Extensive natural variation for cellular hydrogen peroxide release is genetically controlled.

Natural variation in DNA sequence contributes to individual differences in quantitative traits. While multiple studies have shown genetic control over gene expression variation, few additional cellular traits have been investigated. Here, we investigated the natural variation of NADPH oxidase-dependent hydrogen peroxide (H(2)O(2) release), which is the joint effect of reactive oxygen species (ROS) production, superoxide metabolism and degradation, and is related to a number of human disorders. We assessed the normal variation of H(2)O(2) release in lymphoblastoid cell lines (LCL) in a family-based 3-generation cohort (CEPH-HapMap), and in 3 population-based cohorts (KORA, GenCord, HapMap). Substantial individual variation was observed, 45% of which were associated with heritability in the CEPH-HapMap cohort. We identified 2 genome-wide significant loci of Hsa12 and Hsa15 in genome-wide linkage analysis. Next, we performed genome-wide association study (GWAS) for the combined KORA-GenCord cohorts (n = 279) using enhanced marker resolution by imputation (>1.4 million SNPs). We found 5 significant associations (p<5.00×10-8) and 54 suggestive associations (p<1.00×10-5), one of which confirmed the linked region on Hsa15. To replicate our findings, we performed GWAS using 58 HapMap individuals and ∼2.1 million SNPs. We identified 40 genome-wide significant and 302 suggestive SNPs, and confirmed genome signals on Hsa1, Hsa12, and Hsa15. Genetic loci within 900 kb from the known candidate gene p67phox on Hsa1 were identified in GWAS in both cohorts. We did not find replication of SNPs across all cohorts, but replication within the same genomic region. Finally, a highly significant decrease in H(2)O(2) release was observed in Down Syndrome (DS) individuals (p<2.88×10-12). Taken together, our results show strong evidence of genetic control of H(2)O(2) in LCL of healthy and DS cohorts and suggest that cellular phenotypes, which themselves are also complex, may be used as proxies for dissection of complex disorders.

Mechanisms of hydrogen peroxide-induced vasoconstriction in the isolated perfused rat kidney.

The vasoconstrictor effect of hydrogen peroxide (H(2)O(2)) on isolated perfused rat kidney was investigated. H(2)O(2) induced vasoconstriction in the isolated rat kidney in a concentration-dependent manner. The vasoconstrictor effects of H(2)O(2) were completely inhibited by 1200 U/ml catalase. Endothelium-removal potentiated the renal response to H(2)O(2). The H(2)O(2) dose-response curve was not significantly modified by administration of the NO inhibitor L-NAME (10(-4) mol/l), whereas it was increased by the non-specific inhibitor of K+-channels, tetraethylammonium (3.10(-3) mol/l). Separately, removal of extracellular Ca(2+), administration of a mixture of calcium desensitizing agents (nitroprusside, papaverine, and diazoxide), and administration of a protein kinase C (PKC) inhibitor (chelerythrine, 10(-5) mol/l) each significantly attenuated the vasoconstrictor response to H(2)O(2), which was virtually suppressed when they were performed together. The pressor response to H(2)O(2) was not affected by: dimethyl sulfoxide (7.10(-5) mol/l) plus mannitol (3.10(-5) mol/l); intracellular Ca(2+) chelation using BAPTA (10(-5) mol/l); calcium store depletion after repeated doses of phenylephrine (10(-5) g/g kidney); or the presence of indomethacin (10(-5) mol/l), ODYA (2.10(-6) mol/l) or genistein (10(-5) mol/l). We conclude that the vasoconstrictor response to H(2)O(2) in the rat renal vasculature comprises the following components: 1) extracellular calcium influx, 2) activation of PKC, and 3) stimulation of pathways leading to sensitization of contractile elements to calcium. Moreover, a reduced pressor responsiveness to H(2)O(2) in female kidneys was observed.

