[Correlation of gut microbiota and ischemic stroke: a review].

With the widespread application of next-generation sequencing(NGS), especially 16 S rRNA and shotgun sequencing, researchers are no longer troubled with massive data on the gut microbiota, and the correlation between the gut microbiota and the brain(central nervous system) has been gradually revealed. Research on the microbiota-gut-brain axis(MGBA) based on the gut microbiota have provided insights into the exploration of the pathogenesis and risk factors of ischemic stroke(IS), a cerebrovascular disease with high disability and mortality rates, and also facilitate the selection of therapeutic targets of this class of drugs. This study reviewed the application of NGS in the study of gut microbiota and the research progress of MGBA in recent years and systematically collated the research papers on the correlation between IS and gut microbiota. Furthermore, from the bi-directional regulation of MGBA, this study also discussed the high-risk factors of IS under the dysregulation of gut microbiota and the pathophysiological changes of gut microbiota after the occurrence of IS and summarized the related targets to provide a reliable reference for the therapeutic research of IS from the gut microbiota.

[Research and application of quercetin-loaded nano drug delivery system].

Quercetin has a variety of biological activities and pharmacological effects, but its clinical application is limited due to its low water solubility, low bioavailability and poor chemical stability. In order to overcome the above shortcomings of quercetin and achieve better clinical efficacy, many new drug delivery systems have been studied and developed in recent years, of which nano drug delivery system has become a research hotspot. Nano drug delivery system includes nanoparticles, nanocapsules, nanoliposomes, nanomicelles, nanosuspensions and nanoemulsions, with the advantages of reducing particle size, enlarging surface area, increasing drug permeability, improving intracorporeal circulation and distribution of the drug, enhancing drug targeting and so on. This article reviewed the latest progress at home and abroad about the studies and application of quercetin in nano drug delivery system, such as increasing solubility and dissolution, elevating bioavailability, raising drug stability, delaying drug release, enhancing skin permeability, promoting antioxidant capacity and improving therapeutic effect, in the hope of providing references for further research of quercetin preparations.

Expression of AQP2, AQP4 and AQP 8 in mouse intestine induced by unprocessed and processed Euphorbia lathyris.

The present research was designed to study expression of AQP2, AQP4 and AQP8 in mouse intestines induced by unprocessed and processed Euphorbia lathyris. KM mice were given by different dose lavage of unprocessed and processed Euphorbia lathyris, Euphorbia factor L1, Euphorbia factor L2, Euphorbia factor L3. Samples of mouse intestine were collected for protein levels of AQP2, AQP 4 and AQP 8 which were assessed by immunohistochemical staining and mRNA expression of AQP2, AQP 4 and AQP 8 which were quantified by Real Time-PCR. Comparing to the normal control group, the protein levels of AQP2, AQP 4 and AQP 8 were significantly decreased (P<0.05)by Semen Euphorbiae group and Semen Euphorbiae Pulveratum group (unprocessed and processed Euphorbia lathyris) induced. Protein expression of AQP2, AQP 4 and AQP 8 in the Euphorbia factor L1, Euphorbia factor L2 and Euphorbia factor L3 group were not significantly lower than normal control group. There had no differences on the levels of AQP2 and AQP 8 mRNA expressions between the high-dose group of semen Euphorbiae group, semen Euphorbiae Pulveratum group and positive control group, while significantly lower than normal control group (P<0.05). Expression of AQP4 mRNA in the Semen Euphorbiae group and Semen Euphorbiae Pulveratum group has not significantly decreased. But levels of AQP2, AQP 4 and AQP 8 mRNA in the Euphorbia factor L1 group had no significant differences in normal control group and positive control group. These findings suggest that semen Euphorbiae could regulate expression of AQP2, AQP 4 and AQP 8 protein and mRNA, which may be the possible one reason of semen Euphorbiae induces diarrhea. The semen Euphorbiae group has more significant effects on the levels of AQP2, AQP 4 and AQP 8 protein and mRNA than semen Euphorbiae Pulveratum group, which may be one of the mechanisms of processing attenuation.

[Determination of endogenous amino acids in brain tissues after cerebral ischemia by RRLC-QQQ].

