Correlation between ELF–PEMF exposure and Human RPE Cell Proliferation, Apoptosis and Gene Expression

Purpose: Emerging evidence implies that electromagnetic fields (EMFs) can negatively affect angiogenesis. In this regard, the effects of extremely low frequency pulsed electromagnetic field (ELF–PEMF) exposure on the relative expression level of angiogenic factors involved in the pathogenesis of ocular disorders were evaluated in human retinal pigment epithelial (hRPE) cells in order to investigate a noninvasive therapeutic method for patients with several ocular diseases associated with neovascularization. Methods: After separating hRPE cells from globes, hRPE cells were exposed to 15 mT of ELF–PEMF (120 Hz) at 5, 10, and 15 min for seven days. Cell proliferation and apoptosis of treated cells were evaluated via ELISA assay. Moreover, relative expression changes of HIF-1α, CTGF, VEGFA, MMP-2, cathepsin D, and E2F3 were performed using real-time RT-PCR. Results: ELF–PEMF exposure had no significant effects on the apoptosis and proliferation rate of hRPE cells. Expression level of HIF-1α, CTGF, VEGFA, MMP- 2, cathepsin D, and E2F3 was downregulated following 5 min of ELF–PEMF exposure. Conclusion: As ELF–PEMF showed inhibitory effects on the expression of angiogenic genes in hRPE cells with no cytotoxic or proliferative side effects, it can be introduced as a useful procedure for managing angiogenesis induced by retinal pathogenesis, although more studies with adequate follow-up in animal models are needed

Molecular Cloning of DARPins G3 in pET28b Expression Vector and Optimization of the Expression of This Protein in Escherichia Coli

Background: Human epidermal growth factor receptor 2 (HER2) is over-expressed in breast, ovarian, gastric, and prostate cancers and is used as a tumor marker in the diagnosis of cancer. Monoclonal antibodies have been used as a diagnostic and therapeutic tool against HER2. Because of the difficulties associated with the stability and complexity of the construct and the high cost of antibody production, we aimed to investigate, cloning, and expression of HER2- binding DARPins genes to identify, HER2-positive tumor markers, we aimed to investigate. Materials and Methods: After synthesis, the DARPins peptide gene was cloned into the M13 vector and sub-cloned into the TOP10 pet28b bacterial vector. After culturing the bacteria on an agarose plate containing antibiotics, the transfected bacteria expressing the DARPins gene were selected. To ensure gene cloning, we used enzymatic digestion and recombinant plasmid delivery for sequencing. Isopropyl β-d-1-thiogalactopyranosideIPTG was used for the induction of recombinant protein expression and the SDS-PAGE method and Western blot for expression confirmation. Results: The polymerase chain reaction (PCR) amplification product of DARPins was analyzed using agarose gel electrophoresis. Plasmid was purified from the positive clone by PCR cloning, sequenced and gene cloning was confirmed. After culturing from competent cells, protein expression was obtained from positive colonies. SDS- PAGE results showed the effect of different conditions including temperature, IPTG concentration, and time on the pET-DARPins expression. Conclusion: We were succeeded to express a new codon-optimized DARPins gene in Escherichia coli and HEK293t system

The hormonal milieu in primary breast cancer: A correlation between steroid receptors and serum estradiol, progesterone and prolactin

Background: The female breast is subjected to a lifetime of hormonal controls, whose effect is evident at the time of menarche and during the menstrual cycle, pregnancy and lactation. Studies have reported multiple risk factors for breast cancer, some of which are a reflection of hormonally mediated events. The steroid receptors are also served as prognostic factors for evaluating status of malignant tumor of the breast. Estrogen receptor (ER) and progesterone receptor (PgR) formation are influenced by Estrogen and progesterone concentration. The aim of this study was to clarify the hormonal milieu of the breast cancer such as Estradiol (E2), Progesterone (Pg), Prolactin (PRL) and their correlation with prognostic factors like ER, PgR. Materials and Methods: In this study we examined fourthy four samples removed from patients with primary breast cancer by radical mastectomy. The specimens include thirty six malignant and eight benign breast tissues. Blood samples were also obtained before surgery. steroid receptors was assayed by the method of single-point dextrane-coated charcoal (DCC), hormones by radioimmunoassay. Results: The results demonstrated that serum prolactin was higher in patients with benign and malignant tumors than that of normal (progesterone1.5 ng/ml and those of <1.5 ng/ml, it was found that frequency of PgR+ tumors was lower in the formers than the latters. Conclusion: It can be concluded that estrogen, up-regulated and progesterone down-requlate the induction of PgR and the breast tumors probably produce high levels of prolactin

