Expressional Localization and Functionally Identifying an RNA Editing Enzyme BmADARa of the Silkworm Bombyx mori.

The most common type of RNA editing in metazoans is the deamination of adenosine into inosine (A-to-I) catalyzed by the adenosine deaminase acting on the RNA (ADAR) family of proteins. The deletion or dysfunction of ADAR enzymes in higher eukaryotes can affect the efficiency of substrate editing and cause neurological disorders. However, the information concerning A-to-I RNA editing and ADAR members in the silkworm, Bombyx mori (BmADAR), is limited. In this study, a first molecular comprehensive cloning and sequence analysis of BmADAR transcripts was presented. A complete open reading frame (ORF) (BmADARa) was obtained using RT-PCR and RACE and its expression pattern, subcellular localization and A-to-I RNA-editing function on the silkworm synaptotagmin I (BmSyt I) were investigated. Subcellular localization analysis observed that BmADARa was mainly localized in the nucleus. To further study the A-to-I RNA-editing function of BmADARa, BmSyt I-pIZ-EGFP was constructed and co-transfected with BmADARa-pIZ-EGFP into BmN cells. The result demonstrates that BmADARa can functionally edit the specific site of BmSyt I. Taken together, this study not only provides insight into the function of the first ADAR enzyme in B. mori, but also lays foundations for further exploration of the functional domain of BmADARa and its editing substrates and target sites.

Hypermethylation and Expression Silencing of PDCD4 Gene in Hepatocellular Carcinoma: A Consort Study.

Programmed cell death 4 (PDCD4) is a novel tumor suppressor, which is involved in the initiation and progression of cancers. However, the role of PDCD4 in hepatocellular carcinoma (HCC) has not been reported. The aim of this study was to investigate the molecular mechanism and clinical significance of PDCD4 inactivation in HCC.The mRNA levels of PDCD4 in HCC tissues and adjacent nontumor tissues were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Bisulfite sequencing PCR was performed to determine the methylation status of PDCD4 promoter. Furthermore, the mRNA expression level and the methylated level of PDCD4 were analyzed with the clinical and pathological characteristics.qRT-PCR analysis showed that PDCD4 mRNA levels in tumor tissues were significantly decreased compared with that in adjacent nontumor tissues. The methylation rate of PDCD4 promoter was significantly higher in HCC tissues than that in adjacent nontumor tissues. PDCD4 mRNA levels and promoter methylation levels were both statistically correlated with metastasis and the degree of differentiation in HCC. In addition, the correlation between PDCD4 hypermethylation, mRNA levels, and overall survival (OS) was statistically significant.Our results indicated that PDCD4 may be a novel candidate of tumor suppressor gene in HCC, and that promoter hypermethylation is an important mechanism for its downregulation and is also a good predictor of OS for HCC.

Molecular and enzymatic characterization of two enzymes BmPCD and BmDHPR involving in the regeneration pathway of tetrahydrobiopterin from the silkworm Bombyx mori.

Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases and nitric oxide synthase so that BH4 plays a key role in many biological processes. BH4 deficiency is associated with numerous metabolic syndromes and neuropsychological disorders. BH4 concentration in mammals is maintained through a de novo synthesis pathway and a regeneration pathway. Previous studies showed that the de novo pathway of BH4 is similar between insects and mammals. However, knowledge about the regeneration pathway of BH4 (RPB) is very limited in insects. Several mutants in the silkworm Bombyx mori have been approved to be associated with BH4 deficiency, which are good models to research on the RPB in insects. In this study, homologous genes encoding two enzymes, pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) involving in RPB have been cloned and identified from B. mori. Enzymatic activity of DHPR was found in the fat body of wild type silkworm larvae. Together with the transcription profiles, it was indicated that BmPcd and BmDhpr might normally act in the RPB of B. mori and the expression of BmDhpr was activated in the brain and sexual glands while BmPcd was expressed in a wider special pattern when the de novo pathway of BH4 was lacked in lemon. Biochemical analyses showed that the recombinant BmDHPR exhibited high enzymatic activity and more suitable parameters to the coenzyme of NADH in vitro. The results in this report give new information about the RPB in B. mori and help in better understanding insect BH4 biosynthetic networks.