Assessment of oxidative metabolism

Oxidative metabolism is one of the central physiological processes that regulate multiple functions in a cell including cell death and survival, proliferation, gene transcription, and protein modification. There are multitudes of techniques that are used to evaluate oxidative activity. Here, we summarize how to measure oxidative activity by flow cytometry. This versatile technique allows the evaluation of the level of oxidative activity within heterogeneous populations of cells and in cell culture. Flow cytometry is a quick method that yields highly reproducible results with small sample volumes. Therefore, it is an ideal technique for evaluating changes in oxidative activity in samples from mice

Detection of autoantibodies against reactive oxygen species modified glutamic acid decarboxylase-65 in type 1 diabetes associated complications

<p>Abstract</p> <p>Background</p> <p>Autoantibodies against glutamate decarboxylase-65 (GAD₆₅Abs) are thought to be a major immunological tool involved in pathogenic autoimmunity development in various diseases. GAD₆₅Abs are a sensitive and specific marker for type 1 diabetes (T1D). These autoantibodies can also be found in 6-10% of patients classified with type 2 diabetes (T2D), as well as in 1-2% of the healthy population. The latter individuals are at low risk of developing T1D because the prevalence rate of GAD₆₅Abs is only about 0.3%. It has, therefore, been suggested that the antibody binding to GAD₆₅in these three different GAD₆₅Ab-positive phenotypes differ with respect to epitope specificity. The specificity of reactive oxygen species modified GAD₆₅(ROS-GAD₆₅) is already well established in the T1D. However, its association in secondary complications of T1D has not yet been ascertained. Hence this study focuses on identification of autoantibodies against ROS-GAD₆₅(ROS-GAD₆₅Abs) and quantitative assays in T1D associated complications.</p> <p>Results</p> <p>From the cohort of samples, serum autoantibodies from T1D retinopathic and nephropathic patients showed high recognition of ROS-GAD₆₅as compared to native GAD₆₅(N-GAD₆₅). Uncomplicated T1D subjects also exhibited reactivity towards ROS-GAD₆₅. However, this was found to be less as compared to the binding recorded from complicated subjects. These results were further proven by competitive ELISA estimations. The apparent association constants (AAC) indicate greater affinity of IgG from retinopathic T1D patients (1.90 × 10^-6M) followed by nephropathic (1.81 × 10^-6M) and uncomplicated (3.11 × 10^-7M) T1D patients for ROS-GAD₆₅compared to N-GAD₆₅.</p> <p>Conclusion</p> <p>Increased oxidative stress and blood glucose levels with extended duration of disease in complicated T1D could be responsible for the gradual formation and/or exposing cryptic epitopes on GAD₆₅that induce increased production of ROS-GAD₆₅Abs. Hence regulation of ROS-GAD₆₅Abs could offer novel tools for analysing and possibly treating T1D complications.</p

Exact Product Formation Rates for Stochastic Enzyme Kinetics

The rate of product formation is an important measure of the speed of enzyme reactions. Classical studies of enzyme reactions have been conducted in dilute solutions and under conditions that justified the substrate abundance assumption. However, such assumption is well-known to break down in the context of cellular biochemistry. Instead, the concentration of available substrate can become rate limiting. Here we use the chemical master equation to obtain expressions for the instantaneous and time averaged rate of product formation without invoking the conventional substrate abundance assumption. The expressions are derived for a broad range of enzyme reaction mechanisms, including those that involve one or many enzyme molecules, require multiple substrates, and exhibit cooperativity and substrate inhibition. Novel results include: (i) the relationship between the average rate of product formation (calculated over the time it takes for the reaction to finish) and the substrate concentration, for a Michaelis–Menten (MM) reaction with one enzyme molecule, is approximately given by a logarithmically corrected MM form; (ii) intrinsic noise decreases the sharpness of cooperative switches but enhances the filtering response of substrate inhibition; (iii) the relationship between the initial average rate of product formation and the initial substrate concentration for a MM reaction with no reversible reaction and with any number of enzyme and substrate molecules is a sum of Michaelis–Menten equations

Caspase-dependent and -independent suppression of apoptosis by monoHER in Doxorubicin treated cells

