[show abstract][hide abstract] ABSTRACT: Glycation is a non-enzymatic process which involves an interaction between the carbonyl groups of reducing sugars and amino groups of proteins, lipids, nucleic acids resulting in the formation of Amadori products (early glycation products). These products then rearrange themselves and get converted to more stable and irreversible advanced end glycation products (AGEs). Glycation products have been implicated in various diseases like cataract, diabetes mellitus, Alzheimers etc. In the present workthe effect of glycation on DNA and proteins was studied. When plasmid DNA (pBR 322) was incubated with lysine andmethylglyoxal in the presence of metal ion a conformational change was observed on the agarose gel electrophoresis which indicated the damage to DNA by glycation. Different sugars were also incubated with plasmid DNA to check their comparative deleterious effects. In the presence of Lysine and metal ion, pentose sugar and sugar phosphates caused maximum damage to DNA as compared to other sugars. Some natural antioxidants like vitamin B derivatives,curcumin and 6 jasadbhasmawere used to check their effect and mechanism in reversing the glycation induced damage to DNA. SDS-PAGE was performed to analyze the glycation-induced damage to proteins and it was found that methyl glyoxal caused more damage than glucose. Curcumin and jasadbhasma prevented the damage to protein in the presence of methyl gloxal. It can be concluded from the results obtained in this study that the mechanism of damage due to glycation is different for different biomolecules. Free radicals were found to play important role in damaging DNA structure. On the hand aggregation and cross-linking by AGEs were found to be the major cause of structural alteration of proteins.
National Seminar on Biotechnology for Sustainable Growth and Development, CKT College Mumbai; 01/2014
[show abstract][hide abstract] ABSTRACT: Several approaches such as molecular genetics, functional genomics, recombinant DNA technology etc. have been used to elucidate the regulation of nitrate assimilation in plants and microbes. In the present study a combination of enzyme inducers and inhibitors were used to examine the modulation of nitrate reductase in the shoots of 12-days hydroponically (nutrient starved for 10-12 days) grown rice seedlings. First the effect of nitrate and light was checked on the activity of nitrate reductase (NR). Nitrate caused an increase in the NR activity by several fold in both light and dark Light augmented the effect of nitrate. However there was no change in the level of NR mRNA after withdrawal of light signals. Tungstate, an analog of molybdate, caused a severe decrease in the activity of nitrate-induced nitrate reductase even at a very low concentration (0.1mM). These results indicate that the nitrate reductase is regulated in a co-ordinated manner by light and nitrate in rice.
[show abstract][hide abstract] ABSTRACT: Spirulina is a photosynthetic, filamentous, spiral-shaped, multicellular, blue-green microalga. The two most important species are Spirulina maxima and Spirulina platensis. Spirulina is considered an excellent food, lacking toxicity and having corrective properties against viral attacks, anemia, tumor growth and malnutrition. We have observed that cultures of Spirulina platensis grow in media containing up to 80 ppm of the organophosphorous pesticide, Chlorpyrifos. It was found to be due to an alkaline phosphatase (ALP) activity that was detected in cell free extracts of Spirulina platensis. This activity was purified from the cell free extracts using ammonium sulphate precipitation and gel filtration and shown to belong to the class of EC 188.8.131.52 ALP. The purified enzyme degrades 100 ppm Chlorpyrifos to 20 ppm in 1 h transforming it into its primary metabolite 3, 5, 6-trichloro-2-pyridinol. This is the first report of degradation of Chlorpyrifos by Spirulina platensis whose enzymic mechanism has been clearly identified. These findings have immense potential for harnessing Spirulina platensis in bioremediation of polluted ecosystems.
[show abstract][hide abstract] ABSTRACT: Nitrate assimilation is an important process for the growth and development of plants. It is regulated
at both transcritptional and posttranscriptional level by various factors including nitrogen
metabolites. In the present study the effect of nitrogen metabolites on the regulation of nitrate
reductase (NR) and nitrite reductase (NiR) in the shoots of 11-days hydroponically grown rice
seedlings were studied. Both ammonium and glutamine caused a decrease in the activity of nitrateinduced
NR while these metabolites had only a partial inhibitory or no effect on NiR. These
metabolites had no effect on the enzyme activities in the absence of nitrate and on the nitrate uptake.
Methionine sulfoximine, (MSX) an inhibitor of glutamine synthetase, was used to check whether the
observed inhibitory effect on NR and NiR is due to ammonium itself or some other downstream
metabolites. The results obtained show that while MSX had a partial inhibitory effect on NR.
However it had no effect on NiR activities. On the other hand it increased the inhibitory effect of
ammonium on nitrate reductase activity. Increase in inhibition to such an extent was not observed
with NiR activity. These results indicate that the inhibition of NR and NiR activities was a direct
effect of ammonium accumulation rather than an effect of its assimilation products.
Journal of Advances in Science & Technology. 01/2010; 13(3-0971-9563):101-104.
[show abstract][hide abstract] ABSTRACT: The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.
