Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Background

Peroxynitrite, the product of the reaction between superoxide radicals and nitric oxide, is an elusive oxidant with a short half-life and a low steady-state concentration in biological systems; it promotes nitroxidative damage.

Scope of review

We will consider kinetic and mechanistic aspects that allow rationalizing the biological fate of peroxynitrite from data obtained by a combination of methods that include fast kinetic techniques, electron paramagnetic resonance and kinetic simulations. In addition, we provide a quantitative analysis of peroxynitrite production rates and conceivable steady–state levels in living systems.

Major conclusions

The preferential reactions of peroxynitrite in vivo include those with carbon dioxide, thiols and metalloproteins; its homolysis represents only < 1% of its fate. To note, carbon dioxide accounts for a significant fraction of peroxynitrite consumption leading to the formation of strong one-electron oxidants, carbonate radicals and nitrogen dioxide. On the other hand, peroxynitrite is rapidly reduced by peroxiredoxins, which represent efficient thiol-based peroxynitrite detoxification systems. Glutathione, present at mM concentration in cells and frequently considered a direct scavenger of peroxynitrite, does not react sufficiently fast with it in vivo; glutathione mainly inhibits peroxynitrite-dependent processes by reactions with secondary radicals. The detection of protein 3-nitrotyrosine, a molecular footprint, can demonstrate peroxynitrite formation in vivo. Basal peroxynitrite formation rates in cells can be estimated in the order of 0.1 to 0.5 μM s^− 1 and its steady-state concentration at ~ 1 nM.

General significance

The analysis provides a handle to predict the preferential fate and steady-state levels of peroxynitrite in living systems. This is useful to understand pathophysiological aspects and pharmacological prospects connected to peroxynitrite. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Trimethylamine N-oxide suppresses the activity of the actomyosin motor

Background

During actomyosin interactions, the transduction of energy from ATP hydrolysis to motility seems to occur with the modulation of hydration. Trimethylamine N-oxide (TMAO) perturbs the surface of proteins by altering hydrogen bonding in a manner opposite to that of urea. Hence, we focus on the effects of TMAO on the motility and ATPase activation of actomyosin complexes.

Methods

Actin and heavy meromyosin (HMM) were prepared from rabbit skeletal muscle. Structural changes in HMM were detected using fluorescence and circular dichroism spectroscopy. The sliding velocity of rhodamine-phalloidin-bound actin filaments on HMM was measured using an in vitro motility assay. ATPase activity was measured using a malachite green method.

Results

Although TMAO, unlike urea, stabilized the HMM structure, both the sliding velocity and ATPase activity of acto-HMM were considerably decreased with increasing TMAO concentrations from 0–1.0 M. Whereas urea-induced decreases in the structural stability of HMM were recovered by TMAO, TMAO further decreased the urea-induced decrease in ATPase activation. Urea and TMAO were found to have counteractive effects on motility at concentrations of 0.6 M and 0.2 M, respectively.

Conclusions

The excessive stabilization of the HMM structure by TMAO may suppress its activities; however, the counteractive effects of urea and TMAO on actomyosin motor activity is distinct from their effects on HMM stability.

General significance

The present results provide insight into not only the water-related properties of proteins, but also the physiological significance of TMAO and urea osmolytes in the muscular proteins of water-stressed animals.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.     

Insight into genetic determinants of resting heart rate

Background

Recent studies suggested that resting heart rate (RHR) might be an independent predictor of cardiovascular mortality and morbidity. Nonetheless, the interrelation between RHR and cardiovascular diseases is not clear. In order to resolve this puzzle, the importance of genetic determinants of RHR has been recently suggested, but it needs to be further investigated.

Objective

The aim of this study was to estimate the contribution of common genetic variations on RHR using Genome Wide Association Study.

Methods

We performed a Genome Wide Association Study in an isolated population cohort of 1737 individuals, the Italian Network on Genetic Isolates — Friuli Venezia Giulia (INGI-FVG). Moreover, a haplotype analysis was performed. A regression tree analysis was run to highlight the effect of each haplotype combination on the phenotype.

