Reports of Biochemistry and Molecular Biology
Volume:6 Issue: 1, Oct 2017

Dyslipidemia is considered an independent risk factor for coronary heart disease (CHD). In the present study, we examined lipid profiles and paraoxonase 1 (PON1) activity and atherogenic indexes status and the relationship of PON1 activity by high-density lipoprotein (HDL) and atherogenic indexes in CHD patients and healthy people.
The aim of the study was to compare PON1, lipid profiles, and atherogenic indexes in CHD patients and healthy people as controls. This study enrolled 50 CHD patients and 50 matched healthy controls. Serum activities of PON1 and levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL), very low density lipoprotein (VLDL), high-density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), and atherogenic indexes were analyzed. Data were analyzed by unpaired Student’s t tests. Coefficients of correlation were calculated using Pearson’s correlation analysis.
Levels of TG, TC, LDL, VLDL, FBG, and atherogenic indexes, atherogenic coefficients, and cardiac risk ratios were significantly greater in CHD patients than in controls. Paraoxonase 1 activity and HDL-C levels were significantly less in CHD patients than in controls. Also, PON1 activity correlated positively with HDLC and negatively with atherogenic coefficient, and cardiac risk ratios 1 and 2 in CHD patients.
This study showed that CHD is associated with high lipid levels and atherogenic indexes, and low PON1 activity and HDL-C concentrations. Coronary heart disease is a pernicious disease requiring prolonged medical management and hypolipidemic drugs.

Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) molecules are expressed on T-cells and inhibit their function by inhibiting activation of subsequent T-cell molecular pathways. Blocking of CTLA-4 inhibits the growth of malignant tumor cells. Anti-CTLA-4 monoclonal antibodies activate the immune system against cancer. Due to several advantages of single-chain antibodies (scFvs) compared to monoclonal antibodies in cancer immunotherapy, specific anti-CTLA-4 scFvs (single-chain variable fragment) were selected in this study.
A phage antibody display library of scFvs was analyzed and a panning process was performed against an immunodominant epitope of CTLA-4. PCR and DNA fingerprinting were used to differentiate the specific clones. The specificity of the selected clones was investigated by phage ELISA (Enzyme-linked immunosorbent assay).
Two specific clones with frequencies of 35 and 20% were identified. The clones reacted with the corresponding epitope on ELISA, while no reactivity was observed with an unrelated peptide, M13KO7 helper phage, unrelated scFvs, or no peptide as negative controls.
Targeted therapy against cancer markers is an ideal treatment strategy. Specific human anti-CTLA-4scFvs were selected in this study. These scFvs bound the related epitope. These antibodies have the potential to be used for targeted therapy, where the blocking of CTLA4 receptor is needed. The study suggests further evaluation of the selected scFvs to reveal the effects of the selected antibodies.

The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system.
An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b () plasmid were digested with the same enzymes and the construct was ligated into pET-21b (). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b ()/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting.
The hspX/esxS gene construct was inserted into pET-21b () and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein.
An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

Natural products have gained importance recently for the treatment of obesity and its complications, partly because of the side effects of modern drugs.Hence, we aimed to study and compare the effect of varying concentrations of Momordicacharantiaon adipogenesis and adipolysis using 3T3-L1 pre-adipocyte cell lines.
3T3-L1 pre-adipocytes were procured from the National Center for Cell Sciences, Pune, and cultured in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 20% fetal bovine serum (FBS) and 1 mM L-glutamine. An ethanolic extract of M. charantia (EEMC)was prepared by the graded ethanol fractionation method and the total phenol content (TPC) determined using the Folin-Ciocalteau (F-C) assay. The cytotoxic dose was determined by the sulforhodamine-B (SRB) assay. The adipogenesis and adipolysis assays (Cayman chemicals, Ann Arbor, USA) were performed according to the manufacturer’s protocols.
The 3T3-L1 pre-adipocytes treated with increasing concentrations of EEMC during (p= 0.012) and after (p= 0.026) differentiation demonstrated significant reduction in lipid droplet accumulation. There was a significant decrease in glycerol release during differentiation (p= 0.018) and a significant increase in glycerol release after differentiation (p= 0.0007) with increasing concentrations of EEMC. However, the effect of EEMC on adipogenesis and adipolysis was greater during 3T3-L1 pre-adipocyte differentiation than after.
The data showed that the 50% EEMC is potent inhibitor of lipogenesis and stimulator of lipolysis in 3T3-L1 pre-adipocytes. Further analyses will be performed to determine the key antioxidant compound(s) in the extract by phenolic acid profiling using high performance liquid chromatography (HPLC). Also, the mechanism of action of EEMC on adipogenesis and adipolysis will be elucidated.

