Glycoxidation of mammalian whole histone generates highly immunogenic aggregates: Sera of SLE patients contain autoantibodies against aggregates.

Non-enzymatic glycation and oxidation of self-proteins, causing formation and accumulation of advanced glycation end products (AGEs), have been reported in an array of pathologies, including systemic lupus erythematosus (SLE). Such modifications may generate neo-epitopes, break immunological tolerance, and induce antibody response. In this study, we have first analysed the structural modifications of whole histone in the presence of deoxyribose followed by oxidation with hydroxyl radicals. Changes in the secondary and tertiary structure of the whole histone were determined by spectroscopic techniques and biochemical assays. Fluorescence spectroscopy and UPLC-MS showed the generation of AGEs such as carboxymethyl lysine and pentosidine, while DLS and TEM indicated the presence of amorphous AGE-aggregates. Moreover, rabbits immunized with these histone-AGEs exhibited enhanced immunogenicity and ELISA and western immunoblot of IgG antibodies from SLE patients' sera showed a significantly higher specificity towards modified histone-AGEs than the native histone.

Ferulic acid reinstates mitochondrial dynamics through PGC1α expression modulation in 6-hydroxydopamine lesioned rats.

Disruption of the tightly regulated mitochondrial dynamics and energy homeostasis leads to oxidative stress and apoptotic cell death, as observed in neurodegenerative disorders such as Parkinson's disease (PD). Polyphenolic plant derivatives have been shown to alleviate such pathological features and have been used in models of neurodegenerative disorders in previous reports. In the current study, we utilized a 6-hydroxydopamine (6-OHDA) lesioned rat model of PD to explore the protective efficacy of polyphenolic phytochemical ferulic acid (FA) against mitochondrial dysfunction and explored its effect on gene and protein expression of mitochondrial dynamics regulators dynamin-related protein 1 (Drp1)/mitofusin 2 (Mfn2) in lesioned animals. We also evaluated its effect on expression of mitochondrial biogenesis regulator PGC1α and apoptotic regulators BAX, cyt c, p53, and cleaved PARP. We found that oral FA supplementation alleviated 6-OHDA induced oxidative stress, DNA fragmentation, morphological changes, and blocked apoptotic cascade. FA also reduced mitochondrial Drp1 expression and increased gene and protein expression of PGC1α, thereby regulating expression of its downstream target Mfn2 and restoring mitochondrial dynamics in lesioned animals. Our data suggest that targeting mitochondrial dynamics through modulation of PGC1α can prove to be a potent preventive strategy against PD pathology.

Evaluation of phytomedicinal potential of perillyl alcohol in an in vitro Parkinson's Disease model.

Preclinical Research & Development Parkinson's disease (PD) is the second most common neurodegenerative disorder that affects approximately 10 million people worldwide. The risk of developing PD and similar neurodegenerative disorders increases with age and an estimated 4% people are diagnosed with the disease before reaching the age of 50. Oxidative stress, cytotoxicity, and mitochondrial dysfunction are common features exhibited in the development of PD. The 6-hyroxydopamine (6-OHDA) model of PD is one of the most well characterized and studied models of the disease. 6-OHDA, a neurotoxin, can induce most characteristic features of the disease, including mitochondrial dysfunction in-vivo and in-vitro. SH-SY5Y is a neuroblastoma cell line of human origin that has been used for dose response studies on PD in the past. Based on previous data, we have used SH-SY5Y cells as an in-vitro model of PD to analyse the phytomedicinal potential of perillyl alcohol (PA), a monoterpenoid obtained from essential oils of various plants such as sage, peppermint and lavender. We have found that pretreatment with PA (10 µM and 20 µM) mitigated 6-OHDA (150 µM) induced cytotoxicity in a dose-dependent manner. We observed marked restoration of cell viability and mitochondrial membrane potential (MMP) as well as reduced reactive oxygen species generation, Cytochrome c immunofluorescence and DNA fragmentation after treatment with PA. On the basis of on our data, we have come to the conclusion that PA demonstrates sufficient neuroprotective activity to provide new avenues in therapy of PD and its apparent target being restoration of MMP can lead to better understanding of the disease.

