Biochemical Activity of PIF1 Helicase from Thermophilic Bacteria / 中国生物化学与分子生物学报

Petite integration frequency 1 (PIF1) helicases are ubiquitous enzymes which play vital roles in nearly all DNA metabolic processes. In recent years, the biochemical activity and three-dimensional structure of several PIF1 helicases have been reported, but there are few reports on the PIF1 helicase of bacteria living in extreme environments. In this paper, a series of biochemical and biophysical techniques were used to study the Thermodesulfovibrio yellowstonii PIF1 (Ty.PIF1) helicase in many aspects. Ty. PIF1 was obtained with a purity of over 90% and good uniformity using the prokaryotic expression and purification system. Ty.PIF1 is a monomer with a calculated molecular weight of 60 kD in solution. Ty. PIF1 has high thermal stability. The secondary structure remains stable when the temperature is below 65 ℃, and the secondary structure changes only when the temperature is above 70 ℃. The optimal unwinding temperature of Ty.PIF1 in vitro is 45 ℃, which is not the optimal temperature for the survival of thermodesulfovibrio yellowstonii. It indicates that when Ty.PIF1 exerts its enzymatic activity in vivo, it may require the participation of other cofactors. Ty.PIF1 can exert unwinding activity in a wide temperature range (20-55 ℃), and the presence of enzyme activity at 55 ℃ indicates that Ty.PIF1 has heat-resistant properties. Ty.PIF1 prefers to bind to substrates containing ssDNA, but there is certain requirement for the length of the ssDNA, which is at least 4 nt in length. Ty.PIF1 can also bind to the G

Effects of bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27 on biological properties of BLM helicase / 中国药理学通报

Aim To investigate the effects of bisbenzyl-isoquinoline alkaloid tetrandrine derivative HL-27 on the biological properties of the BLM642-1290 helicase. Methods Fluorescence polarization technique was used to investigate the effects of bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27 on the DNA bind-ing activity and unwinding activity of the BLM642-1290 helicase. Malachite green-phosphate ammonium molyb-date colorimetry was used to investigate the effects of HL-27 on the ATPase activity of the BLM642-1290 heli-case. Ultraviolet spectral scanning was used to investi-gate the effects of HL-27 on the conformation of the BLM642-1290 helicase. Results When the concentra-tion of HL-27 reached 33.34 μmol·L-1, the inhibi-tion ratio of dsDNA and ssDNA binding activity of the BLM642-1290 helicase was 41.35% and 59.54% , re-spectively. When the concentration of HL-27 reached 50 μmol·L-1, the inhibition ratio of DNA unwinding activity of the BLM642-1290 helicase was 78.68% . When the concentration of HL-27 reached 100 μmol· L-1, the inhibition ratio of ATPase activity of the BLM642-1290 helicase was 43.8% . Conclusion The DNA binding activity, ATPase activity and unwinding activity of the BLM642-1290 helicase can be inhibited by bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27.

Virtual Screening ofα-glycosidase Inhibitors in Cortex mori and Folium mori / 中国药房

OBJECTIVE:To virtually screen potential α-glycosidase inhibitor ingredients from C. mori and F. mori,and to pro-vide reference for finding out new typeα-glycosidase inhibitor ingredient. METHODS:Surflex-Dock module of Sybyl-x 2.0 molecu-lar simulation software was used to perform the docking of small molecule compound,which was from the ingredients of C. mori and F. mori as ligand stated in literatures,with α-glycosidase. Total score of affinity scoring function was equal to 7 as the thresh-old value,to judge potential α-glycosidase inhibitor ingredient in C. mori and F. mori. RESULTS:After 70 small molecule com-pounds docked with α-glycosidase, 10 compounds showed binding activity (Total score≥7.00). Among them, moracin M-3′-O-β-D-glucopyranoside,5,7,2′-trihydroxyflavanone-4′-O-β-D-glucoside,mulberroside A,resveratrol-4,3′-di-O-β-D-gluco-pyranoside and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol had higher binding activity with α-glycosidase(Total score>8.00). CONCLUSIONS:Multi-constituents of C. mori and F. Mori show potential α-glycosidase inhibitory activity. The method is a kind of highly targeted,rapid and efficient approach to discover α-glycosidase inhibitor from traditional Chinese medi-cine.

