Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Mitochondrial DNA structure and colony expansion dynamics of New Zealand fur seals (Arctocephalus forsteri) around Banks Peninsula

New Zealand fur seals are one of many pinniped species that survived the commercial sealing of the eighteenth and nineteenth centuries in dangerously low numbers. After the enforcement of a series of protection measures in the early twentieth century, New Zealand fur seals began to recover from the brink of extinction. We examined the New Zealand fur seal populations of Banks Peninsula, South Island, New Zealand using the mitochondrial DNA control region. We identified a panmictic population structure around Banks Peninsula. The most abundant haplotype in the area showed a slight significant aggregated structure. The Horseshoe Bay colony showed the least number of shared haplotypes with other colonies, suggesting a different origin of re-colonisation of this specific colony. The effective population size of the New Zealand fur seal population at Banks Peninsula was estimated at approximately 2500 individuals. The exponential population growth rate parameter for the area was 35, which corresponds to an expanding population. In general, samples from adjacent colonies shared 4.4 haplotypes while samples collected from colonies separated by between five and eight bays shared 1.9 haplotypes. The genetic data support the spill-over dynamics of colony expansion already suggested for this species. Approximate Bayesian computations analysis suggests re-colonisation of the area from two main clades identified across New Zealand with a most likely admixture coefficient of 0.41 to form the Banks Peninsula population. Approximate Bayesian computations analysis estimated a founder population size of approximately 372 breeding individuals for the area, which then rapidly increased in size with successive waves of external recruitment. The population of fur seals in the area is probably in the late phase of maturity in the colony expansion dynamic.     

Interaction of thrombin with PAR1 and PAR4 at the thrombin cleavage site

Investigations determined the critical amino acids for alpha-thrombin's interaction with protease-activated receptors 1 and 4 (PAR1 and PAR4, respectively) at the thrombin cleavage site. Recombinant PAR1 wild-type (wt) exodomain was cleaved by alpha-thrombin with a Km of 28 microM, a kcat of 340 s-1, and a kcat/Km of 1.2 x 10(7). When the P4 or P2 position was mutated to alanine, PAR1-L38A or PAR1-P40A, respectively, the Km was unchanged, 29 or 23 microM, respectively; however, the kcat and kcat/Km were reduced in each case. In contrast, when Asp39 at P3 was mutated to alanine, PAR1-D39A, Km and kcat were both reduced approximately 3-fold, making the kcat/Km the same as that of PAR1-wt exodomain. Recombinant PAR4-wt exodomain was cleaved by alpha-thrombin with a Km of 61 microM, a kcat of 17 s-1, and a kcat/Km of 2.8 x 10(5). When the P5 or P4 position was mutated to alanine, PAR4-L43A or PAR4-P44A, respectively, there was no change in the Km (69 or 56 microM, respectively); however, the kcat was lowered in each case (9.7 or 7.7 s-1, respectively). Mutation of the P2 position (PAR4-P46A) also had no effect on the Km but markedly lowered the kcat and kcat/Km approximately 35-fold. PAR1-wt exodomain and P4 and P3 mutants were noncompetitive inhibitors of alpha-thrombin hydrolyzing Sar-Pro-Arg-pNA. However, PAR1-P40A displayed a mixed type of inhibition. Mutation of P4, P3, or P2 had no effect on the Ki. All PAR4 exodomains were competitive inhibitors of alpha-thrombin. Mutation of P5, P4, or P2 had no effect on the Ki. These investigations show that Leu at P4 in PAR1 or P5 in PAR4 critically influences the kinetics of alpha-thrombin binding and cleavage of PAR1 and PAR4 exodomains. It also implies that factors other than the hirudin-like binding region on PAR1 exodomain predominate in influencing PAR1 cleavage on cells.     

Modified Bacillus thuringiensis Toxins and a Hybrid B. thuringiensis Strain Counter Greenhouse-Selected Resistance in Trichoplusia ni