The Protein Journal 07/2008; 27(4):205-14. · 1.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Malondialdehyde (MDA), one of the key end products of lipid oxidation is elevated in a variety of diseases. It is now well established that MDA can modify proteins in vivo. This paper describes the effects of modification of albumin by MDA and peroxidized linolenic acid on the reactivity of Cys34, a crucial residue conferring antioxidant properties. BSA (10 mg/ml) was incubated with MDA (1 mM) for 72 h in phosphate buffer (100 mM, pH 7.4). BSA was also incubated for three days with lipid samples, which have already undergone peroxidation for 2, 5, 7, and 9 days respectively. The reactivity of Cys34 after modification was monitored using cystamine and 5,5'-dithiobis(2-nitro benzoic acid) (DTNB). The Kobs for the reaction was found to be different between native and MDA modified protein clearly indicating that modification affects the reactivity of Cys34. The individual rate constant (K1) for reaction with DTNB varied significantly between albumin and modified albumin suggesting that loss in reactivity was due to changes at Cys34. However, (K2), the rate constant for reaction of protein with cystamine, determined from a plot of Kobs versus cystamine concentration did not change. This study further shows that modification results in significant loss of the esterase like activity of albumin. Since albumin plays a crucial role in the antioxidant defence due to its abundance (approximately 0.6 mM) in serum, these findings have implications in disease states where increased levels of MDA and oxidative stress drastically may affect the antioxidant capacity of serum.
Toxicology in Vitro 05/2008; 22(3):618-24. · 2.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Amino acids react with methylglyoxal to form advanced glycation end products. This reaction is known to produce free radicals. In this study, cleavage to plasmid DNA was induced by the glycation of lysine with methylglyoxal in the presence of iron(III). This system was found to produce superoxide as well as hydroxyl radicals. The abilities of various vitamins to prevent damage to plasmid DNA were evaluated. Pyridoxal-5-phosphate showed maximum protection, while pyridoxamine showed no protection. The protective abilities could be directly correlated to inhibition of production of hydroxyl and superoxide radicals. Pyridoxal-5-phosphate exhibited low radical scavenging ability as evaluated by its TEAC, but showed maximum protection probably by interfering in free radical production. Pyridoxamine did not inhibit free radical production. Thiamine and thiamine pyrophosphate, both showed protective effects albeit to different extents. Tetrahydrofolic acid showed better antioxidant activity than folic acid but was found to damage DNA by itself probably by superoxide generation.
[show abstract][hide abstract] ABSTRACT: In this study the induction and regulation of NR and NiR by various N metabolites in excised leave of rice seedlings grown hydroponically for 10-12 days and adapted for 2 days in darkness was examined.
Physiology and Molecular Biology of Plants 01/2007; 13(1):17-25.
[show abstract][hide abstract] ABSTRACT: The aim of the present study was to determine the effects of activators and inhibitors of signaling pathways on nitrate-induced NR activity and
transcript levels in rice leaves. Rice plants (Oryza sativa indica var. Panvel I) were grown hydroponically (without nutrients) on germination paper
under continuous white light for 10–12 days. Leaves, adapted for darkness for 2 days, were treated with signaling agents both in light and dark. The
results presented in this work show that okadaic acid (protein phosphatases 1 and 2A inhibitor) and lithium (inhibitor of IP3 pathway) specifically
inhibit activity and mRNA levels of NR under light conditions with no effect on NiR activity. Both NR and NiR activities are inhibited and
stimulated in the dark by PMA and Bisindolylmaleimide (BIM) (PKC activator and inhibitor), respectively. Cholera toxin specifically enhances NR
activity and steady state levels of mRNA in the dark showing the involvement of G-proteins. Calcium has a stimulatory effect on both the enzymes,
with an increase in NR mRNA levels albeit to a lesser extent. These results suggest that the activities of nitrate-assimilating enzymes in rice are
regulated independently by G-protein and IP3 mediated pathways and co-regulated by PKC and calcium.
[show abstract][hide abstract] ABSTRACT: Glycation, a deleterious form of post-translational modification of macromolecules has been linked to diseases such as diabetes, cataract, Alzheimer's, dialysis related amyloidosis (DRA), atherosclerosis and Parkinson's as well as physiological aging. This review attempts to summarize the data on glycation in relation to its chemistry, role in macromolecular damage and disease, dietary sources and its intervention. Macromolecular damage and biochemical changes that occur in aging and age-related disorders point to the process of glycation as the common event in all of them. This is supported by the fact that several age-related diseases show symptoms manifested by hyperglycemia. Free radical mediated oxidative stress is also known to arise from hyperglycemia. There is evidence to indicate that controlling hyperglycemia by antidiabetic biguanides prolongs life span in experimental animals. Caloric restriction, which appears to prolong life span by bringing about mild hypoglycemia and increased insulin sensitivity further strengthens the idea that glucose via glycation is the primary damaging molecule.
