OBJECTIVES: To study the association of β2-drenergic receptor ( METHODS: A total of 143 children with asthma who attended the hospital from October 2016 to October 2020 were enrolled as the asthma group, among whom 61 children had mild symptoms (mild group) and 82 children had moderate-to-severe symptoms (moderate-to-severe group). A total of 137 healthy children were enrolled as the control group. Peripheral venous blood samples were collected from the two groups. The SNaPshot SNP technique was used to analyze the SNP and haplotypes of the RESULTS: Polymorphisms were observed in the CONCLUSIONS SNP/haplotype of the
Asthma/genetics* ; Case-Control Studies ; Child ; Genetic Predisposition to Disease ; Genotype ; Haplotypes ; Humans ; Polymorphism, Single Nucleotide ; Receptors, Adrenergic, beta-2/genetics* ; Regulatory Sequences, Nucleic Acid

OBJECTIVE: To explore the pathogenesis for a SRY-negative male with 46,XX disorder of sex development (DSD). METHODS: Peripheral blood samples of the patient and his family members were subjected to chromosomal karyotyping, routine PCR, real-time fluorescence quantitative PCR, whole exome sequencing and whole genome sequencing. The data was analyzed with NextGENe software. RESULTS: Both the proband and his brother presented a 46,XX karyotype with negative SRY gene, while their father presented normal phenotype and karyotype with positive SRY gene. No pathogenic variant associated with sex development was detected by whole exome sequencing, while a 243 kb duplication was detected by whole genome sequencing in the 5' upstream region of the SOX9 gene in the proband, his brother and father. The same duplication was not found in his sister and mother. CONCLUSION The 243 kb duplication at the 5' upstream of the SOX9 gene may predispose to the 46,XX DSD in this family. It is speculated that there exist an unknown core regulatory element in the upstream of the SOX9, and its duplication may trigger expression of SOX9 and initiate testicular differentiation in the absence of SRY gene.
Disorders of Sex Development/genetics* ; Female ; Humans ; Male ; Mutation/genetics* ; Regulatory Sequences, Nucleic Acid/genetics* ; Sex-Determining Region Y Protein/genetics* ; Testis ; Whole Exome Sequencing

OBJECTIVETo identify the causative mutations in two Chinese Han families featuring triphalangeal thumbs (TPT) and preaxial polydactyly (PPD).

METHODSBlood samples were collected from 9 members (2 affected) from family 1 and 14 members (7 affected) from family 2. After genomic DNA was extracted, the ZPA regulatory sequence (ZRS) region was analyzed with real-time quantitative PCR (qPCR) and Sanger sequencing. For family 1, haplotypes compassing the ZRS were also analyzed with short tandem repeats (STR) and single nucleotide changes.

RESULTSNo copy number mutation around the ZRS region was found in both families. Two heterogeneous mutations in the ZRS (406A>G and 105C>G) were found to co-segregate with the TPT/PPD malformation in family 1 and 2, respectively. Neither mutation was detected in 200 healthy individuals. Haplotype analysis and Sanger sequencing of family 1 indicated that the first TPT/PPD patient in the family was both germline and somatic mosaic for the 406A>G mutation.

CONCLUSIONTwo pathogenic ZRS mutations, 105C>G and 406A>G, have been identified in two Chinese Han families with TPT/PPD, among which the 406A>G mutation was de novo.

Asian Continental Ancestry Group ; genetics ; Female ; Hand Deformities, Congenital ; genetics ; Haplotypes ; Humans ; Male ; Mutation ; Polydactyly ; genetics ; Regulatory Sequences, Nucleic Acid ; Thumb ; abnormalities

OBJECTIVETo study the function of the chalcone synthese gene introns in Scutellaria baicalensis, and clarify preliminarily their role in abiotic stress.

METHODThe CHS introns with specific primers were cloned and bioinformatic method was applied to predict the cis-elements in the intron of CHS. The introns were subcloned into binary vector, pCAMBIA-1301 before being transferred to tobacco. Then the activity of GUS of the transgenic tobacco seeds was analyzed.

RESULTSeven cis-elements were found in the introns. Under the dark and high temperature GUS expression rose at the first (3 h), but then declined (9 h). ABA and MeJA regulated insignificantly the GUS activity in normal temperature; treatment of 10% PEG induced GUS expression.

