In order to supply sufficient microsatellite loci for high-density linkage mapping, whole genome shotgun (WGS) sequences of the common carp (Cyprinus carpio) were assembled and surveyed for microsatellite identification. A total of 79,014 microsatellites were collected which were harbored in 68,827 distinct contig sequences. These microsatellites were characterized in the common carp genome. Information of all microsatellites, including previously published BAC-based microsatellites, was then stored in a MySQL database, and a web-based database interface (http://genomics.cafs.ac.cn/ssrdb) was built for public access and download. A total of 3,110 microsatellites, including 1,845 from WGS and 1,265 from BAC end sequences (BES), were tested and genotyped on a mapping family with 192 individuals. A total of 963 microsatellites markers were validated with polymorphism in the mapping family. They will soon be used for high-density linkage mapping with a vast number of polymorphic SNP markers.

Ghrelin, a 28-amino acid peptide, is mainly secreted by the stomach. Ghrelin has been shown to have neuroprotective effects. However, whether ghrelin protects the spinal cord from ischemia/reperfusion (I/R) injury is unknown. To investigate this, 60 rats were randomly divided into three different groups: the sham group (n = 20), the vehicle group (n = 20), and the Ghrelin group (100 μg/kg, n = 20). Rats were sacrificed 12, 24, 48 and 72 h after ischemia. After the evaluation of neurologic function (48 h), the spinal cords were immediately removed for the determination of myeloperoxidase (MPO) activity (12-72 h). Apoptosis was quantitatively measured using the terminal transferase UTP nick end-labeling (TUNEL) method (24 h). The expression of bax and bcl-2 were evaluated by Western blot analysis (1 h), and GHSR-1a mRNA expression was detected using reverse transcriptase polymerase chain reaction (24 h). The neurological motor function was evaluated by 'Tarlov's score'. The neurologic outcomes in the ghrelin-group were significantly better than those in the vehicle group (p < 0.05). Serum tumor necrosis factor (TNF-α) levels were assessed in the peripheral venous blood. Ghrelin decreased the serum TNF-α levels and ameliorated the down regulation of spinal cord MPO activity. The expression of ghrelin receptors (GHSR-1a) in the rat spinal cord was decreased by I/R injury and increased by ghrelin. Ghrelin reduced the TUNEL-positive rate. Greater bcl-2, HSP27, HSP70, and attenuated bax expression were observed in the ghrelin-treated rats. Our results suggest that ghrelin administration may inhibit spinal I/R injury. Moreover, the improvement of neurologic function in rats was increased after the ghrelin treatment.

The majority of ovarian cancer patients present with advanced disease and despite aggressive treatment, prognosis remains poor. Significant improvement in ovarian cancer survival will require the development of more effective molecularly targeted therapeutics. Commonly, mouse models are used for the in vivo assessment of potential new therapeutic targets in ovarian cancer. However, animal models are costly and time consuming. Other models, such as the chick embryo chorioallantoic membrane (CAM) assay, are therefore an attractive alternative. CAM assays have been widely used to study angiogenesis and tumor invasion of colorectal, prostate and brain cancers. However, there have been limited studies that have used CAM assays to assess ovarian cancer invasion and metastasis. We have therefore developed a CAM assay protocol to monitor the metastatic properties of ovarian cancer cells (OVCAR-3, SKOV-3 and OV-90) and to study the effect of potential therapeutic molecules in vivo. The results from the CAM assay are consistent with cancer cell motility and invasion observed in in vitro assays. Our results demonstrate that the CAM assay is a robust and cost effective model to study ovarian cancer cell metastasis. It is therefore a very useful in vivo model for screening of potential novel therapeutics.

