iLIR: A web resource for prediction of Atg8-family interacting proteins

Macroautophagy was initially considered to be a nonselective process for bulk breakdown of cytosolic material. However, recent evidence points toward a selective mode of autophagy mediated by the so-called selective autophagy receptors (SARs). SARs act by recognizing and sorting diverse cargo substrates (e.g., proteins, organelles, pathogens) to the autophagic machinery. Known SARs are characterized by a short linear sequence motif (LIR-, LRS-, or AIM-motif) responsible for the interaction between SARs and proteins of the Atg8 family. Interestingly, many LIR-containing proteins (LIRCPs) are also involved in autophagosome formation and maturation and a few of them in regulating signaling pathways. Despite recent research efforts to experimentally identify LIRCPs, only a few dozen of this class of—often unrelated—proteins have been characterized so far using tedious cell biological, biochemical, and crystallographic approaches. The availability of an ever-increasing number of complete eukaryotic genomes provides a grand challenge for characterizing novel LIRCPs throughout the eukaryotes. Along these lines, we developed iLIR, a freely available web resource, which provides in silico tools for assisting the identification of novel LIRCPs. Given an amino acid sequence as input, iLIR searches for instances of short sequences compliant with a refined sensitive regular expression pattern of the extended LIR motif (xLIR-motif) and retrieves characterized protein domains from the SMART database for the query. Additionally, iLIR scores xLIRs against a custom position-specific scoring matrix (PSSM) and identifies potentially disordered subsequences with protein interaction potential overlapping with detected xLIR-motifs. Here we demonstrate that proteins satisfying these criteria make good LIRCP candidates for further experimental verification. Domain architecture is displayed in an informative graphic, and detailed results are also available in tabular form. We anticipate that iLIR will assist with elucidating the full complement of LIRCPs in eukaryotes.     

Ethylene and polyamine interactions in morphogenesis of <Emphasis Type="Italic">Passiflora cincinnata</Emphasis>: effects of ethylene biosynthesis and action modulators,as well as ethylene scavengers

Ethylene is a plant hormone that is of fundamental importance to in vitro morphogenesis, but in many species, it has not been thoroughly studied. Its relationship with polyamines has been studied mainly because the two classes of hormones share a common biosynthetic precursor, S-adenosylmethionine (SAM). In order to clarify whether competition between polyamines and ethylene influences in vitro morphogenetic responses of Passiflora cincinnata Mast., a climacteric species, different compounds were used that act on ethylene biosynthesis and action, or as ethylene scavengers. Treatment with the ethylene inhibitor, aminoethoxyvinylglycine (AVG) caused a greater regeneration frequency in P. cincinnata, whereas treatment with the ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid (ACC) lessened regeneration frequencies. The data suggested that levels of polyamines and ethylene are not correlated with morphogenic responses in P. cincinnata. It was ascertained that neither the absolute ethylene and polyamine levels, nor competition between the compounds, correlated to the obtained morphogenic responses. However, sensitivity to, and signaling by, ethylene appears to play an important role in differentiation. This study reinforces previous reports regarding the requirement of critical concentrations and temporal regulation of ethylene levels for morphogenic responses. Temporal regulation also appeared to be a key factor in competition between the two biosynthetic pathways, without having any effects on morphogenesis. Further studies investigating the silencing or overexpression of genes related to ethylene perception, under the influence of polyamines in cell differentiation are extremely important for the complete understanding of this process.     

Rolling rhythms in front crawl swimming with six-beat kick

The purpose of this study was to establish the rhythm characteristics of skilled front crawl swimmers using a six-beat kick. These included the amplitudes of the first three Fourier harmonics (H1, H2, H3) and their percent contributions to power contained in the angular displacement signals of the shoulders, hips, knees, and ankles with respect to the longitudinal axis in line with the swimming direction. Three-dimensional video data of seven national/international level swimmers were collected during simulated 200 m front crawl races in which swimmers maintained six-beat kicking patterns. Swimmers differed in all variables but had small variability across the four 50 m laps. Modest changes occurred during the 200 m, with the exception of shoulder roll, which remained constant and was represented almost entirely by a single sinusoid (H1). Changes across laps reached significance for swimming speed, stroke rate, hip roll, and H3 wave velocity between the knee and ankle. A H3 body wave of moderate and increasing velocity travelled caudally from hip to ankle. In the light of existing knowledge of aquatic locomotion this was compatible with the goal of generating propulsion in an efficient manner.     

Time-related alterations of superoxide radical levels in diverse organs of bile duct-ligated rats

The time-related alterations of superoxide radical measured in vivo by employing an ultrasensitive fluorescent assay in the liver, intestine, kidney and brain of rats with experimentally induced obstructive jaundice was investigated. Eighteen rats were randomly divided into Group A, rats subjected to sham operation, and Group B, rats subjected to bile duct ligation (BDL). Three rats from each group were subsequently killed at different time points post-operatively (1, 5 and 10 days). As compared to sham-operated, BDL rats showed a gradual increase with time of superoxide radical in the intestine, liver, kidney and brain: for animals sacrificed on the 1^st, 5^th and 10^th day the increase was 45%, 50% and 96% in the liver, 76%, 81% and 118% in the intestine, 64%, 71% and 110% in the kidney and 76%, 95% and 142% in the brain, respectively. This study provides direct evidence of an early appearance of oxidative stress in diverse organs, implying a uniform systemic response to biliary obstruction and emphasizing the need of early bile flow restoration.     