CONCLUSIONCHS introns could be play a role in the regulation of S. baicalensis phenylpropanoid biosynthetic pathway.

Acyltransferases ; genetics ; Base Sequence ; Gene Expression Regulation, Plant ; drug effects ; Gene Order ; Genetic Vectors ; Introns ; Molecular Sequence Data ; Plants, Genetically Modified ; genetics ; metabolism ; Polyethylene Glycols ; pharmacology ; Regulatory Sequences, Nucleic Acid ; genetics ; Scutellaria baicalensis ; enzymology ; genetics ; Sequence Alignment ; Tobacco ; genetics ; metabolism

OBJECTIVE: To explore the relationship between TK gene expression regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells. METHOD: The reformed reconstructed enhanced vector, pGL3-basic-EGFP-TK-hTRETp-CMV enhancer, and hTERT mono-promoter vector, pGL3-basic-EGFP-TK-hTRETp(as controls), were transfected into telomerase(+) nasopharyngeal carcinoma 5-8F cell lines, telomerase(+) human breast cancer MCF-7 cell lines and telomerase(-) normal vascular endothelium cell lines respectively. TK gene green fluorescent protein was observed by fluorescence microscope. The expression of TK gene mRNA was measured by the real-time fluorescent quantified PCR and the telomerase activity was determined by the method of TRAP argentation in malignant tumour cells pre- and post-transfected by enhanced vector. Meanwhile the relationship between TK and telomerase was analyzed. RESULT: (1) A strong TK gene fluorescent show and TK mRNA expression were displayed after the enhanced suicide gene vector was transfected into nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which were more stronger than those of mono-promoter group, pGL3-basic-EGFP-TK-hTRETp, and ECV cells transfected by enhanced suicide gene vector. Meanwhile,real-time fluorescent quantified PCR showed that the A value of enhanced vector group was higher than that of controls. (2) Telomerase activity after transfection of enhanced vector and GCV was lower than those before by the method of TRAP argentation in nasopharyngeal carcinoma cell lines,but no change in normal control cells after transfection of enhanced vector and GCV. (3) After adding GCV, the obvious inhibitory effect of tumour cells growth induced by pGL3-basic-EGFP-TK-hTRETp-CMV enhancer were observed in nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which was higher than those of mono-promoter, pGL3-basic-EGFP-TK-hTRETp, pGL3-basic-EGFP3 and blank controls, but without inhibitory effect in ECV cells transfected by enhanced vector. CONCLUSION TK gene expression is regulated by hTERT promoter and CMV enhancer, and then the telomerase activity is reduced and the cancer cells are specifically killed. But it is unclear how the telomerase are down-regulated by TK gene.
Cell Death ; Cell Line ; Cell Line, Tumor ; Gene Expression Regulation ; Genes, Transgenic, Suicide ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; Promoter Regions, Genetic ; Regulatory Sequences, Nucleic Acid ; Telomerase ; genetics ; metabolism ; Thymidine Kinase ; genetics ; Transfection

A systematic phylogenetic footprinting approach was performed to identify conserved transcription factor binding sites (TFBSs) in mammalian promoter regions using human, mouse and rat sequence alignments. We found that the score distributions of most binding site models did not follow the Gaussian distribution required by many statistical methods. Therefore, we performed an empirical test to establish the optimal threshold for each model. We gauged our computational predictions by comparing with previously known TFBSs in the PCK1 gene promoter of the cytosolic isoform of phosphoenolpyruvate carboxykinase, and achieved a sensitivity of 75% and a specificity of approximately 32%. Almost all known sites overlapped with predicted sites, and several new putative TFBSs were also identified. We validated a predicted SP1 binding site in the control of PCK1 transcription using gel shift and reporter assays. Finally, we applied our computational approach to the prediction of putative TFBSs within the promoter regions of all available RefSeq genes. Our full set of TFBS predictions is freely available at http://bfgl.anri.barc.usda.gov/tfbsConsSites.
Algorithms ; Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites ; genetics ; Cell Line, Tumor ; Computational Biology ; methods ; Conserved Sequence ; Electrophoretic Mobility Shift Assay ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Luciferases ; genetics ; metabolism ; Mice ; Normal Distribution ; Oligonucleotides ; genetics ; metabolism ; Phosphoenolpyruvate Carboxykinase (GTP) ; genetics ; Promoter Regions, Genetic ; genetics ; Protein Binding ; Rats ; Recombinant Fusion Proteins ; genetics ; metabolism ; Regulatory Sequences, Nucleic Acid ; genetics ; Reproducibility of Results ; Sp1 Transcription Factor ; genetics ; metabolism ; Transcription Factors ; metabolism ; Transfection