We aimed to investigate the expression pattern of phosphatase and tensin homolog (PTEN), to evaluate the relationship between PTEN expression and clinicopathological characteristics, including fatty acid synthase (FAS) expression, and to determine the correlations of PTEN and FAS expression with survival in Chinese patients with hepatocellular carcinoma (HCC). The expression patterns of PTEN and FAS were determined using tissue microarrays and immunohistochemistry. The expression of PTEN was compared with the clinicopathological characteristics of HCC, including FAS expression. Receiver operator characteristic curves were used to calculate the clinical sensitivity and specificity of PTEN expression. Kaplan-Meier survival curves were constructed to evaluate the correlations of PTEN loss and FAS overexpression with overall survival. We found that the loss of PTEN expression occurred predominantly in the cytoplasm, while FAS was mainly localized to the cytoplasm. Cytoplasmic and total PTEN expression levels were significantly decreased in HCC compared with adjacent non-neoplastic tissue (both, p < 0.0001). Decreased cytoplasmic and total PTEN expression showed significant clinical sensitivity and specificity for HCC (both, p < 0.0001). Downregulation of PTEN in HCC relative to non-neoplastic tissue was significantly correlated with histological grade (p = 0.043 for histological grades I-II versus grade III). Loss of total PTEN was significantly correlated with FAS overexpression (p = 0.014). Loss of PTEN was also associated with poor prognosis of patients with poorly differentiated HCC (p = 0.049). Moreover, loss of PTEN combined with FAS overexpression was associated with significantly worse prognosis compared with other HCC cases (p = 0.011). Our data indicate that PTEN may serve as a potential diagnostic and prognostic marker of HCC. Upregulating PTEN expression and inhibiting FAS expression may offer a novel therapeutic approach for HCC.

In the present study we describe the synthesis and biological assessment of new tacrine analogs in the course of inhibition of acetylcholinesterase. The obtained molecules were synthesized in a condensation reaction between activated 6-BOC-hydrazinopyridine-3-carboxylic acid and 8-aminoalkyl derivatives of 2,3-dihydro-1H-cyclopenta[b]quinoline. Activities of the newly synthesized compounds were estimated by means of Ellman's method. Compound 6h (IC(50) = 3.65 nM) was found to be most active. All obtained novel compounds present comparable activity to that of tacrine towards acetylcholinesterase (AChE) and, simultaneously, lower activity towards butyrylcholinesterase (BChE). Apart from 6a, all synthesized compounds are characterized by a higher affinity for AChE and a lower affinity for BChE in comparison with tacrine. Among all obtained molecules, compound 6h presented the highest selectivity towards inhibition of acetylcholinesterase. Molecular modeling showed that all compounds demonstrated a similar binding mode with AChE and interacted with catalytic and peripheral sites of AChE. Also, a biodistribution study of compound 6a radiolabeled with (99m)Tc was performed.

Helicobacter pylori (H. pylori), the human stomach pathogen, lives on the inner surface of the stomach and causes chronic gastritis, peptic ulcer, and gastric cancer. Plasma membrane repair response is a matter of life and death for human cells against physical and biological damage. We here test the hypothesis that H. pylori also causes plasma membrane disruption injury, and that not only a membrane repair response but also a cell proliferation response are thereby activated. Vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA) have been considered to be major H. pylori virulence factors. Gastric cancer cells were infected with H. pylori wild type (vacA+/cagA+), single mutant (ΔvacA or ΔcagA) or double mutant (ΔvacA/ΔcagA) strains and plasma membrane disruption events and consequent activation of membrane repair components monitored. H. pylori disrupts the host cell plasma membrane, allowing localized dye and extracellular Ca(2+) influx. Ca(2+)-triggered members of the annexin family, A1 and A4, translocate, in response to injury, to the plasma membrane, and cell surface expression of an exocytotic maker of repair, LAMP-2, increases. Additional forms of plasma membrane disruption, unrelated to H. pylori exposure, also promote host cell proliferation. We propose that H. pylori activation of a plasma membrane repair is pro-proliferative. This study might therefore provide new insight into potential mechanisms of H. pylori-induced gastric carcinogenesis.

