Firoz Akhter

Glycoxidation of biological macromolecules: A critical approach to halt the menace of glycation

Glycation is the result of covalent bonding of a free amino group of biological macromolecules with a reducing sugar, which results in the formation of a Schiff base that undergoes rearrangement, dehydration and cyclization to form a more stable Amadori product. The final products of nonenzymatic glycation of biomacromolecules like DNA, proteins and lipids are known as advanced glycation end products (AGEs). AGEs may be generated rapidly or over long times stimulated by distinct triggering mechanisms, thereby accounting for their roles in multiple settings and disease states. Both Schiff base and Amadori glycation products generate free radicals resulting in decline of antioxidant defense mechanisms and can damage cellular organelles and enzymes. This critical review primarily focuses on the mechanistic insight of glycation and the most probable route for the formation of glycation products and their therapeutic interventions. Furthermore, the prevention of glycation reaction using therapeutic drugs such as metformin, pyridoxamine and aminoguanidine (AG) are discussed with special emphasis on the novel concept of the bioconjugation of these drugs like, AG with gold nanoparticles (GNPs). At or above 10 mM concentration, AG is found to be toxic and therefore has serious health concerns, and the study warrants doing this novel bioconjugation of AG with GNPs. This approach might increase the efficacy of the AG at a reduced concentration with low or no toxicity. Using the concept of synthesis of GNPs with abovementioned drugs, it is assumed that toxicity of various drugs which are used at high doses can be minimized more effectively.

Bio-physical characterization of ribose induced glycation: a mechanistic study on DNA perturbations.

The non-enzymatic addition of carbonyl group of sugar moiety to the amino group of DNA macromolecule leads to the formation of early glycation products (Amadori products) which undergoes rearrangement, cyclization and dehydration to form advanced glycation end products (AGEs). While the formation of glucose derived glycated DNA has been previously demonstrated, however no extensive studies have been performed to assess the glycation of DNA using D-ribose as glycating agent. D-ribose, an important monosaccharide, is a highly reactive pentose sugar which results in the rapid formation of AGEs. To the best of our knowledge, this is the first study to characterize the d-ribose-induced changes in calf thymus DNA, as well as calf thymus DNA Amadori and calf thymus DNA-AGEs. The main objective of the study is to investigate the non-enzymatic glycation of calf thymus DNA by using different concentrations of d-ribose at increasing time period. The obtained Amadori products, AGEs were characterized with respect to the extent of DNA strand break and base modifications. Additionally, their nitroblue tetrazolium (NBT) reduction assay, absorbance, agarose gel electrophoresis, fluorescence, circular dichroism (CD) and thermal denaturation (Tm) characteristics were extensively studied. We found significant changes in the modification of DNA and in AGE-specific fluorescence, using different concentration of modifiers (D-ribose). The results provide the mechanistic insight of D-ribose induced glycation in calf thymus DNA. The bio-availability of D-ribose makes this carbonyl species quite reactive and damaging, therefore having direct implication in diabetes. This is the preliminary study done on D-ribose glycation and warrants further study to probe the role ribose-DNA glycation in different disease state including diabetes.

Studies on glycation of human low density lipoprotein: A functional insight into physico-chemical analysis

Glycation of biomolecules leads to the establishment of advanced glycation end products (AGEs), which have significant role in the pathophysiology of diabetes leading to disorders, such as atherosclerosis. Ribose appears to be the most reactive among the naturally occurring sugars and contribute significantly to the glycation reactions in vivo, however, no report has been published to discuss D-ribose induced glycation of low density lipoprotein (LDL). The present study initially focuses on the glycation of commercially available LDL using different concentrations of D-ribose for varied incubation time intervals. The structural perturbations induced in LDL were analyzed by UV-vis, fluorescence & circular dichroism spectroscopy, and thermal denaturation studies. The ketoamine moieties, carbonyl content and HMF content were also quantitated in native and glycated LDL. We report structural perturbations, increased carbonyl content, ketoamine moieties and HMF content in D-ribose induced glycated LDL (LDL-AGEs) as compared to native analogue. These results provide evidence that LDL-AGEs could interfere in normal physiological functions and might contribute in the initiation of diabetes induced atherosclerosis and other secondary cardiovascular problems in diabetic patients. Though ours is a preliminary study, we are sure it would open the topic for further studies.