Carbamylation of human serum albumin generates high-molecular weight aggregates: fine characterization by multi-spectroscopic methods and electron microscopy.

Carbamylation is the non-enzymatic reaction between isocyanic acid and macromolecules (mainly proteins) which results in carbamylation-derived products (CDPs) generation, wherein the macromolecules show altered structure and function. In this study, we examined the modifications caused in human serum albumin (HSA) upon interaction with potassium cyanate (KCNO). HSA was incubated with varying concentrations of KCNO for 6 h at 37 °C. The resultant product was characterized by biochemical and biophysical techniques. Among other changes, the carbamylated-HSA showed homocitrulline generation (LC-MS), increase in mass (DLS), and amyloidogenic aggregate formation (Congo red, SEM, TEM). The Gibb's free energy was calculated to be -2.91 to -3.95 kcal mol-1, suggesting that the binding was spontaneous and energetically favourable. The results indicate that in chronic kidney disease patients, elevated levels of isocyanic acid (formed from urea) may modify the albumin structure and lead to its conversion into amyloidogenic aggregates, thus accelerating kidney damage.

Perillyl Alcohol Mitigates Behavioural Changes and Limits Cell Death and Mitochondrial Changes in Unilateral 6-OHDA Lesion Model of Parkinson's Disease Through Alleviation of Oxidative Stress.

In this study, we aim to assess the phytomedicinal potential of perillyl alcohol (PA), a dietary monoterpenoid, in a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of Parkinson's disease (PD). We observed that PA supplementation alleviated behavioural abnormalities such as loss of coordination, reduced rearing and motor asymmetry in lesioned animals. We also observed that PA-treated animals exhibited reduced oxidative stress, DNA fragmentation and caspase 3 activity indicating alleviation of apoptotic cell death. We found reduced mRNA levels of pro-apoptotic regulator BAX and pro-inflammatory mediators IL18 and TNFα in PA-treated animals. Further, PA treatment successfully increased mRNA and protein levels of Bcl2, mitochondrial biogenesis regulator PGC1α and tyrosine hydroxylase (TH) in lesioned animals. We observed that PA treatment blocked BAX and Drp1 translocation to mitochondria, an event often associated with the inception of apoptosis. Further, 6-OHDA exposure reduced expression of electron transport chain complexes I and IV, thereby disturbing energy metabolism. Conversely, expression levels of both complexes were upregulated with PA treatment in lesioned rats. Finally, we found that protein levels of Nrf2, the transcription factor responsible for antioxidant gene expression, were markedly reduced in cytosolic and nuclear fraction on 6-OHDA exposure, and PA increased expression of Nrf2 in both fractions. We believe that our data hints towards PA having the ability to provide cytoprotection in a hemiparkinsonian rat model through alleviation of motor deficits, oxidative stress, mitochondrial dysfunction and apoptosis.

Impact of endogenous stress on albumin structure in systemic lupus erythematosus (SLE) patients.

Systemic lupus erythematosus (SLE) is an inflammatory, autoimmune disorder of unknown etiology. The inflammatory stress in SLE patients may modify macromolecules and produce structural/functional abnormalities. The present study is aimed at examining the consequences of stresses on the structure of albumin in SLE patients. Albumin was isolated from the sera of SLE/healthy subjects. Multiple physicochemical techniques were used to elucidate, structure of albumin. Advanced glycation end products in SLE patients' albumin were identified by the AGE specific fluorescence. Quenching of tryptophan, tyrosine fluorescence and surface protein hydrophobicity was observed in SLE patients' albumin. Protein-bound carbonyls were elevated while free thiol, lysine, arginine, and alpha helicity was found to be decreased in SLE albumin. Furthermore, changes in the secondary structure of SLE albumin were observed as shift in the position of amide I/II bands. Functionality of SLE albumin was also compromised as its cobalt-binding ability was substantially declined. Adduction of moieties was detected by dynamic light scattering (DLS) and confirmed by matrix assisted laser desorption/ionization. DLS, thioflavin T and transmission electron microscopy results confirmed aggregates in SLE patients' albumin. This study may be helpful in understanding the role of modified albumin in the cofounding pathologies associated with SLE.