This study examined the effects of rice bran oil (RBO) on mouse intestinal microbiota and urinary isoflavonoids. Dietary RBO affects intestinal cholesterol absorption. Intestinal microbiota seem to play an important role in isoflavone metabolism. We hypothesized that dietary RBO changes the metabolism of isoflavonoids and intestinal microbiota in mice. Male mice were randomly divided into two groups: those fed a 0.05% daidzein with 10% RBO diet (RO group) and those fed a 0.05% daidzein with 10% lard control diet (LO group) for 30 days. Urinary amounts of daidzein and dihydrodaidzein were significantly lower in the RO group than in the LO group. The ratio of equol/daidzein was significantly higher in the RO group (p < 0.01) than in the LO group. The amount of fecal bile acids was significantly greater in the RO group than in the LO group. The composition of cecal microbiota differed between the RO and LO groups. The occupation ratios of Lactobacillales were significantly higher in the RO group (p < 0.05). Significant positive correlation (r = 0.591) was observed between the occupation ratios of Lactobacillales and fecal bile acid content of two dietary groups. This study suggests that dietary rice bran oil has the potential to affect the metabolism of daidzein by altering the metabolic activity of intestinal microbiota.

The aim of this study was to determine if there are sex-related differences in the acute kidney injury induced by HgCl(2) since female rats express lower levels of renal Oat1 and Oat3 (transporters involved in renal uptake of mercury) as compared with males. Control males and females and Hg-treated male and female Wistar rats were employed. Animals were treated with HgCl(2) (4 mg/kg body weight (b.w.), intraperitoneal (i.p.)) 18 h before the experiments. HgCl(2) induced renal impairment both in male and female rats. However, female rats showed a lower renal impairment than male rats. The observed increase in kidney weight/body weight ratio seen in male and female rats following HgCl(2) treatment was less in the female rats. Urine volume and creatinine clearance decreased and Oat5 urinary excretion increased in both males and females, but to a lesser degree in the latter. Urinary alkaline phosphatase (AP) activity and histological parameters were modified in male but not in female rats after HgCl(2) administration. These results indicate that the lower Oat1 and Oat3 expression in the kidney of females restricts Hg uptake into renal cells protecting them from this metal toxicity. These gender differences in renal injury induced by mercury are striking and also indicate that Oat1 and Oat3 are among the main transporters responsible for HgCl(2)-induced renal injury.

Mytilus coruscus (family Mytilidae) is one of the most important marine shellfish species in Korea. During the past few decades, this species has become endangered due to the loss of habitats and overfishing. Despite this species' importance, information on its genetic background is scarce. In this study, we developed microsatellite markers for M. coruscus using next-generation sequencing. A total of 263,900 raw reads were obtained from a quarter-plate run on the 454 GS-FLX titanium platform, and 176,327 unique sequences were generated with an average length of 381 bp; 2569 (1.45%) sequences contained a minimum of five di- to tetra-nucleotide repeat motifs. Of the 51 loci screened, 46 were amplified successfully, and 22 were polymorphic among 30 individuals, with seven of trinucleotide repeats and three of tetranucleotide repeats. All loci exhibited high genetic variability, with an average of 17.32 alleles per locus, and the mean observed and expected heterozygosities were 0.67 and 0.90, respectively. In addition, cross-amplification was tested for all 22 loci in another congener species, M. galloprovincialis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 22 newly developed microsatellites will be useful in future conservation genetic studies of this species.

