REPORTS OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
Year:2018 | Volume:7 | Issue:1|Papers Count:15

Background: Nephrotic syndrome is a disorder caused by kidney damage that results in severe leakage of protein from blood into urine. Hyperlipidemia is one complication of nephrotic syndrome. L-carnitine and genistein can control cardiovascular diseases by causing changes in lipid metabolism and cytokine production. This study was designed to examine the effects of genistein and L-carnitine on serum lipid and cytokine profiles in experimental nephrotic syndrome. Methods: In this study, 50 male Sprague– Dawley rats were randomly divided into five groups of 10 animals each with similar mean body weights (300± 50 g). The five groups were NC (normal-control), PC (patientcontrol), LC (L-carnitine), G (genistein), and LCG (L-carnitine-genistein). Serum HDL-cholesterol (HDL) LDL-cholesterol (LDL), triglyceride, cholesterol, IL-6, and TNF-α were measured. Statistics were analyzed using SPSS 18. 0. Results: At the end of the study, of the patient groups, HDL was significantly greater in the LC than in the PC or G groups (P<0. 001). LDL was significantly less in the G than in the PC, LC, or LCG groups (P<0. 001). Interleukin-6 was significantly greater in the PC than in the LC, G, or LCG groups, and significantly greater in the LC than in the G group. (P<0. 001), but no significant differences were found for triglyceride, cholesterol, or TNF-α between the patient groups. Conclusions: Genistein had less effect on HDL and triglyceride levels than LC or LCG. Regarding inflammatory cytokines, genistein and L-carnitine had less effect on TNF-α than on IL-6.

Background: The purification of Schwann cells has proven to be a difficult process, with most methods requiring the use of special equipment. However, obtaining a sufficient number and high purity of Schwann cells is an integral aspect in their use for clinical application. Therefore, the aim of this study was to establish a simple and effective protocol for the isolation and purification of Schwann cells from the sciatic nerve of C57BL/6 mice. Furthermore, we aimed to provide a protocol for the isolation of exosomes from these cells. Methods: To purify Schwann cells, we used a combination of in situ nerve pre-degeneration and fetal bovine serum. To determine the most effective method of cell purification, we treated the culture with varying concentrations of fetal bovine serum and examined which concentration provided the highest Schwann cell purity. Exosomes were then isolated from Schwann cells through a process of repeated centrifugation and filtration steps. Results: We were able to increase the purified population of Schwann cells from C57BL/6 mice by reducing the concentration of FBS. The purity of Schwann cells at FBS concentrations of 10%, 5%, and 2% were 93. 42%, 91. 25%, and 97. 83%, respectively. Conclusions: When using a concentration of 2% FBS, we obtained the highest purification yield of Schwann cells. Our protocol does not require special equipment or materials. We have created a protocol that is simple, fast, and safe while providing a high yield of purified Schwann cells. The exosome isolation method described in this paper is an appropriate approach with a high quality and yield.

Background: Inflammatory bowel diseases (IBDs), which include ulcerative colitis (UC) and Crohn’ s disease (CD), are inflammatory disorders that affect the gastrointestinal tract. A combination of inflammatory cytokines has an important role in IBD development. Genome-wide association studies have shown that polymorphisms in the interleukin-23R gene (IL-23R) increase susceptibility to IBD. The aim of this study was to investigate the IL-23R 3' UTR SNP to determine a potential association between genotype distribution and IBD. Methods: The case group included 102 IBD patients and the control group included 107 healthy individuals. IL-23R polymorphisms rs10889677 were genotyped using PCR-RFLP analysis. RFLP results were confirmed by direct sequencing. Results: The allele and genotype frequencies in patients and controls were evaluated and compared, and no significant association between this functional rs10889677 polymorphism and risk of IBD was observed (P=0. 587; adjusted OR: 0. 89; 95% CI: 0. 597-1. 339). We also found no significant association between CD (14. 71%) and UC (85. 29%) patients in allele or genotype levels (P>0. 05). Conclusions: Our results suggest that the rs10889677 A>C polymorphism is not a potential prognostic marker in Iranian patients with IBD.