Immunogenicity of DNA-advanced glycation end product fashioned through glyoxal and arginine in the presence of Fe3+: Its potential role in prompt recognition of diabetes mellitus auto-antibodies

Glyoxal, methylglyoxal and 3-deoxyglucosones are reactive dicarbonyl compounds, which transform free amino groups of proteins and lipoproteins macromolecule into advanced glycation end-products (AGEs). AGEs play a significant role in the pathophysiology of aging and diabetic complications because of their genotoxic effect. Glyoxal also reacts with free amino group of nucleic acids resulting in the formation of DNA-AGEs. The present study reports the genotoxicity and immunogenicity of AGEs formed by Glyoxal-Arginine-Fe(3+) (G-Arg-Fe(3+)) system as a glycating agent. Immunogenicity of native and G-Arg-Fe(3+)-DNA was probed in female rabbits. Immunofluorescence suggests the presence of immune complex deposition in the kidney section of immunized rabbits. Spectroscopic analysis and melting temperature indicates the structural modification in the human DNA. The modified human DNA is found to be highly immunogenic, whereas unmodified form was simply non-immunogenic. This study shows the presence of auto-antibodies against G-Arg-Fe(3+) modified human DNA in the sera of diabetes type 1 and in few cases type 2 patients due to secondary complications of nephropathy. The glyco-oxidative lesions have also been detected in the lymphocyte DNA isolated from patients having type 1 and type 2 diabetes. The results show structural perturbations generating new epitopes in G-Arg-Fe(3+)-DNA rendering it pretty immunogenic.

An Immunohistochemical Analysis to Validate the Rationale behind the Enhanced Immunogenicity of D-Ribosylated Low Density Lipo-Protein

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease in patients with diabetes and renal failure. D-ribose is one of the naturally occurring pentose monosaccharide present in all living cells and is a key component of numerous biomolecules involved in many important metabolic pathways. Formation of D-ribose derived glycated low density lipoprotein (LDL) has been previously demonstrated but no studies have been performed to assess the immune complex deposition in the kidney of rabbits immunized with glycated LDL. In this study, LDL was glycated with D-ribose, and it was further used as an immunogen for immunizing NZW female rabbits. The results showed that female rabbits immunized with D-ribose modified LDL induced antibodies as detected by direct binding and competitive ELISA. The modified LDL was found to be highly immunogenic eliciting high titer immunogen-specific antibodies, while the native forms were moderately immunogenic. The induced antibodies from modified LDL exhibited wide range of heterogeneity in recognizing various proteins and amino acids conformers. Furthermore, our histopathological results illustrated the deposits of immune complex in glomerular basement membrane in rabbits immunized with D-ribose-LDL.

Chemotherapeutic potential of Boerhaavia diffusa Linn: A review.

Boerhaavia diffusa Linn. has been shown to exhibit a wide range of medicinal properties for the treatment of diabetes, inflammation, stress, hepatotoxicity, jaundice and heart failure. The extraordinary antioxidant, hepatoprotective, antibiotic, antidiabetic and anticarcinogenic properties of B. diffusa have attracted pioneers in the field of science and medicine. Moreover, the therapeutic importance of this plant, which is due to presence of polyphenols and flavanoids, makes this plant medically more important to be exploited by clinicians and scientists to gain more insight into its biological and medicinal properties. The present review on B. diffusa focuses over the chemical compositions and its ethno-medicinal uses, linked from ancient times to the present with a scope of development in future. Furthermore, a recent update on mechanistic approaches of B. diffusa has also been discussed, which could be helpful for the researchers working in this field. Eventually, based on its antioxidant and antidiabetic characteristics, it is hypothesized that B. diffusa might exhibit antiglycating properties as well.