Physicochemical characterization of carbamylated human serum albumin: an in vitro study.

Carbamylation is an ubiquitous process in which cyanate (OCN-) reacts with the N-terminal amino or ε-amino moiety and generates α-carbamyl amino acids and ε-carbamyl-lysine (homocitrulline). The process leads to irreversible changes in protein charge, structure and function. In this study, we have investigated the effect of carbamyl (generated from potassium cyanate) on human serum albumin (HSA) structure and function. The carbamylated-HSA (c-HSA) showed various modifications when examined by UV, fluorescence, FT-IR and far-UV CD spectroscopies. c-HSA exhibited hypochromicity, loss in α-helical content, changes in the amide I and amide II band, etc. Native-PAGE showed increase in the mobility of c-HSA compared to native-HSA. Aggregate(s) formation in c-HSA was detected by thioflavin T dye. The biochemical investigations carried out on c-HSA suggested increase in carbonyl content and decreased binding of TNBS (trinitrobenzenesulphonic acid) and Sakaguchi reagent. The attachment of the carbamyl moiety to HSA was confirmed from MALDI-TOF results. The functional defects in c-HSA were confirmed from the low binding of bilirubin. Taken together, carbamylation of albumin caused changes in the structural and functional properties of HSA. To the best of our knowledge, this is the first report on detailed biophysical characterization of carbamylated-HSA.

Hyperglycemia induced reactive species trigger structural changes in human serum albumin of type 1 diabetic subjects.

Chronic oxidative stress fuels pathogenesis of a large set of diseases. Oxidative stress is the cause and consequence of numerous diseases including type 1 diabetes mellitus (T1DM), in which there is selective destruction of insulin producing pancreatic ß-cells. Studies have documented that hyperglycemia produces profound stress. In vivo production of numerous reactive oxygen, nitrogen, chlorine species and lipid/sugar oxidation products in T1DM patients may be the result of persistent hyperglycemia. Post-translational modifications by reactive species may create new antigenic epitopes and play a role in the development of autoimmune response. In this paper our main focus was to establish the effect of existing hyperglycemia induced oxido-nitrosative stress in T1DM patients on the integrity of human serum albumin. Raised nitric oxide, carbonyl, RBC hemolysis, lowered ferric reducing antioxidant power (FRAP), thiol and deformed RBC in T1DM are all highly suggestive of persistent oxido-nitrosative stress. Hyperglycemia induced generation of advanced glycation end products (AGEs) was established by LCMS. Chronic oxido-nitrosative stress can modify HSA in T1DM patients, producing immunologically active albumin. Therefore, it is speculated that the aberrant HSA may play a role in the initiation/progression of T1DM.

SLE autoantibodies are well recognized by peroxynitrite-modified-HSA: Its implications in the pathogenesis of SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disorder where the role of inflammatory processes in the etiopathogenesis is well documented. Despite extensive research, the trigger for initiation of the disease has not been identified. Peroxynitrite, a strong nitrating/oxidizing agent has been reported in SLE and other autoimmune diseases. In this study, human serum albumin (HSA) was exposed to peroxynitrite for 30min at 37°C. The structure of HSA was grossly perturbed when examined by various physico-chemical techniques. Peroxynitrite mediated nitration of HSA was confirmed by LCMS/MS. Furthermore, increase in hydrodynamic radius of peroxynitrite-modified-HSA suggests the attachment of nitro group(s). Aggregation in peroxynitrite-modified-HSA was evident in a TEM scan. Nitration, oxidation, cross linking, aggregation etc conferred immunogenicity on peroxynitrite-modified-HSA. High titre antibodies were elicited in rabbits immunized with peroxynitrite-modified-HSA. Induced antibodies were highly specific for peroxynitrite-modified-HSA but showed considerable binding with other nitrated molecules. Direct binding/inhibition ELISA carried out with autoantibodies in SLE sera showed preferential binding with peroxynitrite-modified-HSA. Anti-nDNA positive IgG from SLE sera showed preference for peroxynitrite-modified-HSA when subjected to immunoassay (direct binding and inhibition) and mobility shift assay. Our results reinforce the role of augmented inflammation in SLE progression.