Background: The First apoptosis signal (FAS) and First apoptosis signal ligand (FASL) genes initiate the apoptosis pathway, playing a central role in the tumor growth and metastasis. Gene polymorphisms including-1377 G/A in the promoter region of FAS and-844 C/T in the promoter region of FASL have shown to change the transcription activities of these genes. Methods: In this study we evaluated association of these polymorphisms with risk of metastasis of breast cancer, in a population selected from Mashhad, Iran. A total of 115 patients with breast cancer and 115 controls were recruited in this case-control study. Polymerase Chain Reaction-based Restriction Fragment Length Polymorphism (PCR-RFLP) was applied for genotyping on extracted DNA from participant’ s blood. Unconditional logistic regression was used to estimate cancer risk by calculating odds ratios (OR) and their 95% confidence intervals (95% CIs). Results: There was no significant association between these genetic polymorphisms and breast cancer risk. Additionally, our results showed no significant influence from the above mentioned gene polymorphisms on metastasis of breast cancer. Conclusions: These results suggest that the FAS-1377G/A and FASL-844 C/T gene polymorphism don’ t have much influence on the susceptibility to metastasis of breast cancer in northeastern Iranian population. Therefore, we suggest to investigate impact of other candidate gene polymorphisms on metastasis of breast cancer for future research.

Interleukin (IL)-10, a multifunctional immune-regulatory cytokine with both immunosuppressive and antiangiogenic functions, is produced by immune cells including macrophages, T lymphocytes, and natural killer cells. Among other effects, IL-10 promotes tumor cell proliferation and metastasis via immunosuppression. Interleukin-10-mediated immunosuppression is aided by synthesis of tumor necrosis factor, IL-1, IL-12, and chemokines, and down regulation of the surface co-stimulatory molecules CD80 and CD86 on tumors. Interleukin-10 also promotes IL-6 expression and synthesis, which causes cell proliferation via B cell lymphoma-2 (Bcl-2) upregulation and changes the proliferation/apoptosis equivalence toward neoplastic cell proliferation. Moreover, IL-10 inhibits tumorigenesis via down-regulation of VEGF, IL-1b, TNF-α , IL-6, and MMP-9. Interleukin-10 also inhibits nuclear factor-κ B (NF-κ B) translocation. Interleukin-10 has been reported to have both tumor-promoting and-inhibiting properties. It seems that IL-10 agonists and antagonists may have therapeutic effects via different mechanisms. Moreover, IL-10 gene polymorphisms may determine breast cancer susceptibility.

Background: Pseudomonas aeruginosa, an opportunistic pathogen, is a common cause of healthcare-associated infections in immunocompromised individuals. The rapid emergence of multidrug-resistant strains has made P. aeruginosa infections progressively difficult to treat. In this study we evaluated the effect of a chimeric protein containing a P. aeruginosa PilQ fragment and the PilA disulfide loop (PilA-DSL) on the humoral immune response in BALB/c mice. Methods: A chimeric gene encoding an immunogenic region of PilQ and the PilA-DSL was synthesized. Following bacterial expression and purification, the protein was administered to mice and the humoral immune response analyzed. The resulting antibodies were analyzed using an opsonophagocytic killing assay. Results: The anti-recombinant protein antibody titer was significantly greater in immunized mice than in controls. In addition, antibody titers were significantly increased after booster immunizations, and the immunizations induced opsonophagocytosis of P. aeruginosa PAO1. Conclusions: These results suggest that an anti-adhesion-based vaccination may be effective in preventing P. aeruginosa infections. Further studies are needed to evaluate the abilities of such bivalent proteins to induce strong immune responses.

