Mechanism underlying berberine's effects on HSP70/TNFα under heat stress: Correlation with the TATA boxes / 中国天然药物

Heat stress can stimulate an increase in body temperature, which is correlated with increased expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNFα). The exact mechanism underlying the HSP70 and TNFα induction is unclear. Berberine (BBR) can significantly inhibit the temperature rise caused by heat stress, but the mechanism responsible for the BBR effect on HSP70 and TNFα signaling has not been investigated. The aim of the present study was to explore the relationship between the expression of HSP70 and TNFα and the effects of BBR under heat conditions, using in vivo and in vitro models. The expression levels of HSP70 and TNFα were determined using RT-PCR and Western blotting analyses. The results showed that the levels of HSP70 and TNFα were up-regulated under heat conditions (40 °C). HSP70 acted as a chaperone to maintain TNFα homeostasis with rising the temperature, but knockdown of HSP70 could not down-regulate the level of TNFα. Furthermore, TNFα could not influence the expression of HSP70 under normal and heat conditions. BBR targeted both HSP70 and TNFα by suppressing their gene transcription, thereby decreasing body temperature under heat conditions. In conclusion, BBR has a potential to be developed as a therapeutic strategy for suppressing the thermal effects in hot environments.

Clinical features and gene mutation profiles of patients with chronic hepatitis B and Gilbert's syndrome / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the clinical features and gene mutation profiles of patients with chronic hepatitis B (CHB) and Gilbert's syndrome.</p><p><b>METHODS</b>Thirty-three patients with CHB and Gilbert's syndrome were enrolled in the study. Serum markers of liver function and histological features of disease-related liver injury were assessed by standard methods. Gene mutations were detected by PCR and direct DNA sequencing.Statistical analysis was carried out with the chi-square and t tests.</p><p><b>RESULTS</b>Sequencing of the Gilbert syndrome-associated gene, UGT 1A 1, revealed mutations in the upstream promoter phenobarbital-responsive element module (PBREM) (-3279 mutation, 23 cases), in the promoter TATA box (a TA insertion mutation, 21 cases), and in the coding region of exon 1 (a GGA-AGA Gly71Arg mutation, 18 cases); there was no statistical difference found for any of the three mutations among this patient population (x2 =1.640, P more than 0.05).</p><p><b>CONCLUSION</b>The traditional methods of diagnosis for patients with CHB and Gilbert's syndrome remain a technical challenge in the clinic, and gene detection may represent a more favorable method for diagnosing this patient population.</p>

Mutations in UGT1A1 gene in neonates with hyperbilirubinemia of Guangxi Heiyi Zhuang nationality / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the distribution of mutations of UDP-glucuronosyltransferase 1A1 (UGT1A1) gene and its relationship with hyperbilirubinemia among neonates with hyperbilirubinemia of Guangxi Heiyi Zhuang nationality.</p><p><b>METHODS</b>Total genomic DNA was extracted from the blood of 100 neonates with hyperbilirubinemia (case group) and 100 neonates without hyperbilirubinemia (control group), all of whom were selected from Guangxi Heiyi Zhuang population. TATA box and all exons of UGT1A1 gene were amplified by PCR and directly sequenced.</p><p><b>RESULTS</b>(TA)7 insertion mutation in TATA box, G71R missense mutation in exon 1, and 4 single nucleotide polymorphisms (SNPs) (rs199539868, rs114982090, rs1042640 and rs8330) in exon 5 were observed. The allele frequency of G71R mutation in the case group was significantly higher than that in the control group (P<0.01). There were no significant differences in the genotype distribution and allele frequency of TATA box mutation and SNPs (rs1042640 and rs8330) between the two groups (P>0.05). The logistic regression analysis showed that the odds ratios (95% confidence intervals) of UGT1A1 TATA box mutation, G71R mutation, and SNPs (rs1042640 and rs8330) associated with the development of neonatal hyperbilirubinemia were 0.846 (0.440, 1.629), 3.932 (1.745, 8.858), 0.899 (0.364, 2.222), respectively.</p><p><b>CONCLUSIONS</b>(TA)7 insertion mutation and G71R missense mutation of UGT1A1 gene are common mutation types in neonates with hyperbilirubinemia of Guangxi Heiyi Zhuang nationality. Four SNPs (rs199539868, rs114982090, rs1042640, and rs8330) was first reported in China. UGT1A1 G71R missense mutation is a risk factor for hyperbilirubinemia in neonates of Guangxi Heiyi Zhuang nationality.</p>

