A Multiscale Framework for Deconstructing the Ecosystem Physical Template of High-Altitude Lakes

An ecosystem is generally sustained by a set of integrated physical elements forming a functional landscape unit—ecotope, which supplies nutrients, microclimate, and exchanges matter and energy with the wider environment. To better predict environmental change effects on ecosystems, particularly in critically sensitive regions such as high altitudes, it is imperative to recognise how their natural landscape heterogeneity works at different scales to shape habitats and sustain biotic communities prior to major changes. We conducted a comprehensive survey of catchment physical, geological and ecological properties of 380 high-altitude lakes and ponds in the axial Pyrenees at a variety of scales, to formulate and test an integrated model encompassing major flows and interactions that drive lake ecosystems. Three composite drivers encompassed most of the variability in lake catchment characteristics. In order of total percentage of variance explained, they were (i) hydrology/hydrodynamics—responsible for type and discharge of inlets/outlets, and for waterbody size; (ii) bedrock geomorphology, summarising geology, slope and fractal order—all dictating vegetation cover of catchment slope and lake shore, and the presence of aquatic vegetation; and (iii) topography, that is, catchment formation type—driving lakes connectivity, and the presence of summer snow deposits. Although driver (i) appeared to be local, (ii) and (iii) showed gradient changes along altitude and latitude. These three drivers differentiated several lake ecotopes based on their landscape similarities. The three-driver model was successfully tested on a riparian vegetation composition dataset, further illustrating the validity and fundamental nature of the concept. The findings inform on the relative contribution of scale-dependent catchment physical elements to lake ecotope and ecosystem formation in high-altitude lakes, which should be considered in any assessment of potentially major deleterious effects due to environmental/climate change.     

Inhibition of heat shock protein 90, a novel RET/PTC1-associated protein, increases radioiodide accumulation in thyroid cells

RET/PTC1 is a rearranged form of the RET tyrosine kinase commonly seen in papillary thyroid carcinomas. It has been shown that RET/PTC1 decreases expression of the sodium/iodide symporter (NIS), the molecule that mediates radioiodide therapy for thyroid cancer. Using proteomic analysis, we identify hsp90 and its co-chaperone p50cdc37 as novel proteins associated with RET/PTC1. Inhibition of hsp90 function with 17-allylamino-17-demothoxygeldanamycin (17-AAG) reduces RET/PTC1 protein levels. Furthermore, 17-AAG increases radioiodide accumulation in thyroid cells, mediated in part through a protein kinase A-independent mechanism. We show that 17-AAG does not increase the total amount of NIS protein or cell surface NIS localization. Instead, 17-AAG increases radioiodide accumulation by decreasing iodide efflux. Finally, the ability of 17-AAG to increase radioiodide accumulation is not restricted to thyroid cells expressing RET/PTC1. These findings suggest that 17-AAG may be useful as a chemotherapeutic agent, not only to inhibit proliferation but also to increase the efficacy of radioiodide therapy in patients with thyroid cancer.     

Effect of Substrate Moisture and Potassium on Water Relations and C, N and K Distribution in Vigna Radiata

The Vigna radiata L. plants were grown in greenhouse at moisture content of sand (SMC) of 12.0 ± 0.5 %. At flower bud initiation stage, i.e. 45 - 50 d after sowing, the SMC was decreased to 3.5 ± 0.5 %, and the effects of applied potassium (0, 2.56 and 3.84 mmol dm-3) were studied. During water stress, K-fed plants maintained higher leaf water potential and relative water content (RWC) of leaves and nodules and lower osmotic potential as compared to untreated plants. The proline content was higher in nodules than in leaves showing their difference in degree of stress. A partial recovery was found after re-irrigation. When subjected to drought, carbon was accumulated in the leaves and declined in nodules and roots. K-fed plants showed higher C and N content in stem, roots and nodules than untreated plants. The content of K significantly increased in stem and nodules in K-fed plants, irrespective of SMC. Dry masses of different plant parts were also increased in K-fed plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequence analysis and homology modeling of peroxidase from Medicago sativa

Plant peroxidases are one of the most extensively studied group of enzymes which find applications in the environment, health, pharmaceutical, chemical and biotechnological processes. Class III secretary peroxidase from alfalfa (Medicago sativa) has been characterized using bioinformatics approach Physiochemical properties and topology of alfalfa peroxidase were compared with that of soybean and horseradish peroxidase, two most popular commercially available peroxidase preparations. Lower value of instability index as predicted by ProtParam and presence of extra disulphide linkages as predicted by Cys_REC suggested alfalfa peroxidase to be more stable than either of the commercial preparations. Multiple Sequence Alignment (MSA) with other functionally similar proteins revealed the presence of highly conserved catalytic residues. Three dimensional model of alfalfa peroxidase was constructed based on the crystal structure of soybean peroxidase (PDB Id: 1FHF A) by homology modelling approach. The model was checked for stereo chemical quality by PROCHECH, VERIFY 3D, WHAT IF, ERRAT, 3D MATCH AND ProSA servers. The best model was selected, energy minimized and used to analyze structure function relationship with substrate hydrogen peroxide by Autodock 4.0. The enzyme substrate complex was viewed with Swiss PDB viewer and one residue ASP43 was found to stabilize the interaction by hydrogen bonds. The results of the study may be a guiding point for further investigations on alfalfa peroxidase.     