Background: CD90, a membrane-associated glycoprotein is a marker used to identify mesenchymal stem cells (MSCs). Recent studies have introduced CD90, which induces tumorigenic activity, as a cancer stem cell (CSC) marker in various malignancies. Blocking CD90 activity with anti-CD90 monoclonal antibodies enhanced antitumor effects. To date, highly specific antibody single-chain variable fragments (scFvs) have been isolated against various targets and showed promising results in cancer immunotherapy. Methods: A phage antibody was produced from a scFv library using M13KO7 helper phage. The phage library was panned against a CD90 epitope. To select specific clones, PCR and DNA fingerprinting were performed and common patterns were identified. The panning results were confirmed by phage ELISA. Results: Of 20 clones selected after panning, 16 shared identical fingerprints. One clone from this group reacted specifically with the epitope in phage ELISA. The average absorbance of wells coated with the CD90 peptide was significantly greater than that of wells containing no peptide (p=0. 03). Conclusions: Currently, recombinant antibodies are used not only as highly specific detection tools, but due to their specific characteristics, are applied in targeted cancer therapies. The anti-CD90 scFv selected in this study has the potential to be used to detect MSCs and target CSCs and offers promising strategies for treatment of various cancers.

Background: Chronic low-grade inflammation may be a cardinal pathophysiologic feature in the pathogenesis of frailty. Interferon-gamma (INF-γ ) is an understudied proinflammatory cytokine in frailty that induces many inflammatory pathways including the guanosine triphosphate cyclohydrolase 1 (GTP-CH1) pathway. Our aim was to evaluate the GTP-CH1 pathway in Egyptian frail elderly subjects. Methods: INF-γ , neopterin, and nitric oxide (NO) levels were measured in 80 participants. Results: Both pre-frail and frail subjects had significantly higher levels of INF-γ , neopterin and lower levels of NO than the control group. These biomarkers were associated with the risk of frailty with significant odds ratio. Conclusions: Elevated INF-γ levels in frail subjects may activate the GTP-CH1 pathway. Elevated neopterin and reduced NO levels correlated with an active GTP-CH1 pathway. The risk of frailty increased with elevated INF-γ and neopterin and decreased with elevated NO levels.

Background: Cardiovascular disease (CVD) is the leading cause of mortality worldwide. Omega-3 fatty acids have been shown to have both anti-atherogenic and anti-inflammatory effects through inducing the expression and production of adipokines. Adipokines such as apelin, have been observed to play a protective role in the incidence and progression of CVD. The aim of this study was to assess the influence of omega-3 fatty acids supplementation on the serum apelin levels in patients with cardiovascular disease. Methods: Forty-six male patients with CVD participated in the study. Patients were randomly allocated into two groups receiving either omega-3 fatty acids or a placebo. Participants received 4 g of omega-3 fatty acids (EPA: 720 mg, DHA: 480 mg) or a placebo (edible paraffin) for 8 weeks. Serum apelin levels, high sensitive C-reactive protein (hs-CRP), and lipid profiles were measured. Dietary intake, anthropometric parameters, body composition, systolic and diastolic blood pressure were evaluated before and after the 8 weeks of intervention. Statistical analyses were performed using SPSS version 22. Results: Two participants from the placebo group withdrew from the study. Prior to the intervention, no significant differences were present between the two groups in age, body mass index, body composition, dietary intakes, lipid profiles and blood pressure. Compared to placebo, the intake of omega-3 fatty acids increased serum apelin levels (p= 0. 018), decreased the levels of LDL cholesterol, and decreased serum hs-CRP concentrations (p= 0. 007, p= 0. 011 respectively). Additionally, the concentrations of VLDL, TG and hs-CRP (p= 0. 037, p= 0. 037 and p= 0. 016 respectively) declined compared to baseline and final values in the omega-3 fatty acids group. Conclusions: Omega-3 fatty acid supplementation increases serum apelin and HDL concentrations, while decreasing serum LDL-C and hs-CRP levels.

Background: Cutaneous leishmaniasis (CL) is a serious public health problem in many tropical countries. The infection is caused by a protozoan parasite of Leishmania genus transmitted by Phlebotominae sandflies. In the present study, we constructed a eukaryotic expression vector to produce a fusion protein containing LmSTI1 from Leishmania major (L. major) and PpSP42 from Phlebotomus papatasi (Ph. papatasi). In future studies we will test this construct as a DNA vaccine against zoonotic CL. Methods: The nucleotide sequences encoding the LmSTI1 protein and a fragment encoding 79% of PpSP42 were amplified using L. major and Ph. papatasi genomic DNA, respectively. The amplicons were cloned into the pcDNA3. 1(+) eukaryotic expression vector. The recombinant plasmid pcDNA-LmSTI1Pp42 was propagated in Escherichia coli (E. coli) and used to transfect HEK-293T cells. The expressed fusion protein was analyzed by Western blotting using anti-LmSTI1 mouse serum. Results: Sequences encoding LmSTI1 and partial PpSP42 were cloned into pcDNA3. 1(+). Production of the recombinant LmSTI1Pp42 fusion protein was confirmed by Western blotting. Conclusions: An LmSTI1Pp42 fusion protein was expressed in HEK-293T cells. This construct may be an effective DNA vaccine against CL.

