[The Study of PI3K-â ¢ Like Functional Polypeptide on Leukemia Cell K562 during the Process of Programmed Cell Death].

OBJECTIVE: To study the molecular mechanism of PI3K-Ⅲ like functional domain inducing programmed cell death of leukemia cell line K562. METHODS: The purified PI3K-Ⅲ like functional domain protein was obtained by Pichia pastoris expression system. MTT assay and colony-forming assay were used to detect the effects of PI3K-Ⅲ like functional domain protein on K562 cell proliferation. The effects of PI3K-Ⅲ like functional domain protein on apoptosis and cell cycle of on K562 cells were detected by flow cytometry. The ultrastructural changes were detected by transmission electron microscopy. The expression of caspase-3 was detected by ELISA. The protein expressions of ATG4B, Beclin-1, Bcl-2 and LC3-II were evaluated by Western blot. RESULTS: PI3K-Ⅲ like functional domain protein could inhibit the proliferation and clony formation of K562 cells, which was significantly higher than the control group (P<0.05). In the experimental group, apoptosis and autophagosome were shown in K562 cells. The proportion of cells in G0/G1 phase increased significantly, while in S phase decreased significantly. Cell growth mostly stagnated in G0/G1 phase, which was significantly different from the control group (P<0.05). With the increase of concentration, the expression of caspase-3 protein increased significantly compared with the control group (r=0.966, P<0.05). The expression of ATG4B and beclin-1 appeared from increase to decrease, LC3-II increased while Bcl-2 decreased at different time points. CONCLUSION: PI3K-Ⅲ like functional polypeptide could induce programmed cell death of leukemia cell K562. Beclin-1/Bcl-2 and caspase pathway may be involved in this way, which suggesting meant autophagy and apoptosis may work together at the same time.

The Inhibitory Effects of Recombinant Hespintor Combined with Sorafenib on Transplanted Human Hepatoma in Nude Mice, and Transcriptional Regulation of Hespintor Based on RNA-Seq.

Objective: As targeted drugs, exogenous serpins could be introduced to patients to restore body balance. This study aimed to observe further the inhibitory effects of recombinant Hespintor (a Kazal-type serpin) combined with Sorafenib on transplanted human hepatoma tumors in nude mice specimens and to explore the possible transcriptional regulation by Hespintor. Methods: A model of human hepatoma tumors transplanted in nude mice was established, and the medication was administrated to observe the growth of the tumors. Four weeks after the drug administration, the tumors were removed to evaluate the inhibition effects of Hespintor on in-situ tumor growth and liver metastasis. The expression levels of MMP2, MMP9, Bax, Bcl-2, and caspase-3 in the tumor organizations were detected with Western blot. The target genes of the Hespintor were screened based on tissue RNA-Seq, and the regulatory network was constructed. Results: It was found that the recombinant Hespintor displayed a significant antitumor effect on the subcutaneous growth of MHCC97-H cells. Moreover, the therapeutic effects of the combination therapy were significantly better than those of single therapy. 10 target genes with significantly different expression by Hespintoron tumor tissue were identified. Finally, a visual regulatory networkwas constructed for target mRNA-pathway. Conclusions: The antitumor effect of Hespintor combined with Sorafenib in treating the subcutaneously implanted hepatocellular carcinoma tumors in nude mice was significant. The possible transcriptional regulation by Hespintor involved multiple signaling pathways, and it was not just the antitumor effect of uPA via its extracellular inhibitions.

The peptide encoded by a novel putative lncRNA HBVPTPAP inducing the apoptosis of hepatocellular carcinoma cells by modulating JAK/STAT signaling pathways.