OBJECTIVE: To establish a method to determine underivatized endogenous amino acids in brain tissues after cerebral ischemia based on RRLC-QQQ. METHOD: Diamonsil chromatographic column C18 (4.6 mm x 250 mm, 5 microm) was adopted to determine 12 amino acids in 15 min, with acetonitrile-0.1% formic acid for gradient elution. The flow rate was set at 0.5 mL x min(-1). With ESI as the ion source, positive ion scanning mode was adopted for multi-reaction monitoring. RESULT: Each amino acid standard curve (AAs) showed good linear relationship within the detection range (r > 0.996), with the limit of detection of less than 11%, the limit of quantitation of less than 3.09 microg x L(-1). The RSD of intra- and inter-day precisions at high, middle and low concentrations were less than 11%. CONCLUSION: The determination results of actual samples showed that compared with the levels of AAs of the sham operation group, all of the remaining amino acids apart from N-acetyl-aspartate increased in brain tissues. Some amino acids showed significant changes in a time-dependent manner after the operation. The method is so simple, rapid and sensitive that it can be used for finding biological metabolite markers of cerebral ischemia, and exploring cerebral ischemia molecular mechanisms and synergistic mechanism of combined administration of multi-component traditional Chinese medicines.

[Advance of studies on metabolic fingerprinting analytical techniques and data processing methods].

Metabolomics is an emerging discipline subsequent to genomics, transcriptomics and proteomics, aiming for systematically studying the regularity of changes in metabolite to revealing organism's nature of movement and metabolism. It is especially important in modern pharmacological studies. Metabolic fingerprinting analysis is a method for metabolic analysis on high throughput of all metabolites, studying changes in drugs, organisms and endogenic metabolites caused by drugs and finding out related biomarkers to reflect dynamic changes inside organisms more directly and explain the mechanism of drugs and their effects on diseases. This essay summarizes some new metabolic fingerprint analytical methods and data processing methods used for metabolic fingerprint, elaborates their advantages and disadvantages and looks ahead to their combination with studies on traditional Chinese medicines, providing room for the development of new methods and new approaches for studies on complexity theory system of traditional Chinese medicines.

Xylomexicanins K-N: limonoids from the leaves and twigs of Xylocarpus granatum.

Six new compounds, xylomexicanins K-N (1-4), granasteroid (5) and 5-methoxy-2-pentylbenzofuran-7-ol (6), along with nine known compounds were isolated from the leaves and twigs of Xylocarpus granatum. Among them, 1 was a biogenetic precursor of 1,8,9-phragmalin limonoid, and 4 represent the first example of degraded A-ring limonoid. The structures of them were elucidated on the basis of one- and two-dimensional NMR spectroscopic data (including 1H, 13C-NMR, DEPT, 1H-1H COSY, HSQC, HMBC, and NOESY) and confirmed by high-resolution mass spectrometry.[Formula: see text].

Application of a mechanically responsive, inflammatory macrophage-targeted dual-sensitive hydrogel drug carrier for atherosclerosis.

Atherosclerotic lesions create obvious vascular stenosis due to the presence of plaque, and a large number of inflammatory macrophages are enriched in the thrombus. In this study, we develop a composite hydrogel drug delivery system that is capable of both mechanically-sensitive drug release and of targeting inflammatory macrophages at the thrombus. The hydrogel is a high molecular weight hyaluronic acid (HA) modified with glycidyl methacrylate as a hydrogel precursor; a cross-linkable block copolymer (CBC) is used as the drug coating material and a microscopic cross-linking agent. The difference in drug release rate of the composite hydrogel (HACBC) in simulated blood vessels with 0 % and 75 % occlusion was as high as 49.3 %. Under long-term cycling conditions in stenotic vessels, dynamic shear rheometry revealed that the HACBC still maintained the hydrogel properties. However, the micelles were deformed and recombined to produce smaller sized micelles. An in vitro cell culture demonstrated precise targeting of the HACBC to inflammatory macrophages, and our rabbit experiments with simvastatin-coated HACBC confirmed the effective release of simvastatin in the plaque of the drug carrier. Moreover, we demonstrated the precise targeting of HACBC in vivo in apoE-/- mice by using HACBC coated with cy7. The mechanical stress-sensitive and CD44 receptor-targeted dual-response drug delivery system prepared by micellar composite hydrogel is the first application in the field of atherosclerosis, which provides a new method for diagnosing and treating atherosclerosis.