Results

A significant level of association (p < 5 × 10^− 8) was detected for Single Nucleotide Polymorphisms (SNPs) in two genes expressed in the heart: MAML1 and CANX. Founding that the three different variants of the haplotype, which encompass both genes, yielded a phenotypic correlation. Indeed, a haplotype in homozygosity is significantly associated with the lower quartile of RHR (RHR ≤ 58 bpm). Moreover no significant association was found between cardiovascular risk factors and the different haplotype combinations.

Conclusion

Mastermind-like 1 and Calnexin were found to be associated with RHR. We demonstrated a relation between a haplotype and the lower quartile of RHR in our populations. Our findings highlight that genetic determinants of RHR may be implicated in determining cardiovascular diseases and could allow a better risk stratification.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity

Aims

Aristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and N^ε-(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger.

Methods

Mice were orally administered water or metformin for 15 days (12 or 24 mg kg^− 1 day^− 1), and injected AA (5 mg kg^− 1 day^− 1) intraperitoneally for 8 days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed.

Key findings

Compared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p < 0.05). Further, compared to control treatment, administration of 12 mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33 ± 9.78 vs. 5.89 ± 2.64 μg/mg of protein, respectively, p < 0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity.

Significance

This study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

Regulation of post-translational protein arginine methylation during HeLa cell cycle

Background

Post-translational arginine methylation which modifies protein-arginyl residues by protein arginine methyltransferase (PRMT) was investigated during synchronized HeLa cell cycle.

Methods

The lysates of cells synchronized at each stage were subjected to one and/or two dimensional electrophoresis followed by Western immunoblot using against anti-asymmetric-dimethyl-arginine (ASYM24), anti-symmetric-dimethyl-arginine (SYM10), and subclasses of PRMTs, including PRMT1, PRMT3, PRMT4 (CARM1), PRMT5, PRMT6, and PRMT7 antibodies.

Results

Proteins with approximate molecular masses of 80 kDa, 68 kDa, and 64 kDa, containing asymmetric-dimethyl-arginine (aDMA) were increased at G0/G1 to G1, which lasted until S phase. In addition, 25 kDa protein of symmetric-dimethyl-arginine (sDMA) was also markedly up-regulated from G0/G1 to G1. The levels of PRMT3, PRMT6 and PRMT7 were concurrently increased during the cell cycle. Two-dimensional gel electrophoresis followed by MALDI-TOF-MS was identified as aDMA-80 kDa and aDMA-68 kDa proteins as heterogeneous nuclear ribonucleoprotein R (hnRNPR), aDMA-64 kDa proteins as cleavage stimulation factor 64 kDa subunit (CstF-64), and sDMA-25 kDa protein as triosephosphate isomerase (TPI). The levels of increased aDMA of hnRNPR were reduced, when HeLa cells were transfected with siRNA for PRMT1, and the aDMA of CstF-64 with siRNA for PRMT3, while depletion of PRMT5 down-regulated sDMA of TPI.

Conclusion

Protein arginine dimethylations of hnRNPR, CstF-64, and TPI were regulated during HeLa cell cycle by respective PRMTs.

General significance

These results suggest that regulation of arginine dimethylation of hnRNPR, CstF-64, and TPI at G0/G1 to G1 are most likely to modulate the cellular growth and proliferation in HeLa cell cycle.     

A rational,non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Context

Molecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward.

Objective

To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis.

Patients and methods

We studied 99 patients from 90 families with salt-wasting (SW; n = 32), simple-virilizing (SV; n = 29), and non-classical (NC; n = 29) CAH-21OHD. Molecular analysis was sequentially performed by detecting the most frequent point mutations by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), large rearrangements by MLPA, and rare mutations by direct sequencing. Parental segregation was evaluated.

