DNA microarray SNP associations with clinical efficacy and side effects of domperidone treatment for gastroparesis.

BACKGROUND: Domperidone treatment for gastroparesis is associated with variable efficacy as well as the potential for side effects. DNA microarray single nucleotide polymorphism (SNP) analysis may help to elucidate the role of genetic variability on the therapeutic effectiveness and toxicity of domperidone. AIM: The aim of this study was to identify SNPs that are associated with clinical efficacy and side effects of domperidone treatment for gastroparesis from DNA microarray experiments. This will help develop a strategy for rational selection of patients for domperidone therapy. METHODS: DNA samples extracted from the saliva of 46 patients treated with domperidone were analyzed using Affymetrix 6.0 SNP microarrays. Then least angle regression (LARS) was used to select SNPs that are related to domperidone efficacy and side effects. Decision tree based prediction models were constructed with the most correlated features selected by LARS. RESULTS: Using the most stable SNP selected by LARS a prediction model for side effects of domperidone achieved (95 ± 0)% true negative rate (TN) and (78 ± 11)% true positive rate (TP) in nested leave-one-out tests. For domperidone efficacy, the prediction based on five most stable SNPs achieved (85 ± 7)% TP and (61±4)% TN. Five identified SNPs are related to ubiquitin mediated proteolysis, epithelial cell signaling, leukocyte, cell adhesion, and tight junction signaling pathways. Genetic polymorphisms in three genes that are related to cancer and hedgehog signaling were found to significantly correlate with efficacy of domperidone. CONCLUSION: LARS was found to be a useful tool for statistical analysis of domperidone-related DNA microarray data generated from a small number of patients.

Probing the role of CXC motif in chemokine CXCL8 for high affinity binding and activation of CXCR1 and CXCR2 receptors.

All chemokines share a common structural scaffold that mediate a remarkable variety of functions from immune surveillance to organogenesis. Chemokines are classified as CXC or CC on the basis of conserved cysteines, and the two subclasses bind distinct sets of GPCR class of receptors and also have markedly different quaternary structures, suggesting that the CXC/CC motif plays a prominent role in both structure and function. For both classes, receptor activation involves interactions between chemokine N-loop and receptor N-domain residues (Site-I), and between chemokine N-terminal and receptor extracellular/transmembrane residues (Site-II). We engineered a CC variant (labeled as CC-CXCL8) of the chemokine CXCL8 by deleting residue X (CXC → CC), and found its structure is essentially similar to WT. In stark contrast, CC-CXCL8 bound poorly to its cognate receptors CXCR1 and CXCR2 (K(i) > 1 µm). Further, CC-CXCL8 failed to mobilize Ca(2+) in CXCR2-expressing HL-60 cells or recruit neutrophils in a mouse lung model. However, most interestingly, CC-CXCL8 mobilizes Ca(2+) in neutrophils and in CXCR1-expressing HL-60 cells. Compared with the WT, CC-CXCL8 binds CXCR1 N-domain with only ∼5-fold lower affinity indicating that the weak binding to intact CXCR1 must be due to its weak binding at Site-II. Nevertheless, this level of binding is sufficient for receptor activation indicating that affinity and activity are separable functions. We propose that the CXC motif functions as a conformational switch that couples Site-I and Site-II interactions for both receptors, and that this coupling is critical for high affinity binding but differentially regulates activation.

Glyceraldehyde 3-phosphate dehydrogenase depletion induces cell cycle arrest and resistance to antimetabolites in human carcinoma cell lines.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that acts at the intersection of energy metabolism and stress response in tumor cells. To elucidate the role of GAPDH in chemotherapy-induced stress, we analyzed its activity, protein level, intracellular distribution, and intranuclear mobility in human carcinoma cells A549 and UO31 after treatment with cytarabine, doxorubicin, and mercaptopurine. After treatment with cytosine arabinoside (araC), enzymatically inactive GAPDH accumulated in the nucleus. Experiments on fluorescence recovery after photobleaching with green fluorescent protein-GAPDH fusion protein in the live cells treated with araC demonstrated reduced mobility of green fluorescent protein-GAPDH inside the nucleus, indicative of interactions with nuclear macromolecular components after genotoxic stress. Depletion of GAPDH with RNA interference stopped cell proliferation, and induced cell cycle arrest in G(1) phase via p53 stabilization, and accumulation of p53-inducible CDK inhibitor p21. Neither p21 accumulation nor cell cycle arrest was detected in GAPDH-depleted p53-null NCI-H358 cells. GAPDH-depleted A549 cells were 50-fold more resistant to treatment with cytarabine (1.68 +/- 0.182 microM versus 0.03 +/- 0.015 microM in control). Depletion of GAPDH did not significantly alter cellular sensitivity to doxorubicin (0.05 +/- 0.023 microM versus 0.035 +/- 0.0154 microM in control). Induction of cell cycle arrest in p53-proficient carcinoma cells via GAPDH abrogation suggests that GAPDH-depleting agents may have a cytostatic effect in cancer cells. Our results define GAPDH as an important determinant of cellular sensitivity to antimetabolite chemotherapy because of its regulatory functions.

