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Introduction: Previous studies have shown that the increase of carbamylated LDL (cLDL), a product of nonenzymatic modification of LDL in human serum by urea-derived cyanate, may cause cardiovascular complications in patients with chronic renal insufficiency. This study examined the inhibitory effect of ascorbic acid, α-tocopherol and lycopene on LDL carbamylation in an in vitro model system. Methods: After isolation of LDL from plasma using an ultracentrifuge technique, cyanate was added to it and then LDL carbamylation was measured in both the absence and presence of ascorbic acid, α-tocopherol and/or lycopene by the colorimetric method at 530 nm Results: The findings indicated that these vitamins inhibit LDL carbamylation and the most effective vitamin of the three is lycopene. Moreover, the effect of lycopene on this process increased in the presence of ascorbic acid and α-tocopherol. Conclusion: This study indicated that ascorbic acid, α-tocopherol and lycopene with antioxidant activity can probably inhibit LDL carbamylation and therefore may have a role in ameliorating atherosclerotic risk of patients with kidney failure. However in vitro and in vivo investigations are required to confirm the exact effects of these vitamins on patients suffering from uremic disorders. [ABSTRACT FROM AUTHOR]

Background: Medullary thyroid cancer (MTC) is a rare type of neuroendocrine tumor. This study aimed to investigate the gene and protein expression of RAP1GAP and DNA methylation patterns of its CpG74a, CpG74b, and CpG24 in an Iranian population with MTC. Methods: In this case-control study, we selected 55 individuals who underwent thyroidectomy in Erfan hospital, Tehran, between 2018 and 2020. Samples were divided into normal thyroid tissues (control; n = 20), benign nodule (n = 20), and MTC (n = 15). DNA methylation patterns were investigated using MSP (methylation-specific PCR). The protein level and mRNA expression of RAP1GAP were also evaluated using western blotting and real-time PCR, respectively. Results: The hyper-methylation rates of CpG24 and CpG74a in the MTC samples were considerably higher than the controls (83% versus 15% and 74% versus 17%, respectively; P < 0.001). The methylation/unmethylation ratio of CpG74a, and CpG24 was considerably higher than the controls (P < 0.001). The methylation/unmethylation ratio of CpG24 in the benign nodules was also considerably greater than the controls (P < 0.001). The mRNA expression and the protein level of RAP1GAP in the MTC group were considerably lower than the controls (P = 0.005 and P = 0.035, respectively). In the MTC group, aberrant methylation of CpG74a and CpG24 was significantly correlated with decreasing expression of the RaplGap gene (R2: 0.23; P = 0.032 and R²: 0.56; P = 0a.001, respectively). Conclusion: Hyper-methylation in CpG24 and CpG74a and decreasing expression of RAP1GAP can be considered as diagnostic biomarkers for MTC. [ABSTRACT FROM AUTHOR]

Vitamin D deficiency is now a recognised problem affecting multiple physiological functions. The aim of the present study was to evaluate the effect of a single dose of vitamin D3 injection on the inflammatory, muscular damage, metabolic and cardiovascular responses to an acute bout of resistance exercise (RE) in vitamin D-deficient resistance-trained males. Blood samples from fourteen vitamin D-deficient resistance-trained males were obtained during two separate trials: lower vitamin D (LVD) and higher vitamin D (HVD, after vitamin D3 injection). Metabolic, inflammatory, muscle damage and cardiovascular markers were evaluated at baseline, immediately and 1 h after RE. There were significant trial-by-time interactions for insulin and homeostatic model assessment of insulin resistance (HOMA-IR) which significantly (P < 0·05) declined for 1 h after RE in the HVD trial compared with the LVD trial. Homeostasis model assessment of β-cell function (HOMA-β) declines at 1 h post-RE in the HVD trial. There was also a time effect for blood sugar which significantly (P < 0·05) decreased and for creatine kinase, lactate dehydrogenase and IL-6 which increased significantly 1 h post-RE in both trials. There were no significant changes in other inflammatory and cardiovascular markers following both trials. A single injection of vitamin D3 improved insulin resistance and β-cell function following RE in previously vitamin D-deficient resistance-trained males. Conversely, the injection did not change muscle damage and the inflammatory response to acute RE. Intramuscular vitamin D replacement may have key implications for the promotion of glucose metabolism and lowering the risk of diabetes in vitamin D-deficient individuals.

