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Biomedical subjects

Biji T Kurien

Publications and source records attributed to Biji T Kurien.

At least 19 recordsLinked to original sources
PubMed2006

Curcumin/turmeric solubilized in sodium hydroxide inhibits HNE protein modification--an in vitro study.

Free radical mediated lipid peroxidation has been implicated in multiple diseases. A major oxidation by-product of this deleterious process is 4-hydroxy-2-nonenal (HNE). HNE is cytotoxic, mutagenic and genotoxic and is involved in disease pathogenesis. Curcumin, a non-steroidal anti-inflammatory agent (occurring as the yellow pigment found in the rhizomes of the perennial herb Curcuma longa known as turmeric), has emerged as the newest "nutraceutical" agent that has been shown to be efficacious against colon cancer and other disorders, including correcting cystic fibrosis defects. Since curcumin has been reported to have anti-oxidant properties we hypothesized that it will inhibit HNE-modification of a protein substrate. Using an ELISA that employed HNE-modification of solid phase antigen following immobilization, we found that the curcumin solubilized in dilute alkali (5mM sodium hydroxide, pH 11) inhibited HNE-protein modification by 65%. Turmeric also inhibited HNE-protein modification similarly (65%) but at a much lower alkali level (130muM sodium hydroxide, pH 7.6). Alkali by itself (5mM sodium hydroxide, pH 11) was found to enhance HNE modification by as much as 267%. Curcumin/turmeric has to inhibit this alkali enhanced HNE-modification prior to inhibiting the normal HNE protein modification induced by HNE. Thus, inhibition of HNE-modification could be a mechanism by which curcumin exerts its antioxidant effects. The pH at which the inhibition of HNE modification of substrate was observed was close to the physiological pH, making this formulation of curcumin potentially useful practically.

Aldehydes↗
PubMed2006

Oxidatively modified autoantigens in autoimmune diseases.

Free radical-mediated oxidative damage and consequent protein modification by the end products of oxidative damage are important mediators of cell toxicity and disease pathogenesis. Aldehydic products, mainly the 4-hydroxy-2-alkenals, form adducts with proteins and make them highly immunogenic. Oxidative modification of proteins has been shown to elicit antibodies in a variety of diseases including systemic lupus erythematosus (SLE), alcoholic liver disease, diabetes mellitus (DM), and rheumatoid arthritis (RA). Oxidatively modified DNA (8-oxodeoxyguanine) and low-density lipoproteins (LDL) occur in SLE, a disease in which premature atherosclerosis is a serious problem. In addition, immunization with 4-hydroxy-2-nonenal (HNE)-modified 60-kDa Ro autoantigen elicits an accelerated epitope spreading in an animal model of SLE. Advanced glycation end product (AGE) pentosidine and AGE-modified IgG have been shown to correlate with RA disease activity. Oxidatively modified glutamic acid decarboxylase is important in type 1 DM, while autoantibodies against oxidized LDL are prevalent in Behcet's disease. The fragmentation of scleroderma-specific autoantigens occurs as a result of oxidative modification and is thought to be responsible for the production of autoantibodies through the release of cryptic epitopes. In the face of overwhelming evidence for the involvement of oxidative damage in autoimmunity the administration of antioxidants is a viable untried alternative for preventing or ameliorating autoimmune disease, although results in cardiovascular disease are disappointing.

Autoantigens↗
PubMed2006

A nonsense mutation of PEPD in four Amish children with prolidase deficiency.

Encoded by the peptidase D (PEPD) gene located at 19q12-q13.11, prolidase is a ubiquitous cytosolic enzyme that catalyzes hydrolysis of oligopeptides with a C-terminal proline or hydroxyproline. We describe here four Amish children with a severe phenotype of prolidase deficiency in the Geauga settlements of Ohio as the first report of prolidase deficiency in the Amish population as well as in the United States. The patients presented with infection, hepatosplenomegaly, or thrombocytopenia, in contrast to most cases previously reported in the literature, presenting with skin ulcers. All four patients had typical facial features, classic skin ulcers, and multisystem involvement. Recurrent infections, asthma-like chronic reactive airway disease, hyperimmunoglobulins, hepatosplenomegaly with mildly elevated aspartate transaminase (AST), anemia, and thrombocytopenia were common and massive imidodipeptiduria was universal. Prolidase activity in our patients is nearly undetectable. Direct sequencing of PCR-amplified genomic DNA for all of the exons from the four patients revealed the same homozygous single nucleotide mutation c.793 T > C in exon 11, resulting in a premature stop-codon at amino acid residue 265 (p.R265X). It is speculated that the severe phenotype in these patients might be associated with the type of the PEPD gene mutation.