Molecular dynamics simulations of class C beta-lactamase from Citrobacter freundii: insights into the base catalyst for acylation

Herein, we present results from molecular dynamics (MD) simulations of the class C beta-lactamase from Citrobacter freundii and its Michaelis complex with aztreonam. Four different configurations of the active site were modeled in aqueous solution, and their relative stability was estimated by means of quantum mechanical energy calculations. For the free enzyme, the energetically most stable configurations present a neutral Lys67 residue or an anionic Tyr150 side chain. Our calculations predict that these two configurations are quite close in terms of free energy, the anionic Tyr150 state being favored by approximately 1 kcal/mol. In contrast, for the noncovalent complex formed between the C. freundii enzyme and aztreonam, the energetic analyses predict that the configuration with the neutral Lys67 residue is much more stable than the anionic Tyr150 one (approximately 20 kcal/mol). Moreover, the MD simulations reveal that the neutral Lys67 state results in a proper enzyme-aztreonam orientation for nucleophilic attack and in a very stable contact between the nucleophilic hydroxyl group of Ser64 and the neutral amino side chain of Lys67. Thus, both the computed free energies and the structural analyses support the assignation of Lys67 as the base catalyst for the acylation step in the native form of the C. freundii enzyme.     

Estimating dung decay rates of roe deer (<Emphasis Type="Italic">Capreolus capreolus</Emphasis>) in different habitat types of a Mediterranean ecosystem: an information theory approach

For elusive species living in concealing habitats (e.g. deer in a forest habitat), indirect methods such as faecal pellet counts are considered more practical means of estimating population density and abundance. Accurate estimation of deer density using the faecal standing crop (FSC) method necessitates the reliable estimation of the mean time to decay of pellet groups present during the survey. Mean time to decay is generally habitat specific, and separate estimations should be made for each habitat type in the study area. In a confined mountainous area of Greece, the habitat-specific mean time to decay of roe deer pellet groups was estimated by locating and marking fresh pellet groups on several dates in the lead up to an FSC survey and returning to the marked signs at the time of the survey to record whether or not each pellet group had survived. Several logistic models were fitted to the data, and estimations were based on a multi-model inference (MMI) approach according to information theory. The highest mean time to decay was estimated in coniferous forests, while mid-ranged values were found in maquis shrubs, and the lowest mean time to decay was observed in open areas. MMI by model averaging, based on Akaike weights, is recommended for making robust parameter estimations and for dealing with uncertainty in model selection.     

Cysteine, thiourea and thiocyanate interactions with clays: FT-IR, Mössbauer and EPR spectroscopy and X-ray diffractometry studies

The present study examined the adsorption of cysteine, thiourea and thiocyanate on bentonite and montmorillonite at two different pHs (3.00, 8.00). The conditions used here are closer to those of prebiotic earth. As shown by FT-IR, Mössbauer and EPR spectroscopy and X-ray diffractometry, the most important finding of this work is that cysteine and thiourea penetrate into the interlayer of the clays and reduce Fe³⁺ to Fe²⁺, and as consequence, cystine and c,c′-dithiodiformamidinium ion are formed. This mechanism resembles that which occurs with aconitase. This is a very important result for prebiotic chemistry; we should think about clays not just sink of molecules, but as primitive vessels of production of biomolecules. At pH 8.00, an increasing expansion was observed in the following order for both minerals: thiourea > thiocyanate > cysteine. At pH 3.00, the same order was not observed and thiourea had an opposite behavior, being the compound producing the lowest expansion. Mössbauer spectroscopy showed that at pH 8.00, the proportion of Fe²⁺ ions in bentonite increased, doubling for thiourea, or more than doubling for cysteine, in both clays. However, at pH 3.00, cysteine and thiourea did not change significantly the relative amount of Fe²⁺ and Fe³⁺ ions, when compared to clays without adsorption. For thiocyanate, the amount of Fe²⁺ produced was independent of the pH or clay used, probably because the interlayers of clays are very acidic and HSCN formed does not reduce Fe³⁺ to Fe²⁺. For the interaction of thiocyanate with the clays, it was not possible to identify any potential compound formed. For the samples of bentonite and montmorillonite at pH 8.00 with cysteine, EPR spectroscopy showed that intensity of the lines due to Fe³⁺ decreased because the reaction of Fe³⁺/cysteine. Intensity of EPR lines did not change when the samples of bentonite at pH 3.00 with and without cysteine were compared. These results are in accordance with those obtained using Mössbauer and FT-IR spectroscopy.     

Purification and characterization of the ouabain-sensitive H/K-ATPase from guinea-pig distal colon

Distal colon absorbs K⁺ through a Na⁺-independent, ouabain-sensitive H⁺/K⁺-exchange, associated to an apical ouabain-sensitive H⁺/K⁺-ATPase. Expression of HKα2, gene associated with this ATPase, induces K⁺-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K⁺-ATPase activities have been described in colonocytes. However, native H⁺/K⁺-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H⁺/K⁺-ATPase from guinea-pig distal colon is described. H⁺/K⁺-ATPase is Mg²⁺-dependent and activated by K⁺, Cs⁺ and NH₄⁺ but not by Na⁺ or Li⁺, independently of K⁺-accompanying anion. H⁺/K⁺-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [³²P]-γ-ATP, forming an acyl-phosphate bond, in an Mg²⁺-dependent, vanadate-sensitive process. K⁺ inhibited phosphorylation, effect blocked by ouabain. H⁺/K⁺-ATPase is an α/β-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKα2 and Na⁺/K⁺-ATPase β1-subunit, respectively. Thus, colonic ouabain-sensitive H⁺/K⁺-ATPase is a distinctive P-type ATPase.