The gastrointestinal tract is the largest digestive organ and the largest immune organ and detoxification organ, which is vital to the health of the body. Drosophila is a classic model organism, and its gut is highly similar to mammalian gut in terms of cell composition and genetic regulation, therefore can be used as a good model for studying gut development. target of rapmaycin complex 1 (TORC1) is a key factor regulating cellular metabolism. Nprl2 inhibits TORC1 activity by reducing Rag GTPase activity. Previous studies have found that nprl2 mutated Drosophila showed aging-related phenotypes such as enlarged foregastric and reduced lifespan, which were caused by over-activation of TORC1. In order to explore the role of Rag GTPase in the developmental defects of the gut of nprl2 mutated Drosophila, we used genetic hybridization combined with immunofluorescence to study the intestinal morphology and intestinal cell composition of RagA knockdown and nprl2 mutated Drosophila. The results showed that RagA knockdown alone could induce intestinal thickening and forestomach enlargement, suggesting that RagA also plays an important role in intestinal development. Knockdown of RagA rescued the phenotype of intestinal thinning and decreased secretory cells in nprl2 mutants, suggesting that Nprl2 may regulate the differentiation and morphology of intestinal cells by acting on RagA. Knockdown of RagA did not rescue the enlarged forestomach phenotype in nprl2 mutants, suggesting that Nprl2 may regulate forestomach development and intestinal digestive function through a mechanism independent of Rag GTPase.
Animals ; Drosophila/genetics* ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Mammals/metabolism* ; Carrier Proteins ; Tumor Suppressor Proteins/metabolism* ; Drosophila Proteins/genetics*

OBJECTIVETo investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A).

METHODSYeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods.

RESULTSAmong twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase.

CONCLUSIONSGenes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.

Carrier Proteins ; genetics ; Cloning, Molecular ; Hepacivirus ; genetics ; Hepatocytes ; metabolism ; Humans ; Two-Hybrid System Techniques ; Viral Nonstructural Proteins ; Viral Proteins ; genetics

To detect the expression of telomerase subunits (human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA) in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect telomerase subunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25%, respectively. The former was significantly higher than the latter (chi2 = 26.4, P < 0.01). The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 91.7%, respectively and no significant difference was found between them (chi2 = 2.1, P > 0.05). The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100% and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.
Carrier Proteins/biosynthesis ; Carrier Proteins/genetics ; RNA/biosynthesis ; RNA/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms/*metabolism ; Telomerase/*biosynthesis ; Telomerase/genetics

Animals ; Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Insulinoma ; genetics ; metabolism ; pathology ; Mice ; Neoplasm Proteins ; genetics ; metabolism ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; Phosphoproteins ; genetics ; metabolism

OBJECTIVETo explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells.

METHODSLuciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3'-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay.

RESULTSThe expressions of PKM2 mRNA and protein were upregulated (5.12 +/- 0.35) times and (4.05 +/- 0.20) times respectively in the PC3 cells as compared with those in the RWPE-1 cells (P < 0.05). Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3'-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated (0.16 +/- 0.04) times (P < 0.05), while the PKM2 mRNA level was not changed significantly (P > 0.05), as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were (66.20 +/- 5.10)% vs (82.10 +/- 6.35)% at 24 hours (P < 0.05) and (49.34 +/- 2.37)% vs (70.10 +/- 5.80)% at 48 hours (P < 0.05).

CONCLUSIONmiR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.

Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; MicroRNAs ; genetics ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Thyroid Hormones ; genetics ; metabolism ; Transfection

OBJECTIVE: To detect the expression of RA SAL2 in patients with hepatocellular carcinoma (HCC), and to investigate the association of RASAL2 expression with pathological characteristics and prognosis. METHODS: Immunohistochemical SP method was used to detect the expression of RA SAL2 in 164 samples of HCC tissue and the adjacent tissue. Th e association of RA SAL2 expression with clinical features and prognosis was analyzed. RESULTS: The expression of RASAL2 in adjacent tissue was significantly increased compared to that in HCC tissue (P<0.001). The expression level of RASAL2 was associated with the degree of differentiation, tumor TNM stage and vascular invasion (P<0.001), but not associated with the level of AFP, tumor size, or the number of nodules (P>0.05). The 5 years recurrence-free survival (RFS) in patients with low expression of RASLA2 was significantly reduced compared with that in patients with high expression of RASLA2 (P<0.001). Cox analysis showed that low expression of RASLA2 was the independent factor for recurrence and death in HCC patients after surgery (P<0.001). CONCLUSION Low expression of RRASAL2 is significantly associated with the poor prognosis of HCC, which is an independent factor for HCC prognosis.
Carcinoma, Hepatocellular ; genetics ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Neoplasm Recurrence, Local ; Prognosis

OBJECTIVETo express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein.

