Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

Specific glycanforms of type IX collagen accumulate in embryonic chick sterna after 17 days of development

Type IX collagen is a key component of the extracellular matrix of cartilage where it occurs at the surfaces of type II collagen fibrils as a glycanated molecule. The function of the glycosaminoglycan (GAG) side chain of the molecule is, however, unknown. We have shown that type IX collagen in chicken sternal cartilage is synthesized with a unimodal distribution of GAG chain size, but at post 17 days of development three predominant glycanforms of type IX collagen accumulate. Such accumulation did not occur in sterna from day 15 embryos. In day 17 embryos predominant glycanforms were found in the caudal region of the sternum. By day 19 of development the three predominant glycanforms are widespread throughout the caudal and cephalic regions. The results indicate that developmental and anatomical changes occur to type IX collagen that depend on the size of the GAG chain attached to the alpha2(IX) chain of the molecule.      

Study of deglutition using various sound signals]

In man quantitative information on deglutition may be obtained with the aid of various sounds recorded by way of microphone and magnetic tapes. The different tapes, microphonic, dynamic and piezoelectric, can be compared through the use of a Doppler signal in high middle position, thus showing the backward movements of the mylohyo?deus. The sound of the deglutition consists of 1 to 3 peaks of great amplitude: 350 +/- 180 microV or 14 +/- 3.3 mV according to the type of microphone used (the piezo gives the greatest amplitude, specially in low frequency range); duration for the main phoneburst: 48 +/- 24 ms with a prolongation with the smaller noises: so total duration amounts to 400 ms. Each sound impulsion repeated in 10 Hz range includes frequency up to 6 KHz. The direct recording on a polygraph falls into 10-60 range; upper frequencies may be eliminated by filtering. In front, the inspiratory relative time increases of 57 +/- 12% without deglutition whereas it reaches 62 +/- 16% with deglutition. In clinical situation, a magnetic tape recorder can be used. The main components in normal situation of deglutition of saliva, water or yogurt show a frequent variability. Nevertheless, a certain number of recurring impulsions can be defined according to the type of deglutition, thus further defining several basic types of deglutition. Amplitude and duration deglutition type 1 (= one impulsion) was observed in young subjects for saliva and yogurt; deglutition type 2-3 (greater than or equal to 200 ms) more frequently for water and also in oldest subjects, whatever the mode of deglutition. In some pathological situations, a significant increase of amplitude and duration was observed. Technical conditions are discussed for application in clinical situations and their control in laboratory.     

Antimicrobial activity of silymarin mediated zinc oxide and hydroxy apatite nanoparticles against oral pathogens

The nanoparticles such as hydroxyapatite, zinc oxide, titanium dioxide and zirconium nanoparticles have application in dentistry. Therefore, it is of interest to document the antimicrobial activity of silymarin mediated zinc oxide and hydroxy apatite nanoparticles against oral pathogens. Hence, we synthesized hydroxyapatie and zinc oxide nanoparticles with silymarin and characterized by UV-visible spectrophotometer. Data shows that silymarin mediated HAP and ZnO nanoparticles have antimicrobial activity against oral pathogens such as Pseudomonas sp, Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis and Candida albicans.     

The Diversity of Flower Flies (Diptera: Syrphidae) in Colombia and Their Neotropical Distribution

In Colombia, like most Neotropical countries, faunistic studies on flower flies have been occasional and most of them have been primarily focused on taxonomy. Colombia is the second-most species-rich country in flower fly diversity in the Neotropics after Brazil, and has one of the highest numbers of species per unit area (2.49 per 10,000?km²), based on a review of literature and national collections. Including new data presented here, a total of 47 genera and 300 species are recorded in Colombia. The genera Scaeva Fabricius and Lycastrirhyncha Bigot, as well as 101 species are recorded here for the first time. The altitudinal range and the distribution of the flower fly genera in Colombia are presented. A preliminary comparison of the fauna of Colombia with that of other Neotropical countries is given. A historical perspective is also provided in order to illustrate how Colombian Syrphidae knowledge has progressed over the last 168?years. Information presented here will be useful for ongoing and future biodiversity research as well as conservation projects on Syrphidae in the Neotropical region.     

Cloning and expression of an anti-LDL(-) single-chain variable fragment,and its inhibitory effect on experimental atherosclerosis

The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr^-/- mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr^-/- mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).     

Impact of transgenic Bt-cotton on the diversity of pink-pigmented facultative methylotrophs

Pink-pigmented facultative methylotrophs (PPFMs) are one of the beneficial proteobacteria commonly found in phyllosphere, rhizosphere and as endophytes in cotton. To assess the impact of transgenic Bt-cotton on changes in the diversity and community profile of PPFMs by comparing with its non-transgenic cotton, a polyphasic approach including differential carbon-substrate utilization profiling and DNA fingerprinting techniques like ARDRA, RISA, BOX-PCR and ERIC-PCR were studied. PPFMs from phyllosphere, rhizoplane and internal tissues of the stem of both Bt-cotton and non-Bt-cotton were isolated and analysed in this study. All the results suggested that the diversity richness of PPFMs present in the phyllosphere, rhizoplane and internal tissues did not differ between Bt- and non-Bt-cotton. In this study, there was no evidence to indicate any adverse effects of Bt-cotton on the diversity of plant-associated methylobacteria.