Cloning and analysis of reverse transcriptase(RT) of Ty1-copia retrotransposons in Dendrobium officinale / 中国中药杂志

Using universal primer Ty1-copia retrotransposon RT,43 Ty1-copia like retrotransposon RT with high heterogeneity, stop codon mutation and frameshift mutation were amplified by PCR from genomic DNA of Zhejiang Lin'an (C15) and Yunnan Guangnan (A39) of Dendrobium officinale. The length of these sequences varied from 260 to 266 bp, and was rich in AT and consistency ranged from 47.1% to 97.7%. Different c/s-acting regulatory elements induced by low temperature, heat, light, all kinds of plant growth regulating substances and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. When being translated into amino acids, ten sequences presented stop codon mutation, five sequences presented frameshift mutation, and thirty-seven sequences presented conserved sequence "SLYGKQ" mutation. Six categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with other plants (eg. Triticum aestivum, Eleocharis quinqueflora) having high homology, which indicated that horizontal transmission of retrotransposon occurred among the plants in the past.

Genetic factors in the occurrence of neonatal unconjugated hyperbilirubinemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study association of uridine-diphosphate-glucuronosyltransferase1A1 (UGT1A1) Gly71Arg, UGT1A1 promoter TATA-box and glucose-6-phosphate dehydrogenase (G6PD) gene mutations with the occurrence of neonatal unconjugated hyperbilirubinemia.</p><p><b>METHODS</b>The TATA-box, exon 1 and exon 5 of the UGT1A1 gene and the exon 12 of G6PD gene were amplified by PCR. The products of PCR were analyzed by direct DNA sequencing. Clones for the mutations of the UGT1A1 gene and the G6PD gene were constructed in order to identify the results of the products of PCR. Seventy-two neonates with unconjugated hyperbilirubinemia (case group) and 65 healthy neonates (control group) were enrolled. The genotypes and allele frequencies of the polymorphisms of UGT1A1 Gly71Arg and UGT1A1 TATA-box were compared between the two groups. The effects of UGT1A1 Gly71Arg, UGT1A1 promoter TATA-box and G6PD gene mutations on the development of neonatal unconjugated hyperbilirubinemia were estimated using logistic regression models.</p><p><b>RESULTS</b>There were significant differences in the genotype distribution of Gly71Arg polymorphism of UGT1A1 gene between the case and control groups (P<0.01). The Arg allele frequency of the polymorphisms of UGT1A1 gene in the case group was significantly higher than in the control group (P<0.01). There were no significant differences in the genotype distribution of the UGT1A1 promoter TATA-box between the two groups (P>0.05). The OR and 95%CI values of UGT1A1 Gly71Arg, UGT1A1 TATA-box and G6PD gene mutations associated with the development of neonatal unconjugated hyperbilirubinemia were 5.468 (2.274, 12.818), 0.688 (0.266, 1.778) and 5.081 (1.070, 24.133) respectively.</p><p><b>CONCLUSIONS</b>UGT1A1 Gly71Arg and G6PD gene mutations may be involved in the development of neonatal unconjugated hyperbilirubinemia.</p>

The relationship between Gly71Arg and TATA box polymorphism of UGT1A1 gene and prolonged hyperbilirubinemia of breast milk feeding infant in Korean / 소아과

PURPOSE: It has been known that breast milk cause prolonged unconjugated hyperbilirubinemia. UGT1A1 is a important gene of uridine diphosphate glucuronosyltransferase (UGT) which has a major role of bilirubin metabolism. These findings suggest that there is a relationship between UGT1A1 gene mutation and prolonged jaundice of breast feeding infant. The aim of study was to investigate whether a polymorphism of the UGT1A1 gene exist in prolonged hyperbilirubinemia of breast milk feeding Korean infant. METHODS: The genomic DNA was isolated from 50 full term Korean neonates, who had greater than a 10 mg/dL of serem bilirubin after 2 weeks of birth with no significant cause, and the other genomic DNA was isolated from 162 full term Korean neonates of the control population. Both group fed breast milk. We performed direct sequencing of TATA box and Gly71Arg polymorphism of the UGT1A1 gene. RESULTS: Two of the 50 neonates with hyperbilirubinemia had AA polymorphism, and 40 had GA polymorphism. Five of the 129 neonates of the control group had AA polymorphism, and 4 had GA polymorphism. The allele frequency of G>A polymorphism in the hyperbilirubinemia group was 44.0%; it was significantly higher than 5.4% of the control group. TATA box polymorpism was not different both group significantly. CONCLUSION: Our result indicated that Gly71Arg polymorphism is associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean, while TATA box polymorphism is not associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean.