The total phenolic content, total flavonoid content, vitamin C content, and antioxidant activities of ethanol extracts from different kiwifruit varieties (Actinidia kolomikta, Actinidia arguta, Actinidia chinensis) were determined in this study. Multiple scavenging activity assays including the hydroxyl radical, O(2) (-)·radical, DPPH, and the ABTS(+) radical scavenging activity assays were used to identify the antioxidant activities of Actinidia extracts. The cell viability of HepG2 and HT-29 cells was also examined in this study. The results demonstrated that the Actinidia kolomikta extract had a higher antioxidant activity than the other two Actinidia extracts. There is a positive correlation between antioxidant activity and the polyphenols and vitamin C content in all three extracts (R(2) ≥ 0.712, p < 0.05). The Actinidia arguta extract had the highest inhibitory effect on HepG2 and HT-29 cell growth. These results provide new insight into the health functions of fruit and demonstrate that Actinidia extracts can potentially have health benefits.

The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV(+) broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.

The polymorphisms of Waxy (Wx) microsatellite and G-T single-nucleotide polymorphism (SNP) in the Wx gene region were analyzed using simplified techniques in fifteen rice varieties. A rapid and reliable electrophoresis method, MetaPhor agarose gel electrophoresis (MAGE), was effectively employed as an alternative to polyacrylamide gel electrophoresis (PAGE) for separating Wx microsatellite alleles. The amplified products containing the Wx microsatellite ranged from 100 to 130 bp in length. Five Wx microsatellite alleles, namely (CT)(10), (CT)(11), (CT)(16), (CT)(17), and (CT)(18) were identified. Of these, (CT)(11) and (CT)(17) were the predominant classes among the tested varieties. All varieties with an apparent amylose content higher than 24% were associated with the shorter repeat alleles; (CT)(10) and (CT)(11), while varieties with 24% or less amylose were associated with the longer repeat alleles. All varieties with intermediate and high amylose content had the sequence AGGTATA at the 5'-leader intron splice site, while varieties with low amylose content had the sequence AGTTATA. The G-T polymorphism was further verified by the PCR-AccI cleaved amplified polymorphic sequence (CAPS) method, in which only genotypes containing the AGGTATA sequence were cleaved by AccI. Hence, varieties with desirable amylose levels can be developed rapidly using the Wx microsatellite and G-T SNP, along with MAGE.

In the present study, we firstly compared rat intestinal α-glucosidase inhibitory activity by different ethanol-aqueous extractions from the dried fruits of Terminalia chebula Retz. The enzymatic assay showed that the 80% ethanol extract was more potent against maltase activity than both 50% and 100% ethanol extracts. By HPLC analysis, it was determined that the 80% ethanol extract had a higher content of chebulagic acid than each of 50% or 100% ethanol extract. Next, we investigated how efficiently chebulagic acid could inhibit sugar digestion by determining the glucose level on the apical side of the Caco-2 cell monolayer. The result showed that the maltose-hydrolysis activity was down-regulated by chebulagic acid, which proved to be a reversible inhibitor of maltase in Caco-2 cells. On the other hand, chebulagic acid showed a weak inhibition of sucrose-hydrolysis activity. Meanwhile, chebulagic acid did not have an obvious influence on intestinal glucose uptake and was not effective on glucose transporters. Further animal studies revealed that the oral administration of chebulagic acid (100 mg/kg body weight) significantly reduced postprandial blood glucose levels by 11.1% in maltose-loaded Sprague-Dawley (SD) rats compared with the control group, whereas the oral administration of chebulagic acid did not show a suppressive effect on postprandial hyperglycemia in sucrose- or glucose-loaded SD-rats. The results presented here suggest that chebulagic acid from T. chebula can be used to control blood glucose and manage type 2 diabetes, although clinical trials are needed.