Production of TNF-α, nitric oxide and hydrogen peroxide by macrophages from mice with paracoccidioidomycosis that were fed a linseed oil-enriched diet

Omega-3 polyunsaturated fatty acids (n-3 PUFA) can modulate the immune system and their primary effect is on macrophage function. Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America that is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). Macrophages are the main defence against this pathogen and have microbicidal activity that is dependent on interferon-Γ and tumour necrosis factor (TNF)-α. These cytokines stimulate the synthesis of nitric oxide (NO) and hydrogen peroxide (H2O2), leading to the death of the fungus. To study the effect of n-3 PUFA on the host immune response during experimental PCM, macrophages that were obtained from animals infected with Pb18 and fed a diet enriched by linseed (LIN) oil were cultured and challenged with the fungus in vitro. The macrophage function was analysed based on the concentrations of TNF-α, NO and H2O2. LIN oil seems to influence the production of TNF-α during the development of disease. A diet enriched with LIN oil influences the microbicidal activity of the macrophages by inducing the production of cytokines and metabolites such as NO and H2O2, predominantly in the chronic phase of infection.

The level of ascorbate peroxidase is enhanced in benznidazole-resistant populations of Trypanosoma cruzi and its expression is modulated by stress generated by hydrogen peroxide

Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide.

Stool sample storage conditions for the preservation of Giardia intestinalis DNA

Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.

Dual control of hydrogen cyanide biosynthesis by the global activator GacA in Pseudomonas aeruginosa PAO1.

The global response regulator GacA of Pseudomonas aeruginosa PAO1 positively controls the production of the quorum sensing signal molecule N-butanoyl-homoserine-lactone (C4-HSL) and hence the synthesis of several C4-HSL-dependent virulence factors, including hydrogen cyanide (HCN). This study presents evidence that GacA positively influences the transcription of the rhlI gene, specifying C4-HSL synthase, explaining the quorum sensing-dependent transcriptional control of the HCN biosynthetic genes (hcnABC). In addition, GacA was found to modulate hcn gene expression positively at a post-transcriptional level involving the hcnA ribosome-binding site. Thus, the activating effect of GacA on cyanogenesis results from both transcriptional and post-transcriptional mechanisms.

Intraovarian sperm storage in Helicolenus dactylopterus dactylopterus fertilitzation, crypt formation and maintenance of stored sperm

The females of the bluemouth rockfish, Helicolenus dactylopterus dactylopterus (DelaRoche, 1809), store sperm within their ovaries for periods of up to 10 months. Twenty six females with standard lengths between 152 and 257 mm and six males with standard lengths between 253 and 209 mm were caught storage crypts with stored spermatozoa and to describe their evolution over the year. After internal fertilization and once sperm reaches the ovary, a crypt forms probably by an epithelial inclusion at the base of the lamellae of one or several spermatozoa groups that are floating freely in the interlamellar space of the ovarian lumen. Stored spermatozoa have a large cytoplasm bag surrounding their heads. This bag could serve as a nutritive reservoir during the long storage period. Many desmosonal and tight junctions between the crypt cells ensure tha male sex cells are protected against the female immune system

Reliable and persistent storage for CoDeS a distributed collaborative system

This research is aimed to find a solution for a distributed storage system adapted for CoDeS. By studying how DSSs work and how they are implemented, we can conclude how we can implement a DSS compatible with CoDeS requirements.

Iowa Petroleum Underground Storage Tank Board, June 30, 2002

State Agency Audit Report

Basis set superposition error-counterpoise corrected potential energy surfaces : application to hydrogen peroxide ... X (X=F-, Cl-, Br-, Li+, Na+) complexes

Møller-Plesset (MP2) and Becke-3-Lee-Yang-Parr (B3LYP) calculations have been used to compare the geometrical parameters, hydrogen-bonding properties, vibrational frequencies and relative energies for several X- and X+ hydrogen peroxide complexes. The geometries and interaction energies were corrected for the basis set superposition error (BSSE) in all the complexes (1-5), using the full counterpoise method, yielding small BSSE values for the 6-311 + G(3df,2p) basis set used. The interaction energies calculated ranged from medium to strong hydrogen-bonding systems (1-3) and strong electrostatic interactions (4 and 5). The molecular interactions have been characterized using the atoms in molecules theory (AIM), and by the analysis of the vibrational frequencies. The minima on the BSSE-counterpoise corrected potential-energy surface (PES) have been determined as described by S. Simón, M. Duran, and J. J. Dannenberg, and the results were compared with the uncorrected PES

How does basis set superposition error change the potential surfaces for hydrogen-bonded dimers?