Molecular analysis of beta-thalassemia intermedia in Guangdong Province / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To determine the molecular defects of beta-thalassemia intermedia in Guangdong Province and to provide basis for gene diagnosis and gene therapy of this disorder.</p><p><b>METHODS</b>DNA analysis of the alpha, beta and gamma globin genes was performed in 18 children with beta-thalassemia intermedia from Guangdong Province using polymerase chain reaction (PCR), microarray technique, Southern blot and direct sequencing.</p><p><b>RESULTS</b>Of the 18 patients,one was identified as the homozygote of TATA box-28 (A-->G) change, one as the homozygote of betaE26 (G-->A) mutation, ten as compound heterozygotes of TATA box- 28 (A-->G) mutation with other beta-globin mutations, two as compound heterozygotes of betaE26 (G-->A ) mutation with other beta globin mutations, and four as double heterozygotes for beta globin and alpha globin mutations including -SEA and -alpha(4.2).</p><p><b>CONCLUSIONS</b>The molecular defects of beta- thalassemia intermedia in Guangdong Province were highly heterogeneous and its spectrum was different from the reports from other provinces of China.</p>

UDP-glucuronosyltransferase 1A1 Gene Polymorphism in Severe Neonatal Hyperbilirubinemia

PURPOSE: TATA box mutation/polymorphism in the promoter region of the bilirubin uridinediphosphoglucuronate glucuronosyltransferase 1A1 (UGT-1A1) gene is known to be an etiology of hyperbilirubinemia. This study examined if a TATA box mutation/polymorphism in UGT-1A1 gene promoter could be associated with the development of severe early neonatal jaundice in Korean infants. METHODS: Thirty-nine neonatal jaundice patients and 40 controlled infants were analyzed for UGT-1A1 promoter genotypes by using DNA sequencing. RESULTS: The homozygote for (TA)7TAA mutation was not found in this study. Comparison of the prevalence of UGT-1A1 promoter (TA)7TAA heterozygotes revealed no difference between the group with jaundice and the controlled group (15.4% vs. 10%). The peak bilirubin level was higher and the onset of jaundice was earlier in the jaundice group with (TA)7TAA heterozygote compared to the jaundice group without (TA)7TAA heterozygote (23.2+/-1.0 mg/dL vs. 19.7+/-2.4 mg/dL, P=0.004, 5.0+/-1.5 days vs. 8.3+/-4.1 days, P= 0.057). CONCLUSION: The results of this study showed that TATA box polymorphism in UGT-1A1 gene promoter did not increase the prevalence of severe early neonatal jaundice in Korean infants.

Bioinformatics scan analysis for predicting drug targeted modulation on ID4 gene expression / 中国实验血液学杂志

Low expression of ID4 gene is tightly related with carcinogenesis and high expression shows a definite anti-leukemia effect, though little expression in some leukemia cells. The main purpose of this preliminary work was to analyze the construction of ID4 gene promoter and to predict the cis elements in the ID4 promoter region by scanning the drug candidate with bioinformatics method. All these work are the primary part for finding effective drugs in the treatment of leukemia via the way of ID4 expression regulation. According to the data in GenBank and Internet platform, the 5'-untranslated sequence just upstream of ID4 ORF was virtually cloned. TESS, Genomatix and GenBank databank were used to analyze the cis elements in this area. RSA was used to find the distribution patterns for all these possible elements. SAGE and GEO datasets were used to find active substances which have the effect on the ID4 expression. The rsults indicated that ID4 had a type II promoter with a typical TATA box-45 bp upstream the transcriptional original site. There were a lot of various cis elements in the 5'-untranslated region upstream, including both positive element candidates such as Sp1, c-Myb, abaA, GR, ER, Zeste and C/EBPalpha and negative element candidates such as CCAAT-binding factor, GCF, WT1-KTS, HiNF-C and EGR2. It is concluded that estrogen, dexamethasone, thyroid hormone and follicle stimulating hormone may participate in the regulation of ID4 gene expression in both positive and negative manners.