Triose phosphate isomerase, a novel enzyme-crystallin, and tau-crystallin in crocodile cornea. High accumulation of both proteins during late embryonic development.

Several enzymes are known to accumulate in the cornea in unusually high concentrations. Based on the analogy with lens crystallins, these enzymes are called corneal crystallins, which are diverse and species-specific. Examining crystallins in lens and cornea in multiple species provides great insight into their evolution. We report data on major proteins present in the crocodile cornea, an evolutionarily distant taxon. We demonstrate that tau-crystallin/alpha-enolase and triose phosphate isomerase (TIM) are among the major proteins expressed in the crocodile cornea as resolved by 2D gel electrophoresis and identified by MALDI-TOF. These proteins might be classified as putative corneal crystallins. tau-Crystallin, known to be present in turtle and crocodile lens, has earlier been identified in chicken and bovine cornea, whereas TIM has not been identified in the cornea of any species. Immunostaining showed that tau-crystallin and TIM are concentrated largely in the corneal epithelium. Using western blot, immunofluorescence and enzymatic activity, we demonstrate that high accumulation of tau-crystallin and TIM starts in the late embryonic development (after the 24th stage of embryonic development) with maximum expression in a two-week posthatched animal. The crocodile corneal extract exhibits significant alpha-enolase and TIM activities, which increases in the corneal extract with development. Our results establishing the presence of tau-crystallin in crocodile, in conjunction with similar reports for other species, suggest that it is a widely prevalent corneal crystallin. Identification of TIM in the crocodile cornea reported here adds to the growing list of corneal crystallins.

High concentration of antioxidants N-acetylcysteine and mitoquinone-Q induces intercellular adhesion molecule 1 and oxidative stress by increasing intracellular glutathione.

In endothelial cells, the intracellular level of glutathione is depleted during offering protection against proinflammatory cytokine TNF-alpha-induced oxidative stress. Administration of anti-inflammatory drugs, i.e., N-acetylcysteine (NAC) or mitoquinone-Q (mito-Q) in low concentrations in the human pulmonary aortic endothelial cells offered protection against depletion of reduced glutathione and oxidative stress mediated by TNF-alpha. However, this study addressed that administration of NAC or mito-Q in high concentrations resulted in a biphasic response by initiating an enhanced generation of both reduced glutathione and oxidized glutathione and enhanced production of reactive oxygen species, along with carbonylation and glutathionylation of the cellular proteins. This study further addressed that IkappaB kinase (IKK), a phosphorylation-dependent regulator of NF-kappaB, plays an important regulatory role in the TNF-alpha-mediated induction of the inflammatory cell surface molecule ICAM-1. Of the two catalytic subunits of IKK (IKKalpha and IKKbeta), low concentrations of NAC and mito-Q activated IKKalpha activity, thereby inhibiting the downstream NF-kappaB and ICAM-1 induction by TNF-alpha. High concentrations of NAC and mito-Q instead caused glutathionylation of IKKalpha, thereby inhibiting its activity that in turn enhanced the downstream NF-kappaB activation and ICAM-1 expression by TNF-alpha. Thus, establishing IKKalpha as an anti-inflammatory molecule in endothelial cells is another focus of this study. This is the first report that describes a stressful situation in the endothelial cells created by excess of antioxidative and anti-inflammatory agents NAC and mito-Q, resulting in the generation of reactive oxygen species, carbonylation and glutathionylation of cellular proteins, inhibition of IKKalpha activity, and up-regulation of ICAM-1expression.