We describe a simple method to automate the geometric optimization of molecular orbital calculations of supermolecules on potential surfaces that are corrected for basis set superposition error using the counterpoise (CP) method. This method is applied to the H-bonding complexes HF/HCN, HF/H2O, and HCCH/H2O using the 6-31G(d,p) and D95 + + (d,p) basis sets at both the Hartree-Fock and second-order Møller-Plesset levels. We report the interaction energies, geometries, and vibrational frequencies of these complexes on the CP-optimized surfaces; and compare them with similar values calculated using traditional methods, including the (more traditional) single point CP correction. Upon optimization on the CP-corrected surface, the interaction energies become more negative (before vibrational corrections) and the H-bonding stretching vibrations decrease in all cases. The extent of the effects vary from extremely small to quite large depending on the complex and the calculational method. The relative magnitudes of the vibrational corrections cannot be predicted from the H-bond stretching frequencies alone

Iowa Petroleum Underground Storage Tank Board State of Iowa, Independent Auditor's Reports Financial Statements and Supplementary Information, June 30, 2003

State Agency Audit Report

Spermiogenesis particularities of a sperm storage species: Helicolenus dactylopterus (Teleostei: Scorpaenidae)

A study of the spermiogenesis and spermatozoa of Helicolenus dactylopterus was conducted. Females of this species have the capacity to store sperm within their ovaries, and male gametes have a considerable cytoplasmic mass surrounding their heads to survive the long period of intraovarian sperm storage. Our observations show that early spermatids are round-shaped cells and have a spherical nucleus with diffuse chromatin. The nuclear volume decreases as a result of progressive chromatin condensation during spermiogenesis, causing the nucleus to take on a U-shape. Flagellar insertion is not central to the nucleus but consistently occurs at an oblique angle towards one side of it. The flagellum is inserted into the nuclear fossa, without subsequent nuclear rotation. In mature spermatozoa, the flagellum is adjacent to the nucleus. A comparison of the spermatozoa in the testicular lobules and those in the intraovarian storage structures suggests that the increase in volume of the cytoplasmic mass may occur in the posterior region of the testis, in the testicular duct. Spermatozoa enter the ovary in groups that reach the ovarian lumen and are surrounded by the ovarian epithelium for storage in sperm storage crypts

The hospital pharmacist: an important contributor to improved patient safety in the hospital.

Injectable drugs are high-risk products and their reconstitution in hospital wards is a potential source of errors. Thus, in order to secure the reconstitution process and thereby improve safety, the pharmacy department of Lausanne University Hospital is focusing on developing ready-to-use forms (CIVAS). These preparations are compounded in controlled clean rooms and are analyzed prior to release. In the intensive care unit, amiodarone 12.5 mg/mL in glucose 5% is one of the high-risk preparations, which has led the pharmacy to develop a ready-to-use solution. To this end, a one-year stability study was initiated, and the preliminary results (after six months) are illustrated here. A stability-indicating HPLC method was developed and validated for monitoring the concentration of amiodarone. Batches were stored at 5 °C and 30 °C, which were analyzed immediately after preparation, after one, two, four and six months of storage. The pH and osmolality values were monitored at the respective time intervals. It was observed that after six months, all the results were within specifications. However, the pH values started to decrease after two months when amiodarone was stored at 30 °C. After six months, a degradation peak appeared on the chromatogram of these solutions, which suggested that amiodarone is more stable at 5 °C. The preliminary results obtained in this study indicated that injectable amiodarone solutions are stable for six months under refrigerated storage conditions. The study is ongoing.

Iowa Petroleum Underground Storage Tank Board, State of Iowa, Independent Auditor's Reports, Financial Statements and Supplementary Information, June 30, 2004

State Audit Reports