Resistance of greenhouse-selected strains of the cabbage looper, Trichoplusia ni, to Bacillus thuringiensis subsp. kurstaki was countered by a hybrid strain of B. thuringiensis and genetically modified toxins Cry1AbMod and Cry1AcMod, which lack helix α-1. Resistance to Cry1AbMod and Cry1AcMod was >100-fold less than resistance to native toxins Cry1Ab and Cry1Ac.Insecticidal proteins from Bacillus thuringiensis are used widely for pest control, but evolution of resistance by pests can reduce their efficacy (3, 4, 6, 14). Resistance to B. thuringiensis toxins has been reported in field populations of four species of Lepidoptera, one species in response to sprays (3, 14) and three species in response to transgenic crops (10, 15, 16). Here, we focus on understanding and countering resistance to sprays of Bacillus thuringiensis subsp. kurstaki that evolved in commercial greenhouse populations of the cabbage looper, Trichoplusia ni (7, 17).We compared responses to single toxins and formulations of B. thuringiensis by two resistant strains (GipBtR and GlenBtR) and two related susceptible strains (GipS and GlenS) of T. ni. All four strains were started by the collection of larvae in 2001 from commercial greenhouses near Vancouver in British Columbia, Canada (7). Resistance evolved in the greenhouses in response to repeated sprays of DiPel (7), a formulation of B. thuringiensis subsp. kurstaki strain HD1 containing Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa (9). Previously reported concentrations required to kill 50% of larvae (LC₅₀s) indicated that, relative to a susceptible laboratory strain, initial resistance to DiPel was 113-fold in the Gip population (labeled T2c in reference 7) and 24-fold in the Glen population (labeled P5 in reference 7).We reared larvae on a wheat germ diet (5) at 26°C on a light-to-dark schedule of 16 h:8 h. GipS and GlenS were reared on diet without B. thuringiensis toxins, which allowed resistance to decline (7). To maintain resistance, GipBtR and GlenBtR were reared each generation on a diet treated with 5 or 10 mg of DiPel WP (Abbott Laboratories, Ontario, Canada) per milliliter of diet (7). In bioassays, groups of five third-instar larvae were put in 60-ml plastic cups containing diet, and mortality was assessed after 3 days by gently probing larvae for movement.We used diet overlay bioassays to evaluate the toxicity to GipBtR and GipS of the protoxin forms of Cry1Ab, Cry1Ac, Cry1AbMod, and Cry1AcMod produced in B. thuringiensis strains (12). Cry1AbMod and Cry1AcMod are genetically engineered variants of Cry1Ab and Cry1Ac, respectively, each lacking 56 amino acids from the amino-terminal region, including helix α-1 (12). An 80-μl aliquot containing distilled water and toxin was dispensed evenly over the surfaces of 2 ml of diet (a mean surface area of 7.1 cm²) and allowed to dry. Fifty to 200 larvae from each strain were tested at five to eight concentrations of each toxin.We used diet incorporation bioassays (7) to evaluate the toxicities of DiPel and Agree WG (Certis, Columbia, MD) to GipS, GipBtR, GlenS, and GlenBtR. Agree is a formulation of hybrid strain GC91, which was created from the conjugation-like transfer of a plasmid from B. thuringiensis subsp. kurstaki strain HD191 into B. thuringiensis subsp. aizawai strain HD135, and it contains Cry1Ac, Cry1C, and Cry1D (1, 8). DiPel and Agree were diluted in distilled water and mixed into diet (7). Twenty-five to 50 larvae from each strain were tested at six to seven concentrations of DiPel and Agree.We used probit analysis (13) to estimate the LC₅₀s and their 95% fiducial limits (FL), as well as the slopes of concentration-mortality lines and their standard errors. The mortality of larvae fed treated diet was not adjusted for the mortality of control larvae on untreated diet, because the control mortality was low (mean, 3.6%; range, 0 to 16%). LC₅₀s with nonoverlapping 95% FL are significantly different. Resistance ratios were calculated as the LC₅₀ of a resistant strain (GipBtR or GlenBtR) divided by the LC₅₀ of its susceptible counterpart (GipS or GlenS).The genetically modified toxins Cry1AbMod and Cry1AcMod were much more effective than the native toxins Cry1Ab and Cry1Ac against larvae of T. ni from the resistant GipBtR strain (Table ​(Table1).1). Resistance ratios of GipBtR were 580 for Cry1Ab and 1,400 for Cry1Ac but only 5.5 for Cry1AbMod and 9.3 for Cry1AcMod (Table ​(Table1).1). Against GipBtR, the LC₅₀ was 53-fold higher for Cry1Ab than for Cry1AbMod and 11-fold higher for Cry1Ac than for Cry1AcMod (Table ​(Table1).1). Against GipS, however, the LC₅₀ was 2-fold higher for Cry1AbMod than for Cry1Ab and 14-fold higher for Cry1AcMod than for Cry1Ac (Table ​(Table11).

TABLE 1.