Results

ASO-PCR detected microconversions in 164 alleles (91.1%). MLPA identified CYP21A1P large conversions to CYP21A2 in 7 of the remaining 16 (43.7%), 30-kb deletions including the 3′-end of CYP21A1P, C4B, and the 5′-end of CYP21A2 in 3 of the 16 (18.7%), and a complete CYP21A2 deletion in one (6.3%). Five alleles (2.7%) required direct sequencing; three mutations located in the CYP21A2 gene and two derived from CYP21A1P were found. No parental segregation was observed in patients with the c.329_336del and/or the CL6 cluster mutations. These cases were not diagnosed by ASO-PCR, but MLPA detected deletions in the promoter region of the CYP21A2 gene, explaining the genotype/phenotype dissociation.

Conclusion

Using the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD.     

Whole blood,flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion

Background

Thymosin beta 4 (Tβ₄) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.

Methods

In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ₄, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.

Results

The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s^− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ₄ (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ₄-potentiating effect was diminished to near control levels. Tβ₄ did interact with fibrinogen with an estimated K_D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.

Conclusion

These results suggest that Tβ₄ could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ₄ could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.

General significance

This work suggests that Tβ₄ might have a dual role in platelet function.     

Polymorphisms of the TLR4 gene and risk of gastric cancer

Objective

Toll-like receptor 4 (TLR4) is an important lipo-polysaccharide (LPS) receptor in gastric epithelial cell signaling transduction and plays critical roles in the development and progression of gastric cancer (GC). We investigated the effects of TLR4 gene polymorphisms and gene–environmental interactions on the risk of GC in Northeastern China.

Methods

We genotyped two single-nucleotide polymorphisms (SNPs) in TLR4 (rs10116253 and rs1927911) in 217 GC patients and 294 cancer-free controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio (OR) and 95% confidence intervals (CIs) were estimated by unconditional logistic-regression models.

Results

Individuals carrying CC genotype of rs10116253 and TT genotype of rs1927911 had a significantly decreased risk of GC (adjusted OR = 0.33, 95% CI 0.18–0.60, P < 0.001 and adjusted OR = 0.37, 95% CI 0.21–0.67, P = 0.001 respectively), compared with TT genotype of rs10116253 and CC genotype of rs1927911. In addition, the SNP effects were additive to the effects of some known environmental factors without any interaction between them in the susceptibility to GC.

Conclusion

Our data suggested that TLR4 gene polymorphisms may be associated with a decreased risk of GC in Chinese population. And these SNPs and their combined effects with environmental factors may be associated with the risk of GC.     

The impact of GGH -401C > T polymorphism on cisplatin-based chemoradiotherapy response and survival in cervical cancer

Aims

Cervical cancer is the third most frequent cancer in women worldwide, mostly treated with cisplatin-based chemoradiotherapy. Since it is known that folate metabolism might interfere with cisplatin effectiveness, we intended to study the influence of the Gamma Glutamyl Hydrolase -401C > T polymorphism in treatment response in cervical cancer.

Methods

We retrospectively reviewed the clinical data of 167 patients with bulky cervical cancer submitted to cisplatin-based chemoradiotherapy. The genotypes of GGH -401C > T SNP were determined by real-time PCR and statistical analysis was performed by χ² test and survival analysis.

Results

The genotypes of GGH-401C > T were significantly associated with the response to platinum-based chemoradiotherapy. Treatment response was higher in patients carrying the CC genotype, who presented a significant increased chance of treatment response (survival time in months/genotype: 91 for CC Vs 72 for CT/TT; p = 0.035, log rank test). A Cox regression analysis accordingly showed that the presence of the T allele was significantly linked to a worse treatment response (HR = 3.036; CI 95% 1.032-8.934, p = 0.044).

Conclusions

The results of our study suggested the potential interest of GGH -401C > T as a predictive factor of the outcome of cervical carcinoma treated with cisplatin-based chemoradiotherapy.     