Identification of a lens-specific cis-acting element within the basal promoter of the human lens intrinsic membrane protein MP19 gene (LIM2) / 生物工程学报

Using overlapping and mutant oligonucleotides as probes, gel mobility assays and competition experiments identified a sequence from -47 to -32 bp upstream of the LIM2 CAP site, which a lens protein complex bound with high affinity which appeared to bind only to the "sense" strand of the double-stranded DNA molecule. This sequence consisted of a string of four guanine residues followed by seven other nucleotides (AACCTAA) and followed by another four guanines, i.e. GGGGAACCTAAGGGG, called the Hsu element. Promoter-CAT constructs containing this sequence or mutations of the sequence indicated that the Hsu element is located within the basal promoter, and is essential for expression of the LIM2 gene. The trans factors binding to the Hsu element are present throughout development, and appear to be lens-specific. Since the LIM2 gene promoter does not contain a classic TATA box, the Hsu element may serve as the site for binding the RNA polymerase complex.

Regulation of activity of the yeast TATA-binding protein through intra-molecular interactions.

Dimerization is proposed to be a regulatory mechanism for TATA-binding protein (TBP) activity both in vitro and in vivo. The reversible dimer-monomer transition of TBP is influenced by the buffer conditions in vitro. Using in vitro chemical cross-linking, we found yeast TBP (yTBP) to be largely monomeric in the presence of the divalent cation Mg2+, even at high salt concentrations. Apparent molecular mass of yTBP at high salt with Mg2+, run through a gel filtration column, was close to that of monomeric yTBP. Lowering the monovalent ionic concentration in the absence of Mg2+, resulted in dimerization of TBP. Effect of Mg2+ was seen at two different levels: at higher TBP concentrations, it suppressed the TBP dimerization and at lower TBP levels, it helped keep TBP monomers in active conformation (competent for binding TATA box), resulting in enhanced TBP-TATA complex formation in the presence of increasing Mg2+. At both the levels, activity of the full-length TBP in the presence of Mg2+ was like that reported for the truncated C-terminal domain of TBP from which the N-terminus is removed. Therefore for full-length TBP, intra-molecular interactions can regulate its activity via a similar mechanism.

Finding signals for plant promoters / 基因组蛋白质组与生物信息学报·英文版

The strongest signal of plant promoter is searched with the model of single motif with two types. It turns out that the dominant type is the TATA-box. The other type may be called TATA-less signal, and may be used in gene finders for promoter recognition. While the TATA signals are very close for the monocot and the dicot, their TATA-less signals are significantly different. A general and flexible multi-motif model is also proposed for promoter analysis based on dynamic programming. By extending the Gibbs sampler to the dynamic programming and introducing temperature, an efficient algorithm is developed for searching signals in plant promoters.

Quasispecies and variations of hepatitis B virus: core promotor region as an example / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the HBV quasispecies groups in the patients with chronic HBV infection.</p><p><b>METHODS</b>A set of specific primers was synthesized according to DNA sequence of HBV strain found in China. The whole core promoter (CP) region was amplified by PCR method from the sera of 3 patients with chronic HBV infection, and the PCR products were subcloned into pGEM Teasy vectors. The clones were randomly selected to be sequenced. Sequence comparison of the selected clones was made to find the difference.</p><p><b>RESULTS</b>By comparison, it was found that each sequence of selected clones was different. The point mutation always occurred in TATA-like boxes, especially from T to C replacement on 184 site. There is a hot region (33.3% 5/15) in basic core promoter where deletion mutation frequently happened.</p><p><b>CONCLUSIONS</b>There is a hot deletion region near DR I in CP. The replacement at 184 nt (T to C) in the third TATA-like box may influence the expression of pre-C/C protein. The sequencing results suggest that there are HBV quasispecies groups in chronically infected patients.</p>

A Human Immunodeficiency Virus Type 1 (HIV-1) Tat Cofactor Absent in Rodent Cells is a TAR-associated Factor

BACKGROUND: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. METHODS: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. RESULTS: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. CONCLUSION: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.

A Human Immunodeficiency Virus Type 1 (HIV-1) Tat Cofactor Absent in Rodent Cells is a TAR-associated Factor

BACKGROUND: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. METHODS: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. RESULTS: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. CONCLUSION: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.