Gelam honey exerts anti-inflammatory and antioxidant activities and is thought to have potent effects in reducing infections and healing wounds. The aim of this study was to investigate the effects of intravenously-injected Gelam honey in protecting organs from lethal doses of lipopolysaccharide (LPS). Six groups of rabbits (N = 6) were used in this study. Two groups acted as controls and received only saline and no LPS injections. For the test groups, 1 mL honey (500 mg/kg in saline) was intravenously injected into two groups (treated), while saline (1 mL) was injected into the other two groups (untreated); after 1 h, all four test groups were intravenously-injected with LPS (0.5 mg/kg). Eight hours after the LPS injection, blood and organs were collected from three groups (one from each treatment stream) and blood parameters were measured and biochemical tests, histopathology, and myeloperoxidase assessment were performed. For survival rate tests, rabbits from the remaining three groups were monitored over a 2-week period. Treatment with honey showed protective effects on organs through the improvement of organ blood parameters, reduced infiltration of neutrophils, and decreased myeloperoxidase activity. Honey-treated rabbits also showed reduced mortality after LPS injection compared with untreated rabbits. Honey may have a therapeutic effect in protecting organs during inflammatory diseases.

Japanese encephalitis (JE), a mosquito-borne viral disease, is endemic to the entire east and southeast Asia, and some other parts of the world. Currently, there is no effective therapeutic available for JE; therefore, finding the effective antiviral agent against JEV replication is crucial. In the present study, the in vitro antiviral activity of baicalein and quercetin, two purportedly antiviral bioflavonoids, was evaluated against Japanese encephalitis virus (JEV) replication in Vero cells. Anti-JEV activities of these compounds were examined on different stages of JEV replication cycle. The effects of the compounds on virus replication were determined by foci forming unit reduction assay (FFURA) and quantitative RT-PCR. Baicalein showed potent antiviral activity with IC(50) = 14.28 µg/mL when it was introduced to the Vero cells after adsorption of JEV. Quercetin exhibited weak anti-JEV effects with IC(50) = 212.1 µg/mL when the JEV infected cells were treated with the compound after virus adsorption. However, baicalein exhibited significant effect against JEV adsorption with IC(50) = 7.27 µg/mL while quercetin did not show any anti-adsorption activity. Baicalein also exhibited direct extracellular virucidal activity on JEV with IC(50) = 3.44 µg/mL. However, results of quantitative RT-PCR experiments confirmed the findings from FFURA. This study demonstrated that baicalein should be considered as an appropriate candidate for further investigations, such as the study of molecular and cellular mechanism(s) of action and in vivo evaluation for the development of an effective antiviral compound against Japanese encephalitis virus.

Massive evidence suggests that genetic abnormalities contribute to the development of lung cancer. These molecular abnormalities may serve as diagnostic, prognostic and predictive biomarkers for this deadly disease. It is imperative to search these biomarkers in different tumorigenesis pathways so as to provide the most appropriate therapy for each individual patient with lung malignancy. Phosphoproteomics is a promising technology for the identification of biomarkers and novel therapeutic targets for cancer. Thousands of proteins interact via physical and chemical association. Moreover, some proteins can covalently modify other proteins post-translationally. These post-translational modifications ultimately give rise to the emergent functions of cells in sequence, space and time. Phosphoproteomics clinical researches imply the comprehensive analysis of the proteins that are expressed in cells or tissues and can be employed at different stages. In addition, understanding the functions of phosphorylated proteins requires the study of proteomes as linked systems rather than collections of individual protein molecules. In fact, proteomics approaches coupled with affinity chromatography strategies followed by mass spectrometry have been used to elucidate relevant biological questions. This article will discuss the relevant clues of post-translational modifications, phosphorylated proteins, and useful proteomics approaches to identify molecular cancer signatures. The recent progress in phosphoproteomics research in lung cancer will be also discussed.

