Isolation and screening of potential actinobacteria for rapid composting of rice straw

Rice straw is produced as a by-product from rice cultivation, which is composed largely of lignocellulosic materials amenable to general biodegradation. Lignocellulolytic actinobacteria can be used as a potential agent for rapid composting of bulky rice straw. Twenty-five actinobacteria isolates were isolated from various in situ and in vitro rice straw compost sources. Isolates A2, A4, A7, A9 and A24 were selected through enzymatic degradation of starch, cellulose and lignin followed by the screening for their adaptability on rice straw powder amended media. The best adapted isolate (A7) was identified as Micromonospora carbonacea. It was able to degrade cellulose, hemicelluloses and carbon significantly (P ≤ 0.05) over the control. C/N ratio was reduced to 18.1 from an initial value of 29.3 in 6 weeks of composting thus having the potential to be used in large scale composting of rice straw.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

Oil palm (Elaeis guineensis) protoplasts: isolation, culture and microcallus formation

Procedures are deseribed for the efficient isolation of protoplasts from a variety of oil palm (Elaeis guineensis Jacq.) tissues. Various factors including donor source, composition of enzyme mixture and culture medium affected the yield and viability of the protoplasts Polyembryogenic cultures of oil palm were the most suitable starting material in terms of yield, viability and metabolic competence. Pectolyase Y-23 in association with cellulase and hemicellulase was required for the efficient release of protoplasts from the oil palm tissues. Limited cell division to form microcallus was observed at very low frequency (<0.01%) when glutathione and catalase were incorporated in the culture medium.Abbreviations 2,4-d dichlorophenoxyacetic acid - DTT dithiothreitol - MES 2[N-morpholino] ethanesulphonic acid - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone     

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.     

Enzymatic characterization of Catalase from Bacillus anthracis and prediction of critical residues using information theoretic measure of Relative Entropy

In order to cope up with the reactive oxygen species (ROS) generated by host innate immune response, most of the intracellular organisms express Catalase for the enzymatic destruction/detoxification of hydrogen peroxide, to combat its deleterious effects. Catalase thus, scavenges ROS thereby playing a pivotal role in facilitating the survival of the pathogen within the host, and thus contributes to its pathogenesis. Bacillus anthracis harbors five isoforms of Catalase, but none of them has been studied so far. Thus, this study is the first attempt to delineate the biochemical and functional characteristics of one of the isoforms of Catalase (Cat1.4) of B. anthracis, followed by identification of residues critical for catalysis. The general strategy used, so far for mutational analysis in Catalases is structure based, i.e. the residues in the vicinity of heme were mutated to decipher the enzymatic mechanism. However, in the present study, protein sequence analysis was used for the prediction of catalytically important residues of Catalase. Essential measures were adopted to ensure the accuracy of predictions like after retrieval of well-annotated sequences from the database with EC 1.11.1.6, preprocessing was done to remove irrelevant sequences. The method used for multiple alignment of sequences, was guided by structural alignment and thereafter, an information theoretic measure, Relative Entropy was used for the critical residue prediction. By exploiting this strategy, we identified two previously known essential residues, H55 and Y338 in the active site which were demonstrated to be crucial for the activity. We also identified six novel crucial residues (Q332, Y117, H215, W257, N376 and H146) located distantly from the active site. Thus, the present study highlights the significance of this methodology to identify not only those crucial residues which lie in the active site of Catalase, but also the residues located distantly.     

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.     

Mutations in the chloroplast inner envelope protein TIC100 impair and repair chloroplast protein import and impact retrograde signaling

Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle’s envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100^cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100^cue8 identified a second mutation within the same gene, tic100^soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100^soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100^cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling.

Complementary mutations in TIC100 of the chloroplast inner envelope membrane cause reductions or corrective improvements in chloroplast protein import, and highlight a signaling role.

IN A NUTSHELLBackground: Plants harvest energy from the sun and CO₂ from the air and convert them into the energy-rich molecules they, and eventually us, are made of. Plants do this, photosynthesis, in bodies called chloroplasts inside their cells. Chloroplasts, made of protein and membrane material, were, before plants evolved, free-living bacteria, but the synthesis of most of their proteins occurs outside them, using information carried by the cell’s nuclear DNA, so most proteins have to be brought into developing chloroplasts, across the double membrane surrounding them, through dedicated, selective channels, formed by TOC (outer) and TIC (inner envelope) proteins. The identity of those channels matters as it helps determine versions of chloroplasts suited for particular environments. Which TIC proteins constitute the inner envelope channel has been a matter of controversy.Question: A mutant Arabidopsis plant called cue8 is slow-to-green (young leaves begin almost white) and shows delayed chloroplast and plant development. We looked for the molecular identity of the CUE8 gene. We also caused further mutations in this mutant and searched whether any corrected the defects in cue8.Findings: We found the mutated gene causing the cue8 defects is the TIC100 gene. This is one essential component of the “TIC 1-MDa complex,” one of the two versions of the TIC import complex under debate. That complex is made of several proteins, all present at reduced levels in cue8. In laboratory assays in which proteins are imported into isolated chloroplasts, cue8 performed worse than normal plants for a photosynthetic and a housekeeping chloroplast protein. A corrective, “suppressor” mutant was identified, and it carried a second mutation in TIC100, one physically complementary to the first one. Both the single and the double (suppressed) mutant still were slow-to-green, which evidences a signaling role for import defects to the nucleus, making photosynthetic genes active or not.Next steps: Surprisingly the grasses, including the cereals, have one core protein of the TIC 1 MDa complex but not the rest (including TIC100). We don’t know how their TIC channels operate. We also need to learn how the information on the defect in protein import, which occurs at the chloroplast envelope, is relayed to the cell’s nucleus (but we do have some clues).