[High throughput screening for glutathione S-transferase inhibitors].

To identify the inhibitor of glutathione S-transferase (GST), a high-throughput screening method was established in a 384-well microplate with total 35 microL volume, and the absorbance at 340 nm is detected. The concentrations of substrates, CDNB and GST were determined by chromatometry. The optimal enzyme kinetics reaction time and temperature are 2 h and 30 degrees C , respectively. The established model was evaluated by NaOCl, a known GST inhibitor, and the parameter Z' was 0.77, which showed a high feasibility and stability of the assay. A total of 31,098 compounds were screened, of which 4 compounds were shown to inhibit GST activity, high inhibiting activity for their IC50 of GST inhibition was 3.94, 4.05, 74.85, and 77.41 mg x L(-1), separately. The results indicated that the colorimetric method by using CDNB and GSH as substrate is stable, sensitive, reproducible and also suitable for high throughput screening.

A male-specific expression gene, encodes a novel anionic antimicrobial peptide, scygonadin, in Scylla serrata.

Scygonadin is a novel antimicrobial peptide, which was originally isolated from the seminal plasma of the mud crab, Scylla serrata. Based on the partial 20-residue NH(2)-terminal sequence of the peptide, H-Gly-Gln-Ala-Leu-Asn-Lys-Leu-Met-Pro-Lys-Ile-Val-Ser-Ala-Ile-Ile-Tyr-Met-Val-Gly-OH, scygonadin was cloned from the gonads of S. serrata using a degenerated reverse transcriptase (RT)-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA sequence contains an open reading frame of 539 bases (excluding polyA) with a coding capacity of 126 amino acids, which constitutes a putative NH(2)-terminal signal sequence (1-24) and a mature peptide (25-126). Analysis of the genomic DNA sequence revealed that scygonadin consists of 2300 bp containing two introns (1569 and 120 bp) and three exons (187, 131 and 218 bp) and this sequence is different from any other reported antimicrobial peptide. The theoretical pI of the mature peptide is 6.09, which suggests that it is an anionic molecule. The sex and tissue-specific expression of the scygonadin gene was revealed using RT-PCR and Northern-blot analysis of multiple tissues of S. serrata males and females and this demonstrated that the scygonadin gene was predominantly expressed in the male reproductive tract of S. serrata and was restricted to the ejaculatory duct. This suggests that scygonadin might be one of the antibacterial peptides responsible for protection of the male crab reproductive tract from invading pathogenic microorganisms, so as to maintain a sterile environment leading to successful fertilization.

[Effect of chitosan gene nanoparticles on L02 cells].

OBJECTIVE: To investigate the effect of the gene nanoparticles using chitosan (CNP), arginine modified chitosan (ANP), or hexadecylated chitosan (HNP) as carriers on the human normal liver cell line L02. METHODS: CNPs, ANPs, and HNPs were prepared using complex coacervation method. The size and zeta potential of the gene nanoparticles were measured using Zetasizer nanoZS. The nanoparticles at concentrations of 5, 10, 30, and 50 microg/ml (based on the content of DNA) were incubated with L02 cells, respectively. The cell viability was evaluated by MTT assay, and the effect of the gene nanoparticles on the cell apoptosis was analyzed by flow cytometry. RESULTS: The zeta potential of the gene nanoparticles ranged from 12.10 to 14.63 mV, and their diameters ranged from 148.07 to 179.47 nm. MTT assay showed that the viability of L02 cells began to decrease when the concentration of CNPs reached 30 microg/ml and higher. Furthermore, the CNPs could induce cell apoptosis as the concentration of CNPs reached 30 microg/ml and higher. CONCLUSION: CNPs can induce L02 cell apoptosis at relatively higher concentrations.

The novel mechanism of recombinant human ciliary neurotrophic factor on the anti-diabetes activity.

