Overexpression of ribonucleotide reductase small subunit, RNRM, increases cordycepin biosynthesis in transformed Cordyceps militaris / 中国天然药物

Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn't been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases (RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR (qRT-PCR) and western blotting results showed that the mRNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wild-type strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn't remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine (3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.

Advances in nucleotide reductase inhibitors for antiviral therapy / 中华临床感染病杂志

Ribonucleotide reductase ( RR ) is a rate-limiting enzyme, and it is responsible for reducing ribonucleotides to their corresponding deoxyribonucleotides , which are the building blocks required for DNA replication and repair .Recent studies have revealed that RR activity is associated with DNA replication in virus , and RR inhibitors have been used for clinical antiviral treatment .This paper reviews research progress on RR and its inhibitors , including the classification , structure and function of RR; the classification, mechanism and clinical application of RR inhibitors in antiviral therapy and the future prospects of RR inhibitors .

Detection of expression of RRM1 mRNA by real-time fluorescent quantitative PCR in non-small cell lung cancer tissues and peripheral blood / 肿瘤

Objective: We tired to develop a method of detecting the expression level of RRM1 mRNA in tumor tissues and peripheral blood by real-time fluorescent quantitative PCR and compare the detection methods and results between the two different samples. We aimed to provide the basis for predicting clinical outcome from the expression of RRMI gene. Methods: The plasmid standard of RRM1 gene was constructed. Real-time quantitative analysis was performed using ABI7000 PCR kit. The PCR condition was optimized and the standard curve was established to detect the clinical samples. Results: The RRMI gene expression was detected in lung cancer tissues and normal lung tissues from 18 patients and in peripheral blood samples from 17 patients. The relative expression of RRM1 normalized by β-actin was 4.46 × 10-3 in lung cancer tissues, 3.43 × 10-3 in normal tissues, 2.54 × 10-3 in peripheral blood. The linear correlation between Ct value and the logarithm of original concentration was favorable. Conclusion: The expression of RRM1 mRNA in non-small cell lung cancer tissues and peripheral blood was successfully detected by using SYBR Green I fluorescence real-time quantitative PCR assay. There is no significant difference between the two sources of samples. The detection method has relatively higher sensitivity and specificity and can be used in clinical analysis.

Effect of antisense oligodeoxynucleotide of small subunit component of human ribonucleotide reductase on human choriocarcinoma cell line in vitro / 中华妇产科杂志

Objective To study effect of the antisense oligodeoxynucleotide (ASODN) of small subunit of human ribonucleotide reductase (RRM2) mRNA on cell line of human choriocarcinoma in vitro. Methods Two 20-mer gapmer ASODNs with a full phosphorothioate backbone were artificially synthesized, which were complementary to nucleotides 626-645 (a coding region) and 1572-1591 (a 3′untranslated region) of RRM2, respectively. ASODNs were transfected into JAR cells through oligofectamine. The survival rate was assessed by methyl thiazolyl tetrazolium (MMT) assay, and RRM2 expression was detected by immunoblot and reverse transcriptase polymerase chain reaction (RT-PCR) methods. Results Antisense oligodeoxynucleotide one (ASODN1) targeting the coding region significantly inhibited growth of JAR cells in a dose- and time-dependent manner and downregulated RRM2 expression in a time-dependent manner. ASODN1 at 100 nmol/L could inhibit significantly cell growth ( P =0.000), and the effects of ASODN1 on JAR cell proliferation were enhanced with increase of ASODN1 concentration and reached the peak point at 400 nmol/L concentration ( P =0.000). Cell growth was significantly inhibited by 200 nmol/L of ASODN1 after 24 h of treatment ( P =0.000). The effect of ASODN1 was at the maximum at 48 h ( P =0.000), and began to decrease at 72 h of treatment. RRM2 expression started to reduce after ASOND1 treatment for 12 hours, and was obviously downregulated at 24 h of treatment, and decreased to the lowest level at 48 h ( P