The kinetics of oxidation of ferroperoxidase by molecular oxygen. A model of a terminal oxidase

1. The decay of oxyferroperoxidase to ferriperoxidase was studied by rapidly mixing solutions of ferroperoxidase with various amounts of oxygen and following the time-course of appearance of oxyferroperoxidase and its subsequent decay to ferriperoxidase by reaction with ferroperoxidase. 2. The scheme can be accommodated by and [Formula: see text] and occurs without detectable intermediates being observed in the millisecond time-range. 3. Analogue-computer simulation of the reaction is in agreement with the initial rate-limiting reaction being an intermolecular, not intramolecular, electron-donating process, and analysis of the data leads to quantitative values for the rate constants of the overall process. 4. The reaction of oxyferroperoxidase with ferroperoxidase is a model of a terminal oxidase, and the results are discussed in terms of the possible importance of this reaction in peroxidase function, and also in the light it throws on autoxidation of haem compounds.     

Molecular cloning,expression in Escherichia coli and production of bioactive homogeneous recombinant human granulocyte and macrophage colony stimulating factor

Human granulocyte and macrophage colony stimulating factor (hGM-CSF) is a glycoprotein that activates and enhances the differentiation and survival of neutrophils, eosinophils and macrophages, which play a key role in the innate immune response. Here we describe the construction of the hGM-CSF encoding gene, cloning, expression in Escherichia coli, purification of recombinant hGM-CSF, N-terminal amino acid sequencing, and biological activity assay using TF-1 cells. The results presented show that the combination of experimental strategies employed to obtain recombinant hGM-CSF can yield biologically active protein, and may be useful to scaling-up production of biosimilar protein.     

The effect of water quality in the life cycle and in the attraction for the egg oviposition of Aedes aegypti (L.) (Diptera: Culicidae)

The present research aimed at evaluating the influence of the water quality in the life cycle and attraction of Aedes aegypti (L.) females to oviposit using different sources of water (raw sewage, effluent of UASB reactor, effluent of polishing lagoon, effluent of anaerobic filter, rain water and de-chlorinated water). The immature development time and survivorship were evaluated on a daily basis in two distinct feeding systems (with and without food). The quality of the water was shown to affect the egg and larval stages, but not the pupal or the adult. In the absence of food, no development was observed in rain water and de-chlorinated water. Immature development was faster in water sources from raw sewage, although with the lowest survivorship (37.3%). Free-choice tests indicated that females preferred to lay most of their eggs on water collected from the effluent of a UASB reactor, achieving the highest oviposition activity index (OAI) of 0.57. In non-choice tests, females laid larger batches of eggs in water collected from anaerobic filters (204.8 eggs), with the lowest number of eggs being laid on de-chlorinated water (37.3 eggs). It can be concluded that A. aegypti does not demonstrate any particular preference to lay eggs on clean water. This has serious implications for developing strategies to manage populations of this important vector in urban areas as it was shown to lay eggs and successfully develop on several different sources of water.     

Occurrence of four‐spined Gasterosteus aculeatus in an isolated Croatian river population

An isolated population of the three‐spined stickleback Gasterosteus aculeatus in Croatia was found to have a high incidence of specimens either having a fourth dorsal spine or showing remnants of a fourth spine. Juvenile individuals showed a 9·4% incidence of a fourth spine. The population was examined for asymmetry of the skeletal defensive complex in order to determine whether the additional spine could be the result of developmental instability, a response to predation or environmental conditions.     

Early development of the endemic dace Telestes karsticus (Leuciscidae,Cypriniformes) in a Dinaric karst stream in Croatia

Early development of the endemic dace Telestes karsticus is described in a Dinaric karst stream in Croatia. Larvae were acquired from successful rearing in an aquarium. Basic morphometric and meristic measures of larvae were taken during 55 days of observation. The mean total length of larvae at the time of hatching was 6.45 mm. The yolk sac was completely absorbed in all larvae by day 9 post-hatching (PH), when the mouth also opened and independent feeding and intestinal function were observed. Flexion of the urostyle was completed between days 12–16 PH. The caudal fin became completely forked on day 23 PH. Formation of fin rays was complete on day 48 PH when fish fully resembled juvenile specimens. The number of soft ray fins corresponds to the adult. Understanding the early development is particularly valuable in assessing environmental impacts and recovering of this endangered species and it is essential for effective monitoring and its conservation. This is the first report of the early development for the endemic Telestes karsticus, pointing out the importance of the obtained results for the effective conservation of the species.     

Flight responses of Sitotroga cerealella (Lepidoptera: Gelechiidae) to corn kernel volatiles in a wind tunnel

The flight behavioral responses of males and females of Sitotroga cerealella (Lepidoptera: Gelechiidae) to corn kernel volatiles, in a wind tunnel, and the determination and identification of the active compounds of corn kernel volatiles were investigated. A wind tunnel bioassay was carried out to test the response of males and females to 15, 30, 60, and 120 g equivalents of corn kernel volatiles. Gas chromatography-electroantennography was used to determine which components in corn kernel volatiles elicited an EAD response using male and female antennae. Gas chromatography-mass spectrometry technique was used to identify the active compounds of corn kernel volatiles. A 60-g equivalent of corn kernel volatiles elicited attractant behavior of unmated males to landing near the source. Mated males and virgin females were not attracted to corn kernel volatiles. The number of mated females landing was significantly higher in 60- and 120-g equivalents of corn kernel volatiles, compared to the other concentrations and the control. The antennae of males and females responded to four active compounds of corn kernel volatiles. Three of these compounds were identified, namely nonanal, decanal, and geranyl acetone. It is suggested that host finding and selection by S. cerealella is associated with its host volatiles.     