Acquired immunogenicity of calf thymus DNA and LDL modified by d-ribose: A comparative study

Glycation of biologically important macromolecules leads to the establishment of advanced glycation end products (AGEs) having significant role in the pathophysiology of various diseases. d-Ribose, is a highly reactive pentose sugar resulting in the rapid formation of AGEs. Formation of d-ribose derived glycated DNA and LDL has been previously demonstrated; however no comparative, extensive studies have been performed to assess the immunogenicity of d-ribose glycated calf thymus DNA (CT-DNA) and LDL. In the present study, the results showed that animals immunized with d-ribose modified CT-DNA and LDL induced antibodies as detected by direct binding and competition ELISA. The modified CT-DNA and LDL were found to be highly immunogenic, eliciting high titer immunogen-specific antibodies, while the native forms of DNA was almost non-immunogenic. The induced antibodies from modified CT-DNA and LDL exhibited wide range of heterogeneity in recognizing various nucleic acid conformers, DNA bases and amino acids. Furthermore, Serum antibodies from diabetes and diabetes atherosclerosis patients were screened for their binding to native CT-DNA, LDL and glycated CT-DNA, LDL. Glycated CT-DNA showed almost equivalent binding to both diabetes and diabetic atherosclerosis group while high recognition was observed when glycated LDL was used as an antigen.

Antigenic role of the adaptive immune response to d-ribose glycated LDL in diabetes, atherosclerosis and diabetes atherosclerotic patients.

AIMS Glycation of proteins leads to the formation of advanced glycation end products (AGEs, which have significant role in the pathophysiology of diabetes complications. d-ribose appears to be the most reactive among the naturally occurring sugars and contribute significantly to the generation of AGEs. Glycation also results in the generation of free radicals causing structural modification which leads to the generation of neoantigenic epitopes. The aim of the present study was to investigate whether LDL modification results in auto-antibodies formation against its glycated conformer in diabetes and atherosclerosis patients. METHODS The binding characteristics of circulating auto-antibodies in patients against native and modified LDL were assessed. T2D (n=105), ATH (n=106) and T2D-ATH patients (n=72) were examined by direct binding ELISA as well as inhibition ELISA, compared with healthy age-matched controls (n=50). Furthermore, ketoamine moieties, HMF and carbonyl content were also estimated in these patients and healthy subjects. KEY FINDINGS High degree of specific binding was observed by 41.91% of T2D, 54.72% of ATH and 70.83% T2D-ATH patients sera towards d-ribose glycated LDL, in comparison to its native analog (P<0.05). Normal human sera showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. The higher concentration of HMF, ketoamine and carbonyl content was observed in patients sera than healthy subjects. SIGNIFICANCE LDL glycation results in structural perturbation causing generation of neoantigenic epitopes that are better antigens for antibodies in T2D, ATH and T2D-ATH patients where T2D-ATH subjects showed higher prevalence in auto-antibodies against ribosylated LDL.

Detection of Circulating Auto-Antibodies Against Ribosylated-LDL in Diabetes Patients

This study analyzes effect of glycation on ApoB‐100 residues by D‐ribose as D‐ribosylated‐glycated LDL might be responsible for the cause of diabetes mellitus because of its far higher antigenic ability. The binding characteristics of circulating auto‐antibodies in type 1 and type 2 diabetes patients against native and modified LDL were assessed.

Determination of multiple antibiotic resistance patterns and indexing among metal tolerant -lactamase-producing Escherichia coli

The antibiotic resistance profiles of Escherichia coli isolated from three different sampling sites of the Gomti River at Lucknow city was evaluated. Water samples were collected and then analyzed for the presence of E. coli, using standard methods. Antibiotic susceptibility testing was performed by the disc diffusion method. Of the 77 E. coli isolates tested, marked antibiotic resistances (over 70%) were observed for amoxicillin, nitrofurozone, chloramphenicol, polymixin B, methicilin, ampicillin, nalidixic acid, cefpodoxime, erythromycin, penicillin, rifampicin and ofloxacin depending upon the sampling sites. All E. coli isolates also showed multiple resistance patterns in different combination of antibiotics. The MAR index ranges were found very high indicating the high risk of environmental contamination. The findings indicated that pollution of aquatic environments from different sources of the city may have a potential impact on the dissemination and survival of E. coli, as well as other pathogenic bacteria in the Gomti River water for public and animal health. This may result to a negative effect on antibiotic therapy for infectious diseases. Key words: Gomti River, coliforms, antibiotic susceptibility, multiple antibiotic resistance (M.A.R).