XRCC3 Thr241Met polymorphism and risk of acute myeloid leukemia in a Romanian population

Background

DNA repair systems have a critical role in maintaining the genome integrity and stability. DNA repair gene polymorphisms may influence the capacity to repair DNA damage, and thus lead to an increased cancer susceptibility. X-ray repair cross-complementing groups 3 (XRCC3), a DNA repair gene, may be involved in acute myeloid leukemia susceptibility. The objective of the current study was to investigate the association of Thr241Met polymorphism of XRCC3 gene with the risk of acute myeloid leukemia (AML).

Methods

This study included 78 AML patients and 121 healthy individuals without cancer. We used polymerase chain reaction-restriction fragment length polymorphism assay to determine XRCC3 genotypes.

Results

The XRCC3 variant genotype (Thr/Met+Met/Met) was more frequent in AML patients than in healthy controls (OR = 2.76, 95% CI: 1.52-4.98, P = 0.001). Our study revealed a statistically significant association between variant genotype (Thr/Met+Met/Met) and AML de novo compared to secondary AML (P = 0.007). No significant associations were found between any genotype and age at diagnosis, number of white blood cells and subtype of AML. Overall survival of patients with Thr/Thr genotype was better than those of variant Thr/Met and Met/Met genotypes.

Conclusions

Our findings indicate that the XRCC3 Thr241Met polymorphism may be a genetic risk factor for AML, particularly in male patients with de novo AML from the central part of Romania.     

The contribution of platelet glycoproteins (GPIa C807T and GPIba C-5T) and cyclooxygenase 2 (COX-2G-765C) polymorphisms to platelet response in patients treated with aspirin

Objective

Aspirin is an antiplatelet agent commonly used in treatment of patients with high risk to develop stroke and myocardial infarction. However, inter-individual variability regarding the inhibition of platelet function by aspirin is well documented. In this study, the correlation between platelet glycoproteins (GPIa C807T and GPIba C-5T) and cyclooxygenase 2 (COX-2G-765C) polymorphisms and antiplatelet response in patients treated with aspirin was investigated.

Methods

Jordanian adult patients (n = 584) who are taking aspirin as an antiplatelet agent participated in the study. Platelet aggregation response was measured using Multiplate Analyzer® system. Polymerase chain reaction–restriction fragment length polymorphism assay (PCR–RFLP) was used for genotyping of the examined polymorphisms.

Results

Aspirin resistance was found in 15.8% of patients. Response to aspirin was significantly associated with GPIba C-5T polymorphism (P < 0.05). However, the GPIa C807T and COX-2G-765C polymorphisms were not related to aspirin resistance (P > 0.05).

Conclusion

A considerable fraction of the Jordanian population is resistant to the antiplatelet effect of aspirin, which might be related to GPIba C-5T polymorphism.     

Association between fibromyalgia syndrome and polymorphism of the IL-4 gene in a Turkish population

Purpose

Fibromyalgia (FM) syndrome is a form of non-articular rheumatism characterized by long term and widespread musculoskeletal pain, morning stiffness, sleep disturbance, paresthesia, and pressure hyperalgesia at characteristic sites, called soft tissue tender points. The etiology of FM is still obscure. Genetic factors may predispose individuals to FM. Cytokines may play a role in the pathophysiology of FM. The aim of this study was to investigate the interleukin-4 (IL-4) 70 bp VNTR variations in Turkish patients with FM and evaluate if there was an association with clinical features, especially between these polymorphisms.

Methods

The study included 300 patients with FM and 270 healthy controls. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR) for the IL-4 gene 70 bp VNTR polymorphisms.

Results

There was statistically significant difference between the groups with respect to IL-4 genotype distribution and allele frequencies (p < 0.0001). The homozygous P₁P₁ genotype and P₁ allele were significantly higher in FM patients than in healthy controls (p = 0.04; OR: 3.25, 95% CI: 1–10, p < 0.0001; OR:4.84, 95% CI:3–7.7). There was not any difference between the groups respect to IL-4 genotype distribution and allele frequencies (p > 0.05) and clinical characteristics.

Conclusion

Our findings suggest that there is an association of IL-4 gene 70 bp VNTR polymorphism with susceptibility of a person for development of FM. As a result, further studies are necessary to determine whether IL-4 may be a genetic marker for FM in the Turkish population.     

The role of calpain in an in vivo model of oxidative stress-induced retinal ganglion cell damage

Purpose

In this study, we set out to establish an in vivo animal model of oxidative stress in the retinal ganglion cells (RGCs) and determine whether there is a link between oxidative stress in the RGCs and the activation of calpain, a major part of the apoptotic pathway.

Materials and methods

Oxidative stress was induced in the RGCs of C57BL/6 mice by the intravitreal administration of 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH, 30 mM, 2 μl). Control eyes were injected with 2 μl of vehicle. Surviving Fluorogold (FG)-labeled RGCs were then counted in retinal flat mounts. Double staining with CellROX and Annexin V was performed to investigate the co-localization of free radical generation and apoptosis. An immunoblot assay was used both to indirectly evaluate calpain activation in the AAPH-treated eyes by confirming α-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100 mg/kg, intraperitoneal administration) in these eyes.

Results

Intravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7 days after treatment (control: 3806.7 ± 575.2 RGCs/mm², AAPH: 3156.1 ± 371.2 RGCs/mm², P < 0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24 h after AAPH injection. An immunoblot assay revealed a cleaved α-fodrin band that increased significantly 24 h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of α-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0 ± 226.9 RGCs/mm², AAPH+SNJ-1945: 3717.1 ± 614.6 RGCs/mm², P < 0.01).

Conclusion

AAPH administration was an effective model of oxidative stress in the RGCs, showing that oxidative stress directly activated the calpain pathway and induced RGC death. Furthermore, inhibition of the calpain pathway protected the RGCs after AAPH administration.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid

Background

Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication.

Methods

In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors.

Results

Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with K_i of 17 ± 5 μM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with K_i of 32 ± 5 μM and IC₅₀ of 177 ± 5 nM, respectively.

Conclusions

This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups.

General significance

This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.     

Impaired cardiac mitochondrial function and contractile reserve following an acute exposure to environmental particulate matter

Background

It has been suggested that mitochondrial function plays a central role in cardiovascular diseases associated with particulate matter inhalation. The aim of this study was to evaluate this hypothesis, with focus on cardiac O₂ and energetic metabolism, and its impact over cardiac contractility.

Methods

Swiss mice were intranasally instilled with either residual oil fly ash (ROFA) (1.0 mg/kg body weight) or saline solution. After 1, 3 or 5 h of exposure, O₂ consumption was evaluated in heart tissue samples. Mitochondrial respiration, respiratory chain complexes activity, membrane potential and ATP content and production rate were assessed in isolated mitochondria. Cardiac contractile reserve was evaluated according to the Langendorff technique.

Results

Three hours after ROFA exposure, tissue O₂ consumption was significantly decreased by 35% (from 1180 ± 70 to 760 ± 60 ng-at O/min g tissue), as well as mitochondrial rest (state 4) and active (state 3) respiration, by 30 and 24%, respectively (control state 4: 88 ± 5 ng-at O/min mg protein; state 3: 240 ± 20 ng-at O/min mg protein). These findings were associated with decreased complex II activity, mitochondrial depolarization and deficient ATP production. Even though basal contractility was not modified (control: 75 ± 5 mm Hg), isolated perfused hearts failed to properly respond to isoproterenol in ROFA-exposed mice. Tissue O₂ consumption rates positively correlated with cardiac contractile state in controls (r² = 0.8271), but not in treated mice (r² = 0.1396).

General Significance

The present results show an impaired mitochondrial function associated with deficient cardiac contractility, which could represent an early cardiovascular alteration after the exposure to environmental particulate matter.