When the hepatitis B virus (HBV) enters target cells, there are complex trans-regulatory mechanisms involved in the interactions between the virus and the target cells. In the present study, a new gene screened from the hepatoblastoma cell line HepG2 using suppression subtractive hybridization, referred to as lncRNA HBVPTPAP, was used to study the trans-regulation of HBV DNA polymerase. According to the structural characteristics of the full-length sequences, it was classified as long non-coding RNA. However, a unique and complete open reading frame (ORF) was still present. Therefore, to further identify the lncRNA HBVPTPAP gene's encoding potential, this study used several online tools to analyze and verify its encoding polypeptide authenticity. On that basis, the effects of the lncRNA HBVPTPAP gene on the biological behaviors of HepG2 cells and its molecular regulatory mechanism were investigated. It was found that the lncRNA HBVPTPAP subcellular was mainly located in the cytoplasm, and possibly activated the downstream JAK/STAT signaling pathway through the interaction between the encoding polypeptide and PILRA intracellular domain. Then, the mitochondrial apoptosis pathway may have been initiated to induce apoptosis. These results provided a basis for further study of the biological functions of the lncRNA HBVPTPAP gene.

[Identification of Differentially Expressed Gene Core Genes in Early T-Cell Precursor Acute Lymphoblastic Leukemia and Its Regulatory Network Analysis].

OBJECTIVE: To identify the differentially expressed gene (DEG) core genes in early T-cell precursor acute lymphoblastic leukemia (ETP ALL) and to analyze their interactions with upstream miRNAs, lncRNAs and involved pathways; to clarify the regulatory mechanism of ETP ALL development; and to explore the molecular targets for clinical diagnosis and treatment. METHODS: The DEG of ETP ALL were screened based on the intersection of GEO database and TCGA database. The functional enrichment analysis and interaction analysis were carried out for DEG. Next, MCODE algorithm was used to screen core genes of DEG, and the mirDIP online tool and starBase online tool were utilized to predict upstream miRNA and lncRNA of the core genes. RESULTS: A total of 424 DEG with a high credibility were identified, which were mainly enriched in the biological activity, such as transcriptional regulation, signaling pathway and protein function activation according to GO function, and the KEGG pathway was enriched in hematopoiesis, anoxic stress response, transcriptional misregulation, immunity and other functions, which interrelated each other 7 core genes were identified. Subsequently, 7 miRNAs and 19 lncRNAs were predicted to meet screening criteria. Finally, a lncRNA-miRNA-mRNA-pathway regulatory network was constructed. CONCLUSION: The DEG in ETP ALL has been identified based on data mining methods; the core genes have been gained by co-expression analysis, and their upstream miRNA and lncRNA can be predicted for the early diagnosis of ETP ALL, thus providing a theoretical basis for the early diagnosis and reasonable treatment of ETP ALL, and helping to look for new tumor biomarkers of ETP ALL different from classical T-ALL.

Application of F0F1­ATPase immuno­biosensors for detecting Escherichia coli O157:H7.

Escherichia coli (E. coli) O157:H7 is an important food­borne pathogen with a low infective threshold and high resistance to treatment. There are currently a number of detection methods available, however, the majority are time­consuming, complex and expensive, thus it is hard for these methods to be applied in routine detection. Therefore, there is urgent requirement to develop more sensitive, rapid and specific detective techniques. In the present study, an immuno­biosensor based on the interference of load to the F0F1­ATPase rotation, indicated by the fluorescence fluctuation, was constructed to detect O157:H7. The results demonstrated a good linear relationship (R2=0.9818) between antigen concentration (range, 102 cfu to 104 cfu) and the fluorescence intensity. The detection signals of the samples containing 102 cfu/well and 104 cfu/well E. coli O157:H7 were significantly stronger than the signal produced by the control sample (P<0.01). Due to its higher sensibility and simplicity when compared with the current methods applied, the results of the present study indicate a promising future for the application of this technique in detecting food source pathogens.

Expression and clinical significance of Sirt1 in colorectal cancer.