METHODSThe fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors. After digestion by EcoR I and Hind III, the fragment was subcloned into PQE-30, a prokaryotic expression vector with 6×His tag. The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15. The expression of his-tagged fusion protein was induced by IPTG. The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads.

RESULTSThe recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15. The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting. The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained.

CONCLUSIONThe recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.

Carrier Proteins ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Humans ; Male ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

Enterovirus 71 (EV71) is a neurotropic pathogen that can induce hand, foot and mouth disease in children. There is an appreciable mortality rate after EV71 infections. The mechanism of action of EV71 replication is not known. Recent work has identified some of cell factors of the host that participate in the synthesis of the RNA and proteins of EV71 (e.g., hnRNP K, reticulon 3 (RTN 3)). In that work, researchers used a competitive assay to show that hnRNP K can interact with EV71 5' UTR, which is required for efficient synthesis of viral RNA. Using a yeast two-hybrid system, other researchers demonstrated that RTN 3 interacts with the N-terminal domain of EV71 2C, which is crucial for replication of viral RNA. Here, we discuss recent work focusing on the molecular mechanisms of hnRNP K and RTN 3 in the synthesis of the RNA and proteins of EV71.
Animals ; Carrier Proteins ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; physiology ; Enterovirus Infections ; genetics ; metabolism ; virology ; Heterogeneous-Nuclear Ribonucleoprotein K ; Host-Pathogen Interactions ; Humans ; Membrane Proteins ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Ribonucleoproteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

Failure of mitotic checkpoint machinery leads to the chromosomal missegregation and nuclear endoreduplication, thereby driving the emergence of aneuploidy and tetraploidy population. Although abnormal nuclear ploidy and the resulting impairment of mitotic checkpoint function are typical physiological event leading to human hepatocellular carcinoma, any mutational change of mitotic checkpoint regulators has not yet been discovered. Therefore, we investigated the mutation of p31(comet), a recently identified mitotic checkpoint regulator, in human hepatocellular carcinoma. Of 51 human hepatocellular carcinoma tissue and 6 cell lines tested, five samples exhibited nucleotide sequence variations dispersed on four sites within the entire coding sequence. Among these sites with sequence substitutions, three were found to be missense mutation accompanied with amino acid change but one was a silent mutation. Of these sequence substitutions, two were present in both tumor and non-tumor liver tissues, suggesting the possibility of polymorphism. The present findings indicate that p31(comet) does not have an impact on the formation of aneuploidy and tetraploidy found in human hepatocellular carcinoma.
Adaptor Proteins, Signal Transducing ; Calcium-Binding Proteins/*metabolism ; Carcinoma, Hepatocellular/genetics/*metabolism ; Carrier Proteins/*genetics/metabolism ; Cell Cycle Proteins/*genetics/*metabolism ; Cell Line, Tumor ; Humans ; Liver Neoplasms/genetics/*metabolism ; *Mutation ; Nuclear Proteins ; Polyploidy ; Repressor Proteins/*metabolism

OBJECTIVETo construct the eukaryotic expression vector of HBEBP1 gene and express HBEBP1 recombinant protein in yeast.

METHODSPCR was performed to amplify the gene of HBEBP1 from the cDNA template origining from HepG2, and the gene was cloned into pGEM-T vector. After sequencing, the correct DNA fragment was cut from pGEM-T-HBEBP1 and inserted into yeast expression plasmid pGBKT7. The reconstructed plasmid pGBKT7-HBEBP1 was transformed into yeast cell AH109 and screened on the synthetic dropout nutrient medium (SD/-Trp/Kana). The yeast protein was isolated and analyzed with SDS-PAGE and Western Blot.

RESULTSThe eukaryotic expressive vector was constructed successfully. The results of Western Blot showed HBEBP1 protein was existed within yeast cells and the molecular weight of it was about 33 x 10(3).

CONCLUSIONSThe successful expression of HBEBP1 protein in yeast cells lay the foundation for studying biological function of HBEBP1.

Blotting, Western ; Carrier Proteins ; genetics ; Hepatitis B e Antigens ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; Saccharomyces cerevisiae ; genetics