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Background: At present, there is no report that the intestinal flora of pregnant women with mild thalassemia is different from that of healthy pregnant women. Objectives: This study compared the composition and changes of the intestinal flora of pregnant women with mild thalassemia to those of healthy pregnant women using metagenomic sequencing technology and evaluated the potential microecological risk for pregnant women and the fetus. Methods: The present study was carried out on 14 mild thalassemia pregnant women with similar backgrounds in the Affiliated Hospital of Putian University, Fujian, China. In the same period, 6 healthy pregnant women were selected as the control group. The genomic deoxyribonucleic acid was extracted from the sable stool samples of pregnant women. Illumina HiSeq sequencing technology was adopted after library preparation. Prodigal software (ver 2.6.3), Salmon software (ver 1.6.0), and Kraken software (ver 2) were used to analyze the sequence data. Moreover, analysis of variance and Duncan’s multiple-comparison test or Wilcoxon rank-sum test were used as statistical methods. Results: The characteristics of the intestinal flora of pregnant women with mild thalassemia differed significantly from those of healthy pregnant women, showing an increase in some conditionally pathogenic bacteria (e.g., Prevotella stercorea rose and Escherichia coli) and a decrease in some probiotic bacteria, which might affect pregnant women and cause physiological function damage to their offspring by changing metabolic pathways; however, further validation is needed. Conclusions: The diversity and composition of intestinal flora in pregnant women with mild thalassemia vary significantly from those in healthy pregnant women, especially at the genus and species levels, representing more profound alterations in intestinal microecology.

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.

Escherichia coli (E. coli) O157:H7 is an important food-borne pathogen with a low infective threshold and high resistance to treatment. There are currently a number of detection methods available, however, the majority are time-consuming, 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 immuno-biosensor based on the interference of load to the F0F1-ATPase 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

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.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.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.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.急性早期前体T淋巴细胞白血病核心基因的筛选及其调控网络分析.筛选急性早期前体T淋巴细胞白血病（ETP ALL）核心基因并分析其与上游互作miRNA、lncRNA和参与通路的相互作用，探讨ETP ALL发生发展过程中的调控机制并寻找可用于临床诊疗的分子靶点.利用基因表达公共数据库（Gene Expression Omnibus, GEO）和癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库的2者交集，筛选ETP ALL差异表达基因集（differentially expressed genes, DEG）分别进行功能富集和相互作用的分析；再利用网络模块划分方法筛选DEG核心基因，并利用mirDIP、starBase在线工具对核心基因上游miRNA、lncRNA进行预测.获得高可信度的差异表达基因424个，基因本体（gene ontology,GO）功能富集于转录调控、信号通路及蛋白功能活化等生物学活性，KEGG通路富集于造血、缺氧应激反应、转录失调、免疫等功能；通过两者相互关联分析。获得7个核心基因，并先后预测到7个miRNA和19个lncRNA符合筛选标准，最后构建出1个lncRNA-miRNA-mRNA-pathway调控网络.基于数据挖掘方法筛选获得ETP ALL中的差异表达基因集，通过共表达分析寻找到其中的核心基因，并对核心基因上游miRNA，lncRNA进行预测，为ETP ALL的早期诊断和合理治疗提供了理论依据，有助于寻找新的区别于经典T-ALL的ETP ALL肿瘤标志物.

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.

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

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.

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 nicotinamideN-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.

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 PILRA‑mediated 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 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.

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.

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.

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.

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 β-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, Hespintor‑Kazal, 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.

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 (nicotinamideN-methyltransferase and DNA methyltransferase 1), for homocysteine metabolism (betaine–homocysteineS-methyltransferase, methionine synthase and cystathionine β-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.

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).

The purpose of this study is to establish a new method for detecting nicotinamide concentration in plasma. In the experiment, the high performance liquid chromatography (HPLC) method was used, with a fluorescence detector. The nicotinamide in the plasma was first converted to N1- methylnicotinamide, then reacted with acetophenone under certain conditions to produce fluorescent derivatives for testing. The method is a kind of highly sensitive detection, of which the lower limit is 10 ng/mL, the recovery rate is between 92.75% and 105.13%, and the relative standard deviation (RSD) is between 3.76% and 4.43%. The results showed that this measurement method is accurate, sensitive and rapid. It meets the requirements of the experiment, and applies to the detection of nicotinamide concentration in plasma.

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.

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.

To investigate the biological functions of TTG1A in liver fibrosis.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.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.This study may help to elucidate the molecular function of TTG1A.