The objective of the present study was to examine the expression of Silent information regulator 1 (Sirt1) in colorectal cancer and peritumoral normal mucosa tissue, and therefore analyze the role and molecular mechanism of Sirt1 in the pathogenesis of colorectal cancer. Colorectal cancer tissue specimens were employed as the experimental group, and adjacent normal mucosa tissues >5 cm from tumor lesions were used as the control group. The expression of Sirt1 was detected by the immunohistochemical streptavidin peroxidase detection method in paraffin-embedded sections, whilst Sirt1 protein expression was examined by western blot analysis in the fresh tissues. Sirt1 protein was primarily expressed in the nuclei of the tumor cells, and positive staining was brownish-yellow in color. The relative expression quantities of Sirt1 in the peritumoral normal rectal mucosa and rectal carcinoma were 1.15 and 2.62, and the differences between the two groups were statistically significant (P<0.05). The expression level of Sirt1 in colorectal carcinoma was significantly associated with the depth of tumor invasion, differentiation and tumor size (P<0.05). Sirt1 expression was also found to be associated with tumor tissue type, lymph node metastasis, Duke's stage and patient age. These characteristics combined may therefore be used as markers for the early diagnosis of colorectal cancer pathogenesis.

Inhibitory effects of the recombinant human serine protease inhibitor Hespintor on the proliferation, migration and invasion of hepatocellular carcinoma cells.

Hespintor is a new Kazal-type serine proteinase inhibitor (Serpin) screened from the HepG2 hepatoblastoma cell line using the suppression subtractive hybridization (SSH) technique. Seprin is closely associated with the progression and remission of malignant tumors, and has certain significance in the diagnosis and treatment of tumors. Investigations on the antitumor activity of Serpin are expected to aid in the development of a new method for tumor treatment based on the serine protease inhibitor. Although the Hespintor prokaryotic expression strain and recombinant Hespintor protein (recombinant fusion protein of Hespintor and rHespintor) have already been obtained, the protein extraction efficiency is low due to the low initial amount of extracted protein and large number of purification steps, which affect the study of the protein function. The aim of the present study was to improve the purification method of rHespintor, increase the protein extraction efficiency, and investigate its effects on the proliferation, migration and invasion of the HepG2 hepatoblastoma cell line. The results demonstrated that the application of urea gradient washing of inclusion body of the protein may effectively remove the majority of impure proteins from the targeted protein. After one-step purification, the target protein rHespintor exhibited a high inhibitory effect of Trypsin Hydrolysis, which was exhibited in a dose-dependent manner. Hoechst 33258 staining was used to determine cell apoptosis. After treating HepG2 hepatoblastoma cells with rHespintor, the cell growth was inhibited, the proliferation ability was reduced, and the number of migrated and invaded cells were significantly decreased. Hoechst 33258 staining and flow cytometry assay results showed clear cell apoptosis. The results reveal showed that rHespintor significantly inhibited proliferation, migration and invasion of the HepG2 hepatoblastoma cell line in vitro, and induced cell apoptosis to a certain extent, indicating that the recombinant protein Hespintor exerts an antitumor effect in vitro, and has the potential and feasibility to become an antitumor drug.

Expression and characterization of protein Latcripin-3, an antioxidant and antitumor molecule from Lentinula edodes C91-3.

In this study, an anti-oxidant and anti-tumor protein Latcripin-3 of Lentinula edodes C91-3 was expressed in Escherichia coli. for the first time. According to the cDNA library, the full-length gene of Latcripin-3 was cloned by the methods of 3'-full rapid amplification of cDNA Ends (RACE) and 5'-full RACE. The structural domain gene of Latcripin-3 was inserted into the pET32 a(+). The functional protein of Latcripin-3 was expressed in Rosetta-gami (DE3) E. coli, evaluated by Western blotting and mass spectrometry. DPPH testing showed that the protein Latcripin-3 can scavenge free radicals remarkably well. The activity of functional protein Latcripin-3 on A549 cells was studied with flow cytometry and the MTT method. The MTT assay results showed that there was a decreases in cell viability in a dose-dependent and time-dependent manner in protein Latcripin-3 treated groups. Flow cytometry demonstrated that Latcripin-3 can induce apoptosis and block S phase dramatically in human A549 lung cancer cells as compared to the control group. At the same time, the cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. This research offers new insights and advantages for identifying anti-oxidant and anti-tumor proteins.

Transactivation of proto-oncogene c-Myc by hepatitis B virus transactivator MHBst167.