Background: Naringenin is a bioactive flavonoid found in grapes and citrus fruits including tangelo, blood orange, lemons, and tangerines. The aims of this study were to investigate the ability of naringenin to scavenge free radicals and determine its ability to protect animals from streptozotocin (STZ)-induced liver damage. Methods: The free radical-scavenging activity of naringenin was evaluated by in vitro cell-free assay systems. In animals, the antioxidant potential of orally administered 50 and 100 mg/kg body weight naringenin for 45 days was assessed by measuring TBARS, lipid hydroperoxides, SOD, catalase, GST, GPx, and glutathione levels in liver homogenates prepared from animals injected intraperitoneally with multiple low dose streptozotocin at 50 mg/kg for five consecutive days. The extent of cellular damage caused by STZ administration was analyzed using H & E staining. Results: Naringenin showed potent free radical scavenging activity in vitro. Naringenin effectively neutralized (a) hydroxyl radicals, (b) superoxide, (c) hydrogen peroxide, (d) nitric oxide radical, (e) DPPH, and (f) lipid peroxidation. In animals, administration of naringenin reduced lipid peroxidation and increased antioxidant levels. Analysis of liver sections showed the restoration of normal morphology upon treatment with naringenin. Conclusions: Naringenin helps to mitigate STZ-induced liver complications by promoting antioxidant defence enzyme activities and increasing glutathione levels.

Background: Cancer treatment methods can lead to male infertility. in this regard, cryopreservation of spermatogonial stem cells (SSC) and cell-to-person transplantation after the course of treatment to resolve the problem of infertility is a good one. The cryopreservation of SSC is an important process as it can help on the return of spermatogenesis. However, during this process, the stem cells often become damaged which degrades their value for experiments and treatments. Caffeic acid (CA) is an antioxidant that has been shown to increase the viability of cells under stress. The aim of this study was to investigate the effect of CA has on spermatogonial stem cell (SSC) cryopreservation. Methods: Spermatogonial stem cells isolated from the testes of Balb/c mice pups were cultured in laminincoated dishes, purified using CD90. 1 microbeads, then cryopreserved in vitrification media supplemented with 10 μ M CA either through a slow or rapid freezing process. After thawing, cell viability was evaluated. Expression of Bax, Fas, Bcl-2 and P53 genes was determined by real-time PCR. Gel electrophoresis was used to confirm the results of the real-time PCR. Results: The viability of the SSCs that were rapidly frozen and treated with CA was observed to be significantly reduced compared to the control group (p < 0. 003). The viability SSCs that received CA and underwent the slow freezing treatment was significantly reduced compared to controls (p < 0. 002). The expression levels of BAX, BCL-2, and Fas in the rapid freeze-thaw group didn’ t significantly change. However, the levels of P53 expression were shown to increase. In the group of SSCs that underwent the slow freezing process, the BAX gene expression levels increased, while the levels of BCL-2 gene expression decreased. No significant changes in the level of Fas and P53 expression were detected. When comparing the groups that received CA treatment, SSCs that were rapidly frozen showed an up-regulation of Fas and P53 expression and a down-regulation of Bcl-2 and Bax expression. Conclusions: Caffeic acid may protect intact SCCs during the cryopreservation process through stimulating the induction of apoptosis in injured SSCs. Supplementing the vitrification media with CA has a superior effect on the preservation of SSCs.