In a previous study, the ciliary neurotrophic factor (CNTF) were demonstrated to lead to weight-loss partly by up-regulating the energy metabolism and the expression of uncoupling protein-1, mitochondrial transcription factor A and nuclear respiratory factor-1 in adipose tissues or muscle. To investigate the up-stream regulators of the expression, recombinant human CNTF (rhCNTF) (0.1, 0.3 and 0.9 mg/kg/day subcutaneously) were administered to KK-Ay mice for 30 days, resulting in reduction of perirenal fat mass, serum free fatty acids and islet triacylglycerol; furthermore, the values of oral glucose tolerance test were found improved. In brown adipose tissues, the gene expressions of peroxisome proliferator-activated receptor alpha (PPARalpha) and peroxisome proliferator-activated receptor coactivator-1 alpha (PGC-1alpha) were found to be up-regulated by rhCNTF. To the best of our knowledge, the changes of gene expression of PPARalpha and PGC-1alpha represent new insights into the mechanisms of anti-diabetes by rhCNTF. In addition, the activity of mitochondrial complexII was found to be increased by rhCNTF. Stimulation of PPARalpha, PGC-1alpha, uncoupling protein-1 and enhanced activity of mitochondrial complex II may be associated with the effects of anti-diabetes. The present study indicates new mechanisms of the activity and mechanisms on anti-diabetes of rhCNTF, which may be a novel anti-diabetes reagent partly acting by enhancing energy metabolism.

Heavy metal exposure reduces hatching success of Acartia pacifica resting eggs in the sediment.

The potential effects of three heavy metals (Cu, Pb, and Cd) on hatching success of Acartia pacifica resting eggs in the sediment of Xiamen Bay were experimentally investigated. The number ofA. pacifica nauplii hatched from the sediment sharply decreased with the increase of metal concentration and exposure time from 3 to 30 d. An increase of the Cu concentration from 34.8 to 348 mg/kg, reduced the number of hatched nauplii by 46.6%-100%. An increase of the Pb concentration from 75.2 to 752 mg/kg, reduced the number of hatched nauplii by 21.4%-78.9%. An increase of the Cd concentration from 0.68 to 6.8 mg/kg, reduced the number of hatched nauplii by 31.6%-94.7%. The number of nauplii also significantly decreased with the increase of mixed-metal concentration and exposure time in the mixed-metal test. Trimmed Spearman-Karber analysis gave sediment metal 72-h LC50 values of 1.25 mmol Cu/kg, 1.73 mmol Pb/kg, and 0.054 mmol Cd/kg, which suggested that Cd was the most toxic to A. pacifica resting eggs in the three tested metals. The results indicate that heavy metals with higher concentrations can reduce recruitment of A. pacifica nauplii from benthic resting eggs to planktonic population.

In Silico Prediction of Drug-Induced Liver Injury Based on Adverse Drug Reaction Reports.

Drug-induced liver injury (DILI) is a major cause of drug attrition. Currently existing Quantitative Structure-Activity Relationship models have limited predictive capabilities for DILI. Furthermore, their practical applications were limited by lack of new hepatotoxicity data. In this study, we first collected and curated a novel set of 122 DILI-positive and 932 DILI-negative drugs from online adverse drug reports using proportional reporting ratios as the signal detection method. Second, three strategies (under-sampling the majority class, synthetic minority over-sampling technique, and adjusting decision threshold approach) were employed to develop predictive classification models to cope with the unbalanced dataset. Random forest (RF) models using CDK, MACCS, and Mold2 descriptors based on the under-sampling and over-sampling strategies afforded correct classification ratio (CCR) of ∼0.77 and 0.78, respectively. Recursive RF models based on the last strategy tremendously reduced modeling descriptors (at most 95.4% for Mold2) while apparently improved the predictability with a consensus CCR of 0.84 (sensitivity of 0.88 and specificity of 0.79). Structural analysis showed that pyrimidine derivatives, purine derivatives, and halogenated hydrocarbon were critical for drugs' hepatotoxicity. The reporting frequency of many drugs was gender-dependent (eg, antiviral and anti-cancer drugs for males and antibacterial drugs for females) as well as age-dependent (eg, antiviral and anti-cancer drugs for the middle age group of 20-29, 30-39, and 40-49). Approximately 84% of total cases were reported during the first 6 months of administration. The curated hepatotoxicity dataset along with the predictive classification models presented here should provide insight into future studies of DILI.