[show abstract][hide abstract] ABSTRACT: The small intestine exhibits numerous morphological and functional alterations during diabetes. Oxidative stress, a factor implicated in the pathogenesis of diabetic complications may contribute towards some of these alterations. We therefore investigated the occurrence of oxidative stress in the small intestine during diabetes by measuring the extent of oxidative damage as well as the status of the antioxidant defense system. Significant increases in lipid peroxidation (four-fold) as measured by TBARS and protein oxidation (38%) as measured by protein carbonyl content were observed after 6 weeks of diabetes. A distinct elevation in the activities of catalase (123.9%) and superoxide dismutase (71.9%) and a decline in the activity of glutathione peroxidase (67.7%) were also observed. The steady state mRNA levels of these enzymes measured by RT-PCR were, however, unchanged suggesting the absence of transcriptional control. In contrast, no changes in the levels of protein and non-protein thiols as well as the activities of glutathione reductase and glutathione-S-transferase were detected. Interestingly, decreases in the activities of xanthine oxidase (XO; 25.7%) and xanthine dehydrogenase (XDH; 42.6%) indicate that they do not contribute significantly to oxidative damage. The results thus reveal the occurrence of oxidative stress in the small intestine during diabetes and suggest its possible involvement in some of the accompanying functional alterations.
The International Journal of Biochemistry & Cell Biology 02/2004; 36(1):89-97. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Momordica charantia (L.) (Cucurbitaceae) commonly known as bitter gourd or karela is a medicinal plant, used in Ayurveda for treating various diseases, one of which is diabetes mellitus. In this study, various extract powders of the fresh and dried whole fruits were prepared and their blood glucose lowering effect compared by administrating them orally to diabetic rats. The aqueous extract powder of fresh unripe whole fruits at a dose of 20mg/kg body weight was found to reduce fasting blood glucose by 48%, an effect comparable to that of glibenclamide, a known synthetic drug. This extract was tested for nephrotoxicity, hepatotoxicity and biochemical parameters such as SGOT, SGPT and lipid profile. The extract did not show any signs of nephrotoxicity and hepatotoxicity as judged by histological and biochemical parameters. Thus the aqueous extract powder of Momordica charantia, an edible vegetable, appears to be a safe alternative to reducing blood glucose.
Journal of Ethnopharmacology 10/2003; 88(1):107-11. · 2.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: The physical state (fluidity) of lipids modulates the activities of several membrane bound enzymes and transport proteins. Alteration of brush border membrane (BBM) fluidity is one of the several changes exhibited by the small intestine during diabetes. In the present study, an investigation of the diabetes induced regional changes in fluidity, oxidative damage, non-enzymatic glycation as well as the activities and the kinetic parameters of the enzymes alkaline phosphatase and gamma-glutamyl transpeptidase was carried out on the intestinal BBM. At the end of 6 weeks of diabetes, significant increases in the extent of both oxidative damage and non-enzymatic glycation were observed along the length of the intestine along with a simultaneous decrease in membrane fluidity. A significant correlation between the decrease in BBM fluidity and increase in non-enzymatic glycation was observed in the duodenum and jejunum. Additionally regional variations in the activities and kinetic parameters of both the enzymes were observed.
Molecular and Cellular Biochemistry 10/2003; 252(1-2):125-32. · 2.33 Impact Factor
[show abstract][hide abstract] ABSTRACT: The present study was aimed at addressing the effect of hyperglycemia on the renal cortical brush border membrane. The fluidity and the functionality of the renal cortical brush border membrane have been evaluated after 6 weeks of streptozotocin-induced diabetes in rats. Lipid peroxidation and protein oxidation were first performed to confirm a state of oxidative stress. The fluidity of the brush border membrane of diabetic rats decreased significantly by 15.76%. There was an increase in the amount of early (19.39%) and advanced (42.23%) glycation end-products suggesting the accumulation of significant amount of non-enzymic glycation products at 6 weeks of diabetes. Although, the activities of both gamma-glutamyl transpeptidase and alkaline phosphatase of the brush border membrane decreased, that of the latter decreased to a significant extent with an increase in K(m) (81%) and no change in the V(max). A study of the activities of glutathione-dependent antioxidant enzymes in the renal cortical homogenates showed that the activities of glutathione peroxidase and glyoxalase II were altered significantly. Our study seems to suggest that increased free radical generation accompanied by non-enzymic glycation may be responsible for oxidative stress and an increased rigidity of the diabetic brush border membrane. Alkaline phosphatase may thus serve as a potentially useful marker of free radical induced damage to the renal cortical brush border membrane. The results also suggest that enhanced susceptibility to oxidative stress during early stages may be an important factor in the development of secondary complications of diabetes.
The International Journal of Biochemistry & Cell Biology 09/2003; 35(8):1163-9. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diabetes related complications, if not treated, can be lethal. The basis of diabetes treatment is management of these complications by different approaches with the aim of providing a healthy life to diabetics. This article gives an overview of the various approaches currently in use to control hyperglycemia like pharmacological compounds and natural products. Many natural products have been used in traditional medicine, but only a few of them are discussed here. A combination therapy appears more useful for the treatment of diabetes rather than the use of a single compound.
Cellular and molecular biology 07/2003; 49(4):635-9. · 0.81 Impact Factor