Background: Pre-eclampsia (PE) is a pregnancy disorder characterized by hypertension and proteinuria. The evidence has suggested that microRNAs (miRs) are associated with pre-eclampsia pathogenesis; however, these results are inconsistent. The aim of this study was to assess the association between miR-210 expression and PE risk. Methods: Previous studies were selected using PubMed, Scopus, MEDLINE, EMBASE, Science Direct, Google Scholar, Directory of Open Access Journals (DOAJ), and Scientific Information Database (SID). This metaanalysis includes 12 studies associated with miR-210 and pre-eclampsia and necessary information was extracted. Results: The standardized mean differences [(SMD (0. 32) 95% CI (014– 0. 49), p=0. 97] and heterogeneity were determined with the chi-square test (Q=3. 63 df =11 p= 0. 97), which found no heterogeneity between these studies. Additionally, publication bias was evaluated by Egger’ s and Begg´ s tests. Visual inspection of the funnel plot graphically, and statistically with Egger’ s weighted regression [(p= 0. 35) (95% CI-0. 90 – 2. 29)] and Begg’ s rank correlation (p= 0. 21), found no important publication bias between studies within the meta-analysis. Conclusions: Our findings suggest that miR-210 contributes to the pathogenesis of PE; therefore, miR-210 could serve as a novel biomarker to predict PE pathophysiology. Further studies are required in this field to characterize the mechanism involved in this process.

Background: Triple-negative breast cancer (TNBC) is treated with highly aggressive non-targeted chemotherapies. Safer and more effective therapeutic approaches than those currently in use are needed. Natural pomegranate peel extract (PPE) has recently been found to inhibit breast cancer progression; however, its mechanisms of action remain unclear. We hypothesized that transcriptional changes in the genes encoding the adherence proteins of intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF), may explain, at least in part, the anti-metastatic properties of PPE. Recently, the tumor microenvironment has been recognized as a key contributor to cancer progression. We speculated that PPE acts by modulating matrix glycoproteins including MMP9 and fibronectin. Moreover, we hypothesized that VEGF, which is required for tumor development, may contribute to the antimetastatic effects of PPE. Methods: To address these possibilities, MDA-MB-231 cells were treated with different doses of PPE at different time points. Apoptosis was detected by flow cytometry using annexin V and propidium iodide. Cell migration was detected with a transwell assay. Gene expression changes were analyzed by real-time PCR. Results: Exposure to PPE resulted in TNBC cell death and markedly inhibited PPE-resistant cell migration. Moreover, PPE up-regulated the expression of ICAM-1, a protein essential for cell adhesion, and down-regulated the expression of MMP9, fibronectin, and VEGF, the products of which contribute to cancer cell migration. Conclusions: Transcriptional changes in ICAM-1, MMP9, fibronectin, and VEGF may contribute to PPEmediated antimetastatic effects in TNBC.

Background: Arsenic is a well-documented human carcinogen widely distributed in the environment. Chronic exposure of humans to inorganic arsenicals causes many adverse health effects. The present work was conducted to evaluate the protective effect of Syzygium cumini seed extract (SCE) on arsenic-induced genotoxicity and hepatotoxicity in Wistar albino rats. Methods: Rats were randomly divided into five groups of six animals each. Group 1 served as normal control, Group 2 received SCE, 200 mg/kg daily, and Group 3 received arsenic, 100 ppm in drinking water. Groups 4 and 5 received SCE, 200 mg/kg and 400 mg/kg, respectively, daily, simultaneously with 100 ppm arsenic in drinking water. After 60 days, blood samples were collected and comet assay was performed using isolated lymphocytes. Activities of serum marker enzymes were assayed and lipid peroxidation (LPO) levels were estimated. Serum catalase (CAT) and superoxide dismutase (SOD) activities, and blood reduced glutathione (GSH) were measured. Results: Exposure to arsenic caused a significant increase in serum aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and bilirubin, accompanied by a decrease in total protein levels as well as CAT and SOD activities, and GSH. Enhanced LPO and lymphocyte DNA damage was also observed in arsenic-administered rats. The arsenic-induced toxicity was significantly reversed by the simultaneous administration of SCE at both the lower and higher dosages. Conclusions: This investigation offers strong evidence for the hepato-protective and antioxidative effects of SCE against arsenic-induced oxidative stress.