Effect of Disulfide Bond Incorporation on the Structure and Activity of Endostatin Peptide.

The structure and function of a 27-a.a. fragment of the N-terminal sequence of human endostatin (ES-Zn) were compared to those of the mutant peptide (ES-SSZn) obtained by adding Cys-Pro-Ala to the endostatin N-terminus and substituting Asn16 for Cys ensuring formation of a disulfide bond. Structural comparison of ES-Zn and ES-SSZn by far-UV circular dichroism (CD), intrinsic fluorescence, and molecular dynamics simulation methods revealed significant structural perturbations in ES-SSZn, such as elimination of the ß-sheet conformer, modification of the N-terminal loop structure, and reorganization of dynamic properties of the entire peptide backbone. ES-SSZn was approximately 2 and 3 times less efficient than ES-Zn and the full-length human endostatin, respectively, in the induction of caspase-3-dependent apoptosis in human umbilical vein endothelial cells (HUVECs) in vitro (p < 0.05). In contrast, treatment of metastatic 4T1 breast tumors in mice with ES-Zn and ES-SSZn (5 mg/kg body weight daily) for 14 days resulted in similar regression of tumor size, comparable downregulation of angiogenesis (CD31 and CD34) and cell proliferation (Ki67), and therefore, the same extent of apoptosis induction (TUNEL, p53, and Bcl-2) for both peptides (as compared to the untreated controls). Western blot analysis of HUVEC and 4T1 tumor lysates revealed the same levels of suppression of key signaling mediators Akt and ERK1/2 by ES-Zn and ES-SSZn. Contrary to the earlier studies, our results showed that the function of the 1-27 endostatin fragment is independent of its overall structure. Stabilization of the N-terminal loop structure by the disulfide bond incorporation causes relief from structural deviations.

ASSOCIATION OF TESTOSTERONE WITH COLORECTAL CANCER (HT29), HUMAN GLIOBLASTOMA (A172) AND HUMAN EMBRYONIC KIDNEY (HEK293) CELLS PROLIFERATION.

Testosterone influences cancer development. This in vitro experiment was exerted to determine the association of testosterone with human colorectal cancer(HT29), glioblastoma (A172) and human embryonic kidney(HEK293) cells proliferation. HT-29, A172 and HEK293 cell lines were cultured in standard growth medium, then randomly divided into control group (not exposed to testosterone) and groups exposed to 1, 10, 100 and 1000 µg/mL of testosterone. Cell viability was quantified by MTT assay. Statistical analysis was performed using ANOVA. Viability of HEK293 cells significantly increased in groups exposed to 1 µg/mL and decreased in groups exposed to 100 and 1000 µg/mL of testosterone compared to control group (P<0.05, P<0.05 and P<0.001, respectively). Viability of HT29 cells significantly increased in groups exposed to 10 and 100 µg/mL of testosterone and significantly decreased when exposed to 1000 µg/mL of testosterone compared to control group (P<0.05, P<0.001 and P<0.001, respectively). Viability of A172 cells significantly decreased in groups exposed to 100 and 1000 µg/mL of testosterone compared to control group (P<0.001). In conclusion, different doses of testosterone have enhancing or suppressive effects on HEK293, HT29 and A172 cells proliferation; according to which, considering clinical use of testosterone therapy for cancer treatment is a highly controversial issue.

Comparative effects of thymol and vitamin E on nonalcoholic fatty liver disease in male Wistar rats.

Following the obesity epidemics, nonalcoholic fatty liver disease (NAFLD) has grown in prevalence and become a main cause of morbidity and death, intimately linked to cardiovascular disease, cancer, and cirrhosis. The key factor in the evolution of NAFLD is thought to be oxidative stress. Because most patients cannot change their lifestyle or dietary habits, a pharmaceutical strategy is now required to treat NAFLD. Nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis, is treated with vitamin E. (NASH). Vitamin E is also a powerful antioxidant that has been demonstrated to lower oxidative stress in people with NAFLD. Thymol is a monoterpene phenol with a variety of pharmacological effects, however its anti-fatty liver properties have yet to be investigated. Despite the fact that oxidative stress is thought to have a role in the etiology of nonalcoholic steatohepatitis, antioxidant therapies have not been well studied in the treatment of nonalcoholic steatohepatitis. The goal was to learn more about vitamin E and thymol's biological activities, with a particular emphasis on their therapeutic effectiveness in NAFLD. Four groups of thirty-two adult male rats were formed (healthy control, thymol, Vit E, and fatty liver). For 28 days, rats were given either oral vitamin E (200 mg/kg) or thymol (50 mg/kg) randomly. The levels of ALT, AST, TNF- α, Ferritin, CK-MB enzymes, and MAPK gene expression were then determined in the serum. Based on a random effect model analysis, at the end of 28 days of therapy, ALT (41.43 U/L), AST (47.91 U/L), Ferritin (1.13 pg/dl), CK-MB (251.22 IU/L), TNF-α (95.39 pg/mL) (p≤0.001), and MAPK gene expression levels (p≤0.05) significantly reduced in both experimental groups compared with the fatty liver group. Vitamin E and thymol therapy is a safe, affordable, and effective therapeutic option in the fatty liver group. Patients with fatty liver disease should be encouraged to take vitamin E and Thymol supplements, which are both safe and affordable, because more effective new therapeutic options are lacking.