Cloning and function analysis of high mobility group box 1(HMGB1)protein of Schistosoma japonicum(Mainland strain) / 中国血吸虫病防治杂志

Objective To clone and express a high mobility group box 1(HMGB1)protein of Schistosoma japonicum(Main-land strain)and analyze its function. Methods The DNA fragment of open reading frame encoding Sj HMGB1 protein was ampli-fied by RT-PCR from the mRNA of S. japonicum worms,then it was subcloned into the expression vector pET28a(+)to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3),and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombi-nant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding ca- pacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 pro-tein and infected with 45±2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection,the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue,respectively. The worm and egg reduction rates were calculat-ed respectively. Results A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR,which was the open reading frame(ORF)encoding SjHMGB1protein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA,and the recombinant protein immu-nized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abun-dantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effec-tive immune protection against S. japonicum. Conclusion The gene encoding HMGB1 from S. japonicum and the soluble recombi-nant SjHMGB1 protein with natural functional activity are obtained,and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

Effect of nimodipine on DNA-binding activity of cAMP response element binding protein and CCAAT enhancer binding protein in hippocampus in vascular dementia rats / 中华行为医学与脑科学杂志

ObjectiveTo observe the effect of nimodipine on hippocampal DNA-binding activity change of cAMP response element binding protein ( CREB )and CCAAT enhancer binding protein (C/EBP) in a rat model of vascular dementia(VD),and to explore the treatment mechanism of nimodipine.Methods66 healthy adult male SD rats were assigned to the following three groups of 22 each:VD model group,Sham-operated group,Nimodipine group.VD rat model was prepared by four-vessel occlusion.Physiological saline solution( 8 ml · kg-1 · d-1 )and Nimodipine (20 mg· kg-1 · d-1 )were administered by gavage respectively.The Morris maze was adopted to detect the changes of spatial learning and memorizing capacity,while HE straining was adopted to observe the changes of pathological characteristics in hippocampal CA1 area,and electrophoretic mobility shift assay(EMSA) were adopted to observe DNA-binding activity changes of CREB and C/EBP in hippocampus tissue.ResultsThe Morris maze showed:the learning and memory ability of nimodipine group rats ( escape latency period ( 26.63 ± 1.31 )s,the times of cross-platform(7.25 ±0.92) times) was higher than that of VD model group(escape latency period (41.25 ± 1.83 ) s,the times of cross-platform ( 5.33 ± 0.64 ) times ),with difference of statistical significance (P ＜0.05).HE results:in VD model group,neurons in CA1 were scaltered and boundaries were unclear,nuclei region was stained,coagulation necrosis appeared,obviously cells lost.The CA1 neurons of nimodipine group returned to be normal,nuclear membrane's profile and nudeolus were clear,regularly arranged; the number of hippocampal normal neurons in nimodipine group (43.19 ± 2.87 ) was more than that of VD model group( 16.33 ± 1.09 ),with difference of statistical significance(P＜0.05 ).EMSA:both CREB and C/EBP DNA-binding activity in rat hippocampus of nimodipine group ( ( 369.75 ± 13.22 ),( 428.25 ± 17.69 ) respectively ) were higher than those of VD model group ( ( 142.25 ± 27.86 ),(97.00 ± 5.88 ),respectively),with difference of statistical significance (P ＜0.01 )).ConclusionNimodipine can improve VD rats hippocampal neuronal injuries and their learning and memory impairment may be involved in the upregulating CREB and C/EBP DNA-binding activity.