Background: Expression of human epidermal growth factor receptor type 2 (HER2) in head and neck squamous cell carcinoma (HNSCC) cell line HN5 can be employed with great opportunities of success for specific targeting of anti-cancer chemotherapeutic agents., Objective: In the current study, HER2-specific affibody molecule, ZHER2:342 (an engineered protein with great affinity for HER2 receptors) was selected for conjugation to idarubicin (an anti-neoplastic antibiotic)., Method: ZHER2:342 affibody gene with one added cysteine code at the its 5' end was synthesized de novo and then inserted into pET302 plasmid and transferred to E. Coli BL21 hosting system. After induction of protein expression, the recombinant ZHER2 affibody molecules were purified using Ni- NTA resin and purity was analyzed through SDS-PAGE. Affinity-purified affibody molecules were conjugated to idarubicin through a heterobifunctional crosslinker, sulfosuccinimidyl 4-(Nmaleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC). Specific toxicity of idarubicin-ZHER2 affibody conjugate against two HER2-positive cells, HN5 and MCF-7 was assessed through MTT assay after an exposure time of 48 hours with different concentrations of conjugate., Results: Idarubicin in the non-conjugated form showed potent toxic effects against both cell lines, while HN5 cells were significantly more sensitive compared to MCF-7 cells. Dimeric ZHER2 affibody showed a mild decreasing effect on growth of both HN5 and MCF-7 cells at optimum concentration. Idarubicin-ZHER2 affibody conjugate at an optimum concentration reduced viability of HN5 cell line more efficiently compared to MCF-7 cell line., Conclusion: In conclusion, idarubicin-ZHER2 affibody conjugate in optimum concentrations can be used for specific targeting and killing of HN5 cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Objective: Calpains are a family of Ca(2+) dependent proteases. There is some evidence that calpains involved in fusion process that occurs between spermatozoa and the oocyte. The current study aimed to investigate the association of smoking with semen quality and µ-calpain level., Materials and Methods: This case-control study was conducted on 117 normospermia males between June 2013 and march 2014 in Jahad Laboratory in ahvaz, Iran. The semen samples were collected from male smokers (n = 50) and non-smokers (n = 67). We divided these participants as light, moderate, or heavy smokers based on their cigarettes per day (CPD). ELISA assays were used to measure µ-calpain concentration. All semen samples were analyzed according to World Health Organization guidelines., Results: The analysis of semen showed the volume, concentration, motility and morphology of semen were significantly lower among the smoker men than the non-smoker men. Also this significant difference was observed based on the number (light, moderate and heavy smokers) and duration (short term and long term smoker) of smoking. Although, showed no significant difference between µ-calpain of smoker men and non-smoker men. CPD showed negatively correlation with semen volume, concentration, motility and morphology of sperm., Conclusion: Sperm quality was negatively correlated with CPD and duration of smoking. However, there is no significant correlation between smoking and µ-calpain concentration.

Human epidermal growth factor receptor 2 (HER2) contributes to the development of breast cancers and malignancies. On the other hand, engineered affibody Z(HER2:342) that binds to HER2 can be successfully used for both diagnostic purposes and specific ablation of malignant HER2-positive cell lines. In the current study, electrostatics-based prediction was applied for improving Z(HER2:342) binding affinity using computational design. The affibody Z(HER2:342) alone and in complex with HER2 was energetically minimized, solvated in explicit water, and neutralized. After heating and equilibration steps, the system was studied by isothermal-isobaric (NPT) MD simulation. According to trajectories, Z(HER2:342) specifically binds to HER2 through hydrogen bonds and salt bridges. Based on the electrostatic binding contributions, two affinity-matured variants namely V1 (Tyr35Arg) and V2 (Asn6Asp and Met9Glu) were rationally designed. More investigations through MD simulation show that V1 interacts with HER2 receptor more strongly, compared to Z(HER2:342) and V2.