Determination of the impact of angiogenesis on tumor development and progression is essential. This study aimed to determine the serum levels of Vascular endothelial growth factor (VEGF) and Interleukin 6 (IL-6) in breast carcinoma, and to correlate them with tumor size, lymph node involvement, and cancer stage.
Under aseptic precautions 5 ml of venous blood was collected from 37 breast cancer patients and 20 healthy females after obtaining due consent and ethical committee clearance. Serum levels of VEGF and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA).
Serum IL-6 and VEGF levels were both significantly greater in patients than controls (P = 0.001, P = 0.001, respectively). The serum IL-6 and VEGF levels also significantly correlated with TNM staging (P = 0.001, P = 0.001). Serum IL-6 and VEGF positively correlated with each other (r2 = 0.668, P = 0.01). Serum IL-6 and VEGF levels did not correlate with tumor size (P = 0.45, P = 0.17) or lymph node metastasis (P = 0.95, P = 0.68).
Serum IL-6 and VEGF were greater in breast cancer patients than controls. The levels increased with advanced tumor, nodes, metastasis (TNM) staging, thus correlating with the patients’ prognoses. Serum IL-6 and VEGF levels can be used as diagnostic tools and prognostic factors in breast cancer.

Organic acids refer to a family of compounds that are intermediates in a variety of metabolic pathways. Many organic acids are present in urine from clinically normal individuals. Elevated levels of urine organic acids cause to the organic acidurias, disorders in which some metabolic pathways in organic acid metabolism are blocked. The present work identified major and minor urinary acidic metabolites in normal subjects, and their quantitative ranges in a pediatric population of Iran.
Two hundred and fifty-one healthy subjects, including 132 males and 119 females, from 2 days to 15 years of age were enrolled. Urinary organic acids were extracted from urine with organic solvents and identified and quantified by gas chromatography-mass spectrometry.
The results provide a foundation on which to check results for patients with potentially abnormal organic acidurias. By this method 98 organic acids were identified in a pediatric population of Iran.
The present work identifies and quantifies major and minor urinary metabolites excreted by normal subjects. We also analyzed urine from 30 patients with organic acid metabolism abnormalities and compared the concentrations of specific organic acids with those from urines of normal individuals.

The prevalence of hepatitis C virus (HCV) infection is increasing worldwide. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) may play a role in the intensity of the disease. The aim of this study was to evaluate the association between genetic variants of the CTLA-4 and HCV infection.
Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) was performed as the genotyping assay at four different positions ( A>G, -318 C>T, -1722 T>C, and -1661 A>G). Haplotypes were analyzed using PHASE software. Sixty-five HCV patients and 65 healthy individuals as controls who were referred to the hepatitis clinic in Mashhad, Iran, were recruited. Genomic DNA was extracted from whole blood of participants.
In a dominant analysis model of the -1661 position (GG vs. AA), the AA genotype was more common in controls than in patients (adjusted P = 0.0003; OR = 0.15, 95% CI = 0.051 -0.42). The GCAT haplotype was also more prevalent in controls than in patients (adjusted P = 0.01; OR = 0.40, 95% CI = 0.20-0.81). Furthermore, the ACGT/ACGT diplotype was more common in controls than in patients (P = 0.0037; OR = 0.15, 95% CI = 0.04-0.54). In addition, the ACGT/ACAT diplotype was more frequent in patients than controls (adjusted P =0.003; OR = 2.48, 95% CI = 1.37- 4.50).
Our results indicated that polymorphisms in CTLA-4 and certain haplotypes may affect the risk of HCV infection in our population, although a larger sample size may be required to confirm this association.

Disorders of sex development (DSDs) belong to uncommon pathologies and result from abnormalities during gonadal determination and differentiation. Various gene mutations involved in gonadal determination and differentiation have been associated with gonadal dysgenesis. Despite advances in exploration of genes and mechanisms involved in sex disorders, most children with severe 46,XY DSDs have no definitive etiological diagnoses; therefore, the possibility that other genes or loci might play important roles in these disorders needs to be explored.
Patients (37) clinically suspicious for 46,XY gonadal dysgenesis (46,XY GD) of unknown etiology were studied. SRY, encoding the sex-determining region Y protein, NR5A1, encoding a transcription factor called steroidogenic factor 1, and DHH, encoding the desert hedgehog protein, were directly sequenced. Multiplex ligation-dependent probe amplification (MLPA) was used to detect deletions in NR0B1, encoding the DAX1 protein, and WNT4, encoding the WNT4 protein, and real-time PCR (qPCR) confirmed the MLPA data. Other potential loci have been investigated in the complete genome using Array-Comparative Genomic Hybridization, (Array CGH).
The SRY deletion was found in five patients. One each of previously described NR5A1, DHH, and AR (androgen receptor) allelic variants were identified. A pathogenic c.2522G>A AR mutation was found in two affected brothers. A heterozygous partial deletion was found in NR5A1 and heterozygous partial duplications were found in WNT4. These deletions and duplications (del/dup) were confirmed by qPCR. The Array CGH result demonstrated one partial deletion in SOX2-OT, which encodes a member of the SOX family of transcription factors, and the exact region of the rearrangements.
According to our study, del/dup mutations could be checked prior to point mutations, SOX2-OT has a potential role in gonadal dysgenesis, and Array CGH has a prominent role in gonadal dysgenesis diagnosis.

