Evaluation of intravenous T-61 as a euthanasia method for birds

The use of T-61 as a sole euthanasia agent for birds was investigated. Nine broiler chickens (Gallus gallus domesticus) were euthanized by intravenous T-61 and assessed for insensibility [brainstem reflexes: nictitating membrane reflex (NIC), palpebral blink reflex (PAL)], brain death [isoelectric electroencephalogram activity (EEG)], cessation of audible heartbeat, and abnormal electrocardiogram. Birds were considered dead when the heart rate was less than 180 beats/minute with an isoelectric EEG. No vocalization or wing flapping occurred. Both NIC and PAL were lost 10.5 s from start of injection and audible heartbeat ceased at 24.5 s. Latency to isoelectric activity was 16.6 s. All but 1 bird died within 60 s. Rapid induction of insensibility meant birds did not experience pain and distress within 10.5 s from start of injection and birds were not conscious during cardiac and circulatory arrest. Intravenous injection of T-61 is an effective and efficient euthanasia method for birds.     

Inhibition of inducible nitric oxide synthase expression by an acetonic extract from Feijoa sellowiana Berg. fruits

Feijoa sellowiana Berg. fruits and especially the acetonic extract have been shown to possess biological activities, although the responsible compounds have never been identified. The present study was designed to evaluate the anti-inflammatory activity of an acetonic extract from F. sellowiana Berg. fruits on the nitric oxide (NO) pathway, which plays an important role in inflammation. To this aim the J774 cell line, which expresses inducible nitric oxide synthase (iNOS) following stimulation with lipopolysaccharide (LPS), has been utilized, and the effects of this extract and its fractions on NO production, iNOS protein expression, and signal pathways involved in its regulation have been evaluated. This study demonstrates that at least some part of the anti-inflammatory activity of the acetonic extract is due to the suppression of NO production by flavone and stearic acid. The mechanism of this inhibition seems to be related to an action on the expression of the enzyme iNOS through the attenuation of nuclear factor kappaB (NF-kappaB) and/or mitogen-activated protein kinase (MAPK) activation.     

Litter-fall and litter decomposition in a low Mediterranean shrubland

Annual production of litter by Cistus incanus (L.) and Myrtus communis (L.) and decomposition dynamics of leaf litter of these species was studied in a Mediterranean shrubland. Myrtus and Cistus produced 472 and 429 g dry weight litter m^-2 year^-1, respectively. Leaves were the predominant litter component for both species. The average decay constant of Myrtus and Cistus litters enclosed in litter bags, calculated over the whole study period (38 months), was 0.71 year^-1 and 0.31 year^-1 respectively. In green leaves the N content differed during growth seasons for both species, whereas the content of Ca, Mg, P, K, and Na did not show significant changes. Abscised leaves had lower N, P and K contents than green leaves, evidencing that a nutrient translocation before abscission occurred from senescent leaves. The nutrient contents of the leaves at abscission time, generally higher in Cistus than in Myrtus, allowed us to estimate the annual nutrient input to the soil. Phosphorus and K more than N were rapidly released by the decomposing litters after exposure. Nutrient limitation, in particular P, might be considered the main growth limiting factor for Myrtus and Cistus. Both species were adapted to recovery and rapidly recycle P more than N and K in the living biomass through retranslocation from green leaves before abscission and/or a high release rate from the decomposing litter. The former strategy was better used by Cistus, the latter by Myrtus.     

Kiwellin, a modular protein from green and gold kiwi fruits: evidence of in vivo and in vitro processing and IgE binding

Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.     