Responses of resistant (GipBtR and GlenBtR) and susceptible (GipS and GlenS) strains of T. ni to native toxins (Cry1Ab and Cry1Ac), modified toxins (Cry1AbMod and Cry1AcMod), and formulations (DiPel and Agree)

Effect of hydrocarbon concentration of pasture production (<i>Brachiaria humidicola</i>) in Texistepec,Veracruz

In this study, the relationship between the concentration of extra-heavy crude petroleum in a clayey material and the toxicity, field capacity, temperature, and growth of a tropical forage grass (Brachiara humidicola) was determined empirically. For this type of petroleum the acute toxicity (Microtox^®) was slight (CE₅₀ = 63200 - 76400 mg/kg) even at high hydrocarbon concentrations (29279 mg/kg). Nonetheless, serious impacts were encountered in terms of an increase in soil temperature (+ 1.3 °C), reduction in field capacity (-10.7%) and reduction in aerial biomass (-97%). The relationship between hydrocarbon concentration and biomass resulted in a typical dose-response curve (r = 0.99), where a concentration of 2626 mg/kg of hydrocarbons corresponds to a maintenance of 90% biomass. Furthermore, during the duration of this study (one year) the biodegradation was proportional to the pasture biomass production (r = 0.997) indicating a synergistic relationship between the petroleum biodegrading microorganisms in the rhizosphere and the pasture.     

Influence of Pistachios on Performance and Exercise-Induced Inflammation,Oxidative Stress,Immune Dysfunction,and Metabolite Shifts in Cyclists: A Randomized,Crossover Trial

Objectives

Pistachio nut ingestion (3 oz./d, two weeks) was tested for effects on exercise performance and 21-h post-exercise recovery from inflammation, oxidative stress, immune dysfunction, and metabolite shifts.

Methods

Using a randomized, crossover approach, cyclists (N = 19) engaged in two 75-km time trials after 2-weeks pistachio or no pistachio supplementation, with a 2-week washout period. Subjects came to the lab in an overnight fasted state, and ingested water only or 3 oz. pistachios with water before and during exercise. Blood samples were collected 45 min pre-exercise, and immediately post-, 1.5-h post-, and 21-h post-exercise, and analyzed for plasma cytokines, C-reactive protein (CRP), F₂-isoprostanes (F₂-IsoP), granulocyte phagocytosis (GPHAG) and oxidative burst activity (GOBA), and shifts in metabolites.

Results

Performance time for the 75-km time trial was 4.8% slower under pistachio conditions (2.84±0.11 and 2.71±0.07 h, respectively, P = 0.034). Significant time effects were shown for plasma cytokines, CRP, F₂-IsoP, GPHAG, and GOBA, with few group differences. Metabolomics analysis revealed 423 detectable compounds of known identity, with significant interaction effects for 19 metabolites, especially raffinose, (12Z)-9,10-Dihydroxyoctadec-12-enoate (9,10-DiHOME), and sucrose. Dietary intake of raffinose was 2.19±0.15 and 0.35±0.08 mg/d during the pistachio and no pistachio periods, and metabolomics revealed that colon raffinose and sucrose translocated to the circulation during exercise due to increased gut permeability. The post-exercise increase in plasma raffinose correlated significantly with 9,10-DiHOME and other oxidative stress metabolites.

Conclusions

In summary, 2-weeks pistachio nut ingestion was associated with reduced 75-km cycling time trial performance and increased post-exercise plasma levels of raffinose, sucrose, and metabolites related to leukotoxic effects and oxidative stress.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01821820","term_id":"NCT01821820"}}NCT01821820     

Effect of increased alveolar surface tension on segmental pulmonary vascular resistance

The site of change in pulmonary vascular resistance (PVR) after surfactant displacement with the detergent diocytl sodium sulfosuccinate (OT) was studied in the isolated canine left lower lobe preparation. Changes in PVR were assessed using the arterial and venous occlusion technique and the vascular pressure-flow relationship. Changes in alveolar surface tension were confirmed from measurements of pulmonary compliance as well as from measurements of surface tension of extracts from lung homogenates. After surfactant depletion (the perfusion rate constant) the total pressure gradient (delta PT) across the lobe increased from 13.4 +/- 1 to 17.1 +/- 0.8 mmHg. This increase in delta PT was associated with a significant increase in the arterial and venous gradients (3.7 +/- 0.3 to 4.9 +/- 0.4 and 5.7 +/- 0.5 to 9.4 +/- 0.6 mmHg, respectively) and a decrease in middle pressure gradient (4.1 +/- 0.8 to 2.9 +/- 0.6 mmHg). The vascular pressure-flow relationship supported these findings and showed that the mean slope increased by 52% (P less than 0.05), whereas the pressure intercept decreased slightly but not significantly (3.7 +/- 0.7 to 3.2 +/- 0.8 mmHg). These results suggest that the resistance of arteries and veins increases, whereas the resistance of the middle segment decreases after surfactant depletion. These effects were apparently due to surface tension that acts directly on the capillary wall. Direct visualization of subpleural capillaries supported the notion that capillaries become distended and recruited as alveolar surface tension increases. In the normal lung (perfused at constant-flow rate) changes in alveolar pressure (Palv) were transmitted fully to the capillaries as suggested by equal changes in pulmonary arterial pressure.(ABSTRACT TRUNCATED AT 250 WORDS)