of an annual herbaceous plant, Kochia scoparia (L.) Schrad (Macrophomina), were bioassayed to determine their acaricidal activities against Tetranychus urticae Koch, Tetranychus cinnabarinus (Boisduval), and Tetranychus viennensis Zacher (Acari: Tetranychidae) in the laboratory. Extracts had both contact and systemic toxicity to these mites. Three solvents were tested for preparing crude extracts: petroleum ether, chloroform, and methanol. Methanol was the most effective solvent, extracting 3.11-4.53% of the acaricide. Petroleum ether was the least effective solvent, extracting 1.25-1.54%. However, extracts with chloroform resulted in the highest mite mortality (78.86%), and ultrasound-assisted extraction required the least time (10 min). Concentrated extracts were prepared using chloroform, methyl acetate, or distilled water as a solvent. Mite mortalities from the concentrated extracts by methyl acetate or distilled water were significantly lower than those by chloroform. The mean lethal concentrations (LC50) of the extracts by chloroform, methyl acetate, and distilled water to the mites were 0.71 +/- 0.06, 2.08 +/- 0.16 and 8.75 +/- 0.062 mg/ml, respectively. After liquid chromatography and thin layer chromatography, the concentrated extracts by chloroform were separated into seven groups of isolated fractions and tested for acaricidal activity.

Acaricidal activities of extracts of Stellera chamaejasme against Tetranychus viennensis (Acari: Tetranychidae).

Extracts of a perennial and poisonous weed, Stellera chamaejasme L. (Thymelaeaceae) were bioassayed to determine their acaricidal activities against Tetranychus viennensis Zacher (Acari: Tetranychidae) in the laboratory. Extracts had both contact and systemic toxicity to the mite. After liquid chromatography and thin layer chromatography, the extract was further concentrated, separated, and bioassayed. This study is the first to report the acaricidal property of S. chamaejasme and its potential as a botanical pest control agent.

Identification of inhibitors of bacterial transcription/translation machinery utilizing a miniaturized 1536-well format screen.

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC(50) values derived for this assay in a 1536-well format and 384-well format.

[Identification of notoginseng(Panax notoginseng) and its adulterants using DNA sequencing].

OBJECTIVE: To analyze the nuclear ribosomal RNA small subunit (18S rRNA) and chloroplast matK gene sequence of notoginseng (Panax notoginseng) in order to provide molecular evidence for its genuine origin identification. METHODS: To sequence 18S rRNA and matK genes of Panax notoginseng and its four adulterants such as P. japonicus, Curcuma phaeocaulis, C. wenyujin, C. kwangsiensis using PCR direct sequencing and to detect their variation of sequences. RESULTS: The sequence length of notoginseng and its adulterants is 1809-1811 bp for 18S rRNA gene and 1259-1548 bp for matK gene, respectively. Multiple sequence alignment shows that there are much sequence variation between notoginseng and its adulterants. CONCLUSION: DNA sequencing is an accurate and reliable method in origin identification of the genuine notoginseng.

Development of a high throughput equilibrium dialysis method.

The identification of large numbers of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with drug-like properties early during the lead prioritization and development processes. One of the major steps in lead prioritization is an assessment of compound binding to plasma proteins, because it affects both the pharmacokinetics and pharmacodynamics of the compound in vivo. Equilibrium dialysis is the preferred method to determine the free drug fraction, because it is less susceptible to experimental artifacts. However, even low-volume standard equilibrium dialysis is currently not amenable to the HTS format. Those considerations dictate the development of a high throughput equilibrium dialysis device, without compromising the analytical quality of the data. The present paper demonstrates successful development of a 96-well format equilibrium dialysis plate. Plasma protein binding of three drugs, propranolol, paroxetine, and losartan, with low, intermediate, and high binding properties, respectively, were chosen for assay validation. The data indicate that the apparent free fraction obtained by this method correlates with the published values determined by the traditional equilibrium dialysis techniques.

[Nuclear ribosomal RNA small subunit (18S rRNA) nucleotide sequencing and characterization of sailonggu(whole bone of Myospalax baileyi Thomas)].

OBJECTIVE: Sequencing the nuclear ribosomal RNA small subunit (18S rRNA) gene of Myospalax baileyi (Cricetidae) to develop an ultimate and definitive means for origin identification of genuine Sailonggu. METHODS: The total DNA was prepared from dried tail tissues. The nuclear 18S rRNA gene region was amplified by PCR using a consensus primer set and its nucleotide sequence was determined by PCR direct sequencing. The characteristic analysis of 18S rRNA sequences was generated using software program Genetyx-SV/R Version 10.1. RESULTS: The entire 18S rRNA gene region of M. baileyi spanded 1851 bp in length. Although multiple alignment of sequence indicates that there are only lower homology (72.04%-72.18%) comparing with its two alias Mus musculus (GenBank Accession number X00686) and Rattus norvegicus (M11188) (Muridae), their highly conservative domain is located in 1020-1509 nt. There are many variable sites from upstream of 5'-end, which could provide a novel information for molecular recognition of Sailonggu. CONCLUSION: DNA sequencing could be a useful and reliable tool in the origin identification of genuine Sailonggu.