Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.     

De novo formation of plant endoplasmic reticulum export sites is membrane cargo induced and signal mediated

The plant endoplasmic reticulum (ER) contains functionally distinct subdomains at which cargo molecules are packed into transport carriers. To study these ER export sites (ERES), we used tobacco (Nicotiana tabacum) leaf epidermis as a model system and tested whether increased cargo dosage leads to their de novo formation. We have followed the subcellular distribution of the known ERES marker based on a yellow fluorescent protein (YFP) fusion of the Sec24 COPII coat component (YFP-Sec24), which, differently from the previously described ERES marker, tobacco Sar1-YFP, is visibly recruited at ERES in both the presence and absence of overexpressed membrane cargo. This allowed us to quantify variation in the ERES number and in the recruitment of Sec24 to ERES upon expression of cargo. We show that increased synthesis of membrane cargo leads to an increase in the number of ERES and induces the recruitment of Sec24 to these ER subdomains. Soluble proteins that are passively secreted were found to leave the ER with no apparent up-regulation of either the ERES number or the COPII marker, showing that bulk flow transport has spare capacity in vivo. However, de novo ERES formation, as well as increased recruitment of Sec24 to ERES, was found to be dependent on the presence of the diacidic ER export motif in the cytosolic domain of the membrane cargo. Our data suggest that the plant ER can adapt to a sudden increase in membrane cargo-stimulated secretory activity by signal-mediated recruitment of COPII machinery onto existing ERES, accompanied by de novo generation of new ERES.     

Neutrophil effector functions triggered by Fc-gamma and/or complement receptors are dependent on B-ring hydroxylation pattern and physicochemical properties of flavonols

Tissue damage in autoimmune diseases involves excessive production of reactive oxygen species (ROS) triggered by immune complexes (IC) and neutrophil (PMN) interactions via receptors for the Fc portion of IgG (FcgammaR) and complement receptors (CR). Modulation of both the effector potential of these receptors and ROS generation may be relevant to the maintenance of body homeostasis. In the present study, the modulatory effect of four flavonols (myricetin, quercetin, kaempferol, galangin) on rabbit PMN oxidative metabolism, specifically stimulated via FcgammaR, CR or both classes of receptors, was evaluated by luminol- and lucigenin-dependent chemiluminescence assays. Results showed that flavonol inhibitory effect was not dependent on the cell membrane receptor class stimulated but related to the lipophilicity of the compounds (their apparent partition coefficient values were obtained by high-performance liquid chromatography), and was also inversely related to the number of hydroxyl groups in the flavonol B ring and the ROS-scavenger activity (assessed by the luminol--H2O2--horseradish peroxidase reaction). Under the experimental conditions the flavonols tested were not toxic to PMNs (evaluated by lactate dehydrogenase release and trypan blue exclusion) and did not interfere with IC-induced phagocytosis (evaluated by transmission electron microscopy). Our results suggested that inhibition of IC-stimulated PMNs effector functions by the flavonols tested herein was the result of cooperation of different cellular mechanisms.     

Fermentation of cashew apple juice to produce high added value products

The use of agriculture substrates in industrial biotechnological processes has been increasing because of its low cost. Cashew apples are considered an agriculture low cost product in the Brazilian Northeast because the cashew cultivation is done mainly to produce cashew nuts. About 90% of the cashew apples production is lost in the field after removing the nut. In this work, the use of clarified cashew apple juice as substrate for microbial cultivation was investigated. The results showed that cashew apple juice is a good source of reducing sugars and can be used to grow Leuconostoc mesenteroides to produce high added value products such as dextran, lactic acid, mannitol and oligosaccharides.     