We studied the influence of the first intron and 3'-regulatory region of ovalbumin gene (ov) on oviduct-specific transgene expression. The 3'-regulatory region in the oviduct-specific expression vector containing human tissue kallikrein (hK1) cDNA was replaced with bovine growth hormone (BGH) poly A, and the first intron was deleted by restriction enzyme digestion, resulting in five new vectors pOV2K, pOV3K, pOV4K, pOV5K and pOV6K. After mixing with polyethylenimine, we injected same copies of the five vectors via wing vein route into laying hens and compared their expression levels by quantitative assay for enzymatic activities in the egg whites. Among the five vectors tested, the pOV2K containing both the 5'- and 3'-regulatory regions expressed highest level of rhK1 activity, followed by pOV3K with the 3'-regulatory region replaced with BGH poly A, and then by the first intron-shortened vectors pOV4K, pOV5K and pOV6K. These data suggest that both the first intron and 3'-regulatory region of ov gene have enhancing effect on transgene expression in oviduct cells, which should be included in oviduct-specific expression vectors.
Animals ; Animals, Genetically Modified ; Cattle ; Chickens ; Cloning, Molecular ; Female ; Gene Transfer Techniques ; Growth Hormone ; genetics ; Humans ; Introns ; genetics ; Ovalbumin ; genetics ; Oviducts ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Regulatory Sequences, Nucleic Acid ; genetics ; Tissue Kallikreins ; genetics ; Transgenes ; genetics

OBJECTIVESTo identify the host single nucleotide polymorphisms (SNP) of myxovirus resistance A (MxA) protein and eukaryote initiation factor 2alfa regulatory region 2(eIF-2a-reg2) and to predict interferon (IFN) treatment responses in patients with chronic hepatitis B.

METHODSTwo hundred sixty-two patients with chronic hepatitis B (CHB) were treated with interferon alfa (IFN ) for 12 months. Six months later the therapeutic effectiveness was evaluated. All the patients had signed a formal consent form. The patients were grouped into a sustained response (SR) group and a non-sustained response (NSR) group according to their responses to the IFNa treatment. Single nucleotide polymorphisms of the antiviral protein MxA promoter -88,-123 and protein kinase(PKR) activated eIF-2a-reg2 sites were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and were compared with the responsiveness to IFN treatment of these CHB patients.

RESULTSAmong the 262 patients, 212 (80.9%) were non-sustained responders to IFNa and 50 (19.1%) were sustained responders. The rate of sustained responders with GT heterozygote at MxA promoter -88 was higher than that of the GG genotype (OR: 5.3, 95% CI: 2.46-11.43, P less than 0.01) and also higher than that of the TT genotype (OR: 4.1, 95% CI: 1.86-9.09, P less than 0.01). There were no statistically significant differences in IFN therapeutic effectiveness among the patients with different genotypes at MxA promoter -123, eIF-2a-reg2 and haplotypes made by MxA promoter -88 G/T, and -123 C/A alleles (P more than 0.05).

CONCLUSIONPatients with GT genotype at MxA promoter -88 responded well to IFN treatment. SNP as a potential marker could be used to predict IFN treatment responses of patients with chronic hepatitis B.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; Eukaryotic Initiation Factor-2 ; genetics ; Female ; GTP-Binding Proteins ; genetics ; Genotype ; Hepatitis B virus ; Hepatitis B, Chronic ; drug therapy ; genetics ; Humans ; Interferon-alpha ; therapeutic use ; Male ; Middle Aged ; Myxovirus Resistance Proteins ; Polymorphism, Single Nucleotide ; Regulatory Sequences, Nucleic Acid ; genetics ; Young Adult

OBJECTIVETo establish chromosome conformation capture (3C) strategy and to use this method for exploring the effect of chromosome conformation on human alpha-globin gene expression in the human alpha-globin transgenic mouse.