Response of an agricultural soil to pentachlorophenol (PCP) contamination and the addition of compost or dissolved organic matter

The response of a fresh, agricultural soil when contaminated with pentachlorophenol (PCP) and supplemented with compost (C) or dissolved organic matter (DOM) was studied in the laboratory. The concentration of PCP and the changes in various functionally related properties (i.e. microbial biomass, basal respiration, soil hydrolase and oxidoreductase activity) were measured over 150 d. Variations in the main physical and chemical properties of the soils were also monitored. Two different doses of compost (C1 = 0.27% and C2 = 0.83%, corresponding to 10 and 30 t ha⁻¹, respectively) or DOM (D1 = 0.07% and D2 = 0.2%) equivalent to the carbon content of the two compost doses C1 and C2 were used and the following five systems were investigated: soil (S), soil–compost (S-C1 and S-C2) and soil–DOM (S-D1 and S-D2). PCP concentrations declined progressively and significantly with time. This effect was most pronounced for the soils amended with the lower compost dose C1 (S-C1) and with the two DOM (S-D1 and S-D2) amounts. Significantly reduced amounts of PCP were extracted after its 500-d residence in the various systems. Higher amounts of the residual PCP were extracted from the humic acids (HA), fulvic acids (FA) and humin–mineral (HU) fractions of the 500 d aged samples than from the same unfractionated samples, indicating that the residual PCP preferentially accumulated in the organic fractions of soil. The soil showed an endogenous microbial activity as indicated by basal respiration, microbial biomass and all the enzymatic activities tested (dehydrogenase, glucosidase, phosphatase, arylsulphatase and urease). Addition of the PCP severely depressed some of the tested biochemical properties suggesting an inhibitory effect on microbial activity. Conversely, higher basal respiration, and similar β-glucosidase and phosphatase activities were measured in comparison with the controls. No significant effects were observed following the addition of two doses of the compost or the DOM. Fungal colonies belonging to the taxonomic group of Ascomycetes and identified as Byssochlamys fulva developed with time in all the PCP-contaminated samples. Growth of B. fulva in vitro in the presence of PCP showed that the isolate was tolerant to 12.5 and 25 mg l⁻¹ PCP and degraded 20% of its initial concentration in 8 d. Overall, the results indicate that many complex processes occurred in the contaminated soil and combinations of these determined the response to PCP contamination. The sorption of PCP to the soil matrix (which increased with time) and its degradation/transformation by indigenous soil microbial activity were likely involved. Both the processes appeared to be favoured by the presence of dissolved organic matter.     

Interaction of alkylsulfonate ligands with beta-lactoglobulin AB from bovine milk.

The interaction of alkyl sulfonate ligands (AL) with bovine beta-lactoglobulin AB was measured using Trp fluorescence enhancement. One binding site per protein molecule was observed. The location of this site was related with the dimer formation and could be coincident with the fatty acids and SDS binding site. The apparent binding constants for AL were in the range of 10(-)(6) M, at pH 6.8. At pH 3.0 no binding was observed by this fluorescence method. The strength of the interaction was decreasing in the following way: AL16 > AL12 > AL14 > AL10. Other sites on the monomer were evidenced by the protective action of the AL toward the urea unfolding of the protein.     

Gas exchange and water-use efficiency of cv. Sangiovese grafted to rootstocks of varying water-deficit tolerance

Testing of new rootstocks for drought tolerance targets traditionally rain-fed districts where supplemental irrigation is more frequently needed due to the pressures of global warming. A seasonal evaluation of gas exchange and water-use efficiency (WUE) of cv. Sangiovese grafted to the new drought-tolerant genotype M4 in a dry-down trial against the commercial SO4 stock is reported. The experiment was conducted in 2014 on twelve 2-year-old, non-fruiting potted Sangiovese grapevines grafted on M4 and SO4 stocks and assigned to SO4-WW (well-watered), SO4-WS (water-stressed), M4-WW and M4-WS treatments. Progressive water deficit was imposed by reducing water supply to 70, 50 and 30 % of whole-canopy demand derived from concurrent measurements of transpiration in WW. Unlike SO4, M4 showed slower stress progression, as highlighted by pre-dawn leaf water potential, and retained higher whole-canopy net CO₂ exchange rates (NCER) and transpiration rates per unit of leaf area at all replenishment levels as well as exhibiting higher canopy WUE at both 50 and 30 % WW. Although single-leaf assessment was in partial disagreement with data recorded on the whole-canopy basis, robust data were acquired to confirm that the M4 stock performs better than SO4 at any water replenishment level in terms of higher NCER/leaf area and/or canopy WUE. Findings feed expectations that grafting Sangiovese to the M4 rootstock should result in a problem-solving tool for rain-fed areas subject to temporary summer drought.     

Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.