TRPA1 channels are non-selective cation channels that could be activated by plant-derived pungent products, including gingerol, a main active constituent of ginger. Ginger could improve the digestive function; however whether ginger improves the digestive function through activating TRPA1 receptor in gastrointestinal tract has not been investigated. In the present study, gingerol was used to stimulate cell lines (RIN14B or STC-1) while depletion of extracellular calcium. TRPA1 inhibitor (rethenium red) and TRPA1 gene silencing via TRPA1-specific siRNA were also used for mechanistic studies. The intracellular calcium and secretion of serotonin or cholecystokinin were measured by fura-2/AM and ELISA. Stimulation of those cells with gingerol increased intracellular calcium levels and the serotonin or cholecystokinin secretion. The gingerol-induced intracellular calcium increase and secretion (serotonin or cholecystokinin) release were completely blocked by ruthenium red, EGTA, and TRPA1-specific siRNA. In summary, our results suggested that gingerol derived from ginger might improve the digestive function through secretion releasing from endocrine cells of the gut by inducing TRPA1-mediated calcium influx.
Calcium ; metabolism ; Calcium Channels ; genetics ; metabolism ; Catechols ; pharmacology ; Cell Line ; Fatty Alcohols ; pharmacology ; Gastrointestinal Tract ; drug effects ; metabolism ; Ginger ; chemistry ; Humans ; Nerve Tissue Proteins ; genetics ; metabolism ; Plant Extracts ; pharmacology ; TRPA1 Cation Channel ; Transient Receptor Potential Channels ; genetics ; metabolism

OBJECTIVETo investigate the expression of nesfatin-1/NUCB2 and ghrelin in the gastric mucosa of rats with intrauterine growth retardation (IUGR) and its significance.

METHODSThe IUGR animal model was established by feeding rats low-protein diets during their pregnancy. Newborn rats were divided into catch-up growth, non-catch-up growth and control groups. Protein and mRNA levels of nesfatin-1/NUCB2 and ghrelin in the gastric mucosa of rats were determined by RT-PCR and Western blot, respectively.

RESULTSNesfatin-1/NUCB2 mRNA and protein were expressed in the gastric mucosa of rats immediately after birth, and their expression increased in an age-dependent manner in all three groups. Furthermore, the level of nesfatin-1/NUCB2 in the catch-up growth group was higher than that in the control group before weaning, whereas there was no significant difference in nesfatin-1/NUCB2 expression between the two groups after weaning. The level of nesfatin-1/NUCB2 in the non-catch-up growth group was lower than that in the catch-up growth group during the whole observation period. The level of ghrelin in the catch-up growth group was higher than that in the control group starting from day 12 after birth, whereas there was no significant difference in ghrelin expression between the two groups after weaning. The level of ghrelin in the non-catch-up growth group was lower compared with those in the catch-up growth and control groups from days 12 to 28 after birth.

CONCLUSIONSNesfatin-1 and ghrelin are co-expressed in the gastric mucosa of rats with IUGR after birth and interact with each other to produce long-term nutritional regulation.

Age Factors ; Animals ; Calcium-Binding Proteins ; analysis ; genetics ; DNA-Binding Proteins ; analysis ; genetics ; Female ; Fetal Growth Retardation ; metabolism ; Gastric Mucosa ; chemistry ; Ghrelin ; analysis ; genetics ; Male ; Nerve Tissue Proteins ; analysis ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo construct the recombinant plasmid pcDNA3.0-RGC32 and evaluate the effect of the response gene to complement-32 (RGC32) on cell cytoskeleton in vitro.

METHODSThe full-length cDNA of RGC32 was obtained by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.0 to generate the recombinant plasmid pcDNA3.0-RGC32. After transfection of the recombinant plasmid into SW480 cells, the expression of RGC32 in the cells was detected by Western blotting. The cytoskeleton of SW480 cells was visualized before and after the transfection, and the changes in the cell migration ability was assessed by wound-healing assay.

RESULTSThe recombinant plasmid pcDNA3.0-RGC32 was successfully constructed. The expression of RGC32 was significantly increased in SW480 cells after transfection with pcDNA3.0-RGC32. Before the transfection, the microfilaments of SW480 cells were few and short without obvious polarity, but after the transfection, the microfilaments were increased and elongated with also an obvious polarity, and the invasive structures of lamellae and lamellipodia occurred. The migration ability of the cells was enhanced after transfection with pcDNA3.0-RGC32.

CONCLUSIONOverexpression of RGC32 can cause the reorganization of cytoskeleton and promotes the cell migration, which can be an important mechanism of RGC32 in promoting cancer metastasis.

Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Movement ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Cytoskeleton ; chemistry ; metabolism ; Genetic Vectors ; Humans ; Muscle Proteins ; biosynthesis ; genetics ; Neoplasm Metastasis ; genetics ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Animals ; Globins ; analysis ; chemistry ; genetics ; physiology ; Humans ; Hypoxia-Ischemia, Brain ; metabolism ; prevention & control ; Nerve Tissue Proteins ; analysis ; chemistry ; genetics ; physiology ; Signal Transduction

OBJECTIVETo study the expression pattern of Polycomb gene Nspc1 at the early developmental stage in zebrafish.

METHODSIn situ hybridization probe for Nspc1 was designed according to the GenBank information. Collecting zebrafish embryos at different stages including one cell stage, two-cell stage, bud stage, and somites stage, we hybridized them with the prepared probe. Then the hybridization signals at different intervals were observed and photographed at the right time.

RESULTSNspc1 was expressed globally at the early stage. Its expression specificity began at the somites stage, mainly in the nervous system of the head.

CONCLUSIONNspc1 may play essential roles in the early stage development of zebrafish, especially in the nervous system.

Amino Acid Sequence ; Animals ; Gene Expression Regulation, Developmental ; Humans ; Mice ; Molecular Sequence Data ; Nerve Tissue ; growth & development ; metabolism ; Polycomb Repressive Complex 1 ; Repressor Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Zebrafish ; genetics ; growth & development ; metabolism ; Zebrafish Proteins ; chemistry ; genetics ; metabolism

To date, nearly 28 distinct genetic loci of autosomal dominant cerebellar ataxias have been identified, among them 18 disease-causing genes have been cloned. Of these, Machado-Joseph disease (MJD), also named as spinocerebellar ataxia type 3 (SCA3), is perhaps the most common subtype among different races and origins in the world. It is a neurodegenerative disease caused by the expansion of a CAG repeat in the coding region of the MJD1 gene, with obvious clinical and genetic heterogeneity. In this review, authors covered the recent advances in molecular genetic of SCA3/MJD.
Ataxin-3 ; Humans ; Machado-Joseph Disease ; genetics ; Molecular Biology ; Mutation ; Nerve Tissue Proteins ; chemistry ; genetics ; metabolism ; Nuclear Proteins ; chemistry ; genetics ; metabolism ; Repressor Proteins ; chemistry ; genetics ; metabolism

In the post-genomic era, various computational methods that predict protein-protein interactions at the genome level are available; however, each method has its own advantages and disadvantages, resulting in false predictions. Here we developed a unique integrated approach to identify interacting partner(s) of Semaphorin 5A (SEMA5A), beginning with seven proteins sharing similar ligand interacting residues as putative binding partners. The methods include Dwyer and Root-Bernstein/Dillon theories of protein evolution, hydropathic complementarity of protein structure, pattern of protein functions among molecules, information on domain-domain interactions, co-expression of genes and protein evolution. Among the set of seven proteins selected as putative SEMA5A interacting partners, we found the functions of Plexin B3 and Neuropilin-2 to be associated with SEMA5A. We modeled the semaphorin domain structure of Plexin B3 and found that it shares similarity with SEMA5A. Moreover, a virtual expression database search and RT-PCR analysis showed co-expression of SEMA5A and Plexin B3 and these proteins were found to have co-evolved. In addition, we confirmed the interaction of SEMA5A with Plexin B3 in co-immunoprecipitation studies. Overall, these studies demonstrate that an integrated method of prediction can be used at the genome level for discovering many unknown protein binding partners with known ligand binding domains.
Binding Sites ; genetics ; Cell Line, Tumor ; Cluster Analysis ; Computational Biology ; methods ; Databases, Protein ; Gene Expression Profiling ; Humans ; Hydrophobic and Hydrophilic Interactions ; Immunoprecipitation ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Models, Molecular ; Nerve Tissue Proteins ; chemistry ; genetics ; metabolism ; Neural Cell Adhesion Molecules ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Interaction Mapping ; methods ; Protein Structure, Tertiary ; Reverse Transcriptase Polymerase Chain Reaction

Animals ; Desmin ; analysis ; genetics ; Female ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; genetics ; Kidney Glomerulus ; chemistry ; Nephrosis ; metabolism ; Nerve Tissue Proteins ; analysis ; genetics ; Nestin ; Podocytes ; chemistry ; RNA, Messenger ; analysis ; Rats ; Rats, Inbred WKY ; Vimentin ; analysis ; genetics

OBJECTIVEChinese jianpi herbal recipe Weichangan (WCA) could increase the survival rate of advanced gastric cancer. This study was designed to investigate the molecular mechanism of WCA in treatment of gastric cancer by cDNA array, real-time quantitative PCR and immunohistochemical technique.