Increased oxidized-LDL levels and arylesterase activity/HDL ratio in ESRD patients treated with hemodialysis

Purpose: Investigations, in which oxidized-low density lipoprotein (ox-LDL), serum paraoxonase (PON1) and homocysteine (Hcy) are considered together as important agents involved in the development of oxidative and atherogenic events in non-diabetic hemodialysis (HD) population, are limited. This case-control study was designed to evaluate these parameters in the patients and control subjects and to determine the correlations among the factors. Methods: Forty-nine age- and sex- matched subjects, including 28 non-diabetic HD patients (paired pre-and post-dialysis samples) and 21 control subjects, were enrolled. Ox-LDL and Hcy levels were measured with ELISA and EIA methods, respectively. Arylesterase activity of PON1 was measured by spectrophotometric assay. Results: Compared with the control group, ox-LDL levels were significantly increased both before (p=0.001) and after HD (p=0.036). Arylesterase activity-to-HDL ratio in HD patients was significantly higher than control subjects (p=0.003). Homocysteine levels in the ESRD patients were higher than control subjects both in pre-dialysis and post-dialysis. There was a significant positive correlation (r= 0.25, p= 0.026) between ox-LDL and homocysteine in samples obtained before HD. Logistic regression analysis revealed ox-LDL levels (OR=3.02, p < 0.001) and arylesterase activity/HDL ratio (OR=2.43, p=0.01) to be associated with the increased risk of ESRD. Conclusions: Ox-LDL levels and arylesterase activity/HDL ratio indicated the strongest association with ESRD risk. These factors, especially ox-LDL as an indicator of oxidative stress, may be biomarkers in evaluating the status of non-diabetic ESRD patients. Because of the pathogenic relationship between ox-LDL and homocysteine as nontraditional risk factors of atherosclerosis, therapeutic strategies adopted to reduce them may be useful in decrease of high prevalence of cardiovascular mortality in dialysis patients. In addition, measurement of PON1 activity to HDL ratio is possibly a more valuable biomarker than arylesterase activity alone in non-diabetic ESRD

Association of Uric Acid with Antioxidant Capacity of Plasma in Patients with Type 2 Diabetic Nephropathy

Background: The role of attenuation in antioxidant capacity or expansion of pro-oxidant/oxidant is well known for the development of stress oxidative. The aim of this study was to evaluate the plasma antioxidant capacity along with uric acid in patients with diabetic nephropathy (DN+) and without diabetic nephropathy (DN-). Materials and Methods: The research population included 88 patients with DN, 66 patients without DN and 54 healthy people who were matched for age, gender, and body mass index (BMI). In all groups, total antioxidant capacity of plasma by the ferric reducing antioxidant power (FRAP) assay and serum uric acid by commercial kit, respectively. Results: The mean age of patients in DN+, DN- and control groups were 59.3&plusmn; 9.4, 60 &plusmn;11.2, and 54.6&plusmn;6.9 years, respectively. Plasma antioxidant capacity was higher in patients with DN+, (1589&plusmn;330&mu mol/l Fe2+)&nbsp; and DN- (1344&plusmn; 347 &mu mol/l Fe2+) than that in healthy controls (1187&plusmn;271 &mu mol/l Fe2+ ) (P<0.001). The mean plasma uric acid in patients with DN+ was significantly higher (8.7&plusmn;1.3 mg/dl) compared with DN- (7.3&plusmn;1.2 mg/dl) (P<0.01), and significantly lower in control group (4.1&plusmn;1.4 mg/dl) (P<0.001). Conclusion: According to our results, despite innate antioxidant activity of uric acid and increase of total antioxidant capacity and concentration of uric acid in diabetic patients with or without nephropathy, it cannot compensates the severity of oxidative stress. Further studies are required to determine the value of other antioxidant factors in increasing the power of total antioxidant capacity

Relationship between the clinical scoring and demyelination in central nervous system with total antioxidant capacity of plasma during experimental autoimmune encephalomyelitis development in mice