To clone and sequence variant-specific surface antigen gene from Giardia lamblia isolate SUCH/89/BTMR/2(C2) derived from human in China.Total genomic DNA of G. lamblia was extracted and a full-length variant-specific surface antigen gene fragment was amplified by polymerase chain reaction (PCR). The PCR product was cloned into pMD19-T simple-vector, transformed into an Escherichia coli JM109 strain and then sequenced. The sequence analysis for cloned fragment was finished by Vector NTI 9.0 software for the homology of Giardia variant-specific surface antigen gene to that of sequences published in GenBank.The full-length variant-specific surface antigen gene fragment from G. lamblia was found to be 2 142 bp, encoding a 713 amino acid polypeptide and contained a single open reading frame (ORF). The deduced polypeptide sequence was rich in cysteine (11.8 mol%), most of which occurred with in 29 copies of the 4-amino acid CXXC motif, one GGCY-tetrapeptide motifs and three NXS consensus N-linked glycosylation sites. This polypeptide was also rich in threonine (10.2 mol%), glycine (12.1 mol%) and alanine (10.1 mol%). Like other previously identified VSPs, it contained a highly conserved hydrophobic C-terminal region. The homology of G. lamblia SUCH/89/BTMRI/2(C2) variant-specific surface antigen gene to that of sequence (TSA417) published in GenBank was 99% both at the nucleotide and the amino acid levels.The full length variant-specific surface antigen gene from the isolate of G. lamblia has the common characteristics with other previously identified VSPs.

To construct a cDNA subtractive library of genes transactivated by TGF beta 1 in LX02 hepatic stellate cells (HSC); to screen and to clone the regulated genes transactivated by TGF beta 1; and to elucidate the molecular biological mechanism of hepatic fibrosis mediated by TGF beta 1.mRNA was isolated from HSC treated with TGF beta 1 or with PBS (as controls). Suppression subtractive hybridization (SSH) technique was employed to analyze the differentially expressed DNA sequence between the two groups. After restriction enzyme Rsa I digestion, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent polymerase chain reaction twice it then was subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5a. The cDNA was sequenced and analyzed in GenBank with Blast search.The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC was constructed successfully. The amplified library contained 146 positive clones, which contained 200-1000 bp of inserts. Randomly, thirty clones were analyzed by sequencing and bioinformatics, consisting of 28 known genes and 2 unknown genes.The subtractive cDNA library of genes transactivated by TGF beta 1 in HSC using SSH technique was constructed successfully. Some gene coding proteins are those involved in cell growth regulation, protein synthesis, signal transduction, extracellular matrix metabolism, and anti-lipid peroxidative, which gives us some new clues for the study of the mechanism of liver fibrosis.

Background The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein. Methods The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for alpha-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis. Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2 receptor gamma chain, PALM2-AKAP2 protein (PALM2-AKAP2), eukaryotic translation initiation factor 4A, beta-2-microglobin, solute carrier family 9 (sodium/hydrogen exchanger), calreticulin, asialoglycoprotein receptor 1 (ASGR1), MHC class II lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86 (LY86) and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank (accession number: DQ471327). Conclusions Genes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S1 protein.

AIM: To investigate whether nicotinamide overload plays a role in type 2 diabetes. METHODS: Nicotinamide metabolic patterns of 14 diabetic and 14 non-diabetic subjects were compared using HPLC. Cumulative effects of nicotinamide and N1-methylnicotinamide on glucose metabolism, plasma H2O2 levels and tissue nicotinamide adenine dinucleotide (NAD) contents of adult Sprague-Dawley rats were observed. The role of human sweat glands and rat skin in nicotinamide metabolism was investigated using sauna and burn injury, respectively. RESULTS: Diabetic subjects had significantly higher plasma N1-methylnicotinamide levels 5 h after a 100-mg nicotinamide load than the non-diabetic subjects (0.89 ± 0.13 μmol/L vs 0.6 ± 0.13 μmol/L, P < 0.001). Cumulative doses of nicotinamide (2 g/kg) significantly increased rat plasma N1-methylnicotinamide concentrations associated with severe insulin resistance, which was mimicked by N1-methylnicotinamide. Moreover, cumulative exposure to N1-methylnicotinamide (2 g/kg) markedly reduced rat muscle and liver NAD contents and erythrocyte NAD/NADH ratio, and increased plasma H2O2 levels. Decrease in NAD/NADH ratio and increase in H2O2 generation were also observed in human erythrocytes after exposure to N1-methylnicotinamide in vitro. Sweating eliminated excessive nicotinamide (5.3-fold increase in sweat nicotinamide concentration 1 h after a 100-mg nicotinamide load). Skin damage or aldehyde oxidase inhibition with tamoxifen or olanzapine, both being notorious for impairing glucose tolerance, delayed N1-methylnicotinamide clearance. CONCLUSION: These findings suggest that nicotinamide overload, which induced an increase in plasma N1-methylnicotinamide, associated with oxidative stress and insulin resistance, plays a role in type 2 diabetes.