Abnormalities, Multiple↗
PubMed2006

Western blotting.

Western blotting (protein blotting or immunoblotting) is a powerful and important procedure for the immunodetection of proteins post-electrophoresis, particularly proteins that are of low abundance. Since the inception of the protocol for protein transfer from an electrophoresed gel to a membrane in 1979, protein blotting has evolved greatly. The scientific community is now confronted with a variety of ways and means to carry out this transfer. This review describes the various procedures that have been used to transfer proteins from a gel to a membrane based on the principles of simple diffusion, vacuum-assisted solvent flow and electrophoretic elution. Finally, a brief description of methods generally used to detect antigens on blots is also described.

Animals↗
PubMed2006

Lipid peroxidation in systemic lupus erythematosus.

Lipid peroxidation is an important process in oxygen toxicity. Free radicals inflict this damage by attacking polyunsaturated fatty acids, thus setting off a deleterious chain reaction that ultimately results in their disintegration into malondialdehye, 4 hydroxy-2-nonenal and other harmful by-products. Peroxidation of lipids has been implicated in several diseases including systemic lupus erythematosus (SLE). SLE is an autoimmune disorder with unknown aetiology, characterized by the presence of autoantibodies to self-antigens. There is a significant increase in the production of free radicals like superoxide and hydroxyl radicals in SLE. Indices of lipid peroxidation, like conjugated dienes, malondialdehyde, 8-isoprostaglandin F2 alpha are significantly elevated in SLE. Increased ceruloplasmin levels and decreased transferrin levels in the sera of SLE patients have also been described. The activities of the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase and the amounts of the antioxidant reduced glutathione are also significantly altered in this disease. In addition, there are significant changes in the essential fatty acid profile in the sera of those affected with the disease. In animal models of the disease, immunization of mice with peptides derived from autoantigens induces SLE like disease. Immunization with an oxidatively modified autoantigen led to the rapid development of autoimmunity compared to immunization with the unmodified autoantigen. Thus, oxidative damage appears to play an important role in SLE pathogenesis.

Animals↗
PubMed2005

Immunization with short peptides from the 60-kDa Ro antigen recapitulates the serological and pathological findings as well as the salivary gland dysfunction of Sjogren's syndrome.

Sjögren's syndrome is a poorly understood autoimmune inflammatory illness that affects the salivary and lacrimal glands as well as other organ systems. We undertook the present study to determine whether mice immunized with short peptides from the 60-kDa Ro (or SSA) Ag, which is a common target of the autoimmunity of Sjögren's syndrome, develop an illness similar to Sjögren's syndrome. BALB/c mice were immunized with one of two short peptides from 60-kDa Ro that are know to induce epitope spreading. The animals were analyzed for the presence of anti-Ro and anti-La (or SSB) in the sera by immunoblot and ELISA. Salivary glands were collected and examined by histology after H&E staining. Salivary lymphocytes were purified and studied for cell surface makers by fluorescence-activated cell sorting. Timed stimulated salivary flow was measured. As reported previously, BALB/c mice immunized with 60-kDa Ro peptides developed an immune response directed against the entire Ro/La ribonucleoprotein particle that was similar to that found in humans with lupus or Sjögren's syndrome. Functional studies showed a statistical decrease in salivary flow in immunized mice compared with controls. Furthermore, there were lymphocytic infiltrates in the salivary glands of immunized animals that were not present in controls. The infiltrates consisted of both CD4- and CD8+ T lymphocytes as well as B lymphocytes. BALB/c mice immunized with 60-kDa Ro peptides develop anti-Ro, salivary gland lymphocyte infiltrates, and salivary dysfunction that is highly reminiscent of human Sjögren's syndrome.

Animals↗
PubMed2005

Modification of lupus-associated 60-kDa Ro protein with the lipid oxidation product 4-hydroxy-2-nonenal increases antigenicity and facilitates epitope spreading.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with autoantibodies as a near universal feature of the disease. The Ro ribonucleoprotein particle, composed of a 60-kDa protein noncovalently associated with human cytoplasmic RNA, is the target of antibodies in 25-40% of lupus patients. Purified human 60-kDa Ro was found to be oxidatively modified. Earlier investigations from our laboratory revealed increased oxidative damage in SLE patients. Therefore we hypothesized that oxidation by-products, such as 4-hydroxy-2-nonenal (HNE), could lead to neoantigens like HNE-modified 60-kDa Ro, which could in turn initiate autoimmunity or drive epitope spreading. To test this hypothesis we immunized rabbits with either HNE-modified 60-kDa Ro or the unmodified Ro. Intramolecular epitope spreading within the Ro molecule and intermolecular epitope spreading to La, double-stranded DNA, nRNP, and Sm occurred preferentially in HNE-Ro-immunized animals. Nonspecific anti-HNE antibody, generated by immunization with HNE-keyhole limpet hemocyanin conjugate, did not significantly bind to these autoantigens. These data may suggest a hitherto unappreciated mechanism by which oxidative stress facilitates epitope spreading in SLE.

