Size and shape of two intestinal dipeptidases

Physicochemical parameters were determined on glycyl-L-leucine hydrolase (glycy-leucine dipeptidase, EC 3.4.13.2) and aminoacyl-L-proline hydrolase (proline dipeptidase, EC 3.4.13.9), purified from pig small intestine. The native molecular weights were found to be 115,000 and 113,000, respectively, as determined by a sedimentation equilibrium technique. Under denaturing conditions the molecular weights were found to be 51,000 and 63,200, respectively, using the same technique. It is concluded that each dipeptidase is composed of two subunits of equal molecular weight. The two dipeptidases have the same Stokes radius, 4.2 nm, analysed by gel chromatography. The sedimentation coefficients were found to be 5.8. S and 6.5 S and the intrinsic viscosities 5.4 ml/g and 5.8 ml/g, respectively. For both dipeptidases the measured physicochemical parameters are in accordance with the model of a prolate ellipsoid of revolution, having an axial ratio of about 5.     

Changes of the quaternary structure of microvillus aminopeptidase in the membrane

Microvillus aminopeptidase (EC 3.4.11.2) is an enzyme with a molecular weight around 300 000. Normal preparations contain three different subunits (subunit A, Mr 162 000; subunit B, Mr 123 000; subunit C, Mr 61 000). The relationship between the three subunits was studied by immunoelectrophoresis using specific antibodies against individual denatured subunits and by densitometric scanning of polyacrylamide gels after separation of the three subunits. The results suggest that microvillus aminopeptidase initially appears in the membrane as a symmetric molecule built up to two identical A subunits. These subunits are then split into equimolar amounts of subunit B and subunit C by trypsin. Subunit B cannot generate subunit C but may be further degraded. The reaction sequence described is one which occurs in vivo. Treatment of purified aminopeptidase with trypsin increases the specific activity twofold. This phenomenon does not seem to be correlated to the generation of subunit B and subunit C or to the transformation of amphiphilic form into hydrophilic form.     

Evidence for an apical sorting signal on the ectodomain of human aminopeptidase N.

In polarized epithelial cells aminopeptidase N is targeted to the apical membrane. The aim of this study was to determine whether a sorting signal is necessary for its correct transport to the apical membrane and, if so, to localize this sorting signal to one of the domains of the transmembrane protein. Anchor-minus aminopeptidase N, consisting of the hemagglutinin signal peptide including its cleavage site, and the ectoplasmic domain of human aminopeptidase N were stably expressed in Madin-Darby canine kidney cells cultured on polycarbonate filters. By measurement of the enzymatic activity it was found that the anchor-minus aminopeptidase N was secreted in a polarized manner to the apical side. As a reference the secretion of the secretory granule protein, cystatin C, was likewise studied. Cystatin C was found to be secreted in a nonpolarized manner to both domains. Our data thus show that human aminopeptidase N carries an apical sorting signal and that it is localized on the ectodomain of the enzyme.     

Sulphur mineralization and adsorption in soils

Summary Studies were conducted to determine the comparative sulphur mineralizing capacity of selected Malaysian and Iowan soils and to determine the amounts of available and adsorbed sulphate in a number of Malaysian soils. Results of the mineralization study indicated that more sulphur mineralised from Malaysian soils although their average contents of total sulphur were lower compared to Iowan Soils. For both sets of soils, significant correlations between contents of organic carbon and total sulphur existed indicating that most of the sulphur was in organic combination. Phosphate solution consistently extracted higher quantities of sulphate in comparison to chloride solution in the Malaysian surface soils implying that a portion of the sulphate existed in adsorbed form. Adsorption of sulphate in soils was found to be dependent on concentration of sulphate added and followed the Langmuir adsorption isotherm.     