Complete Sequences of HIV-1 in a Korean Long-term Nonprogressor with HIV-1 Infection

To characterize the molecular nature of human immunodeficiency virus (HIV)-1, we determined the full-length HIV-1 sequences from cultured peripheral blood mononuclear cells (PBMC) of a Korean long-term nonprogressor (LTNP). Without antiretroviral therapy, the individual has maintained CD4+ T counts over 500/microliter from 1989 to 1999. Plasma viral RNA copy was 992 U/ml in 1998. Culture supernatant showed positive from culture days 9.4 series of 9 overlapping PCR products were amplified from cultured PBMC and cloned. About 9.2 kb from R of 5' LTR to R of 3' LTR was determined by automated sequencing. The G-to-A hypermutations were shown throughout the entire region. As a result of G to A hypermutations, premature stop codon was found in integrase coding region. Though there was no recombination between subtypes over all genomes, TATA box in both LTRs was TAAAA which is detected in subtype E instead of TATAA in subtype B. And, there were nucleotide GC insertion between NF- kappaB I and Spl III, and duplication of TCF-lalpha in LTR. We could not find any deletion of amino acid in Nef, Gog, Pol and Euv gene. This study is the first report on molecular nature of full genomes of HIV-1 isolated in Korea.

Complete Sequences of HIV-1 in a Korean Long-term Nonprogressor with HIV-1 Infection

To characterize the molecular nature of human immunodeficiency virus (HIV)-1, we determined the full-length HIV-1 sequences from cultured peripheral blood mononuclear cells (PBMC) of a Korean long-term nonprogressor (LTNP). Without antiretroviral therapy, the individual has maintained CD4+ T counts over 500/microliter from 1989 to 1999. Plasma viral RNA copy was 992 U/ml in 1998. Culture supernatant showed positive from culture days 9.4 series of 9 overlapping PCR products were amplified from cultured PBMC and cloned. About 9.2 kb from R of 5' LTR to R of 3' LTR was determined by automated sequencing. The G-to-A hypermutations were shown throughout the entire region. As a result of G to A hypermutations, premature stop codon was found in integrase coding region. Though there was no recombination between subtypes over all genomes, TATA box in both LTRs was TAAAA which is detected in subtype E instead of TATAA in subtype B. And, there were nucleotide GC insertion between NF- kappaB I and Spl III, and duplication of TCF-lalpha in LTR. We could not find any deletion of amino acid in Nef, Gog, Pol and Euv gene. This study is the first report on molecular nature of full genomes of HIV-1 isolated in Korea.

Cloning of mouse AQP-CD gene

Water transport in highly-permeable membranes is facilitated by some specialized pathways, which are called aquaporins (AQP). AQP1 (AQP-CHIP) is the first recognized aquaporin identified from red cells and renal proximal tubules. Up until now 4 other aquaporin homologs have been reported. Each aquaporin has its unique tissue distribution and regulatory mechanisms. To elucidate molecular mechanisms for their transcription regulation and tissue-specific expression isolation of aquaporin genes is required. To clone promoters of the AQP family mouse genomic library was screened by the 1st exon-specific probe of AQP4, and 5 different plaques were positively hybridized. Phage DNAs were purified and characterized by restriction mapping and sequencing. One of them is the mouse AQP-CD gene. The gene was consisted of 4 exons and the exon-intron boundaries of mouse AQP-CD gene were identified at identical positions in other related genes. The 5'-flanking region of AQP-CD gene contains one classic TATA box, a GATA consensus sequence, an E-box and a cyclic AMP-responsive element. The cloning of the mouse AQP-CD gene, of which product is expressed in the collecting duct and is responsible for antidiuresis by vasopressin, will contribute to understand the molecular mechanisms of tissue-specific expression and regulation of AQP-CD gene under various conditions.

Hormone-mediated positive and negative regulation of a silencer region containing laminin B2 promoter in astrocytes.

Primary astrocytes synthesize only B2 chain (200 Kda) of laminin, which is one of the major components of basement membranes in the central nervous system (CNS). In CNS, B2 laminin functions as a potent neurite growth factor. Laminin B2 promoter contain no TATA or CAAT boxes but is GC rich. By deletion analysis and transient transfection assays of B2 laminin promoter, we have identified a silencer-like activity in the upstream region of the promoter. Thyroid hormone, insulin and phorbol ester mediated the induction of the promoter activity. Induction was repressed by the treatment of astrocytes with synthetic glucocorticoid hormone, dexamethasone. Hormone-mediated regulation through specific positive and negative responsive elements in transactivation of this silencer-containing TATA-less laminin B2 gene has been postulated.