Experimental autoimmune encephalomyelitis (EAE) was induced in a mouse model (C57/BL6) to investigate the antioxidant status of animals at various clinical stages of the disease. For this purpose, blood, brain and spinal cord samples from EAE mice were collected and examined at different scores following post-immunization with myelin oligodendrocyte glycoprotein (MOG). The clinical sign of mobility of animals on different days was associated with gradual increase in lipid peroxidation products (malondialdehyde, i.e. NIDA) in brain and spinal cord. Changes in lipid peroxidation during EAE progression was inversely related to superoxide dismutase (SOD) activity in erythrocyte preparation. However, suppression of catalase in erythrocytes, tissue glutathione (GSH) and plasma total antioxidant capacity (FRAP assay) were the early events in EAE, occurred during scores 1 and 2. Biochemical alterations were corroborated with histopathological observations showing demyelination and inflammatory foci in central nervous system (CNS) of animals suffering from partial hind limb paralysis (score 3). These data suggest that generation of NIDA in CNS is a continuous process during EAE induction and suppression of antioxidant factors are early events of the disease, but crucial in increasing the vulnerability of CNS to demyelinating lesions

Administration of zinc against arsenic-induced nephrotoxicity during gestation and lactation in rat model

Background: Free radicals production by toxicity of arsenic (Ar) is most important in the nephrotoxicity. There is accumulating evidence that zinc (Zn), has anti-oxidant properties. Objectives: The aim of present study was to evaluate protective and ameliorative effects of Zn against Ar-induced nephrotoxicity in rat pups during gestation and lactation. Materials and Methods: Twenty-four adult pregnant wistar rats were randomly divided into four groups (n = 6). Group one was given vehicle only. Group two received Zn (ZnSO4) at 20 mg/kg/d. Group three received Ar at 5 mg/kg/d as sodium meta-arsenite. Group four received Ar + Zn at the same dose that mentioned in groups of two and three. At the end of the study, 24 hours after the last treatment, samples were killed with overdose of sodium pentobarbital and kidneys were harvested for measuring malondialdehyde (MDA), glutathione (GSH) and histopathological assessment. Results: The MDA level in kidney was increased in the Ar group, which was decreased after Zn administration in the Ar + Zn group. The GSH level in kidney was decreased in the Ar group, which were increased after Zn administration in the Ar + Zn group. Also, the histopathological changes which were detected in the Ar group attenuated after Zn consumption. Conclusions: Our findings suggested that administration of Zn during gestation and lactation could have protective and prevent effect in Ar-induced oxidative stress in kidney tissue

Alleviation of cisplatin‐induced hepatotoxicity by gliclazide: Involvement of oxidative stress and caspase‐3 activity

Abstract Aims Cisplatin (CP), as an effective alkylating agent, is widely used in cancer treatment, while hepatotoxicity is one of its side effects. Gliclazide (GLZ), as an oral hypoglycemic drug, has antioxidant and anti‐inflammatory properties. This study was designed to investigate the protective effect of GLZ against CP‐induced hepatotoxicity in mice. Methods In this experimental study, 64 adult male mice randomly were allocated into eight groups (8 mice/group). Control, GLZ (5, 10, and 25 mg/kg, orally), CP (10 mg/kg, single dose, intraperitoneally), and CP+GLZ (in three doses). GLZ was administrated for 10 consecutive days. CP was injected on the 7th day of the study. At the end of the experiment, hepatotoxicity was evaluated by serum and tissue biochemical, histopathological, and immunohistochemical assessments. Results The data were revealed that CP increased oxidative stress (increased MDA and reduced GSH), liver damage enzymes (ALT, AST, and ALP), and immunoreactivity of caspase‐3 in liver tissue of CP‐injected mice. Also, CP induced histopathological changes such as eosinophilic of hepatocytes, dilatation of sinusoids, congestion, and proliferation of Kupffer cells. GLZ administration significantly ameliorated serum functional enzyme and hepatic oxidative stress markers in CP‐injected mice. In addition, the histological and immunohistochemical alterations were ameliorated in GLZ‐treated mice. Of the three doses, 10 and 25 mg/kg were more effective. Conclusions In conclusion, GLZ with its antioxidant, anti‐inflammatory, and anti‐apoptotic activities, can be suggested as a promising drug in the treatment of CP‐induced hepatotoxicity

A novel anti-CD22 scFv–apoptin fusion protein induces apoptosis in malignant B-cells

Abstract CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv–apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv–apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies