In vivo protein tyrosine nitration in S. cerevisiae: Identification of tyrosine-nitrated proteins in mitochondria

Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with pathophysiological conditions. Although yeast cells lack of mammalian nitric oxide synthase (NOS) orthologues, still it has been shown that they are capable of producing nitric oxide (NO). Our studies showed that NO or reactive nitrogen species (RNS) produced in flavohemoglobin mutant (Δyhb1) strain along with the wild type strain (Y190) of Saccharomyces cerevisiae can be visualized using specific probe 4,5-diaminofluorescein diacetate (DAF-2DA). Δyhb1 strain of S. cerevisiae showed bright fluorescence under confocal microscope that proves NO or RNS accumulation is more in absence of flavohemoglobin. We further investigated PTN profile of both cytosol and mitochondria of Y190 and Δyhb1 cells of S. cerevisiae using two-dimensional (2D) gel electrophoresis followed by western blot analysis. Surprisingly, we observed many immunopositive spots both in cytosol and in mitochondria from Y190 and Δyhb1 using monoclonal anti-3-nitrotyrosine antibody indicating a basal level of NO or nitrite or peroxynitrite is produced in yeast system. To identify proteins nitrated in vivo we analyzed mitochondrial proteins from Y190 strains of S. cerevisiae. Among the eight identified proteins, two target mitochondrial proteins are aconitase and isocitrate dehydrogenase that are involved directly in the citric acid cycle. This investigation is the first comprehensive study to identify mitochondrial proteins nitrated in vivo.     

An inductive pulse of hydrogen peroxide pretreatment restores redox-homeostasis and oxidative membrane damage under extremes of temperature in two rice cultivars

Imbibitional heat and chilling stress caused disruption of redox-homeostasis and oxidative damage to newly assembled membrane system by aggravating membrane lipid peroxidation and protein oxidation [measured in terms of thiobarbituric acid reactive substances (TBARS), free carbonyl content (C=O groups) and membrane protein thiol level (MPTL)] along with concomitant increase in accumulation of reactive oxygen species (superoxide and hydrogen peroxide) and significant reduction of antioxidative defense (assessed in terms of total thiol content and activities of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) in both the salt sensitive (Ratna) and resistant (SR 26B) germinating tissues of rice cultivars. When compared, salt resistant cultivar SR 26B found to suffer significantly less oxidative membrane damage as compared to salt sensitive cultivar Ratna. Treatment with low titer of hydrogen peroxide caused significant reversal in oxidative damages to the newly assembled membrane system imposed by imbibitional heat and chilling stress (evident from the data of TBARS, C=O, MPTL, ROS accumulation, membrane permeability status, membrane injury index and oxidative stress index) in seedlings of both the cultivars of rice (Ratna and SR 26B). Imbibitional H₂O₂ pretreatment also caused up-regulation of antioxidative defense (activities of superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and total thiol content) in the heat and chilling stress-raised seedlings of experimental rice cultivars. When the parameters of early growth performances were assessed (in terms of relative growth index, biomass accumulation and vigor index), it clearly exhibited significant improvement of early growth performances of both the rice cultivars. Better response towards H₂O₂-mediated acclamatory performances and restoration of redox- homeostasis under extremes of temperature were noticed in salt sensitive rice cultivar Ratna compared to salt resistant SR 26B. Taken as a whole, the result suggests the significance of the role of ‘inductive pulse’ of H₂O₂ in acclimatizing adverse temperature stress by restoration of redox-homeostasis and mitigation of oxidative membrane protein and lipid damages during the recovery phase of post-germination event.     

RNA and β-Hemolysin of Group B Streptococcus Induce Interleukin-1β (IL-1β) by Activating NLRP3 Inflammasomes in Mouse Macrophages

The inflammatory cytokine IL-1β is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, β-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1β production.     

An erythrocyte vesicle protein exported by the malaria parasite promotes tubovesicular lipid import from the host cell surface

Plasmodium falciparum is the protozoan parasite that causes the most virulent of human malarias. The blood stage parasites export several hundred proteins into their host erythrocyte that underlie modifications linked to major pathologies of the disease and parasite survival in the blood. Unfortunately, most are 'hypothetical' proteins of unknown function, and those that are essential for parasitization of the erythrocyte cannot be 'knocked out'. Here, we combined bioinformatics and genome-wide expression analyses with a new series of transgenic and cellular assays to show for the first time in malaria parasites that microarray read out from a chemical perturbation can have predictive value. We thereby identified and characterized an exported P. falciparum protein resident in a new vesicular compartment induced by the parasite in the erythrocyte. This protein, named Erythrocyte Vesicle Protein 1 (EVP1), shows novel dynamics of distribution in the parasite and intraerythrocytic membranes. Evidence is presented that its expression results in a change in TVN-mediated lipid import at the host membrane and that it is required for intracellular parasite growth, but not invasion. This exported protein appears to be needed for the maintenance of an essential tubovesicular nutrient import pathway induced by the pathogen in the host cell. Our approach may be generalized to the analysis of hundreds of 'hypothetical' P. falciparum proteins to understand their role in parasite entry and/or growth in erythrocytes as well as phenotypic contributions to either antigen export or tubovesicular import. By functionally validating these unknowns, one may identify new targets in host-microbial interactions for prophylaxis against this major human pathogen.     