Antitumor Effect of the Essential Oil from Leaves of Guatteria pogonopus (Annonaceae)

Guatteria pogonopus Martius , a plant belonging to the Annonaceae family, is found in the remaining Brazilian Atlantic Forest. In this study, the chemical composition and antitumor effects of the essential oil isolated from leaves of G. pogonopus was investigated. The chemical composition of the oil was determined by GC‐FID and GC/MS analyses. The in vitro cytotoxicity was evaluated against three different tumor cell lines (OVCAR‐8, NCI‐H358M, and PC‐3M), and the in vivo antitumor activity was tested in mice bearing sarcoma 180 tumor. A total of 29 compounds was identified and quantified in the oil. The major compounds were γ‐patchoulene (13.55%), (E)‐caryophyllene (11.36%), β‐pinene (10.37%), germacrene D (6.72%), bicyclogermacrene (5.97%), α‐pinene (5.33%), and germacrene B (4.69%). The essential oil, but neither (E)‐caryophyllene nor β‐pinene, displayed in vitro cytotoxicity against all three tumor cell lines tested. The obtained average IC₅₀ values ranged from 3.8 to 20.8 μg/ml. The lowest and highest values were obtained against the NCI‐H358M and the OVCAR‐8 cell lines, respectively. The in vivo tumor‐growth‐inhibition rates in the tumor‐bearing mice treated with essential oil (50 and 100 mg/kg/d) were 25.3 and 42.6%, respectively. Hence, the essential oil showed significant in vitro and in vivo antitumor activity.     

Molecular insights on pathogenic effects of mutations causing phosphoglycerate kinase deficiency

Phosphoglycerate kinase (PGK) catalyzes an important ATP-generating step in glycolysis. PGK1 deficiency is an uncommon X-linked inherited disorder, generally characterized by various combinations of non-spherocytic hemolytic anemia, neurological dysfunctions, and myopathies. Patients rarely exhibit all three clinical features. To provide a molecular framework to the different pathological manifestations, all known mutations were reviewed and 16 mutant enzymes, obtained as recombinant forms, were functionally and structurally characterized. Most mutations heavily affect thermal stability and to a different extent catalytic efficiency, in line with the remarkably low PGK activity clinically observed in the patients. Mutations grossly impairing protein stability, but moderately affecting kinetic properties (p.I47N, p.L89P, p.C316R, p.S320N, and p.A354P) present the most homogeneous correlation with the clinical phenotype. Patients carrying these mutations display hemolytic anemia and neurological disorders, and,except for p.A354P variant, no myopaty. Variants highly perturbed in both catalytic efficiency (p.G158V, p.D164V, p.K191del, D285V, p.D315N, and p.T378P) and heat stability (all, but p.T378P) result to be mainly associated with myopathy alone. Finally, mutations faintly affecting molecular properties (p.R206P, p.E252A, p.I253T, p.V266M, and p.D268N) correlate with a wide spectrum of clinical symptoms. These are the first studies that correlate the clinical symptoms with the molecular properties of the mutant enzymes. All findings indicate that the different clinical manifestations associated with PGK1 deficiency chiefly depend on the distinctive type of perturbations caused by mutations in the PGK1 gene, highlighting the need for determination of the molecular properties of PGK variants to assist in prognosis and genetic counseling. However, the clinical symptoms can not be understood only on the bases of molecular properties of the mutant enzyme. Different (environmental, metabolic, genetic and/or epigenetic) intervening factors can contribute toward the expression of PGK deficient clinical phenotypes.     

Meta-analysis of two genome-wide association studies of bovine paratuberculosis

Background

Bovine paratuberculosis (ParaTB) also known as Johne''s disease, is a contagious fatal disease resulting from infection by Mycobacterium avium subspecies paratuberculosis (MAP). Previous studies have identified loci associated with ParaTB using different measurements to define cases and controls. The objective of this study was to combine the data from two recent studies to identify genetic loci associated with MAP tissue infection and humoral immune response, defined by MAP ELISA-positive cattle, by comparing cases and control animals for one or both measures of infection.

Methodology/Principal Findings

The two populations used for the association analyses were a cohort of MAP tissue infected animals and control Holstein cows from the USA and the second cohort composed of ELISA-positive and ELISA-negative Holstein cows from Italy. Altogether 1190 cattle were genotyped with the Illumina BovineSNP50 BeadChip. SNP markers were removed if the minor allele frequency <0.01 or genotyping failure was >5%. Animals were removed with >5% genotyping failure. Whole genome association analyses were conducted with the GRAMMAR-CG method using two different definitions of control populations.

Conclusion/Significance

The analyses identified several loci (P<5 e-05) associated with ParaTB, defined by positive ELISA and presence of bacteria in tissue compared to ELISA and tissue negative animals, on chromosomes 1, 12 and 15 and one unassigned SNP. These results confirmed associations on chromosome 12 and the unassigned SNP with ParaTB which had been found in the Italian population alone. Furthermore, several additional genomic regions were found associated with ParaTB when ELISA and tissue positive animals were compared with tissue negative samples. These loci were on chromosomes 1, 6, 7, 13, 16, 21,23 and 25 (P<5 e-05). The results clearly indicate the importance of the phenotype definition when seeking to identify markers associated with different disease responses.