Rapid Analysis of Components in Coptis chinensis Franch by Ultra-Performance Liquid Chromatography with Quadrupole Time-of-Flight Mass Spectrometry.

BACKGROUND: Coptis chinensis Franch is a traditional Chinese medical herb. OBJECTIVE: In this article, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to rapidly, qualitatively, and comprehensively identify the components in Coptis chinensis Franch. MATERIALS AND METHODS: Chromatographic separation was achieved on an Agilent Zorbax RRHD Eclipse Plus C18 column. The mobile phase consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) with a gradient program. Qualitative analysis was performed on an Agilent 6540 quadrupole time-of-flight mass spectrometer, which was equipped with a Dual AJS ESI source operating in negative mode. RESULTS: A total of 30 alkaloid and non-alkaloid components of Coptis chinensis Franch were identified in only 14 min. CONCLUSION: This study helped to provide a basis for the quality control of Coptis chinensis Franch. SUMMARY: Qualitative analysis method of chlorogenic alkaloids and non-alkaloids in Coptis chinensis Franch is developed by Ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) method.Established UPLC-Q-TOF-MS/MS analysis method is validated with rapidness and accuracy.The developed method was successfully applied for qualitative analysis of Coptis chinensis Franch sample collected from cultivation place in China. Abbreviations used: Q-TOF-MS: quadrupole time-of-flight mass spectrometry, UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. Q-TOF-MS: quadrupole time-of-flight mass spectrometry, UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. UPLC: ultra-performance liquid chromatography, pos: positive, neg: negative. pos: positive, neg: negative. neg: negative.

Reduction of recruitment of Acartia pacifica nauplii from benthic resting eggs due to organochlorine pesticides.

Many estuarine and coastal planktonic copepods depend on the hatching of benthic resting eggs for recruitment of nauplii to the water column population. The potential effects of two organochlorine pesticides, hexchloriobinzene (HCH) and dichlorodiphenyltrichloroethane (DDT), on the recruitment of Acartia pacifica nauplii from benthic resting eggs in the seabed of Xiamen Bay were experimentally investigated. The abundance of A. pacifica nauplii hatched from the sediment significantly decreased with the increase of pesticide concentration. Trimmed Spearman-Karber analysis gave sediment 96-h LC50 values were 84.81 ng/g for HCH, and 157.94 ng/g for DDT. The median AI (AI50) was -0.77, which suggested that the combined effect of HCH and DDT showed a weak effect than individual effects. There was a positive relationship between mortality and exposure time in DDT treatment, while the relationship was not significant in HCH treatment. The results suggest that organochlorine pesticides can reduce recruitment of A. pacifica nauplii from benthic resting eggs to planktonic population.

Synergism and rules of the new combination drug Yiqijiedu formulae (YQJD) on ischemic stroke based on amino acids (AAs) metabolism.

The use of combination drugs is considered to be a promising strategy to control complex diseases such as ischemic stroke. The detection of metabolites has been used as a versatile tool to reveal the potential mechanism of diverse diseases. In this study, the levels of 12 endogenous AAs were simultaneously determined quantitatively in the MCAO rat brain using RRLC-QQQ method. Seven AAs were chosen as the potential biomarkers, and using PLS-DA analysis, the effects of the new combination drug YQJD, which is composed of ginsenosides, berberine, and jasminoidin, on those 7 AAs were evaluated. Four AAs, glutamic acid, homocysteine, methionine, and tryptophan, which changed significantly in the YQJD-treated groups compared to the vehicle groups (P < 0.05), were identified and designated as the AAs to use to further explore the synergism of YQJD. The result of a PCA showed that the combination of these three drugs exhibits the strongest synergistic effect compared to other combination groups and that ginsenosides might play a pivotal role, especially when combined with jasminoidin. We successfully explored the synergetic mechanism of multi-component and provided a new method for evaluating the integrated effects of combination drugs in the treatment of complex diseases.

Differential gene expression profile of the calanoid copepod, Pseudodiaptomus annandalei, in response to nickel exposure.

