[Establishment of drug screening assay and pharmacodynamic evaluation method targeting influenza RNA polymerase].

Influenza virus RNA-dependent RNA polymerase (RdRP) is essential for replication and expression of influenza virus genome. Viral genomic sequences encoding RdRP are highly conservative, thus making it a potential anti-influenza drug target. A cell-based influenza RdRP inhibitor screening assay was established by a luciferase reporter system to analyze the activity of RdRP. Specificity study and statistic analysis showed that the screening assay is sensitive and reproducible.

Further purification of industrial quartz by much milder conditions and a harmless method.

A much "greener" and harmless leaching method for removing impurity aluminum further from industrial quartz sands by very dilute mixed acids has been presented. With the help of supersonic, the percentage of removal aluminum reached up to 52.5%/53%, that is, 17.4 ppm/17.7 ppm at 30 °C/80 °C, respectively. These results are 4.4/4.7 ppm lower than that supplied by a world famous quartz sands supplier, and the leaching conditions are much milder compared with other comparable methods: the concentration of hydrogen chloride in the mixed acid is only 10% of the others, the leaching temperature is much lower; at the same time, the operating time is only 13-20% of the others, thereby pollution of industrial strong acids and thermo-scattering is reduced substantially.

Association between the CDC6 G1321A polymorphism and the risk of cervical cancer.

INTRODUCTION: Cell division cycle protein 6 (CDC6) plays critical roles in DNA replication and carcinogenesis. The biological significance of the CDC6 G1321A polymorphism (V441I, rs13706) on cervical carcinogenesis is still unknown. Here, we examined the potential influence of this polymorphism on cell proliferation and the individual's susceptibility to cervical cancer. METHODS: We genotyped the CDC6 G1321A polymorphism in 87 cervical cancer cases and 110 healthy female subjects. Unconditional logistic regression analysis was used to estimate the association between the genotypes and the risk of cervical cancer. The BrdU incorporation assay was applied to analyze the effect of this polymorphism on cell proliferation. RESULTS: Compared with the GG homozygotes, the cervical cancer risk was significantly reduced in the individuals with the heterozygous AG genotype (odds ratio [OR], 0.53; 95% confidence interval [CI], 0.28-0.98; P = 0.042) or the homozygous AA genotype (OR, 0.29; 95% CI, 0.09-0.89; P = 0.030). Further stratified analyses showed that the decreased risk of cervical cancer was more evident among younger subjects (≤44 years old) with the AG or AA genotypes (OR, 0.44; 95% CI, 0.21-0.92; P = 0.029 and OR, 0.12; 95% CI, 0.03-0.61; P = 0.010, respectively). The BrdU incorporation assay showed that 293T cells transfected with CDC6-441I (1321A) had a lower proliferation rate in comparison with those transfected with CDC6-441V (1321G), although the difference did not reach statistical significance at the 0.05 level. CONCLUSIONS: The CDC6 G1321A polymorphism may contribute to the risk of cervical cancer. Further studies with more subjects and in diverse ethnic populations are necessary to confirm the general validity of our findings.

A small natural molecule CADPE kills residual colorectal cancer cells by inhibiting key transcription factors and translation initiation factors.

Residual disease is the major cause for colorectal cancer (CRC) relapse. Herein, we explore whether and how a natural molecule CADPE killed heterogenic populations in a panel of CRC cell lines with KRAS/BRAF mutations that are natively resistant to EGFR- or VEGFR-targeted therapy, without sparing persistent cells, a reservoir of the disease relapse. Results showed that CADPE killed the tumor bulk and residual cells in the panel of CRC cell lines, rapidly inactivated c-Myc, STAT3, and NF-κB, and then decreased the protein levels of key signaling molecules for CRC, such as ß-catenin, Notch1, and the nodes of mTOR pathways; eukaryotic translation initiation factors (eIF4F); anti-apoptotic proteins (Bcl-xl, Mcl-1, and survivin); and stemness-supporting molecules (CD133, Bim-1, and VEGF). In terms of mechanism of action, concurrent downregulation of Mcl-1, Bcl-xl, and survivin was necessary for CADPE to kill CRC bulk cells, while additional depletion of CD133 and VEGF proteins was required for killing the residual CRC cells. Moreover, the disabled c-Myc, STAT3, NF-κB, and eIF4F were associated with the broadly decreased levels of anti-apoptosis proteins and pro-stemness proteins. Consistently, CADPE suppressed CRC tumor growth associated with robust apoptosis and depleted levels of c-Myc, STAT3, NF-κB, eIF4F, anti-apoptotic proteins, and pro-stemness proteins. Our findings showed the promise of CADPE for treating CRC and suggested a rational polytherapy that disables c-Myc, STAT3, NF-κB, and eIF4F for killing CRC residual disease.