Aldehydes↗
PubMed2005

In vitro modification of solid phase multiple antigenic peptides/autoantigens with 4-hydroxy-2-nonenal (HNE) provide ideal substrates for detection of anti-HNE antibodies and peptide antioxidants.

The role of free radicals in protein modification and the importance of anti-4-hydroxy-2-nonenal (HNE) antibodies as marker of HNE-mediated cell toxicity has been well documented. Proteins modified by HNE in vitro, prior to immobilization on ELISA plates, have served as substrates for assaying these antibodies. We found preferential binding of HNE-modified versus unmodified proteins to ELISA plates and this prompted us to seek a more reliable assay. We report a method to HNE-modify any cysteine/histidine/lysine-containing protein or multiple antigenic peptide (MAP) following their immobilization on an ELISA plate. To a set of wells, HNE (200 microM) dissolved in PBS is added and incubated for 4 h, followed by regular ELISA. Since HNE was supplied dissolved in ethanol, PBS with appropriate amount of ethanol added was used as control. For inhibition experiments, HNE is incubated with or without inhibitors and then added to the wells. The commercial anti-HNE serum bound only to HNE-modified antigens. Sera from rabbits and mice immunized with HNE-modified 60 kDa Ro autoantigen preferentially bound the modified antigens. Modification of solid phase antigens in this manner makes assaying anti-HNE antibodies unambiguous. Lengthy dialysis procedures or the use of spin columns that lead to antigen loss becomes unnecessary for the separation of free HNE. We were able to HNE-modify various antigens (BSA, the autoantigens Ro, La and Sm/nRNP, 60 kDa Ro and Sm MAPs) using this procedure. Using MAPs, we confirmed the importance of histidine, lysine and cysteine residues in HNE modification. In addition, this method allowed identification of inhibitors of HNE-modification. We obtained 61%, 70% and 74% inhibition of HNE-modification of solid phase Ro MAP 166 substrate using BSA, Ro MAP 482 and Ro MAP 166, respectively. Glycyl-proline dipeptide and a MAP from the Sm autoantigen (PPPGMRPP) showed 0% inhibition of HNE-modification.

Aldehydes↗
PubMed2004

Determination of prolidase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Proline-containing peptides of the X-proline type are cleaved by the dipeptidase prolidase. The classical method of prolidase assay relied on the colorimetric estimation of the liberated proline with ninhydrin using acidic media and heat. This method, however, gave inconsistent results due to the nonspecificity of the ninhydrin color reaction. We report here a method for the detection of the liberated proline using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Human sera were incubated with a mixture containing the dipeptide glycyl-proline in Tris-HCl supplemented with manganese at 37 degrees C for 24h. The samples were precipitated with trifluoroacetic acid and centrifuged. An aliquot of the supernatant was mixed with an equal volume of ferulic acid solution. An aliquot from this mixture was spotted on a stainless steel mass spectrometry grid and analyzed using MALDI-TOF mass spectrometry. The activity of the enzyme was determined by the complete disappearance of the glycyl-proline peak with the concomitant appearance of the proline peak and can be expressed in terms of the ratio of the area beneath the proline to the area beneath the glycyl-proline peak. Subjects homozygous for prolidase deficiency had a ratio ranging from 0.006 to 0.04 while obligatory heterozygotes had a ratio ranging from around 1.1 to 2.4. Normal subjects had ratios ranging from 9 to 239. Using this method we have unambiguously identified subjects with homozygous or heterozygous prolidase deficiency. In addition to the advantage of rapid sample preparation time, this method is highly specific, reproducible, and sensitive.

Colorimetry↗
PubMed2004

Interaction of calcium and Ro60: increase of antigenicity.

