Acetoacetate enhancement of glucose mediated DNA glycation.

Acetoacetate (AA) is a ketone body, which generates reactive oxygen species (ROS). ROS production is impacted by the formation of covalent bonds between amino groups of biomacromolecules and reducing sugars (glycation). Glycation can damage DNA by causing strand breaks, mutations, and changes in gene expression. DNA damage could contribute to the pathogenesis of various diseases, including neurological disorders, complications of diabetes, and aging. Here we studied the enhancement of glucose-mediated DNA glycation by AA for the first time. The effect of AA on the structural changes, Amadori and advanced glycation end products (AGEs) formation of DNA incubated with glucose for 4 weeks were investigated using various techniques. These included UV-Vis, circular dichroism (CD) and fluorescence spectroscopy, and agarose gel electrophoresis. The results of UV-Vis and fluorescence spectroscopy confirmed that AA increased the DNA-AGE formation. The NBT test showed that AA also increased Amadori product formation of glycated DNA. Based on the CD and agarose gel electrophoresis results, the structural changes of glycated DNA was increased in the presence of AA. The chemiluminescence results indicated that AA increased ROS formation. Thus AA has an activator role in DNA glycation, which could enhance the adverse effects of glycation under high glucose conditions.

Antiamyloidogenic Effects of Ellagic Acid on Human Serum Albumin Fibril Formation Induced by Potassium Sorbate and Glucose.

Oxidative stress has the main role in protein conformational changes and consequent direct involvement in different kind of diseases. Potassium sorbate as a widespread industrial preservative and glucose are two important oxidants that can be involved in oxidative stress. In this study the effect of ellagic acid as a phenolic antioxidant on amyloid fibril formation of human serum albumin upon incubation of potassium sorbate and glucose was studied using thioflavin T assay, surface tension, atomic force microscopy, Amadori product, and carbonyl content assays. The thioflavin T assay and atomic force microscopy micrographs demonstrated the antiamyloidogenic effect of ellagic acid on the human serum albumin fibril formation. This antioxidant also had the repair effect on surface tension of the modified human serum albumin (amyloid intermediates), which was destructed, caused by potassium sorbate and glucose. This mechanism takes place because of potent carbonyl stress suppression effect of ellagic acid, which was strengthening by potassium sorbate in the presence and absence of glucose.

Inhibition of fluorescent advanced glycation end products (AGEs) of human serum albumin upon incubation with 3-ß-hydroxybutyrate.

Advanced glycation end products (AGEs), which are the final products of glycation, have a major role in diabetic complication and neurodegenerative disorders. The 3-ß-hydroxybutyrate (3BHB), a ketone body which is produced by the liver, can be detected in increased concentrations in individuals post fasting and prolonged exercises and in diabetic (type I) patients. In this study, the inhibitory effect of 3BHB on AGEs formation by glucose from the human serum albumin (HSA) was studied at physiological conditions after 35 days of incubation, using physical techniques such as circular dichroism and fluorescence spectroscopy, as well as differential scanning calorimetry (DSC). The fluorescence intensity measurements of glycated HSA by glucose (GHSA) in the presence of 3BHB indicate a decrease in AGEs formation. The DSC deconvolution profile results also confirm the protective role of 3BHB on incubated with glucose by preventing the enthalpy reduction of the HSA tail segment, compared with the deconvolution profile seen for incubated with glucose alone. The concentration of 3BHB used in this study is in accordance with the concentration detected in the body of individuals post fasting and prolonged exercises.

Energetic domains and conformational analysis of human serum albumin upon co-incubation with sodium benzoate and glucose.

Sodium benzoate (SB), a powerful inhibitor of microbial growth, is one of the most commonly used food preservative. Here, we determined the effects of SB on human serum albumin (HSA) structure in the presence or absence of glucose after 35 days of incubation under physiological conditions. The biochemical, biophysical, and molecular approaches including free amine content assay (TNBSA assay), fluorescence, and circular dichroism spectroscopy (CD), differential scanning calorimetry (DSC), and molecular docking and LIGPLOT studies were utilized for structural studies. The TNBSA results indicated that SB has the ability to bind Lys residues in HSA through covalent bonds. The docking and LIGPLOT studies also determined another specific site via hydrophobic interactions. The CD results showed more structural helicity for HSA incubated with SB, while HSA incubated with glucose had the least, and HSA incubated with glucose + SB had medium helicity. Fluorescence spectrophotometry results demonstrated partial unfolding of HSA incubated with SB in the presence or absence of glucose, while maximum partial unfolding was observed in HSA incubated with glucose. These results were confirmed by DSC and its deconvoluted thermograms. The DSC results also showed significant changes in HSA energetic structural domains due to HSA incubation with SB in the presence or absence of glucose. Together, our studies showed the formation of three different intermediates and indicate that biomolecular investigation are effective in providing new insight into safety determinations especially in health-related conditions including diabetes.

Potassium sorbate as an AGE activator for human serum albumin in the presence and absence of glucose.