METHODA human gastric adenocarcinoma cell line SGC-7901 grafted onto nude mouse was used as the animal model. The mice were divided into 3 groups, one control and the two representing experimental conditions. Animals in the two experimental groups received either WCA over a 34-day period or 5-fluorouracil (5-FU) over 6-day period starting at 8th day after grafting. Control animals received saline on an identical schedule. Animals were killed 41 days after being grafted. To assess the effect of therapy tumor weight was determined by a electron balance immediately after the animals killed. SP immunohistochemical method was used to detect the expression of proliferating cell nuclear antigen (PCNA) in xenografts. For detection of apoptotic cells, apoptotic indices (AI) were examined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method. SP method was also used to detect the expression of cleaved Caspase-3. The expression profiles in paired WCA treated gastric cancer samples and the N. S. control samples were studied by using a cDNA array representing 14, 181 cDNA clusters. The alterations in gene expression levels were confirmed by real-time quantitative PCR. SP method was used to detect the expression of Phospho-Stat3 (Tyr705) and bcl-2.

RESULTWhen compared with controls, tumor growth was significantly inhibited by treatment with the WCA or 5-FU (P < 0.01, respectively). The average of tumor inhibitory rate in WCA group was (44.32 +/- 5.67)% and 5-FU (47.04 +/- 11.33)%. The average labeling index (LI) for PCNA in WCA group and 5-FU group was significantly decreased compared with the control group respectively. AI of human gastric cancer xenografts in nude mice was significantly increased to (9.72 +/- 4.51)% using TUNEL method in WCA group compared with the controls (2.45 +/- 1.37)%. 5-FU group was also found a significantly increased AI compared with the controls. The expression of cleaved Caspase-3 in WCA group and 5-FU group was significantly increased compared with the control group respectively. There were 45 different expression ESTs among the control sample pool and WCA sample pool. There were 24 ESTs up-regulated in WCA samples and 21 ESTs down-regulated. These 45 ESTs contains 35 cloned genes and 11 unknown ESTs. By using Real-time Quantitative PCR, the expression level of Stat3 (2(-deltadeltaCT) = 0.16) , RIPX (2(-deltadeltaCT) = 0.18), ROD1 (2(-deltadeltaCT) = 0.23) and bcl-2 (2 (-deltadeltaCT) = 0.10) was lower in WCA group than that in control group respectively. The expression of Phospho-Stat3 (Tyr705) and bcl-2 in WCA group and 5-FU group was significantly decreased compared with the control group respectively.

CONCLUSIONChinese jianpi herbal recipe WCA could inhibit gastric cancer cell SGC-7901 growth in vivo. WCA could induce gastric cancer cell apoptosis and suppress proliferation. Its mechanisms might be involved in the down-regulation of Stat3, RIPX, ROD1 and bcl-2 gene.

Adenocarcinoma ; drug therapy ; genetics ; pathology ; Animals ; Antimetabolites, Antineoplastic ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; therapeutic use ; Expressed Sequence Tags ; Fluorouracil ; pharmacology ; therapeutic use ; Gene Expression Profiling ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nerve Tissue Proteins ; metabolism ; Plants, Medicinal ; chemistry ; Polypyrimidine Tract-Binding Protein ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA-Binding Proteins ; metabolism ; STAT3 Transcription Factor ; metabolism ; Stomach Neoplasms ; drug therapy ; genetics ; pathology ; Xenograft Model Antitumor Assays ; methods

In order to provide a complete picture of pathogenesis in cerebral ischemia, cerebral cortex in MCAO rats were analysed for alteration in their proteomes. Comparative proteome analysis was used to compare signal corresponding to individual cerebral cortex proteins on a two-dimensional gel between MCAO rats and the normal control (NC) group. After sample preparation, two-dimensional electroghoresis separated proteins were stained with Commassie Brilliant Blue. The image data were analyzed on a Dell computer using Image Master v 3.01 software. In cerebral cortex, 30 proteins were differentially expressed in MCAO rats compared with NC. There were 11 spots significantly increased, 15 spots significantly decreased and Adenylate kinase isoenzyme 1 was detected only in NC group, biliverdin reductase B, small inducible cytokine A4 [Precursor] only in MCAO group. Peroxiredoxin 2 divided into two points in MCAO6h group. In the end, this approach may lay a foundation for the further investigation of pathogenic mechanisms in cerebral ischmic injury.
Animals ; Brain Ischemia ; genetics ; metabolism ; Cerebral Cortex ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Profiling ; Infarction, Middle Cerebral Artery ; genetics ; metabolism ; Nerve Tissue Proteins ; chemistry ; genetics ; metabolism ; Proteome ; Proteomics ; methods ; Random Allocation ; Rats ; Rats, Wistar