The structural and functional integrity of the cell is largely maintained by protein-protein interactions. Recently, we demonstrated that multiple antigenic peptides (MAPs) constructed from 60 kDa Ro sequence could be used to show intramolecular and intermolecular protein-protein interaction within the 60 kDa Ro ribonucleoprotein particle. We were interested in understanding the mechanism of this binding and hypothesized that this interaction might be mediated through divalent metal ions. The 60 kDa Ro-MAPs failed to interact with purified 60 kDa Ro in the presence of EDTA or EGTA when analyzed by Ouchterlony or surface plasmon resonance (SPR) analysis. When purified 60 kDa Ro was incubated with various metal ions such as Cu2+, Mg2+, Zn2+ and Ca2+, and analyzed by Ouchterlony or SPR for binding to specific 60 kDa Ro-MAPs only Ca2+ ions significantly increased the binding. It was interesting to note that recombinant 60 kDa Ro formed precipitin lines with Ro-MAPs only in the presence of Ca2+ ions. Anti-Ro60 containing SLE sera bound to recombinant Ro60 strongly when incubated in the presence of Ca2+ ions but not in the absence of Ca2+ ions. Using SPR analysis we also found that native Ro60 binds to La only in the presence of Ca2+. These data imply that Ca2+ induces a more native tertiary structure to recombinant 60 kDa Ro and makes it more antigenic. Thus, the observed intramolecular and intermolecular interactions and antigen-antibody interactions could be Ca2+ ion mediated conformational interactions, and we propose that 60 kDa Ro is a calcium binding protein.

Animals↗
PubMed2004

Mechanized syringe homogenization of human and animal tissues.

Tissue homogenization is a prerequisite to any fractionation schedule. A plethora of hands-on methods are available to homogenize tissues. Here we report a mechanized method for homogenizing animal and human tissues rapidly and easily. The Bio-Mixer 1200 (manufactured by Innovative Products, Inc., Oklahoma City, OK) utilizes the back-and-forth movement of two motor-driven disposable syringes, connected to each other through a three-way stopcock, to homogenize animal or human tissue. Using this method, we were able to homogenize human or mouse tissues (brain, liver, heart, and salivary glands) in 5 min. From sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometric enzyme assay for prolidase, we have found that the homogenates obtained were as good or even better than that obtained used a manual glass-on-Teflon (DuPont, Wilmington, DE) homogenization protocol (all-glass tube and Teflon pestle). Use of the Bio-Mixer 1200 to homogenize animal or human tissue precludes the need to stay in the cold room as is the case with the other hands-on homogenization methods available, in addition to freeing up time for other experiments.

Animals↗
PubMed2004

Experimental animal urine collection: a review.

Animal urine collection is a vital part of veterinary practice for ascertaining animal health and in scientific investigations for assessing the results of experimental manipulations. Untainted animal urine collection is very challenging, especially with small rodents, and is an almost impossible task under conditions of microgravity. The fundamental aspects of urine collection are: (1) ease of collection, (2) quality of sample, (3) prevention of contamination, (4) severity of procedures used, (5) levels of pain caused to the animal and (6) refinement of methods to reduce stress, pain or distress. This review addresses the collection of urine for qualitative and quantitative purposes from rodents, rabbits, felines, canines, avian species, equines, porcines, ungulates and certain non-human primates, with animal welfare in mind. Special emphasis has been given to rodents, canines and non-human primates, since they are the animals of choice for research purposes. Free catch (voluntary voiding), methods with mild intervention, surgical methods, modified restraint, cage and special requirement methods have been reviewed here. Efforts need to be taken to provide appropriate animal husbandry and to nurture the animals in as natural an environment as possible since experimental results obtained from these research subjects are, to a great extent, dependent upon their well-being. A continuous refinement in the procedures for collecting urine from experimental animals will be the most efficient way of proceeding in obtaining pure urine specimens for obtaining reliable research data.

Animal Husbandry↗
PubMed2003

Free radical mediated peroxidative damage in systemic lupus erythematosus.

Free radicals and damage caused by these molecular species are implicated in the pathogenesis of a variety of diseases, including autoimmune. Here we have examined oxidative damage, SOD activity and autoantibodies against SOD in systemic lupus erythematosus (SLE), a multifactorial disease with autoantibody production as an universal feature. We found significantly increased amounts of conjugated dienes in the SLE patients compared to normals (mean value of 0.917 vs 0.627, p = 0.0001) and MDA formation (6.96 vs 4.17 nmoles/microl, p = 0.0006) as well as decreased SOD activity. In addition, we found autoantibodies binding SOD by both ELISA and immunoblot. The presence of anti-SOD antibodies was associated with increased free radical damage in SLE patients. Heat inactivated anti-SOD autoantibodies were able to inhibit the activity of the enzyme. We propose that the inhibition of SOD by autoantibodies is, in part, responsible for the increased free radical damage seen in the disease.

Autoantibodies↗