Omega-alkoxy analogues of SAHA (vorinostat) as inhibitors of HDAC: a study of chain-length and stereochemical dependence

A series of omega-alkoxy ethers were prepared with variation of the length of the aliphatic chain of suberoylanilide hydroxamic acid (SAHA, vorinostat). Eight carbon long chain analogues showed the best activity, among which several substituted benzyl ether derivatives exhibited inhibitory activity on HDAC comparable to SAHA, and antiproliferative activity on three human cell lines (NB4, H460, and HCT-116) better than SAHA. However, no significant difference in antiproliferative activity was observed between two enantiomers bearing the benzyl ether moiety.     

Oxygen affinity controlled by dynamical distal conformations: the soybean leghemoglobin and the Paramecium caudatum hemoglobin cases

The binding of diatomic ligands, such as O(2), NO, and CO, to heme proteins is a process intimately related with their function. In this work, we analyzed by means of a combination of classical Molecular Dynamics (MD) and Hybrid Quantum-Classical (QM/MM) techniques the existence of multiple conformations in the distal site of heme proteins and their influence on oxygen affinity regulation. We considered two representative examples: soybean leghemoglobin (Lba) and Paramecium caudatum truncated hemoglobin (PcHb). The results presented in this work provide a molecular interpretation for the kinetic, structural, and mutational data that cannot be obtained by assuming a single distal conformation.     

The influence of macrofouling on the corrosion behaviour of API 5L X65 carbon steel

Seawater is a complex corrosive system, and biofouling is one of the factors that influences corrosion processes. The behaviour of corrosion associated with the development of macrofouling was investigated during the first 6 months of the successional process. Three treatments were compared: the 'Control' treatment (absence of macrofouling); 'Community' treatment, and 'Barnacle' treatment, where other macroorganisms were excluded. In the Community treatment, the dominant organisms were filamentous macroalgae (23.73%), barnacles (17.51%), hydroids (16.96%) and encrusting bryozoans (9.58%). In the Barnacle treatment, the cover varied between 39.38% and 62.50%. The corrosion potential ranged from -665.75 to -517.50 mV(Ag/AgC l((KCl))) and could not be associated with fouling development. The highest corrosion rate in the control suggests that macrofouling provides a protection against mass loss. The highest percentage of localised attacks was found in the Community treatment. This may indicate that not only barnacles, but also other organisms induce localised corrosion.     

Response of five lettuce cultivars to root-knot nematode, Meloidogyne incognita

The root-knot nematode, Meloidogyne incognito (Kofoid et White) Chitwood is an important pathogen of vegetables. Five commercial cultivars of lettuce (Lactuca sativa L.) were evaluated under greenhouse conditions for resistance to Meloidogyne incognita, Benguet population. Plants were inoculated with 1000 eggs collected from 'Apollo' tomato (Lycopersicon esculentum) roots. The degree of galling and number of egg masses were assessed 4 and 8 weeks after inoculation. Host plant response was classified as immune, highly resistant, resistant, moderately resistant, intermediate, moderately susceptible, and highly susceptible based on the resistance index of Kouamè et at., 1998 [RI = (gall2 + egg2)]. Inoculation of 1000 eggs/plant significantly affected the growth and yield of the five lettuce cultivars 4 and 8 weeks after inoculation. A significant interaction was observed between treatment and cultivar during the two evaluation periods in terms of marketable and non-marketable yield, plant height, root weight, number of galls and number of egg masses. A reduction in growth and yield was observed in the cultivars Ballon, Lollo Rosa and Red Wave. Significant differences were noted in the number of galls and egg masses among the different cultivars tested. The highest average number of galls was obtained from the cultivars Red Wave, Ballon and Lollo Rosa. Cultivar Ballon had the highest average number of recovered nematode while Gilaben had the lowest with 15 and 4 per roots, respectively after 4 weeks inoculation. After 8 weeks, nematode was highest in cultivar Red Wave (615) and lowest in Great Lakes (70). Based on the host response, cultivars Great Lakes and Gilaben were rated highly resistant and resistant, respectively, while Red Wave, Ballon and Lollo Rosa were rated intermediate.