Preparation and identification of anti-transforming growth factor β1 U1 small nuclear RNA chimeric ribozyme in vitro

AIM: To study the preparation and cleavage activity of antitransforming growth factor (TGF)β1 U1 small nuclear (sn)RNA chimeric hammerhead ribozymesin vitro.METHODS: TGFβ1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. 32p-labeled TGFβ1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFβ1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator.32p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: Active UlsnRNA chimeric ribozyme (U1Rz803)had the best cleavage activity at 50 °C; at 37 °C, it was active, Km=34.48 nmol/L, Kcat=0.14 min-1; while the point mutant ribozyme U1Rz803m had no cleavage activity, so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFβ1in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.

Maxizyme-mediated specific inhibition on mutant-type p53 in vitro

AIM: To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG→AGT)in vitro.METHODS: Two different monomers of anti-mtp53maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G5→A5) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR,pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme).Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEN-T vector under the T7 promoter control.The 32p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with 32p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis.RESULTS: In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 ℃ and 25 mM MgCL2. The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either.CONCLUSION: pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good fundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.

Identification of up-regulated genes in human uterine leiomyoma by sup-pression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.

Non-classical diffusion model for heat and mass transfer in laser drying

A numerical analysis of the laser drying process by employing a generalized,Maxwell-Cattaneo equation to treat both heat and mass transfer was presented. Calculations wereperformed to illustrate the non-classical transport of heat and moisture. The effect of the heatflux density and the initial moisture content on water removal was also investigated. The resultsindicate that the non-equilibrium mass diffusion plays an important role during the very earlystages of moisture removal, especially at the surface of the medium. Away from the surface, thenon-Fickian model shows a delay in the reduction of the moisture content. The calculation resultsalso show that the initial moisture content of the medium has a considerable effect on waterremoval.