Anti-colorectal cancer effects of tripolinolate A from Tripolium vulgare.

Tripolinolate A (TLA) is recently identified as a new compound from a halophyte plant Tripolium vulgare and has been shown to have significant in vitro activity against the proliferation of colorectal cancer and glioma cells. This study was designed to further investigate the effects of TLA on the proliferation of human normal cells, and the apoptosis and cell cycle in colorectal cancer cells, and the growth of tumors in the colorectal cancer-bearing animals. The data obtained from this study demonstrated that: 1) TLA had much less cytotoxicity in the human normal cells than the colorectal cancer cells; 2) TLA remarkably induced apoptosis in the human colorectal cancer cells and blocked cell cycle at G2/M phase, and 3) TLA had significant anti-colorectal cancer activity in the tumor-bearing animals.

Anticonvulsant activity of ginseng on seizures induced by chemical convulsants.

PURPOSE: To test the anticonvulsant activity of three preparations of American ginseng: whole root extract, whole leaves/stems extract, and a partially purified extract that concentrates the Rb ginsenosides (Rb extract). METHODS: One hour after treatment with normal saline, or one of the three ginseng preparations, seizures were induced in adult, male, Sprague-Dawley rats with kainic acid (KA; 10 mg/kg), pilocarpine (300 mg/kg, preceded by methylscopolamine, 1 mg/kg, s.c.), or pentylenetetrazol (PTZ, 50 mg/kg). Time to onset of seizure activity, duration of seizure activity for PTZ, seizure severity, and weight change for KA and pilocarpine were determined for each animal. The brains from animals who had received KA or pilocarpine were examined for severe neuronal stress, by using immunoreactivity for heat-shock protein (HSP)72. RESULTS: The Rb extract had a dose-dependent anticonvulsant effect in all three models of chemically induced seizures: increasing the latency to the seizures; decreasing the seizure score, weight loss, and subsequent neuronal damage after pilocarpine; and shortening the seizure duration and reducing mortality after PTZ. The Rb extract also significantly reduced the effects of KA, including completely blocking behavioral seizures. The root preparation increased the mortality rate after administration of pilocarpine, but had no other significant effects. The leaves/stems preparation, at 120 mg/kg, reduced the weight loss after pilocarpine, but had no other significant effects. CONCLUSIONS: Ginseng extract made from either the root or leaves/stems is ineffective against chemically induced seizures. A partial purification of the whole extract that concentrates the Rb1 and Rb3 ginsenosides has significant anticonvulsant properties.

Expression, purification, and C-terminal amidation of recombinant human glucagon-like peptide-1.

Human glucagon-like peptide-1 (hGLP-1) (7-36) amide, a gastrointestinal hormone with a pharmaceutical potential in treating type 2 diabetes mellitus, is composed of 30 amino acid residues as a mature protein. We report here the development of a method for high-level expression and purification of recombinant hGLP-1 (7-36) amide (rhGLP-1) through glutathione S-transferase (GST) fusion expression system. The cDNA of hGLP-1-Leu, the 31st-residue leucine-extended precursor peptide, was prepared by annealing and ligating of artificially synthetic oligonucleotide fragments, inserted into pBluescript SK (+/-) plasmid, and then cloned into pGEX-4T-3 GST fusion vector. The fusion protein GST-hGLP-1-Leu, expressed in Escherichia coli strain BL21 (DE3), was purified by affinity chromatography after high-level culture and sonication of bacteria. Following cleavage of GST-hGLP-1-Leu by cyanogen bromide, the recombinant hGLP-1-Leu was released from fusion protein, and purified using QAE Sepharose ion exchange and RP C(18) chromatography. After purification, the precursor hGLP-1-Leu was transacylated by carboxypeptidase Y, Arg-NH(2) as a nucleophile, to produce rhGLP-1. Electrospray ionization mass spectrometry showed the molecular weight was as expected. The biological activity of rhGLP-1 in a rat model demonstrated that plasma glucose concentrations were significantly lower and insulin concentrations higher after intraperitoneal injection of rhGLP-1 together with glucose compared with glucose alone (P < 0.001).