Actinobacillus utilizes a binding protein–dependent ABC transporter to acquire the active form of vitamin B6

Bacteria require high-efficiency uptake systems to survive and proliferate in nutrient-limiting environments, such as those found in host organisms. ABC transporters in the bacterial plasma membrane provide a mechanism for transport of many substrates. In this study, we examine an operon containing a periplasmic binding protein in Actinobacillus for its potential role in nutrient acquisition. The electron density map of 1.76 Å resolution obtained from the crystal structure of the periplasmic binding protein was best fit with a molecular model containing a pyridoxal-5′-phosphate (P5P/pyridoxal phosphate/the active form of vitamin B₆) ligand within the protein''s binding site. The identity of the P5P bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from Actinobacillus pleuropneumoniae into an Escherichia coli K-12 strain that was devoid of a key enzyme required for P5P synthesis. The growth of this strain at low levels of P5P supports the functional role of this operon in P5P uptake. This is the first report of a dedicated P5P bacterial uptake system, but through bioinformatics, we discovered homologs mainly within pathogenic representatives of the Pasteurellaceae family, suggesting that this operon exists more widely outside the Actinobacillus genus.     

Water relations and nitrogen fixation in potassium fedVigna radiata nodules

Drought created by withholding the irrigation at 30 and 45 d after sowing significantly decreased relative water content (RWC) and osmotic potential (ψ_s) ofVigna radiata (L.) Wilczek cv. MH-83-30 nodules. Potassium fed plants showed higher RWC, whereas ψ_s was further declined irrespective of soil moisture levels. The nitrogenase activity and leghemoglobin content of nodules markedly decreased under drought and nodules of potassium fed plants showed better recovery after rehydration. The proline content significantly increased under drought but declined upon reirrigation. Also, the C, N and K contents of nodules significantly declined under drought.     

Nonstarch Polysaccharides Modulate Bacterial Microbiota,Pathways for Butyrate Production,and Abundance of Pathogenic Escherichia coli in the Pig Gastrointestinal Tract

