Construction of an eGFP expression plasmid under control of T7 promoter and IRES sequence for assay of T7 RNA polymerase activity in mammalian cell lines

Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site [IRES] sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells. For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity. Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient. Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics

Comparative molecular PCR-RFLP study of native herpes simplex virus type 1 [HSV-1] with KOS strain

Recent research on several DNA fragments covering open reading frames [ORF] 1-37 shows a new genetic marker in ORF 6 which is specific for differentiating wild-type varicella-zoster virus [VZV] strains from Oka varicella vaccine strain. On the other hand, herpes simplex virus [HSV] genome analysis by restriction enzymes is used to differentiate types one and two of the virus and even strains of each type. Previous studies using PCR-sequencing technique have shown that the thymidine kinase [TK] gene of HSV-1 is polymorphic. In this study, TK gene and DNA binding protein [UL29] gene of HSV-1 were selected. Both genes were analyzed with restriction endonucleases in order to identify a genetic marker for differentiating native strains of HSV-1 from the foreign strain. Three isolates of HSV-1 as well as standard strain of KOS were propagated in Vero cells. Initially, a pair of specific primers for each gene was designed to amplify UL29 and TK genes of these isolates. Subsequently, PCR products of these genes were digested separately with five restriction enzymes and subjected to polyacrylamide gel electrophoresis. Using PCR-RFLP [Restriction fragment length polymorphisms] technique, results indicate that the patterns of restriction endonuclease digestion of UL29 and TK genes of the three isolates show no differences when compared to KOS strain. The genotypes of Iranian isolates are the same as KOS genotype and both genotypes are derived from a common ancestor. Hence, it can be postulated that in the process of random population flow among Iran, Europe and USA, the original KOS strain infected the Iranian population at some point in time

Fluoxetine decreased serum total cholesterol and triglyceride levels in a hypercholesterolemic patient with postpartum depression

To report the case of a 28-year old hypercholesterolemic female with postpartum depression, whose triglyceride [TG] and total cholesterol [TC] levels decreased while being treated with fluoxetine. A 28-year old female, with a diagnosis of major depressive disorder with postpartum onset based on DSM-IV criteria, was hospitalized at a mental health hospital. Her past history included another episode of depression 4 months after giving birth to her second child, which was 12 years prior to her recent episode. Her serum total cholesterol and triglyceride levels were measured prior to the initiation of medication. Then fluoxetine was initiated at a daily dose of 20 mg and had been increased to 40 mg per day at the time of discharge. The lipid profile measurements was repeated at week 4 and 8 following treatment. Total cholesterol level was reduced from 242 mg/dL at baseline to 224 mg/dL at week 4 and to 202 mg/dL at week 8; triglyceride level was decreased from 516 mg/dL to 448 mg/dL at week 4 and to 404 mg/dL at week 8. Fluoxetine may be an appropriate treatment for hyperlipidemic women with postpartum depression

[Comparing the effects of 8- week treatment with fluoxetin and imipramine on fasting blood glucose in patients with major depression disorder]

Background: this study was designed to compare the effects of fluoxetine and imipramine on fasting blood glucose [FBG] in patients with major depressive disorder

Methods: non-diabetic patients, with major depressive disorder [based on DSM-IV criteria] entered this randomized, double - blind study. Patients did not receive any medication affecting serum FBG levels at least for 2 weeks prior to the initiation of the study. Patients were assigned to receive 20 to 40 mg/day of fluoxetine or 75 to 200 mglday of imipramine for 8 weeks. Benzodiazepines were allowed when needed for anxiety, agitation or sleep. Pregnant women and patients with diabetes mellitus, and history of major heart diseases were excluded from this study. Additionally, none of the patients should have received electroconvulsive therapy [ECT] within 6 months prior to initiation of the antidepressants. FBG levels were measured at the initiation of study as well as 4 and 8 weeks after starting antidepressants

Results: nineteen patients in the fluoxetine and 24 patients in the imipramine groups completed the study. In the fluoxetine group, FBG level was decreased from 88.5 mg/dL [baseline] to 85.0 mg/dL at week 4 [P=0.73], and to 79.8mg/dL at week 8 [P<0.001]. On the other hand, in the imipramine group, FBG level was increased from 86.96 mg/dL [baseline] to 89.71 mg/dL at week 4 [P=0.079], and to 96.90 mg/dL at week 8 [P<0.001]

Conclusion: this 8-weeks study showed that FBG levels may decrease in depressive patients receiving fluoxetine and may increase in those patients treated with imipramine. Therefore, it is suggested to measure and monitor FBG before initiation and during treatment with fluoxetine and imipramine