Protein Glycation

The aim of the present study was to develop a novel liposomal formulation of thymoquinone (TQ) to treat fluconazole-susceptible and -resistant Candida albicans (C. albicans) infections. The liposomal preparation of TQ (Lip-TQ) was used against a fluconazole-susceptible or -resistant isolate of C. albicans. Various doses of fluconazole (0, 5, 10, 20 and 40 mg/kg) or free TQ or Lip-TQ (0, 1, 2 and 5 mg/kg) were used to treat C. albicans infected mice. Mice were observed for 40 days post C. albicans infection, and their kidneys were assessed for the fungal load. Fluconazole showed anti-fungal activity against the drugsusceptible, but not against the -resistant isolate of C. albicans. Free TQ showed its activity against both fluconazole-susceptible or -resistant C. albicans, however, Lip-TQ was found to be the most effective and imparted ∼100% and ∼90% survival of mice infected with fluconazole-susceptible and -resistant isolates of C. albicans, respectively. Mice treated with Lip-TQ showed highly reduced severity of infection in their tissue homogenates. Therefore, Lip-TQ may effectively be used in the treatment of C. albicans infections, including those which are not responding to fluconazole.

Glycation plays an important role in various oxidative stress related diseases. Superoxide dismutase (SOD) constitutes an essential defense against oxidative stress. The damage caused by oxidative stress is exacerbated if the antioxidant enzymes themselves are inactivated by glycation. Thymoquinone (TQ) has been reported to have various pharmacological activities. Therefore, the glycation of SOD by glucose or methylglyoxal (MG) and its protection by TQ has been investigated. Incubation of SOD with glucose, MG or both at 37 • C resulted in a progressive decrease in the activity of the enzyme, and a parallel decrease in the amount of protein on SDS-PAGE gels for glucose incubated SOD and formation of high molecular weight aggregates for MG or both glucose and MG incubated enzyme. TQ offered protection against glucose or MG induced loss in SOD activity and fragmentation/cross-linking. The antiglycating activity of TQ appears to be better for mild glycating agents. It is also effective in protecting against strong glycating agents, more when the exposure time to the glycating agent is short. TQ has also earlier been reported to have anti-diabetic effects, and this along with the observed antiglycating effect makes it an effective compound against diabetes and its complications.

Accumulation of advanced glycation end products (AGEs) in tissues and serum plays important roles in diabetes-associated complications. Therefore, the identification of antiglycating compounds is attracting considerable interest. In this study, the structural changes associated with the glycation of superoxide dismutase (SOD) and its protection by thymoquinone (TQ) have been investigated by biophysical techniques. Incubation of SOD with glucose, methylglyoxal (MG) or both at 37 • C resulted in progressive hyperchromicity at 280 nm, intrinsic fluorescence quenching at 310 nm, decrease in negative ellipticity at 208 nm, AGE-specific fluorescence enhancement in the wavelength range 400-480 nm and Thioflavin T (ThT) fluorescence enhancement at 480 nm (fibrillar state enhancement). Therefore, glycation by glucose or MG induced both tertiary and secondary structural changes in SOD and formation of AGEs and fibrils. The changes were more and faster with MG than with glucose since MG is a stronger glycating agent than glucose. TQ offered protection against glucose or MG-induced glycation of SOD as observed by a reduction in the structural changes, formation of AGEs and fibrils. Thus, TQ can be used for reducing diabetic complications many of which are due to protein glycation.

The binding of glucose to serum proteins in a non-enzymatic reaction occurs proportionately to the prevailing serum glucose concentration during the lifespan of each protein. The non-enzymatic reaction of proteins with reducing sugars results in the formation of glycated proteins that are indicative of the relative amount of reducing sugars present. Hemoglobin, albumin, lipoproteins and other tissue proteins can be non-enzymatically glycated. Hence, estimation of these glycated proteins can be used for the estimation of average glycemic status. Spectrophotometric, chromatographic and immunoassays techniques are used for the detection and quantitation of these glycated proteins.

Pathological levels of oxidative stress (OS) have been implicated in many diseases including diabetes mellitus, neurodegenerative diseases, inflammatory diseases, atherosclerosis, and cancer. Studies of oxidative stress are however complicated by the low concentration of oxidation products. To resolve this problem, we tested a new derivative of aminoadipic semialdehyde (Fmoc-Aea-OH) in the solid-phase synthesis of carbonylated peptides. We prepared a series of peptides with free and acetylated N-terminal amino groups using the Fmoc-Aea-OH reagent. LC-MS, ESI-MS, and MS/MS spectra confirmed the sequences of the modified peptides, although the LC-MS and ESI-MS spectra were dominated by signals corresponding to dehydration products. NMR studies of acetylated products revealed that the dominant product formed in this reaction contains a 1,2,3,4-tetrahydropyridine-2-carboxylic acid residue. Another side reaction in this system was the cleavage of the amide bond between the Aea residue and the amino acid moiety preceding it resulting in the formation of a side product with a six-membered ring at the N-terminus (2,3,4,5-tetrahydropyridine-2-carboxylic acid residue). We found that, depending on the peptide sequence, one of those side products is predominant. Our work suggests new methods for the solid-state synthesis of peptides containing unnatural amino acids.

In diabetic patients, accelerated glycation process causes increased oxidative stress and chronic hyperglycaemia that play a vital role in the diabetic complications. Extensive intracellular and extracellular generation of these glycated products finally form advanced glycation end products (AGEs). The accumulation of AGEs is related with the intensive risk for microvascular and macrovas-cular injuries for diabetic patients. Therefore, formation of AGEs results from the condensation of reducing sugars with biomolecules like nucleic acids, proteins, and lipids which potentially alter their function. Effect of AGEs formation is also related with the cross-linking that promotes vascular stiffness which modifies the vascular structure and long-life function of proteins. Formation of AGEs may also activate specific receptors, like receptor for AGEs (RAGEs) that induce the intracellular signaling which enhance the oxidative stress and also the amplification of key pro-sclerotic and pro-inflammatory cyto-kines. From last few decades, a huge number of pre-clinical studies related with the AGEs formation in the diabetic patients have been performed. The target for such trials was the formation and degradation of AGEs, and its interaction with RAGEs. This review focuses on the mechanism how these AGEs exert detrimental nuisance in the diabetes, as well as deal with existing strategies to disrupt the action or formation of AGEs. Therefore, the unseen role of both the early and advanced stage glycation in the diabetic Vasculopathy is described. We have also illustrated how the glycation inhibition results in the delay of the development of vascular complications in diabetic patients.