The Recombinant Expression and Receptor-binding Activity of the B Subunit of Shiga-like Toxin Type Ⅱ / 中国生物工程杂志

Objective:To express the B subunit of Shiga-like toxin type Ⅱ,and analyze its expression form and receptor-binding activity.Methods:The slt2b gene was obtained from EHEC O157∶H7 by PCR,and cloned to the expression vector pET22b(+).The genetically engineered bacteria pET22b(+)-stx2B/BL21 expressed the recombinant StxB after induced with IPTG.The renatured inclusion bodies were purified by ion exchange chromatography.The expression form of rStx2B was investigated by denaturing and native electrophoresis.The receptor-binding activity was confirmed by fluorescence detection and flow cytometer.Result:The constructed genetically engineered bacteria expressed the rStx2B at a high level.The purified protein was obtained after denaturation,renaturation and ion exchange chromatography.According to the denaturing and native electrophoresis,the rStx2B was expressed in a dimmer form,which consists of two monomers cross linked with disulfide bridge.The rStx2B showed good receptor-binding activity by Hela-binding assay.Conclusion:The genetically engineered bacteria were constructed successfully.The receptor-binding activity of rStx2B was independent of the pentamers.

The Regulation of AP-1 DNA Binding Activity by Long-term Nicotine Stimulation in Bovine Adrenal Medullary Chromaffin Cells: Role of Second Messengers

The signal pathways involved in the regulation of AP-1 DNA binding activity in long-term nicotine stimulated bovine adrenal medullary chromaffin (BAMC) cells have not been well characterized. To understand the involvement of second messengers in the regulation of AP-1 DNA binding activity, the present study was designed to define the time-course for inhibition of nicotine-induced responses by cholinergic antagonists, Ca2+ and calmodulin (CaM) antagonists, and calcium/calmodulin-dependent protein kinase (CaMK) II inhibitor using electrophoretic mobility shift assay. Nicotine (10microM) stimulation increased AP-1 DNA binding activity at 24 hr after treatment. Posttreatment with hexamethonium (1 mM) plus atropine (1microM) (HA), nimodipine (1microM), or calmidazolium (1microM) at 0.5, 3, and 6 hr after the nicotine treatment significantly inhibited the AP-1 DNA binding activity increased by long-term nicotine stimulation. However, posttreatment with HA, nimodipine, or calmidazolium at 9 or 12 hr after the nicotine treatment did not affect the nicotine-induced increase of AP-1 DNA binding activity. The pretreatment of BAMC cells with various concentrations of KN-62 inhibited the increase of AP-1 DNA binding activity induced by nicotine in a concentration-dependent manner. KN-62 (10microM) posttreatment beginning at 0.5, 3, or 6 hr after the nicotine treatment significantly inhibited the increase of AP-1 DNA binding activity. However, KN-62 posttreatment beginning at 9 or 12 hr after the nicotine treatment did not affect the increase of AP-1 DNA binding activity. This study suggested that stimulation (for at least 6 hr) of nicotinic receptors on BAMC cells was necessary for increase of AP-1 DNA binding activity, and activation of Ca2+, CaM, and CaMK II up to 6 hr at least seemed to be required for the increase of nicotine-induced AP-1 DNA binding activity.

A radioimmunoassay method for detection of DNA based on chemical immobilization of anti-DNA antibody

High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA.

Role of nuciear factor-?B in the ischemic acute renal failure rat / 中华肾脏病杂志

Objective To determine the role of nuclear factor-?B(NF-?B) in ischemic acute renal failure (ARF) rats. Methods Gel mobility shift assay was used to detect the DNA binding activity of NF-KB in ischemic ARF rats and reverse transcription-polymerase chain reaction (RT-PCR) assay was used to study the expression of renal inducible nitric oxide synthase (iNOS) . The relationship between DNA binding activity of NF-?B and expression of iNOS was also analyzed. Results The DNA binding activity of NF-?B in renal cortex increased from 1.00 ?0.17 of controls to 3. 67 ? 1. 94 of 6 hours after ischemia-reperfusion ( P

Colchicine binding activity of rat brain polysomes.

In this communication, we report the presence of a unique colchicinebinding activity in the polysomes of rat brain. This drug-binding property, is somewhat similar to that of tubulin isolated from many sources; however, it differs in several biochemical characteristics such as (i) thermal stability of colchicine-binding site, (ii) protection of binding site by vinblastine and (iii) time required for binding equilibration. Such binding of colchicine to the polysomes is most probably due to the presence of a nascent peptide chain of tubulin in the polysome.