C-terminally truncated hepatitis B virus (HBV) middle size surface proteins (MHBst) has been shown to be a transcriptional activator and may be relevant to hepatocarcinogenesis by transactivating gene expression. In the present study, a pcDNA3.1(-)-MHBst167 vector coding for MHBst truncated at amino acid 167 (MHBst167) was constructed and transfected into the HepG2 hepatoma cell line. mRNA and protein expression of MHBst167 in the cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. A cDNA library of genes transactivated by the truncated protein in HepG2 cells was made in pGEM-T Easy using suppression subtractive hybridization. The cDNAs were sequenced and analyzed with BLAST searching against the sequences in GenBank. The results showed that certain sequences, such as that of human proto-oncogene c-Myc, may be involved in tumor development. An expression vector pCAT3/c-Myc containing the chloramphenicol acetyltransferase (CAT) gene under the control of a c-Myc promoter was generated, and the transcriptional transactivating effect of MHBst167 on the c-Myc promoter was investigated by RT-PCR and western blotting. MHBst167 was found to upregulate the transcriptional activity of the promoter, as well as transcription and translation of c-Myc. MHBst167 was also shown to transactivate SV40 immediate early promoter, and transcriptionally transactivate the expression of human c-Myc. These findings provide new directions for studying the biological functions of MHBst167, and for a better understanding of the tumor development mechanisms of HBV infection.

Purification and identification of the Kazal domain of a novel serine protease inhibitor, Hespintor, through a bacterial (Escherichia coli) expression system.

In this study, Hespintor, a protein with unknown function, was screened and obtained from the hepatoblastoma cell line, HepG2, using suppression subtractive hybridization (SSH). Sequence analysis demonstrated that the protein is a novel secreting member of the Kazal-type serine protease inhibitor (serpin) family, and possesses the basic structure of serpin, which is highly homologous to esophageal cancer-related gene 2 (ECRG2). To further elucidate its biological functions, the Hespintor protein was expressed and purified. The coding sequence of the Hespintor Kazal domain was cloned into the prokaryotic expression vector, pET-40b(+), and was then transformed into host bacteria (Escherichia coli) Rosetta (DE3). The optimally expressed recombinant fusion protein, Hespintor-Kazal, with a molecular weight of 42 kDa was obtained by 0.25 mmol/l isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction at 30˚C for 5 h. Western blot analysis was performed to further confirm the specificity of the recombinant protein, Hespintor-Kazal. The recombinant fusion protein, Hespintor­Kazal, was expressed in the host bacteria in the form of an inclusion body. Two-step metal chelating affinity chromatography and anion exchange chromatography columns were used to purify the recombinant protein. The preliminary activity identification results revealed that the purified recombinant fusion protein, Hespintor-Kazal, specifically inhibited the hydrolysis activity of trypsin, suggesting that Hespintor has potential value as a novel antitumor drug.

Maternal nicotinamide supplementation causes global DNA hypomethylation, uracil hypo-incorporation and gene expression changes in fetal rats.

Recent evidence shows that excess nicotinamide can cause epigenetic changes in developing rats. The aim of the present study was to investigate the effects of maternal nicotinamide supplementation on the fetus. Female rats were randomised into four groups fed a standard chow diet (control group) or diets supplemented with 1 g/kg of nicotinamide (low-dose group), 4 g/kg of nicotinamide (high-dose group) or 4 g/kg of nicotinamide plus 2 g/kg of betaine (betaine group) for 14-16 d before mating and throughout the study. Fetal tissue samples were collected on the 20th day of pregnancy. Compared with the control group, the high-dose group had a higher fetal death rate, and the average fetal body weight was higher in the low-dose group but lower in the high-dose group. Nicotinamide supplementation led to a decrease in placental and fetal hepatic genomic DNA methylation and genomic uracil contents (a factor modifying DNA for diversity) in the placenta and fetal liver and brain, which could be completely or partially prevented by betaine. Moreover, nicotinamide supplementation induced tissue-specific alterations in the mRNA expression of the genes encoding nicotinamide N-methyltransferase, DNA methyltransferase 1, catalase and tumour protein p53 in the placenta and fetal liver. High-dose nicotinamide supplementation increased fetal hepatic α-fetoprotein mRNA level, which was prevented by betaine supplementation. It is concluded that maternal nicotinamide supplementation can induce changes in fetal epigenetic modification and DNA base composition. The present study raises the concern that maternal nicotinamide supplementation may play a role in the development of epigenetic-related diseases in the offspring.