Medium optimization for chitinase production from Trichoderma virens using central composite design

Medium development for chitinase production by Trichoderma virens was first carried out using conventional method of one-factor-at-a-time. The medium was further optimized using Central Composite Design in which response surface was generated later from the derived model. An experimental design of four variables including various initial pH values, chitin, ammonium sulphate, and methanol concentrations were created using Design Expert® Software, Version 6.0. The design consists of 30 experiments, which include 6 replicates at center points. The optimal value for each variable are 3.0 g/L, chitin; 0.1 g/L, ammonium sulphate; 0.4% (v/v), methanol; and initial pH, 4.0 with predicted chitinase activity of 0.1495 U/mL. These predicted parameters were tested in the laboratory and the final chitinase activity obtained was 0.1471 U/mL, which is almost reaching the predicted value. The optimal medium design showed an improvement of chitinase activity of 80.9% compared to activity obtained from the original Absidia medium composition.     

New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus

Baculoviruses are orally infectious to insects and considered to be natural insecticides. To enhance their speed-of-kill these viruses were engineered to express arthropod neurotoxins under the control of various strong promoters. Although this strategy proved to be efficient, it raised recently concerns about safety. We analyzed the speed-of-kill and safety of Autographa californica multiple nucleopolyhedrovirus expressing the insecticidal scorpion neurotoxin AaIT and found that the mortality of Helicoverpa armigera larvae was enhanced significantly when the expression was controlled by the baculovirus delayed-early promoter 39K rather than the very late promoter p10. This improvement was also reflected in better protection of cotton leaves on which these insects were fed. Using lacZ as a sensitive reporter we also found that expression driven by the 39K promoter was detected in insect but not in mammalian cells. These results imply that by selection of an appropriate viral promoter, engineered baculoviruses may comply with the high standard biosafety requirements from a genetically modified organism (GMO). Our results provide further support for the potential use of engineered baculoviruses in insect pest control in a safely manner.     

A porous medium‐chain‐length poly(3‐hydroxyalkanoates)/hydroxyapatite composite as scaffold for bone tissue engineering

Polyhydroxyalkanoates (PHA) are hydrophobic biopolymers with huge potential for biomedical applications due to their biocompatibility, excellent mechanical properties and biodegradability. A porous composite scaffold made of medium‐chain‐length poly(3‐hydroxyalkanoates) (mcl‐PHA) and hydroxyapatite (HA) was fabricated using particulate leaching technique and NaCl as a porogen. Different percentages of HA loading was investigated that would support the growth of osteoblast cells. Ultrasonic irradiation was applied to facilitate the dispersion of HA particles into the mcl‐PHA matrix. The different P(3HO‐co‐3HHX)/HA composites were investigated using field emission scanning electron microscopy (FESEM), X‐ray diffraction (XRD) and energy dispersive X‐ray analysis (EDXA). The scaffolds were found to be highly porous with interconnecting pore structures and the HA particles were homogeneously dispersed in the polymer matrix. The scaffolds biocompatibility and osteoconductivity were also assessed following the proliferation and differentiation of osteoblast cells on the scaffolds. From the results, it is clear that scaffolds made from P(3HO‐co‐3HHX)/HA composites are viable candidate materials for bone tissue engineering applications.     

Eicosanoids in insect immunity: bacterial infection stimulates hemocytic phospholipase A2 activity in tobacco hornworms

Intracellular phospholipase A(2) (PLA(2)) is responsible for releasing arachidonic acid from cellular phospholipids, and is thought to be the first step in eicosanoid biosynthesis. Intracellular PLA(2)s have been characterized in fat body and hemocytes from tobacco hornworms, Manduca sexta. Here we show that bacterial challenge stimulated increased PLA(2) activity in isolated hemocyte preparations, relative to control hemocyte preparations that were challenged with water. The increased activity was detected as early as 15 s post-challenge and lasted for at least 1 h. The increased activity depended on a minimum bacterial challenge dose, and was inhibited in reactions conducted in the presence of oleyoxyethylphosphorylcholine, a site-specific PLA(2) inhibitor. In independent experiments with serum prepared from whole hemolymph, we found no PLA(2) activity was secreted into serum during the first 24 h following bacterial infection. We infer that a hemocytic intracellular PLA(2) activity is increased immediately an infection is detected. The significance of this enzyme lies in its role in launching the biosynthesis of eicosanoids, which mediate cellular immune reactions to bacterial infection.