Construction of heteroduplex DNA and<Emphasis Type="Italic">in vitro</Emphasis> model for functional analysis of mismatch repair

Functional deficiency of mismatch repair (MMR) system is one of the mechanisms of tumorigenesis. With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZα gene have been used to construct two kinds of hetero-duplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G·G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. coli NR9162 (mutS-) without the MMR ability on the indicator plate with x-gal and IPTG, there are three kinds of plaques, mixture plaque as the characteristic phenotype of heteroduplex DNA, blue and clear plaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100× (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functional status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G-G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G-G is less than 20%. Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target, TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (microsatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the func- tion status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumori-genesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.     

Mechanism of skeletal reorganization of 1,6-enynes catalyzed by GaCl<Subscript>3</Subscript>

The mechanism of skeletal reorganization of 1,6-enynes catalyzed by GaCI3 has been studied with the density functional method at the B3LYP/6-31G* level. The structures and energies of the stationary points were calcu-lated to identify the activation barriers. The transition stateswere testified with vibration analysis and IRC calculations.The results of calculation show that the conversion of 1,6-enynes is a step-wise reaction. The whole reaction process is formation and migration of three-membered cycle involvinga three-center and two-electron (3c-2e) bond. High stereose-lectivity of the reaction is in good agreement with experimental results.     

DNA purification and genetyping： Based on multifunc-tional nanobeads

In this report, a universal protocol for extract-ing genomie DNA from whole blood, saliva, and bacterialculture by using magnetic nanobeads as solid-phase absor-bents was presented. The enrichment of target cells and ad-sorption of DNA have been functionally integrated onto thesurfaces of the earboxyl-modified magnetic nano-beads, andthe DNA segments bound on the surface of the beads can bedirectly used as PCR templates to amplify a target geue. ThePCR products were applied to an oligonueleotide array toperform gene typing. The protocol proves to be simple, rapid,biologically and chemically nonhazardous, and promising forthe mierofabrieation of DNA preparation chip.     

Inducible expression pattern of rice Bowman-Birk inhibitor gene<Emphasis Type="Italic">OsWIP1-2</Emphasis> and its protease inhibitory activity

The WIP1-2 gene was cloned from rice. It be-longs to the Bowman-Birk inhibitor gene family. Northernblot showed that expression of this gene was induced bywounding and jasmonic acid (JA). It indicates that the OsWIPI gene plays an important role in the rice defense sys-tem. The OsWIP1-2 was cloned into pET28a and expressed inE. coli. Its expressed product was purified in the form offusion protein and tested for the inhibitory activities againsttrypsin and chymotrypsin. It was found that the fusion pro-tein could inhibit chymotrypsin, but not trypsin. It was alsofound that the His tag at its C-terminal affected its inhibitoryactivity significantly. The fusion protein with a naturalC-terminal had the inhibitory activity, while no inhibitoryactivity was detected in the fusion protein with a (His)6-tag atits C-terminal. This implies that extra amino acid residues atthe C-terminal of OsWIP1-2 may interfere with its correctfolding. The inhibitory assay indicated that the members ofrice Bowman-Birk inhibitor gene family probably differenti-ated both in their structure and function.     

Mantle olivine xenocrysts entrained in Mesozoic basalts from the North China craton：Implication for replacement process of lithospheric mantle

Mesozoic (125 Ma) Fangcheng basalts fromShandong Province contain clearly zoned olivines that arerare in terrestrial samples and provide first evidence for thereplacement of lithospheric mantle from high-Mg peridotitesto Iow-Mg peridotites through peridotite-melt reaction.Zoned olivines have compositions in the core (Mg# = 87.2--90.7) similar to those olivines from the mantle peridotiticxenoliths entrained in Cenozoic basalts from the NorthChina craton and in the rim (Mg# = 76.8--83.9) close to oli-vine phenocrysts of the host basalts (75.7--79.0). Thesecompositional features as well as rounded crystal shapes andsmaller grain sizes (300—800 μm) demonstrate that thesezoned olivines are mantle xenocrysts, i.e. disaggregates ofmantle peridotites. Their core compositions can representthose of olivines of mantle peridotites. The zoned texture ofolivines was formed through rapid reaction between the oli-vine xenocryst and the host basalt. This olivine-basaltic meltreaction could have been ubiquitous in the Mesozoic litho-spheric mantle beneath the North China craton, i.e. an im-portant type of the replacement of lithospheric mantle. Thereaction resulted in the transformation of the Paleozoic re-fractory (high-Mg) peridotites to the late Mesozoic fertile(Iow-Mg) and radiogenic isotope-enriched peridotites, lead-ing to the loss of old lithospheric mantle.     

Characterization of viscoelastic properties of hydrolyzed polyacrylamide using the stress recovery experiment

Polymer solutions have very important applications in the enhanced oil recovery due to their unique viscoelastic properties increasing microscale displacement efficiency. In this paper, the viscoelastic properties of partially hydrolyzed polyacrylamide (HPAM) solution have been investigated. Results show that the viscoelastic properties of HPAM increase with HPAM concentration increasing, and decrease with the increase of sodium dodecyl sulfate (SDS).Meanwhile, adding NaCI can destroy the viscoelastic properties.     

Assembly of gold nanoparticles with H10TTPR （3a, 4, 5, 8, 9, 9a, 10, 11-octa-hydro-2H,3H-1,6,7,12-tetrathia-perylene） as the rigid cross-linker

Gold nanoparticles were assembled with the rigid cross-linker, H10TTPR. The assembly process was studied with the UV-vis spectroscopy and TEM. By adjusting the amount of H10TTPR added to the gold nanoparticle solution,the aggregate form of gold nanoparticles could be controlled.     

Regeneration of fertile plants from isolated tobacco zygotes by<Emphasis Type="Italic">in vitro</Emphasis> culture

Living zygotes of tobacco (Nicotiana Tabacum L.) SR-1 were isolated and cultured in vitro by the microculture technique. Fertile plants were regenerated from the calli derived from cultured zygotes via organogenesis. Ovules were collected 120h after pollination and used as feeder. MS combined with KM8p was selected as basic medium in the experiment. Zygotes isolated from ovules 108h after pollination turned out to be suitable material for in vitro culture.Over 80% such zygotes could divide and around 10% of them could grow into calli and regenerate fertile plants.