Identification of paired immunoglobulin-like type 2 receptor α as hepatitis B virus DNA polymerase transactivated protein 1 interacting proteins.

Hepatitis B Virus (HBV) DNA polymerase transactivated protein 1 (HBVDNAPTP1) is a novel protein transfected by HBV DNA polymerase, which has been screened by a suppression subtractive hybridization technique. In the present study, a yeast two-hybrid system was used to screen the proteins interacting with HBVDNAPTP1 in leukocytes in order to investigate the biological function of HBVDNAPTP1. The HBVDNAPTP1 coding sequence was cloned into a pGEM-T vector. Subsequent to sequencing, the HBVDNAPTP1 was subcloned into the bait plasmid pGBKT7 and transformed into yeast AH109. Western blotting confirmed the presence of HBVDNAPTP1 expression in the AH109 yeast strains. The transformed yeast AH109 cells were mated with Y187 yeast cells containing the leucocyte cDNA library pACT2 plasmids in 2X yeast extract peptone D-glucose adenine (YPDA) medium. For selection and screening, diploid yeast was plated on synthetic dropout medium (SD/-Trp-Leu-His-Ade) containing X-α-gal. Following sequencing and the verification of the open reading frames of positive colonies, four different proteins were obtained. To further confirm the interaction between HBVDNAPTP1 and the screened proteins, paired immunoglobulin-like type 2 receptor α (PILRA), one of the positive colonies, was cloned. The glutathione S-transferase pull-down in vitro assay and a co-immunoprecipitation in vivo assay were used to examine the interaction between HBVDNAPTP1 and PILRA, respectively. HBVDNAPTP1 may be involved in the negative regulation of the PILRA­mediated Janus-activated kinase/signal tranducer and activator of transcription signaling pathway, and exert a positive effect on the initiation of monocyte apoptosis. These results contribute our knowledge of the biological functions of HBVDNAPTP1 and provide novel data to aid in the further analysis of the regulatory mechanism of this protein.

Nicotinamide supplementation induces detrimental metabolic and epigenetic changes in developing rats.

Ecological evidence suggests that niacin (nicotinamide and nicotinic acid) fortification may be involved in the increased prevalence of obesity and type 2 diabetes, both of which are associated with insulin resistance and epigenetic changes. The purpose of the present study was to investigate nicotinamide-induced metabolic changes and their relationship with possible epigenetic changes. Male rats (5 weeks old) were fed with a basal diet (control group) or diets supplemented with 1 or 4 g/kg of nicotinamide for 8 weeks. Low-dose nicotinamide exposure increased weight gain, but high-dose one did not. The nicotinamide-treated rats had higher hepatic and renal levels of 8-hydroxy-2'-deoxyguanosine, a marker of DNA damage, and impaired glucose tolerance and insulin sensitivity when compared with the control rats. Nicotinamide supplementation increased the plasma levels of nicotinamide, N1-methylnicotinamide and choline and decreased the levels of betaine, which is associated with a decrease in global hepatic DNA methylation and uracil content in DNA. Nicotinamide had gene-specific effects on the methylation of CpG sites within the promoters and the expression of hepatic genes tested that are responsible for methyl transfer reactions (nicotinamide N-methyltransferase and DNA methyltransferase 1), for homocysteine metabolism (betaine-homocysteine S-methyltransferase, methionine synthase and cystathionine ß-synthase) and for oxidative defence (catalase and tumour protein p53). It is concluded that nicotinamide-induced oxidative tissue injury, insulin resistance and disturbed methyl metabolism can lead to epigenetic changes. The present study suggests that long-term high nicotinamide intake (e.g. induced by niacin fortification) may be a risk factor for methylation- and insulin resistance-related metabolic abnormalities.