Doxorubicin (DOX) is an antitumour agent for different types of cancer, but the dose-related cardiotoxicity limits its clinical use. To prevent this side effect we have developed the flavonoid monohydroxyethylrutoside (monoHER), a promising protective agent, which did not interfere with the antitumour activity of DOX. To obtain more insight in the mechanism underlying the selective protective effects of monoHER, we investigated whether monoHER (1 mM) affects DOX-induced apoptosis in neonatal rat cardiac myocytes (NeRCaMs), human endothelial cells (HUVECs) and the ovarian cancer cell lines A2780 and OVCAR-3. DOX-induced cell death was effectively reduced by monoHER in heart, endothelial and A2780 cells. OVCAR-3 cells were highly resistant to DOX-induced apoptosis. Experiments with the caspase-inhibitor zVAD-fmk showed that DOX-induced apoptosis was caspase-dependent in HUVECs and A2780 cells, whereas caspase-independent mechanisms seem to be important in NeRCaMs. MonoHER suppressed DOX-dependent activation of the mitochondrial apoptotic pathway in normal and A2780 cells as illustrated by p53 accumulation and activation of caspase-9 and -3 cleavage. Thus, monoHER acts by suppressing the activation of molecular mechanisms that mediate either caspase-dependent or -independent cell death. In light of the current work and our previous studies, the use of clinically achievable concentrations of monoHER has no influence on the antitumour activity of DOX whereas higher concentrations as used in the present study could influence the antitumour activity of DOX

Oxidized and Aggregated Recombinant Human Interferon Beta is Immunogenic in Human Interferon Beta Transgenic Mice

PurposeTo study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNβ-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice.MethodsUntreated rhIFNβ-1a was degraded by metal-catalyzed oxidation, H2O2-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNβ-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured.ResultsAll rhIFNβ-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNβ-1a contained high levels of covalent aggregates as compared with untreated rhIFNβ-1a. H2O2-treated rhIFNβ-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNβ-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNβ-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H2O2-treated rhIFNβ-1a as compared to untreated and guanidine-treated rhIFNβ-1a.ConclusionsOxidation-mediated aggregation increased the immunogenicity of rhIFNβ-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic

Riboflavin Ameliorates Cisplatin Induced Toxicities under Photoillumination

BACKGROUND: Cisplatin is an effective anticancer drug that elicits many side effects mainly due to induction of oxidative and nitrosative stresses during prolonged chemotherapy. The severity of these side effects consequently restricts its clinical use under long term treatment. Riboflavin is an essential vitamin used in various metabolic redox reactions in the form of flavin adenine dinucleotide and flavin mononucleotide. Besides, it has excellent photosensitizing property that can be used to ameliorate these toxicities in mice under photodynamic therapy. METHODS AND FINDINGS: Riboflavin, cisplatin and their combinations were given to the separate groups of mice under photoilluminated condition under specific treatment regime. Their kidney and liver were excised for comet assay and histopathological studies. Furthermore, Fourier Transform Infrared Spectroscopy of riboflavin-cisplatin combination in vitro was also conducted to investigate any possible interaction between the two compounds. Their comet assay and histopathological examination revealed that riboflavin in combination with cisplatin was able to protect the tissues from cisplatin induced toxicities and damages. Moreover, Fourier Transform Infrared Spectroscopy analysis of the combination indicated a strong molecular interaction among their constituent groups that may be assigned for the protective effect of the combination in the treated animals. CONCLUSION: Inclusion of riboflavin diminishes cisplatin induced toxicities which may possibly make the cisplatin-riboflavin combination, an effective treatment strategy under chemoradiotherapy in pronouncing its antineoplastic activity and sensitivity towards the cancer cells as compared to cisplatin alone