Background: The aim of the present study was to investigate the in vitro effects of mercury (Hg+2), lead (Pb+2), silver (Ag+2), tin (Sn+2), bismuth (Bi+3) and indium (In+3) ions on sperm creatine kinase., Methods: creatine kinase was isolated from human sperm homogenates after chromatography on a DEAE cellulose column., Results: At 60 mug ml-1 metal concentration, 70% of the creatine kinase activity was inhibited by Hg+2, while at the same concentration, Pb+2, Ag+2, Sn+2, Bi+3 and In+3 caused 68%, 66.5%, 65.7%, 64.7% and 62.7% inhibition, respectively. All six metal ions displayed a competitive type of inhibition mechanism for the isolated creatine kinase as analyzed by Lineweaver-Burk plot. Ki values of Hg+2, Pb+2, Ag+2, Sn+2, Bi+3 and In+3 were calculated and 8.34 mM, 5 mM, 4.54 mM, 3.45 mM, 3.12 mM and 2.63 mM values were obtained, respectively., Conclusion: All the studied metal ions, at levels of 60 mug ml-1, may reduce normal sperm metabolism by inhibition of sperm creatine kinase, which probably is an important cause of infertility in men. However, further investigations, as in vitro and in vivo, are needed to elucidate the exact mechanism of heavy metals on male reproductive functioning at the molecular level.

Background: This study examined the effect of grape seed proanthocyanidin extract (GSPE) on lipid peroxidation content and activity of tissue antioxidant enzymes, including catalase, superoxide dismutase and glutathione peroxidase in diabetic rats., Methods: Thirty male rats were divided into three groups of 10 rats each: control, diabetic and diabetic groups that received 500 mg/kg GSPE for 6 weeks. Diabetes was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg body weight). Rats with fasting blood glucose levels above 250 mg/dl were used as diabetic animals. The first 24-hour urinary albumin excretion (UAE) was measured two weeks after diabetes induction and then each week until the end of the experimental period in all groups. Lipid peroxidation content and activities of catalase, superoxide dismutase and glutathione peroxidase were measured in kidney homogenate supernatants. Statistical significance of differences was assessed with one-way ANOVA by SPSS followed by Tukey's t-test. P < 0.05 was assumed statistically significant., Results: UAE in diabetic nephropathy rats were significantly higher than in control. In addition, an increase in lipid peroxidation content and decrease in catalase, superoxide dismutase and glutathione peroxidase activities in kidney of diabetic nephropathy rats were observed. The GSPE administration did not affect on body weight, but significantly decreased lipid peroxidation and augmented the activities of antioxidant enzymes studied in kidney of diabetic nephropathy rats as well as reduced UAE and decreased kidney weight., Conclusion: The results suggested that GSPE could ameliorate diabetic nephropathy rats through reduction of oxidative stress and increase in renal antioxidant enzyme activity.

The non-enzymatic carbamylation of low density lipoprotein (LDL) is a naturally occurring chemical modification of apolipoprotein B as a result of condensation between lysine residues and cyanate derived from urea. Carbamylated LDL is poorly recognized by LDL receptors and initiates different processes that can be considered proatherogenic. Thus, LDL carbamylation may contribute to the increased risk of atherosclerosis in patients with kidney failure. The objective of this study was to investigate in vitro effects of flavonoids on LDL carbamylation. LDL was isolated from plasma using ultracentrifuge technique with a single step discontinuous gradient. Then, cyanate was added to LDL and LDL carbamylation level was estimated in absence and presence of flavonoids by a colorimetric method at 530 nm. The results of this study showed that a number of flavonoids including rutin, catechin, morin, myricetin, kaempferol, taxifolin, luteolin, naringin and quercetin decreased LDL carbamylation in a dose dependent manner. Also, it was demonstrated that these nutrients decreased electrophoretic mobility of carbamylated LDL. Based on the results obtained in this study, it is suggested that flavonoids are able to inhibit LDL carbamylation (probably by scavenging cyanate ions) and thus, may have a role in ameliorating atherosclerotic risk of patients with kidney failure.