The critical role of interleukin-7 (IL-7) in homeostatic proliferation and T cell survival has made it a promising cytokine for the treatment of various clinical conditions, especially those associated with lymphopenia.
In the present study we expressed recombinant human interleukin-7 (rhIL-7) in Chinese hamster ovary (CHO)-K1 cells. CHO-K1 cells were stably transfected with both circular and linear forms of the pBud-hIL-7 recombinant by electroporation. Expression of rhIL-7 in CHO-K1 cells was confirmed by enzyme-linked immunosorbent assay (ELISA) and dot and western blots.
On western blots of transformed cells, a single 25 kDa band was observed, consistent with the expected molecular weight of glycosylated hIL-7. No significant expression difference was observed between cells transfected with circular or linear plasmids.
We established a stable CHO-K1 cell line expressing rhIL-7, which we consider to be a promising system for the production of rhIL-7 as a biopharmaceutical.

Several components of metabolic syndrome (MetS) facilitate its diagnosis, including abdominal obesity, hyperlipidemia, high blood pressure, and insulin resistance. The production of tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6) seem to be associated with MetS components. The aim of this study was to evaluate the correlation between the serum levels of TNFα and IL-6 with metabolic syndrome and its components. This case-control study investigated a total of 250 subjects, comprising 125 healthy controls from the Kerman Blood Transfusion Organization and 125 metabolic syndrome patients. Serum IL-6 and TNF-a levels were measured using the ELISA technique. There was a significant increase in the serum levels of TNF-a and IL-6 in patients with metabolic syndrome compared with the controls. However, a lack of correlation between MetS components and TNF-a and IL-6 serum levels was detected. Patients with MetS had significantly raised serum IL-6 and TNF-a levels compared with the controls, supporting the evidence that inflammation plays an important role in the immunopathogenesis of the disease. Additionally, serum levels of IL-6 and TNF-a are suggested as valuable predicting factors for MetS. The lack of association between IL-6 and TNF-a serum levels and MetS components remains to be investigated by further research.

Perilipins are proteins at the external level of the lipid glob in adipocytes steroid-generating cells and play a central role in lipid storage and breakdown. FTO gene is associated with Type-2 diabetes (T2D) and increased fat mass. Association of Perilipin and FTO genes polymorphism with T2D was investigated. Clinical traits in a random sample of 183 Iranian men and women with T2D and 174 controls without any metabolic disease took part in the study. The sample was genotyped using Tetra-ARMS method. In Perilipin (rs1052700) polymorphism tested, a significant association was found between this polymorphism and T2D (P=0.03). In FTO (rs3751812) polymorphism, a significant association was seen between this gene and obesity (P=0.003). But, no significant relation was found between this genotype and T2D. The rs3751812 polymorphism of FTO gene is the major genetic determinant of obesity, but not type-2 diabetes. The rs1052700 polymorphism of Perilipin gene is related to T2D but not obesity.

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T-cell lymphotropic virus type-1 (HTLV-1). HTLV-1 oncogenes can induce malignancy through controlled gene expression of cell cycle checkpoints in the host cell. HTLV-I genes play a pivotal role in overriding cell cycle checkpoints and deregulate cellular division. In this study, we aimed to determine and compare the HTLV-1 proviral load and the gene expression levels of cyclin-dependent kinase-2 (CDK2), CDK4, p53, and retinoblastoma (Rb) in ATLL and carriers groups. A total of twenty-five ATLL patients (12 females and 13 males) and 21 asymptomatic carriers (10 females and 11 males) were included in this study. TaqMan real-time polymerase chain reaction assay was used for evaluation of proviral load and gene expression levels of CDK2, CDK4, p53, and Rb. Statistical analysis was used to compare proviral load and gene expression levels between two groups, using SPSS version 18. The mean scores of the HTLV-1 proviral load in the ATLL patients and healthy carriers were 13067.20±6400.41 and 345.79±78.80 copies/104 cells, respectively (P=0.000). There was a significant correlation between the gene expression levels of CDK2 and CDK4 (P=0.01) in the ATLL group. Our findings demonstrated a significant difference between the ATLL patients and healthy carriers regarding the rate of proviral load and the gene expression levels of p53 and CDK4; accordingly, proviral load and expression levels of these genes may be useful in the assessment of disease progression and prediction of HTLV-1 infection outcomes.