Advanced glycation end products (AGEs) are the predominant intermediates of glycation process, and mediate oxidative stress and complications of diabetes. Potassium sorbate (PS) as a widespread preservative is an oxidative agent and used in different dairy and drug products, which can readily enter biological matrices. Here we studied the PS interference with glycation of human serum albumin (HSA) in the presence of glucose (Glc) using various techniques. These included TNBSA assay, circular dichroism, fluorescence spectroscopy, differential scanning calorimetry (DSC), Th T assay, and atomic force microscopy. Our results indicated that HSA glycation was accelerated in the presence of PS. Furthermore, PS produced AGEs in the absence of glucose. Secondary and tertiary structural changes were also observed in HSA incubated with glucose in the presence or absence of PS through beta-sheet inducing effects. Th T assay demonstrated the role of PS in HSA fibril formation in the presence or absence of glucose. Atomic force microscopy determined different amyloid fibril formation in HSA incubated with PS in the presence or absence of glucose. Together our results indicated that PS has a stimulatory effect on glycation and fibrillation of HSA in the presence or absence of glucose, and could exacerbate complication of diabetes.

Investigation of thermal reversibility and stability of glycated human serum albumin.

Protein glycation, the process by which carbohydrates attach to proteins upon covalent binding, can alter protein thermal reversibility and stability. Protein stability and reversibility have important role in protein behavior and function. Also they are benefit properties for drug produce and protein industrial applications. In this research the thermal reversibility and stability changes in human serum albumin (HSA) were studied upon incubation with glucose (GHSA) under physiological conditions for 21 and 35 days. The thermal reversibility and stability changes in GHSA were evaluated using circular dichroism (CD), UV-vis spectroscopy, fluorescence spectroscopy and differential scanning calorimetry (DSC). Our results showed that the glycation of HSA increased its thermal reversibility and stability, but decreased its conformational entropy compared to fresh native HSA and untreated HSA. Free lysine content assay (TNBSA test) indicated glucose can bind to protein covalently. These alterations were mainly attributed to the formation of crosslink between the lysine residues of HSA upon incubation with glucose.

Thermal inactivation and conformational lock studies on horse liver alcohol dehydrogenase: structural mechanism.

Horse liver alcohol dehydrogenase (HLADH) is a two subunits metal enzyme that has two catalytic sites and two coenzyme domains for each subunit. These subunits are connected together by coenzyme domains. In this study, we investigated the number and sequences of residues that participated in interface locks of HLADH. For this purpose, the kinetics of thermal inactivation of HLADH were studied in a 50 mM pyrophosphate buffer, pH 8.8, using ethanol as a substrate and NAD(+) as a cofactor. The temperature range was between 46°C and 55°C and the conformational lock was developed based on the Poltorak theory and analysis of the curves was done by the conformational lock method for oligomeric enzymes. The conformational lock number of HLADH was 2 when calculated experimentally. The results were confirmed by the Ligplot program computations. Using computational method it was shown that there are two patches binding sites at the interface and they spread over two regions of each chain. In this study we also proposed a thermal denaturation mechanism for HLADH by using different techniques such as UV-Vis fluorescence and circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). The subunits are dissociated and several intermediates appeared during inactivation through increasing the temperature. DLS measurement was performed to study the changes in hydrodynamic radius during thermal inactivation. The three distinct zones that were shown by DLS were also confirmed by fluorescence and CD techniques.

Thermodynamics of a molten globule state of human serum albumin by 3-ß-hydroxybutyrate as a ketone body.

The molten globule (MG) state is an intermediate which is considered as the third thermodynamic state of protein molecules. In this work the effect of incubating human serum albumin (HSA) at physiological condition in the presence of 3-ß-hydroxybutyrate for 7, 14, 21 and 35 days were studied by different techniques such as UV/vis, fluorescence and circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) and dynamic light scattering (DLS). In this paper, we introduce the MG state for HSA upon 21 days incubation with 3-ß-hydroxybutyrate as a ketone body at physiological condition. The results from the HSA sample incubated for 21 days shows a similar secondary structure by CD, more surface hydrophobicity and a little change on tertiary structure by fluorescence, and a larger size by DLS as compared to the native sample or other incubated samples. These results were also confirmed by calculated parameters and DSC deconvoluted thermograms.

Gastric cancer prevalence, according to survival data in iran (national study-2007).

BACKGROUND: Gastric cancer is a common and lethal disease throughout the world. In Iran with 7300 new cases annually, it is the first cause of cancer related death in both sexes. Regarding the high incidence (10.5/100000 individuals) of gastric cancer and priority of prevalence index in cancer management, in this study we tried to determine 1, 2-3 and 4-5 year point prevalence of the disease according to survival data. METHOD: Survival and incidence data were used for determination of cancer prevalence. Incidence data were extracted from cancer registry in Iran and survival data were determined in a descriptive study by following up 3439 gastric cancer patients in Cancer Research Center (CRC). 1, 2-3 and 4-5 year prevalence was estimated from incidence rates in different years and the proportion of patients surviving 0.5, 1.5, 2.5, 3.5 and 4.5 years from the time of diagnosis. RESULTS: patients with survival of 0.5, 1.5, 2.5, 3.5 and 4.5 years from the time of diagnosis were 46.38%, 26 %, 19.36%, 15.47% and 12.8% respectively. The prevalence of 1, 2-3 and 4-5 year was 3392, 3118and 1824 respectively. The cumulative 5 year prevalence was 8334 cases. CONCLUSION: These estimates of the point prevalence of gastric cancer at 1, 2-3 and 4-5 years are applicable to the evaluation of initial treatment, clinical follow-up, and point of cure. Therefore 1, 2-3 and 4-5 year point prevalence estimates, are necessary in health service planning for gastric cancer management and should be considered by public health managers.