We studied the effect of different concentrations of alpha-tocopherol on in vitro cupric ions induced oxidation of low density lipoproteins (LDL). Human native LDL (50 µg protein/ml) oxidation was induced by 10 µmol/L of CuSO4. Conjugated dienes were measured spectrophotometrically for up to 440 minutes. The length of the lag phase (Tlag), maximum velocity of the reaction (Vmax) and the maximum amount of generated dienes were obtained from kinetic data. Alpha-tocopherol increased Tlag and decreased Vmax with a dependence upon concentration (0-100 µmol/L). There was no difference between the Dmax obtained with cupric ions alone or in the presence of the various concentrations of alpha-tocopherol. The results suggest that alpha-tocopherol may decrease free radicals presence in LDL and thus decrease velocity of LDL oxidation by cupric ions. This mechanism may be a reason for alpha-tocopherol effect in ameliorating atherosclerosis.

Background: It is well established that oxidative modification of low density lipoprotein (LDL) plays a causal role in human atherogenesis and the risk of atherosclerosis is increased in patients with diabetes mellitus. We examined the in vitro effect of glucose on native and glycated LDL oxidation using copper ion dependent oxidation system., Methods: In this study, LDL was isolated from plasma by ultracentrifugation using a single step discontinuous gradient. Native LDL preparations were glycated by glucose and also were oxidized by copper ions. LDL glycation and oxidation levels were estimated by sodium periodate assay and thiobarbituric acid reactive substances (TBARS), respectively. Then, native LDL was incubated with glucose and copper and LDL oxidation was estimated by TBARS. Finally, oxidation of glycated LDL was studied in presence of copper ions by TBARS and relative electrophoretic mobility on polyacrylamide gel., Results: This study showed that glucose considerably decreased the oxidation of native LDL by copper ions. But oxidation of glycated LDL elevated with presence of copper ions., Conclusion: The results of this investigation show that LDL glycated in vitro is prone to oxidation by copper ions. Thus, promotion of glycated LDL oxidation by glucose is specific for copper ion dependent oxidation and involves increased copper ion reduction. These results provide one mechanism that may enhanced LDL oxidation in diabetes and thus contribute to the pathogenesis of atherosclerosis in diabetic patients.

Introduction: The non-enzymatic glycation of Low density lipoprotein (LDL) is a naturally occurring chemical modification of apolipoprotein B as a result of condensation between lysine residues and glucose. Glycated LDL is poorly recognized by LDL receptors and initiates different processes that can be considered proatherogenic. Thus, LDL glycation may contribute in the increased atherosclerotic risk of patients with diabetes. The objective of this study was to investigate the effect of naturally occurring flavonols on LDL glycation in vitro., Methods: In this study, LDL was isolated from EDTA-plasma by ultracentrifugation using a single step discontinuous gradient. Then, glucose was added to LDL and LDL glycation level was estimated in absence and presence of flavonols by sodium periodate assay., Results: This study was showed that five flavonols: quercetin, myricetin, kaempferol, rutin and morin decreased LDL glycation in a dose-dependent manner. Also, it was demonstrated this nutrients decreased electrophoretic mobility of glycated LDL., Conclusion: The results of this investigation show that flavonols probably with their antioxidant properties inhibited LDL glycation and thus may have a role in ameliorating atherosclerotic risk of patients with diabetes mellitus.