Vitamin D Binding Protein as screening biomarker candidate for late-onset preeclampsia without intrauterine growth restriction during 16 week of gestation

The aim of this investigation was conducted to proteomic analysis of plasma obtained from pregnant women who destined to develop late-onset preeclampsia without intrauterine growth restriction [IUGR] during 16[th] week of gestation. Plasma was obtained from primiparous women during 16[th] week of gestation. 2-DE proteomic analysis was done for plasma from 11 healthy pregnant women and 11 women who developed preeclampsia later. Using bioinformatic analysis with Progenesis SameSpots ver4.0 software and ANOVA test, expression of 2 spots were statistically different between two groups. In preeclamptic state, expression of both were decreased, one of these spots was vitamin D binding protein [p-value: 0.047], the other one will be discussed in another paper. According to results, we concluded that during 16[th] week of gestation, occurance of late-onset preeclampsia without IUGR is predictable. During this week, pathology of disease is present and may be the process of placental degeneration and impaired placentation are include in disease pathology

Comparison of three routine protein precipitation methods: acetone, TCA/acetone wash and TCA/acetone

The aim of this study was to compare three routine precipitation methods, including: acetone, TCA/acetone wash and TCA/acetone. 30 plasma samples were precipitated using above mentioned three methods. Pellets were dissolved in rehydration solution and protein quantification was done for this solution. According to statistical analysis using SPSS ver 16.0 software and one-way ANOVA test, the protein yield of acetone method was greater than two other methods and was statistically different from TCA/acetone precipitation method [p-value: 0.006]. The acetone method is more efficient than two other methods in protein precipitation

Homocysteine induces heme oxygenase-1 expression via transcription factor Nrf2 activation in HepG2 cells

Background: elevated level of plasma homocysteine has been related to various diseases. Patients with hyperhomocysteinemia can develop hepatic steatosis and fibrosis. We hypothesized that oxidative stress induced by homocysteine might play an important role in pathogenesis of liver injury. Also, the cellular response designed to combat oxidative stress is primarily controlled by the transcription factor Nrf2, a principal inducer of anti-oxidant and phase II-related genes

Methods: hepG2 cells were treated with homocysteine in different time periods. Glutathione content was measured by flowcytometry. Using electrophoretic mobility shift assay [EMSA] and Western-blotting, anti-oxidant response element [ARE]-binding activity of Nrf2 for heme ocygenase-1 [HO-1] was demonstrated. Real time RT-PCR and Western-blotting were performed to evaluate whether homocysteine was able to induce mRNA and protein expression of HO-1

Results: the role of Nrf2 in cellular response to homocysteine is substantiated by the following observations in HepG2 cells exposed to homocysteine [i] Western-blotting revealed that Nrf2 is strongly stabilized and became detectable in nuclear extracts. [ii] EMSA demonstrated increased binding of Nrf2 to oligomers containing HO-1 promoter-specific ARE-binding site. [iii] Real time RT-PCR and Western-blotting revealed increased mRNA and protein expression of inducible gene HO-1 after treatment with Homocysteine

Conclusion: data presented in the current study provide direct evidence that the immediate cellular response to oxidative stress provoked by homocysteine is orchestrated mainly by the Nrf2-ARE pathway. Therefore, induction of Nrf2-ARE-dependent expression of HO-1 could be a therapeutic option for hepatic cells damage induced by homocysteine. Iran. Biomed. J. 17 [2]: 93-100, 2013

Production of recombinant human T lymphotropic virus type 1 tax protein in Rosetta [DE3] bacterial host

HTLV1 is the first detected retrovirus causing disease in human. The physiopathology of HTLV1 related diseases was mainly linked with its Tax protein characteristics. Use of mutant Tax proteins accompanied by immune regulator drugs could help treating HTLV1 associated myelopathy patients as a modulator of potent immune response against this viral protein. Since Tax protein is not commercially available, production of recombinant Tax protein was targeted for this study. Coding sequence of Tax protein [containing R222K mutation] in the pcDNA3.1[+] was digested with BamHI and XhoI restriction enzymes, and then removed and inserted into the expression vector pET32a[+] within the same cutting sites and cloned into E.coli DH5alpha. Recombinant vector was confirmed with enzymatic digestion, colony PCR, and sequencing of cloned gene. E.coli Rosetta [DE3] was transformed with the recombinant plasmid and the expression was induced. The expression of protein was assayed with SDS-PAGE and western blot using monoclonal antibodies against Tax and 5His epitope. Finally, antigenic characteristic of the recombinant protein was evaluated by western blotting against patient sera. Presence of Tax protein band in the SDS-PAGE and western blot was confirmed. Western blotting of the recombinant protein with patient sera showed the band related to Tax protein. The recombinant protein is well produced and could be detected by patients' sera, making it eligible to be used as a recombinant viral antigen for future purposes

Angiotensin Converting Enzyme Gene polymorphism in Iranian Patients with Type 2 Diabetes

Angiotensin I converting enzyme [ACE] is a Zinc metalloproteinase, converts Ang I to Ang- IIa pro-inflammatory agent which may contribute to pathophysiology of some diseases like type 2 diabetes. To investigate the relationship between ACE I/D polymorphism and type 2 diabetes in 261 Iranian casecontrol pairs. 170 patients [85 type 2 diabetics with nephropathy and 85 type 2 diabetics without nephropathy] and 91 healthy control subjects were enrolled in our study. I/D polymorphism of the ACE gene was detected by polymerase chain reaction [PCR] utilizing specific primers. The frequency of DD genotype in the DN group was higher than that of the type 2 diabetic patients [30.6% vs. 20%, P = 0.157] and the control group [30.6% vs. 14.3%, P=0.006]. The frequency of D allele in nephropathic patients was 58.2% as compared to type 2 diabetic patients without nephropathy 50.5% [P=0.19] and control subjects 37.3% [P =0.001]. Therefore, the frequency of DD genotype and D allele significantly increased in DN patients in comparison to healthy controls. It is concluded that the DD genotype and /or D allele of ACE gene may increase the risk for type 2 diabetes but not diabetic nephropathy