Excess nicotinamide increases plasma serotonin and histamine levels.

Methylation, a methyl group-consuming reaction, plays a key role in the degradation (i.e., inactivation) of monoamine neurotransmitters, including catecholamines, serotonin and histamine. Without labile methyl groups, the methylation-mediated degradation cannot take place. Although high niacin (nicotinic acid and nicotinamide) intake, which is very common nowadays, is known to deplete the body's methyl-group pool, its effect on monoamine-neurotransmitter degradation is not well understood. The aim of this article was to investigate the effect of excess nicotinamide on the levels of plasma serotonin and histamine in healthy subjects. Urine and venous blood samples were collected from nine healthy male volunteers before and after oral loading with 100 mg nicotinamide. Plasma N(1)-methylnicotinamide, urinary N(1)-methyl-2-pyridone-5-carboxamide (2-Py), and plasma betaine levels were measured by using high-performance liquid chromatography (HPLC). Plasma concentrations of choline, serotonin and histamine were measured using commercial kits. The results showed that the plasma N(1)-methylnicotinamide level and the urinary excretion of 2-Py significantly increased after oral loading with 100 mg nicotinamide, which was accompanied with a decrease in the methyl-group donor betaine. Compared with those before nicotinamide load, five-hour postload plasma serotonin and histamine levels significantly increased. These results suggest that excess nicotinamide can disturb monoamine-neurotransmitter metabolism. These findings may be of significance in understanding the etiology of monoamine-related mental diseases, such as schizophrenia and autism (a neurodevelopmental disorder).

Excess nicotinamide inhibits methylation-mediated degradation of catecholamines in normotensives and hypertensives.

Nicotinamide and catecholamines are both degraded by S-adenosylmethionine-dependent methylation. Whether excess nicotinamide affects the degradation of catecholamines is unknown. The aim of this study was to investigate the effect of nicotinamide on the methylation status of the body and methylation-mediated catecholamine degradation in both normotensives and hypertensives. The study was conducted in 19 normotensives and 27 hypertensives, using a nicotinamide-loading test (100 mg orally). Plasma nicotinamide, N(1)-methylnicotinamide, homocysteine (Hcy), betaine, norepinephrine, epinephrine, normetanephrine and metanephrine levels before and 5 h after nicotinamide loading were measured. Compared with normotensives, hypertensives had higher baseline (fasting) levels of plasma nicotinamide, Hcy and norepinephrine, but lower levels of plasma normetanephrine, a methylated norepinephrine derivative. Nicotinamide loading induced a significant increase in the levels of plasma N(1)-methylnicotinamide and norepinephrine, and a significant decrease in the levels of O-methylated epinephrine (metanephrine) and betaine, a major methyl donor, in both hypertensives and normotensives. Moreover, nicotinamide-loading significantly increased plasma Hcy levels, but decreased plasma normetanephrine levels in normotensives. The baseline levels of plasma epinephrine in hypertensives were similar to those of normotensives, but the post-nicotinamide-loading levels of plasma epinephrine in hypertensives were higher than those of normotensives. This study demonstrated that excess nicotinamide might deplete the labile methyl pool, increase Hcy generation and inhibit catecholamine degradation. It also revealed that hypertensives had an abnormal methylation pattern, characterized by elevated fasting plasma levels of unmethylated substrates, nicotinamide, Hcy and norepinephrine. Therefore, it seems likely that high nicotinamide intake may be involved in the pathogenesis of Hcy-related cardiovascular disease.

Dietary methyl-consuming compounds and metabolic syndrome.