METHODSHomozygous human alpha-globin transgenic male mouse was crossed with KM female mouse. The 14.5-day post-coitum (dpc) embryos were used for the isolation of fetal liver and fetal brain cells. Homogeneous single-cell suspension was treated with formaldehyde to crosslink the chromatin conformation in the nuclear. The cross-linked chromatin compound was digested with Nco I and then ligated with T4 DNA ligase. The ligated compound was reversely cross-linked and then the ligated genomic DNA was purified for PCR analysis. The primers were designed along the two sides of cut and ligated sites. Semi-quantitative PCR was used to analyze the chromosome conformation of the whole human alpha-globin gene locus in fetal liver and fetal brain cells.

RESULTSWhen HS40 fragment was used as the fixed fragment, in fetal brain cells, the ligation frequencies of HS40 fragment with other fragments were decreased as the linear distances to HS40 fragment were increasing; while in fetal liver cells, two active genes (alpha1 and alpha2) fragments showed higher ligation frequencies with HS40 fragment than other fragments. However, the fragment containing an inactive gene (xi) displayed the comparable low ligation frequency as that in fetal brain. When alpha2 fragment was used as the fixed fragment, similarly, in fetal brain cells the ligation frequencies of alpha2 fragment with other ones were decreased as the linear distances increasing; when in fetal liver cells, it showed higher ligation frequencies with two upstream regulatory elements (HS 40 and 33). However, it showed a little bit lower ligation frequency with another two upstream regulatory elements (HS10 and 8) than those in fetal brain.

CONCLUSIONIn fetal liver cells, the distant regulatory elements are in close proximity to the downstream of the expressed globin genes through looping out, the interval region; however, in fetal brain, they were not in vicinity to the expressed globin genes.

Animals ; Brain ; metabolism ; Chromosomes, Artificial, Bacterial ; Chromosomes, Mammalian ; chemistry ; Female ; Gene Expression Regulation ; Humans ; Liver ; metabolism ; Male ; Mice ; Mice, Transgenic ; Nucleic Acid Conformation ; Regulatory Sequences, Nucleic Acid ; alpha-Globins ; biosynthesis ; genetics

In vascular smooth muscle cells (VSMCs), induction of the heme oxygenase-1 (HO-1) confers vascular protection against cellular proliferation mainly via its up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) that is involved in negative regulation of cellular proliferation. In the present study, we investigated whether the phytochemical curcumin and its metabolite tetrahydrocurcumin could induce HO-1 expression and growth inhibition in rat VSMCs and, if so, whether their antiproliferative effect could be mediated via HO-1 expression. At non-toxic concentrations, curcumin possessing two Michael-reaction acceptors induced HO-1 expression by activating antioxidant response element (ARE) through translocation of the nuclear transcription factor E2-related factor-2 (Nrf2) into the nucleus and also inhibited VSMC growth triggered by 5% FBS in a dose-dependent manner. In contrast, tetrahydrocurcumin lacking Michael-reaction acceptor showed no effect on HO-1 expression, ARE activation and VSMC growth inhibition. The antiproliferative effect of curcumin in VSMCs was accompanied by the increased expression of p21(WAF1/CIP1). Inhibition of VSMC growth and expression of p21(WAF1/CIP1) by curcumin were partially, but not completely, abolished when the cells were co- incubated with the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs), curcumin also inhibited growth triggered by TNF-alpha and increased p21(WAF1/CIP1) expression via HO-1-dependent manner. Our findings suggest that curcumin has an ability to induce HO-1 expression, presumably through Nrf2-dependent ARE activation, in rat VSMCs and HASMCs, and provide evidence that the antiproliferative effect of curcumin is considerably linked to its ability to induce HO-1 expression.
Active Transport, Cell Nucleus ; Animals ; Aorta/cytology ; Cell Nucleus/metabolism ; Cell Proliferation/*drug effects ; Cells, Cultured ; Curcumin/analogs & derivatives/*pharmacology ; Cyclin-Dependent Kinase Inhibitor p21/biosynthesis/metabolism ; Gene Expression Regulation ; Heme Oxygenase (Decyclizing)/biosynthesis/genetics/*physiology ; Heme Oxygenase-1/biosynthesis/genetics/*physiology ; Humans ; Metalloporphyrins/pharmacology ; Muscle, Smooth, Vascular/drug effects/*physiology ; Myocytes, Smooth Muscle/drug effects/*physiology ; NF-E2-Related Factor 2/metabolism ; Protoporphyrins/pharmacology ; Rats ; Regulatory Sequences, Nucleic Acid ; Response Elements ; Tumor Necrosis Factor-alpha/pharmacology