LC-ESI/MS determination of xanthone and secoiridoid glycosides from in vitro regenerated and in vivo Swertia chirayita

A rapid analytical method has been developed to determine xanthone and secoiridoid glycoside in in vitro and in vivo Swertia chirayita extracts. Ultra performance liquid chromatography–electrospray ionization mass spectrometry (LC-ESI/MS) was applied and validated for the analysis of xanthone and secoiridoid glycoside a potential active component isolated from methanolic extracts of in vitro and in vivo Swertia chirayita plantlets. Chromatographic separation was achieved on a RP-C18 column using gradient elution. Mangiferin (Xanthone), Amarogentin and Swertiamarin (Secoiridoid glycosides) were identified in both the extracts. In the LC/ESI-MS spectra, major [M + H] ⁺ and [M + Na] ⁺ ions were observed in positive ion mode and provided molecular mass information. An ultra-performance liquid-chromatography in combination with electrospray ionization tandem mass spectrometry involving metal cationisation was successfully utilized for the rapid identification of xanthone and secoiridoid glycosides. This method is suitable for the routine analysis, as well as for the separation and identification of known and novel secoiridoid glycoside and xanthone.     

Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na(+)) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na(+)/H(+) exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na(+)/H(+) exchange activity by Na(+)-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na(+)/H(+) exchange activity by >30%. Moreover, the sgk2-mediated increase in Na(+)/H(+) exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na(+) transport through NHE3 in the proximal tubule.     

Induction and complementation of lysine auxotrophs in Saccharomyces

Four chemical agents, EMS EMS: Ethyl methanesulfonate; MNNG: N-methyl-N\t'-nitro-N\t'-nitrosoguanidine; NA: Nitrous acid; ICR-170: 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl) aminopropylamino] acridine 2 HCl; UV: Ultra violet radiation. , MNNG, NA, ICR-170, as well as UV were used to induce mutations in the wild-type haploid strain X2180-1B (α) of Saccharomyces. A total of 2053 (EMS, 427; MNNG, 444; NA, 469; ICR-170, 456; UV, 257) lysine-requiring mutant clones were isolated from many independent treatments and by nystatin enrichment technique. Mutants were classified into various functional groups on the basis of complementation analysis with 14 tester strains (lys 1 to lys 15 except lys 3). Of the clones analyzed, the number of isolates unable to complement with a given tester strain ranged from 2 for lys 5 to 918 for lys 4. Three of the mutually complementing lysine loci (lys 1, lys 2, and lys 4) accounted together for over 85% of the mutant clones whereas lys 6, lys 7, lys 8, and lys 14 had less than 10 noncomplementing isolates each. Mutants for lys 4 were most frequent with all of the mutagens tested except with NA in which case the mutants for lys 2 were most frequent. A total of 56 isolates failed to complement with lys 10, lys 11, and lys 12. Similarly, 47 isolates failed to complement with lys 9 and lys 13 simultaneously. Only 44 isolates complemented with all of the tester strains used.     

Tumor T1 Relaxation Time for Assessing Response to Bevacizumab Anti-Angiogenic Therapy in a Mouse Ovarian Cancer Model

Purpose

To assess whether T₁ relaxation time of tumors may be used to assess response to bevacizumab anti-angiogenic therapy. Procedures: 12 female nude mice bearing subcutaneous SKOV3ip1-LC ovarian tumors were administered bevacizumab (6.25ug/g, n=6) or PBS (control, n=6) therapy twice a week for two weeks. T₁ maps of tumors were generated before, two days, and 2 weeks after initiating therapy. Tumor weight was assessed by MR and at necropsy. Histology for microvessel density, proliferation, and apoptosis was performed.

Results

Bevacizumab treatment resulted in tumor growth inhibition (p<0.04, n=6), confirming therapeutic efficacy. Tumor T₁ relaxation times increased in bevacizumab treated mice 2 days and 2 weeks after initiating therapy (p<.05, n=6). Microvessel density decreased 59% and cell proliferation (Ki67+) decreased 50% in the bevacizumab treatment group (p<.001, n=6), but not apoptosis.

Conclusions

Findings suggest that increased tumor T₁ relaxation time is associated with response to bevacizumab therapy in ovarian cancer model and might serve as an early indicator of response.