The risk of prostate cancer has been increasing in men by degrees. To develop a new prostate cancer therapy, we used a stem cell-derived gene directed prodrug enzyme system using human neural stem cells (hNSCs) that have a tumor-tropic effect. These hNSCs were transduced with the therapeutic genes for bacterial cytosine deaminase (CD), alone or in combination with the one encoding human interferon-beta (IFN-β) or rabbit carboxyl esterase (CE) to generate HB1.F3.CD, HB1.F3.CD.IFN-β, and HB1.F3.CE cells, respectively. CD enzyme can convert the prodrug 5-fluorocytosine (5-FC) into the activated form 5-fluorouracil (5-FU). In addition, CE enzyme can convert the prodrug CPT-11 into a toxic agent, SN-38. In our study, the human stem cells were found to migrate toward LNCaP human prostate cancer cells rather than primary cells. This phenomenon may be due to interactions between chemoattractant ligands and receptors, such as VEGF/VEGFR2 and SCF/c-Kit, expressed as cancer and stem cells, respectively. The HB1.F3.CE, HB.F3.CD, or HB1.F3.CD.IFN-β cells significantly reduced the LNCaP cell viability in the presence of the prodrugs 5-FC or CPT-11. These results indicate that stem cells expressing therapeutic genes can be used to develop a new strategy for selectively treating human prostate cancer.

Wild soybean (Glycine soja Sieb. et Zucc) is the most important germplasm resource for soybean breeding, and is currently subject to habitat loss, fragmentation and population decline. In order to develop successful conservation strategies, a total of 604 wild soybean accessions from 43 locations sampled across its range in China, Japan and Korea were analyzed using 20 nuclear (nSSRs) and five chloroplast microsatellite markers (cpSSRs) to reveal its genetic diversity and population structure. Relatively high nSSR diversity was found in wild soybean compared with other self-pollinated species, and the region of middle and lower reaches of Yangtze River (MDRY) was revealed to have the highest genetic diversity. However, cpSSRs suggested that Korea is a center of diversity. High genetic differentiation and low gene flow among populations were detected, which is consistent with the predominant self-pollination of wild soybean. Two main clusters were revealed by MCMC structure reconstruction and phylogenetic dendrogram, one formed by a group of populations from northwestern China (NWC) and north China (NC), and the other including northeastern China (NEC), Japan, Korea, MDRY, south China (SC) and southwestern China (SWC). Contrib analyses showed that southwestern China makes the greatest contribution to the total diversity and allelic richness, and is worthy of being given conservation priority.

When studying the altered expression of genes associated with cartilage regeneration by quantitative real-time RT-PCR (RT-qPCR), reference genes with highly stable expression during different stages of chondrocyte developmental are necessary to normalize gene expression accurately. Until now, no reports evaluating expression changes of commonly used reference genes in rabbit articular cartilage have been published. In this study, defects were made in rabbit articular cartilage, with or without insulin-like growth factor 1 (IGF-1) treatment, to create different chondrocyte living environments. The stability and intensity of the expressions of the candidate reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S Ribosomal RNA (18S rRNA), cyclophilin (CYP), hypoxanthine phosphoribosyl transferase (HPRT1), and beta-2-microglobulin (B2M) were evaluated. The data were analyzed by geNorm and NormFinder. B2M and 18S rRNA were identified to be suitable reference genes for rabbit cartilage tissues.

Tumor progression is a key aspect in oncology. Not even the overexpression of a powerful oncogenic stimulus such as platelet derived growth factor-B (PDGF-B) is sufficient per se to confer full malignancy to cells. In previous studies we showed that neural progenitors overexpressing PDGF-B need to undergo progression to acquire the capability to give rise to secondary tumor following transplant. By comparing the expression profile of PDGF-expressing cells before and after progression, we found that progressed tumors consistently downregulate the expression of the antiproliferative gene Btg2. We therefore tested whether the downregulation of Btg2 is sufficient and necessary for glioma progression with loss and gain of function experiments. Our results show that downregulation of Btg2 is not sufficient but is necessary for tumor progression since the re-introduction of Btg2 in fully progressed tumors dramatically impairs their gliomagenic potential. These results suggest an important role of Btg2 in glioma progression. Accordingly with this view, the analysis of public datasets of human gliomas showed that reduced level of Btg2 expression correlates with a significantly worse prognosis.