Monthly Archives: April 2011

RNA secondary structure prediction

5′-UTR sequence of DHV-1 ZJ strain (GenBank No. EF382778) was aligned with other DHV-1 isolates from GenBank using DNAstar (DNASTAR Inc.) software. Secondary structure elements were predicted in Mfold. Plasmid construction DNA preparations were performed using standard methods as described in manufacturers’ instructions. The reporter plasmids pGEM-CAT/EMC/LUC, containing the EMCV IRES cDNA, and pGEM-CAT/LUC were […]

DHV-1 IRES initiation does not require functional eIF4A. Part 2

Some picornaviruses, such as the AEV, contain HCV-like IRES elements organized in two major secondary structure domains, II and III, in which all the structural elements crucial for initiation of translation are located. Mutations within the putative loop of domain IIIe and stem1, 2 inhibited the DHV-1 IRES function. Moreover, the restoration of these structures […]

DHV-1 IRES initiation does not require functional eIF4A

In order to evaluate the contribution of the remaining component of the eIF4F complex, the RNA helicase eIF4A, two different experimental in vitro approaches were used. Firstly, the effect on DHV-1 translation initiation of two different dominant negative mutants of eIF4A was evaluated. The dominant negative mutant PRRVAA contains a mutation in the conserved Ia […]

DHV-1 IRES activity is modulated by downstream coding sequences

DHV-1 IRES activity is modulated by downstream coding sequences To further characterize the presence of accessory regulatory sequences downstream of the 5′-UTR of the viral genome, 4 additional dicistronic reporter plasmids were constructed, containing DHV-1 5′-UTR and 10, 40 or 60 nucleotides of the coding region adjacent to the viral 5′-UTR (DHV-1 UTR+10nt, UTR+40nt, UTR+60nt, […]

The duck hepatitis virus 5′-UTR possesses HCV-like IRES activity. Part 3

The in vitro translation of the reporter proteins was evaluated measuring 35S-methionine incorporation. Cap-dependent translation from the first ORF was determined by the level of CAT expression and internal initiation activity of the different sequences included between the two reporter genes was estimated by the accumulation of fLUC. All the mRNAs expressed CAT efficiently as […]

The duck hepatitis virus 5′-UTR possesses HCV-like IRES activity. Part 2

According to their secondary structure, picornavirus IRES elements can be divided into four groups that display distinct biological properties. The first group (class I) includes the IRES elements from entero- and rhinoviruses (e.g. poliovirus, PV), while the second includes cardio- and aphthoviruses IRES elements (e.g. encephalomyocarditis virus, EMCV). The IRES element from hepatitis A virus […]

The duck hepatitis virus 5′-UTR possesses HCV-like IRES activity

Duck hepatitis virus (DHV-1) is a worldwide distributed picornavirus that causes acute and fatal disease in young ducklings. Recently, the complete genome of DHV-1 has been determined and comparative sequence analysis has shown that possesses the typical picornavirus organization but exhibits several unique features. For the first time, we provide evidence that the 626-nucleotide-long 5′-UTR […]

In vitro study on human cytomegalovirus affecting early pregnancy villous EVT’s invasion function. Discussion

Discussion HCMV is the most common pathogen in intrauterine infection. Among the new born babies in Europe and America, HCMV infection rate is 2%, while that in developing countries is even higher. HCMV intrauterine infection may cause miscarriage, stillbirth, fetus developmental retardation, central nervous system damage and other serious results, but its specific pathogenesis remains […]

In vitro study on human cytomegalovirus affecting early pregnancy villous EVT’s invasion function. Results

Controls The above immunohistochemical procedures controls were performed replacing the primary antibody by PBS. Further controls were performed omitting the secondary antibody. The controls were always negative. Mexican online pharmacies Western Blot detection It is to detect c-erbB-2, MMP-2 and MMP-9 expression levels. Referring to the instruction of kits, two groups of villous total protein […]

In vitro study on human cytomegalovirus affecting early pregnancy villous EVT’s invasion function. Materials

Specimen and sources of the virus The tissue of villus is from healthy pregnant women whose peripheral blood is HCMV antibody negative and whose gestational age are between 5 to 10 weeks, who are voluntarily conducted abortion because of planned parenthood, from department of gynecology and obstetrics of Tongji Hospital of Tongji Medical College of […]