To better understand the underlying mechanisms of reactions of copepods exposed to elevated level of nickel, the suppression subtractive hybridization (SSH) was used to elucidate the response of the copepod Pseudodiaptomus annandalei to nickel exposure at the gene level. P. annandale is one of a few copepod species that can be cultured relatively easy under laboratory condition, and it is considered to be a potential model species for toxicity study. In the present study, P. annandalei were exposed to nickel at a concentration of 8.86 mgL(-1) for 24h, after which the RNA was prepared for SSH using unexposed P. annandalei as drivers. A total of 474 clones on the middle scale in the SSH library were sequenced. Among these genes, 129 potential functional genes were recognized based on the BLAST searches in NCBI and Uniprot databases. These genes were then categorized into nine groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 129 genes, 127 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains (CD) and proteins using the CD-Search service and BLASTp. Among 129 genes, 119 (92.2%) were annotated to be involved in different biological processes, while 10 genes (7.8%) were classified as an unknown-function gene group. To further confirm the up-regulation of differentially expressed genes, the quantitative real time PCR were performed to test eight randomly selected genes, in which five of them, i.e. α-tubulin, ribosomal protein L13, ferritin, separase and Myohemerythrin-1, exhibited clear up-regulation after nickel exposure. In addition, MnSOD was further studied for the differential expression pattern after nickel exposure and the results showed that MnSOD had a time- and dose-dependent expression pattern in the copepod after nickel exposure. To the best of our knowledge, this is the first attempt to investigate the toxicity effects of nickel on a copepod at molecular level.

[Construction of suppression subtractive hybridization (SSH) library of copepod Pseudodiaptomous annandalei and its ferritin cDNA cloning and differential expression under nickel stress].

To study the molecular response mechanisms of copepod to nickel stress, a suppression subtractive hybridization (SSH) cDNA library of Pseudodiaptomous annandalei under nickel stress was constructed by using SSH technique, and a total of 140 clones were randomly picked from the growing colonies and identified by PCR. The recombinant rate of the library was 98.6%, and the volume of the library was 1.12 x 10(6) cfu. After the recombinant plasmids were sequenced, a partial cDNA fragment of ferritin was recognized based on BLAST searches in NCBI, with a size of 859 bp and continuously encoding 170 amino acid residues. The semi-quantitative PCR results showed that the ferritin cDNA under 24 h nickel stress was distinctly up-regulated. The successful construction of the SSH library and the obtaining of ferritin cDNA fragment would supply basis for the further study of the molecular response mechanisms of copepod to nickel stress.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 October 2009-30 November 2009.

This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.

Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy (Acanthopagrus schlegelii B).

Hepcidin is an antimicrobial peptide and putative iron regulatory hormone previously described in mice and humans. Dozens of fish hepcidins have been isolated and characterized so far. Here we present seven hepcidin-like cDNA sequences named AS-hepc1-7, amplified from the normal commercially cultured fish (black porgy) by RACE-PCR. Sequence analysis reveals that these seven potential hepcidin peptides have highly conserved sequences with other known hepcidins, but they are different from each other in constitution and characteristics of predicted mature amino acids. Based on the study, it is deduced that AS-hepc1-7 represent different variants of a family of hepcidin genes in black porgy. To understand the organization of these hepcidin-like genes, we sequenced AS-hepc2 DNA, AS-hepc3 DNA, AS-hepc4 DNA, AS-hepc7 DNA and AS-hepc2 upstream region; and all of the four genomic DNAs consisted of two introns and three exons, the same organization as other reported hepcidins. The tissue-specific gene expression of hepcidins in normal black porgy was evaluated using RT-PCR and dot blot approaches. RT-PCR showed that transcripts of hepcidin-like mRNAs were present in each tested tissue of normal juvenile black porgy, including liver, spleen, kidney, heart, brain, stomach, intestine, gill, skin and blood, but abundant hepcidin-like mRNA transcripts were only detected in the liver, kidney, spleen, intestine and stomach by dot blot assay. In addition, using dot blot and Northern blot approach, a significant increase of hepcidin mRNA transcription was observed in the liver within 48 h after immersion in a suspension of live bacteria, which suggested that the expression pattern of hepcidin-like genes in black porgy might be different in the liver from the other tissues as previously reported in several hepcidin studies.