The metabolic syndrome, a major risk factor for type 2 diabetes and cardiovascular disease, is a cluster of metabolic abnormalities including obesity, insulin resistance, hypertension and dyslipidemia. Although systemic oxidative stress and aberrant methylation status are known to have important roles in the development of metabolic syndrome, how they occur remains unclear. The metabolism of methyl-consuming compounds generates reactive oxygen species and consumes labile methyl groups; therefore, a chronic increase in the levels of methyl-consuming compounds in the body can induce not only oxidative stress and subsequent tissue injury, but also methyl-group pool depletion and subsequent aberrant methylation status. In the past few decades, the intake amount of methyl-consuming compounds has substantially increased primarily due to pollution, food additives, niacin fortification and high meat consumption. Thus, increased methyl consumers might have a causal role in the development and prevalence of metabolic syndrome and its related diseases. Moreover, factors that decrease the elimination/metabolism of methyl-consuming compounds and other xenobiotics (for example, sweat gland inactivity and decreased liver function) or increase the generation of endogenous methyl-consuming compounds (for example, mental stress-induced increase in catecholamine release) may accelerate the progression of metabolic syndrome. Based on current nutrition knowledge and the available evidence from epidemiological, ecological, clinical and laboratory studies on metabolic syndrome and its related diseases, this review outlines the relationship between methyl supply-consumption imbalance and metabolic syndrome, and proposes a novel mechanism for the pathogenesis and prevalence of metabolic syndrome and its related diseases.

Cloning, expression and identification by immunohistochemistry of humanized single-chain variable fragment antibody against hepatitis C virus core protein.

Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library containing the gene for scFv antibody against Hepatitis C virus core protein was panned with core protein immobilized on microtiter plate wells. After five rounds of panning 60 phage clones specific to core protein were obtained and one selected clone was sequenced. It was found that the specifically detected antigen consists of 774bp and is capable of encoding 257 amino acids in the patients but not in healthy persons.

[Chronic nicotinamide overload and type 2 diabetes].

Type 2 diabetes is a major global health problem. It is generally accepted that type 2 diabetes is the result of gene-environmental interaction. However, the mechanism underlying the interaction is unclear. Diet change is known to play an important role in type 2 diabetes. The fact that the global high prevalence of type 2 diabetes has occurred following the spread of food fortification worldwide suggests a possible involvement of excess niacin intake. Our recent study found that nicotinamide overload and low nicotinamide detoxification may induce oxidative stress associated with insulin resistance. Based on the relevant facts, this review briefly summarized the relationship between the prevalence of type 2 diabetes and the nicotinamide metabolism changes induced by excess niacin intake, aldehyde oxidase inhibitors, liver diseases and functional defects of skin. We speculate that the gene-environmental interaction in type 2 diabetes may be a reflection of the outcome of the association of chronic nicotinamide overload-induced toxicity and the relatively low detoxification/excretion capacity of the body. Reducing the content of niacin in foods may be a promising strategy for the control of type 2 diabetes.

[Function of TTG1A in hepatic stellate cells].

OBJECTIVE: To investigate the biological functions of TTG1A in liver fibrosis. METHODS: Yeast two-hybrid system was used to screen proteins associated with TTG1A. Briefly, the coding sequence of TTG1A was cloned into pGBKT7 vector, and the recombinant plasmid was transformed into yeast cells AH109 ( a type), then these cells were mated with yeast cells Y187 (a type) transformed with human leukocyte cDNA library plasmid pACT2. The obtained diploid yeast cells were plated on synthetic dropout nutrient medium containing X-alpha-gal for double selection. The plasmids from positive colonies were transformed into E.coli and sequenced. RESULTS: The recombinant yeast expression vector pGBKT7-TTG1A was successfully constructed. Nineteen TTG1A binding proteins, including Homo sapiens major histocompatibility complex, class II DP beta 1 (HLA-DPb1), Homo sapiens ribosomal protein L30 (RPL30), Homo sapiens nucleophosmin Homo sapiens nucleobindin 2 (NUCB2), Homo sapiens ash2, variant Gaucher disease and variant metachromatic leukodystrophy, MORF4L1, Homo sapiens ubiquitin-conjugating enzyme E2L3 (UBE2L3), APOA1, Homo sapiens lectin, and galectin 1, were identified. CONCLUSIONS: This study may help to elucidate the molecular function of TTG1A.