Expression and characterization of Escherichia coli derived hepatitis C virus ARFP/F protein.

Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called "ARFP/F", or "core+1", protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11-161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the 1b-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.

Forced Suppression of let-7a-5p in Mouse Blastocysts Improves Implantation Rate.

Embryo implantation requires appropriate communication between the blastocyst and endometrium. Recurrent implantation failure is an essential component of assisted reproductive technology. Also, miRNA-mediated gene expression impacts the implantation process, and the downregulation of some miRs, such as mmu-let-7a, improves this process. In the present study, we evaluated the effect of let-7a forced suppression on the mouse implantation rate. In total, 100 adult female mice and 10 adult male mice were included (Strain CD-1). We analysed the expression of let-7a and its potential mRNAs targets (Igf1, Il1a, Itgb3 and Tgfb1) in control, sham and antagomir-treated blastocysts using quantitative reverse transcription PCR (qRT-PCR). The control and treated blastocysts were transferred to the 20 pseudopregnant mice so that the effect of let-7a suppression on the rate of implantation could be determined. The expression level of let-7a in the treatment group was significantly downregulated (P=0.001) In contrast, no significant expression changes were observed for let-7a or mRNAs targets when the sham and control groups were compared (P>0.05). In comparison to the controls, the antagomir-treated group exhibited significantly upregulated expression levels of Igf1 (0.0167), Itgb3 (0.045) and Tgfb1 (0.0115). Additionally, the implantation rate was significantly higher in the treatment group (78%) than the control group (61%) (P=0.0098). We found that forced suppression of mmu-let-7a-5p through successful transfection of Anti-miR leads to upregulation of downstream genes, Igf1, Itgb3 and Tgfb1, which directly involved in the trophoblast-endometrium attachment and improve the implantation rate.

Antidiabetic effect of Olea europaea L. in normal and diabetic rats.

The antidiabetic effect of an alcohol extract of olive (Olea europaea L.) leaves was investigated in normal and streptozotocin-induced diabetic rats. The oral administration of the olive leaves extract (0.1, 0.25 and 0.5 g/kg body wt) for 14 days significantly decreased the serum glucose, total cholesterol, triglycerides, urea, uric acid, creatinine, aspartate amino transferase (AST) and alanine amino transferase (ALT) while it increased the serum insulin in diabetic rats but not in normal rats (p < 0.05). A comparison was made between the action of olive leaves extract and glibenclamide (600 microg/kg), a known antidiabetic drug. The antidiabetic effect of the extract was more effective than that observed with glibenclamide.

Association between IRF1 Gene Expression and Liver Enzymes in HBV-infected Liver Transplant Recipients with and without Experience of Rejection.

BACKGROUND: Liver function indices and anti-viral immune regulatory markers can both improve graft outcomes, which lead to better post-transplantation management and increase the possibility of surveillance in liver transplant recipients with chronic hepatitis B virus (HBV) infection. OBJECTIVE: To determine the association between the interferon regulatory factor 1 (IRF1) mRNA levels and liver enzymes in HBV-infected liver transplant recipients with and without experience of rejection. METHODS: A total of 46 chronic HBV-infected patients who had undergone liver transplant surgery was divided into 2 groups of recipients "with rejection" and "without rejection.". Blood samples were collected form each patient on days 1, 4, and 7 post-transplantation. A SYBER GREEN real-time PCR was used to evaluate the expression level of IRF1 in liver recipients. Liver enzyme activities were also measured in all patients. RESULTS: The expression of IRF1 in the patients with rejection was up-regulated at all 3 follow-up days compared with those without rejection. The serum levels of ALT and AST were more than normal levels at 3 follow-up times in both study groups. Significant differences were found in IRF1 gene expression levels and also serum ALT levels between those with and without rejection after 7 days post-transplantation. CONCLUSION: The IRF1 expression and serum ALT levels were increased significantly in patient with rejection compared to those without rejection. IRF1, an inflammatory factor, may also intensify induction of inflammatory pathways in engrafted liver and promote liver inflammation and injuries leading to liver enzymes elevation in patients with graft rejection.

Antidiabetic effect of garlic (Allium sativum L.) in normal and streptozotocin-induced diabetic rats.

OBJECTIVE: The antidiabetic effect of garlic ethanolic extract (Allium sativum L.) was investigated in normal and streptozotocin-induced diabetic rats. RESEARCH METHODS AND PROCEDURE: In the present study, oral administration of garlic extract (0.1, 0.25 and 0.5 g/kg body wt.) for 14 days on the level of serum glucose, total cholesterol, triglycerides, urea, uric acid, creatinine, aspartate amino transferase (AST) and alanine amino transferase (ALT) in normal and streptozotocin-induced diabetic rats were evaluated. RESULTS: Oral administrations of the garlic extract significantly decreased serum glucose, total cholesterol, triglycerides, urea, uric acid, creatinine, AST and ALT levels, while increased serum insulin in diabetic rats but not in normal rats (p<0.05). A comparison was made between the action of garlic extract and glibenclamide (600 microg/kg), the known antidiabetic drug. The antidiabetic effect of the extract was more effective than that observed with glibenclamide. CONCLUSION: It is concluded that the plant must be considered as excellent candidate for future studies on diabetes mellitus.