With one-third of the world’s population infected, tuberculosis (TB) is one of the most common infectious diseases and a major public health problem, especially in developing countries. The efficacy of the BCG vaccine for controlling the disease in adults is poor. The development of an effective TB vaccine is a global objective. An effective tuberculosis vaccine should stimulate cellular immunity. DNA vaccines are a new generation of vaccines with the potential to achieve this goal. The aim of this study was to produce a DNA vaccine of Mtb72F. mtb32C, mtb39, and mtb32N were cloned into pcDNA3.1 using restriction enzyme digestion and T4 DNA ligase. Colony-PCR and restriction enzyme digestion were performed to detect transformed bacteria. DNA sequencing confirmed the desired gene insertion into the vector. A Chinese hamster ovary (CHO) cell line was transfected with the recombinant plasmid and RT-PCR was performed to assess gene expression. Gel electrophoresis showed the expected amplified gene fragments of 429, 614, and 1200 base pairs (bps) for mtb32C, mtb32N, and mtb39, respectively. Enzyme digestion and gel electrophoresis showed the expected fragments, indicating the desired gene position and orientation in the recombinant plasmid. This finding was verified by DNA sequencing, and RT-PCR demonstrated gene expression in the CHO cell line. An Mtb72F DNA plasmid was successfully constructed. This plasmid may be a candidate for animal immunizations.

Psoriasis is a T cell-mediated autoimmune disease with elevated level of pro-inflammatory cytokines belonging mainly to Th1 pathway. We investigated whether treatment with micronutrients along with methotrexate (MTX) is able to modulate mRNA expression of Th1 and Th2 patterns and its correlation with disease severity. Thirty plaque type psoriasis patients with Psoriasis Area and Severity Index (PASI) higher than 10 recruited; 15 non- micronutrients taker (NMT) patients, treated by MTX daily (0.2-0.3 mg/kg/week) and 15 micronutrients taker (MT) patients treated by MTX plus micronutrient supplement daily for 12 weeks. Blood samples collected at baseline and after 12 weeks. Taqman quantitative real-time polymerase chain reaction was applied to analyses the expression of Th1 (T-bet, IL-12, IFN- γ) and Th2 (GATA-3, IL-4) pathway. Disease severity measured under PASI scoring system. Significant clinical improvement in MT group was accordance with significant down-regulation of Th1 and up-regulation of Th2 studied markers (P<0.05). Respect to PASI-75 cut-point, expression of IFN-γ in MT group with upper PASI-75 was significantly lower than in related patients in NMT group (P=0.05). Also mRNA expressions of GATA3 and IL-4 in MT group with upper PASI-75 were significantly higher than patients in NMT group respectively (P=0.05, P=0.04) According to significant attenuating of PASI score correlated with upregulation of Th2 pathway in favor of MT group, consumption of micronutrients in combination MTX in psoriasis patients are suggested. Our results contribute to a better understanding of methotrexate immune-pathogenesis mechanisms and its correlation to clinical response in psoriasis.

Breast cancer is one of the most common cancers among women worldwide. Tumor protein 53 (TP53) and its regulator the mouse double murine 2 (MDM2) have important roles in tumorigenesis by playing key roles in cell division and response to DNA damage. MDM2 SNP309 T>G (rs2279744) polymorphism in the promoter region of MDM2 gene can cause dysfunction and inactivation of TP53 which promote tumor progression as a result. The aim of this study was to investigate the association between this polymorphism and risk of breast cancer in northeastern Iranian population. A case-control study with 128 breast cancer patients and 143 healthy women was conducted. PCR-ARMS was performed to assess the MDM2 SNP309 T>G (rs2279744) polymorphism. The GG Genotype frequency between patients and controls showed no significant association between this polymorphism with breast cancer risk (p=0.116, OR [95% CI]: 1.267 [0.616, 2.603]). Also, G allele frequency was not associated with breast cancer risk in studied population (p=0.143, OR [95% CI]: 1.326 [0.908, 1.935]). For this polymorphism, a significant difference of 8.0 years in the average age of cancer diagnosis was observed between TT and TG carriers (40.57 versus 48.15 years, p = 0.029). The results of this study suggest that the SNP309 T>G polymorphism in the MDM2 gene may not be associated with the risk of breast cancer in an Iranian population