Mechanism of two classes of cancer mutations in the phosphoinositide 3-kinase catalytic subunit

Many human cancers involve up-regulation of the phosphoinositide 3-kinase PI3Kalpha, with oncogenic mutations identified in both the p110alpha catalytic and the p85alpha regulatory subunits. We used crystallographic and biochemical approaches to gain insight into activating mutations in two noncatalytic p110alpha domains-the adaptor-binding and the helical domains. A structure of the adaptor-binding domain of p110alpha in a complex with the p85alpha inter-Src homology 2 (inter-SH2) domain shows that oncogenic mutations in the adaptor-binding domain are not at the inter-SH2 interface but in a polar surface patch that is a plausible docking site for other domains in the holo p110/p85 complex. We also examined helical domain mutations and found that the Glu545 to Lys545 (E545K) oncogenic mutant disrupts an inhibitory charge-charge interaction with the p85 N-terminal SH2 domain. These studies extend our understanding of the architecture of PI3Ks and provide insight into how two classes of mutations that cause a gain in function can lead to cancer.     

Application of an Improved Antimony Microelectrode for Measuring pH-Changes at the Soil-Root Interface of Maize

In both buffer solutions and soil-water suspensions linear correlations with highly significant coefficients were found between pH-values (glass electrode) and mV-values measured with an improved Sb-microelectrode. Addition of 20% (w/w) CaCO₃ to the original soil gave an increase in the pH-values (glass electrode) and also mV-values (Sb-electrode). Liming the soil with more than 20% CaCO₃ showed no effect on both pH- and mV-values. Significant correlation coefficients were obtained between pH- and mV-values at all different-percentages of soil water holding capacity. In a pot experiment the pH- and mV-values in maize rhizosphere were lower in localized fertilizer spots than in the bulk soil. The pH-values calculated from mV-measurements in both bulk soils and localized fertilizer spots were lower than pH-values measured directly using the glass electrode. These variations were related to the type and the combinations of applied fertilizers in the localized spots.     

Purification and characterization of three trypsin isoforms from viscera of sardinelle (<Emphasis Type="Italic">Sardinella aurita</Emphasis>)

Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20–70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0–11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K _m and k _cat were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s⁻¹, respectively. The N-terminal sequences of the first 10 amino acids were “I V G G Y E C Q K Y” for trypsin A and “I V G G Y E A Q S Y” for trypsins B and C. These sequences showed highly homology to other fish trypsins.     

Effect of suckling cow's milk or milk replacer on abomasal luminal pH in dairy calves

Abomasal ulceration occurs commonly in suckling calves, and the cause for the high prevalence of abomasal ulceration is unknown. We hypothesized that diet may play a role in the etiopathogenesis of abomasal ulceration. Six male dairy calves with an abomasal body cannula suckled fresh Holstein cow's milk, all milk-protein milk replacer, or combined milk- and soy-protein milk replacer twice daily at 12% of body weight/d. Abomasal luminal pH was measured every second for 24 hours by using a miniature glass pH electrode. Mean 24-hour abomasal luminal pH for all milk-protein milk replacer (3.22) and combined milk- and soy-protein milk replacer (3.27) were similar but significantly (P < .05) higher than that for cow's milk (2.77; standard error = 0.08). Both milk-replacer formulations failed to clot after the addition of chymosin, whereas cow's milk clotted within 2 minutes. The in vitro titration curve of cow's milk and all milk-protein milk replacer were similar, but different to that of combined milk- and soy-protein milk replacer. The osmolalities of all milk-protein milk replacer (375 mOsm/kg) and combined milk- and soy-protein milk replacer (410 mOsm/kg) were greater than that of cow's milk (278 mOsm/kg). The slightly lower mean abomasal luminal pH in calves suckling cow's milk, compared to milk replacer, was probably due to clotting of cow's milk, with extrusion of low pH whey, and a slower rate of abomasal emptying caused by the hyperosmolality of milk replacer. Examination of our results suggests that suckling cow's milk may increase the prevalence of abomasal ulceration by decreasing mean luminal pH, although this remains to be determined.     

Effect of Jasmonic Application on Economically Insect Pests and Yeald in Spring Wheat

Field trials were conducted in spring wheat to observe effects of jasmonic acid (JA) on aphids, thrips and wheat blossom midge (WBM). Two spring wheat varieties (Triso and Kadrilj) were sprayed twice, with two concentrations of JA plus control. Wheat pests and associated natural enemies (Coccinellidae, Empididae and Aphelinidae) were surveyed by direct count and sweep net methods. Thrips larvae and adults and WBM larvae were estimated by dissecting wheat ears. Wheat midge larvae were monitored using white traps in treated and untreated plots. At the end of season, wheat yield was assessed in treated and control plots. There was a significant difference in the number of insect pests and their natural enemies between treatments in both varieties. Plants in control plots had higher numbers of aphids, thrips and midges than in treated plots. There were more aphids, thrips and midges on the variety ??Triso?? than on ??Kadrilj??. JA application enhanced wheat yield in treated plots compared to control plots. The results indicated that JA mediated induction of plant defense in wheat plants can improve resistance against insect herbivores. JA could be used to develop environmentally sound crop management with reduced insecticide applications.     

Lipolytic activity levels and colipase presence in digestive glands of some marine animals

Studies on the digestive secretions in aquatic animals can elucidate certain aspects of their nutritive physiology. The aim of the present study was to compare the digestive lipase and phospholipase activities in ten marine species belonging to four classes following the taxonomic classification of marine organisms. All aquatic digestive tissues tested are equipped with lipase and phospholipase activities, assuming the hydrolysis of fat-rich food. The lipolytic activities determined in the pancreases of cartilaginous fishes were greater than those in bony fishes, molluscs and crustaceans. This finding might be explained by the strong digestive utilization of fat-rich macronutrients by these carnivorous fishes. A trend of activities and stabilities at different pH and temperatures for crude lipases and phospholipases from these aquatic animals suggests that the optimum pH and temperature for marine lipases are species dependent. Interestingly, the sardine caecal lipase and phospholipase were found to be mostly stable in a broad range of acidic pH values. The maximum activities of lipolytic enzymes from the hepatopancreases of Hexaplex trunculus (molluscs) and Carcinus mediterranus (crustaceans) were found to be 50 and 60?°C, respectively, whereas the optimal temperature of lipolytic enzymes for the other species was classically around 40?°C. Thermoactivity of molluscs' lipolytic preparations makes them potential candidates in industrial applications. Among digestive glands studied, only pancreas (cartilaginous fish) contained the classically known colipase. Regarded as the most primitive living jawed vertebrates, cartilaginous fishes represented by sharks and rays could be considered as the oldest vertebrates possessing a complex digestive system like that of mammals.