Interleukin-1 receptor antagonist haplotype associated with prostate cancer risk

IL1-RN is an important anti-inflammatory cytokine that modulate the inflammation response by binding to IL1 receptors, and as a consequence inhibits the action of proinflammatory cytokines IL1α and IL1β. In this study, we hypothesise that sequence variants in the IL1-RN gene are associated with prostate cancer risk. The study population, a population-based case–control study in Sweden, consisted of 1383 prostate cancer case patients and 779 control subjects. We first selected 18 sequence variants covering the IL1-RN gene and genotyped these single-nucleotide polymorphisms (SNPs) in 96 control subjects. Gene-specific haplotypes of IL1-RN were constructed and four haplotype-tagging single-nucleotide polymorphisms (htSNPs) were identified (rs878972, rs315934, rs3087263 and rs315951) that could uniquely describe >95% of the haplotypes. All study subjects were genotyped for the four htSNPs. No significant difference in genotype frequencies between cases and controls were observed for any of the four SNPs based on a multiplicative genetic model. Overall there was no significant difference in haplotype frequencies between cases and controls; however, the prevalence of the most common haplotype (ATGC) was significantly higher among cases (38.7%) compared to controls (33.5%) (haplotype-specific P=0.009). Evaluation of the prostate cancer risk associated with carrying the ‘ATGC' haplotype revealed that homozygous carriers were at significantly increased risk (odds ratio (OR)=1.6, 95% confidence interval (CI)=1.2–2.2), compared to noncarriers, while no significant association was found among subjects heterozygous for the haplotype (OR=1.0, 95% CI=0.8–1.2). Restricting analyses to advanced prostate cancer strengthened the association between the ‘ATGC' haplotype and disease risk (OR for homozygous carriers vs noncarriers 1.8, 95% CI=1.3–2.5). In conclusion, the results from this study support the hypothesis that inflammation has a role of in the development of prostate cancer, but further studies are needed to identify the causal variants in this region and to elucidate the biological mechanism for this association

Assembly of Inflammation-Related Genes for Pathway-Focused Genetic Analysis

Recent identifications of associations between novel variants in inflammation-related genes and several common diseases emphasize the need for systematic evaluations of these genes in disease susceptibility. Considering that many genes are involved in the complex inflammation responses and many genetic variants in these genes have the potential to alter the functions and expression of these genes, we assembled a list of key inflammation-related genes to facilitate the identification of genetic associations of diseases with an inflammation-related etiology. We first reviewed various phases of inflammation responses, including the development of immune cells, sensing of danger, influx of cells to sites of insult, activation and functional responses of immune and non-immune cells, and resolution of the immune response. Assisted by the Ingenuity Pathway Analysis, we then identified 17 functional sub-pathways that are involved in one or multiple phases. This organization would greatly increase the chance of detecting gene-gene interactions by hierarchical clustering of genes with their functional closeness in a pathway. Finally, as an example application, we have developed tagging single nucleotide polymorphism (tSNP) arrays for populations of European and African descent to capture all the common variants of these key inflammation-related genes. Assays of these tSNPs have been designed and assembled into two Affymetrix ParAllele customized chips, one each for European (12,011 SNPs) and African (21,542 SNPs) populations. These tSNPs have greater coverage for these inflammation-related genes compared to the existing genome-wide arrays, particularly in the African population. These tSNP arrays can facilitate systematic evaluation of inflammation pathways in disease susceptibility. For additional applications, other genotyping platforms could also be employed. For existing genome-wide association data, this list of key inflammation-related genes and associated subpathways can facilitate comprehensive inflammation pathway- focused association analyses

Telomere-Mediated Chromosomal Instability Triggers TLR4 Induced Inflammation and Death in Mice

BACKGROUND: Telomeres are essential to maintain chromosomal stability. Cells derived from mice lacking telomerase RNA component (mTERC-/- mice) display elevated telomere-mediated chromosome instability. Age-dependent telomere shortening and associated chromosome instability reduce the capacity to respond to cellular stress occurring during inflammation and cancer. Inflammation is one of the important risk factors in cancer progression. Controlled innate immune responses mediated by Toll-like receptors (TLR) are required for host defense against infection. Our aim was to understand the role of chromosome/genome instability in the initiation and maintenance of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: We examined the function of TLR4 in telomerase deficient mTERC-/- mice harbouring chromosome instability which did not develop any overt immunological disorder in pathogen-free condition or any form of cancers at this stage. Chromosome instability was measured in metaphase spreads prepared from wildtype (mTERC+/+), mTERC+/- and mTERC-/- mouse splenocytes. Peritoneal and/or bone marrow-derived macrophages were used to examine the responses of TLR4 by their ability to produce inflammatory mediators TNFalpha and IL6. Our results demonstrate that TLR4 is highly up-regulated in the immune cells derived from telomerase-null (mTERC-/-) mice and lipopolysaccharide, a natural ligand for TLR4 stabilises NF-kappaB binding to its promoter by down-regulating ATF-3 in mTERC-/- macrophages. CONCLUSIONS/SIGNIFICANCE: Our findings implied that background chromosome instability in the cellular level stabilises the action of TLR4-induced NF-kappaB action and sensitises cells to produce excess pro-inflammatory mediators. Chromosome/genomic instability data raises optimism for controlling inflammation by non-toxic TLR antagonists among high-risk groups