The impact of nonstarch polysaccharides (NSP) differing in their functional properties on intestinal bacterial community composition, prevalence of butyrate production pathway genes, and occurrence of Escherichia coli virulence factors was studied for eight ileum-cannulated growing pigs by use of terminal restriction fragment length polymorphism (TRFLP) and quantitative PCR. A cornstarch- and casein-based diet was supplemented with low-viscosity, low-fermentability cellulose (CEL), with high-viscosity, low-fermentability carboxymethylcellulose (CMC), with low-viscosity, high-fermentability oat β-glucan (LG), and with high-viscosity, high-fermentability oat β-glucan (HG). Only minor effects of NSP fractions on the ileal bacterial community were observed, but NSP clearly changed the digestion in the small intestine. Compared to what was observed for CMC, more fermentable substrate was transferred into the large intestine with CEL, LG, and HG, resulting in higher levels of postileal dry-matter disappearance. Linear discriminant analysis of NSP and TRFLP profiles and 16S rRNA gene copy numbers for major bacterial groups revealed that CMC resulted in a distinctive bacterial community in comparison to the other NSP, which was characterized by higher gene copy numbers for total bacteria, Bacteroides-Prevotella-Porphyromonas, Clostridium cluster XIVa, and Enterobacteriaceae and increased prevalences of E. coli virulence factors in feces. The numbers of butyryl-coenzyme A (CoA) CoA transferase gene copies were higher than those of butyrate kinase gene copies in feces, and these quantities were affected by NSP. The present results suggest that the NSP fractions clearly and distinctly affected the taxonomic composition and metabolic features of the fecal microbiota. However, the effects were more linked to the individual NSP and to their effect on nutrient flow into the large intestine than to their shared functional properties.The porcine intestinal microbiota change in response to dietary carbohydrate composition due to specific substrate preferences of bacteria (6). Therefore, inclusion of specific nonstarch polysaccharides (NSP) in the diet of pigs allows manipulation of the composition of the intestinal microbiota. The NSP can also reduce digestibility of nutrients in the small intestine (8). The resulting changes in nutrient flow alter the availability of fermentable substrate in the different sections of the gut and thus may modify the bacterial community structure. Differences in the fermentability levels of individual NSP may not only affect the kinetics of their degradation by intestinal bacteria but may also change the composition of the fermentation end products (49). Particularly, butyrate is an important metabolite because of its potential to affect gene expression and to improve cellular development in enterocytes (38). The ability of gut microbiota to produce butyrate can vary considerably in response to environmental factors, such as diet composition (3). However, the number of butyrate-producing bacteria in complex fecal samples has been difficult to estimate by targeting the 16S rRNA gene, because these bacteria do not form a homogeneous phylogenetic group, and both butyrate producers and non-butyrate producers are found within the same phylogenetic clusters belonging to Clostridium clusters I, III, IV, XI, XIVa, XV, and XVI (27). Two alternative pathways for butyrate formation in bacteria harboring the rumen and human colon have been described (7, 26). The majority of human colonic butyrate producers use butyryl-coenzyme A (CoA) CoA transferase, whereas soil bacteria mostly utilize the butyrate kinase for the last step of butyrate formation (26, 27). However, information about the butyrate pathways used by intestinal bacteria in pigs is not available.In addition to the effects of the functional properties of NSP on intestinal physiology and fermentation processes, selection of specific NSP fractions may also prevent or stimulate overgrowth of pathogenic bacteria. For instance, dietary inclusion of highly viscous carboxymethylcellulose (CMC) has been shown to increase fecal shedding of enterotoxigenic Escherichia coli in weaned pigs (15). There is a need to identify those dietary NSP fractions that may either increase or reduce the numbers of potential pathogenic bacteria to formulate diets exerting beneficial effects on gut health, which is particularly important in antibiotic-free feeding regimens.Most studies pertaining on the effect of diet composition on the bacterial community in pigs have employed natural NSP sources and cereal-based diets, thereby resulting in a mixture of different soluble and insoluble NSP showing considerable interactions and modification of intestinal bacterial ecophysiology (6, 36, 37). Purified NSP fractions are increasingly available from the bioprocessing industry for use in food preparation and potentially in animal feeds, where economics and possible health benefits warrant this use. However, less is known about the fermentative properties of purified NSP fractions than about those of NSP in the grain matrix (37), which may also differ according to their origins.The aim of the present study was to examine the effects of four purified NSP fractions differing in their functional properties, i.e., viscosity and fermentability, on the ileal and fecal bacterial community, butyrate production pathway genes, and the occurrence of virulence factor genes of swine-pathogenic E. coli, including enterotoxigenic and enteroaggregative E. coli (11, 13).(This study was presented in part at the 11th Digestive Physiology in Pigs Symposium, Reus, Spain, 19 to 22 May 2009.)     

Purification and partial characterization of oxalate oxidase from leaves of forage Sorghum (Sorghum vulgare var. KH-105) seedlings

An oxalate oxidase was purified to apparent homogeneity from the leaves of 10-days old seedlings of forage Sorghum (Sorghum vulgare var. KH-105). The enzyme had a Mr of 124 kDa with two identical subunits, an optimum pH of 4.5, optimum temperature of 37 degrees C and activation energy (Ea) of 2.0338 Kcal/mol. The rate of reaction was linear up to 7 min. K(m) value for oxalate was 0.22 mM. The enzyme was stimulated by Cu2+ and inhibited by EDTA, NaCN, diethyldithiocarbamate, Na2SO4, but unaffected by NaCl at 0.1 mM concentration. Although the enzyme was stimulated by flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), UV and visible spectra of the enzyme did not match with that of a flavoprotein. The positive reaction of the enzyme with orcinol-H2SO4 reagent indicated its glycoprotein nature. The superiority of the purified enzyme over earlier reported oxalate oxidases for determination of urinary oxalate has been demonstrated.     

Optimization and evaluation of gastroretentive ranitidine HCl microspheres by using design expert software

The current study involves the development and optimization of their drug entrapment and ex vivo bioadhesion of multiunit chitosan based floating system containing Ranitidine HCl by ionotropic gelation method for gastroretentive delivery. Chitosan being cationic, non-toxic, biocompatible, biodegradable and bioadhesive is frequently used as a material for drug delivery systems and used to transport a drug to an acidic environment where it enhances the transport of polar drugs across epithelial surfaces. The effect of various process variables like drug polymer ratio, concentration of sodium tripolyphosphate and stirring speed on various physiochemical properties like drug entrapment efficiency, particle size and bioadhesion was optimized using central composite design and analyzed using response surface methodology. The observed responses were coincided well with the predicted values given by the optimization technique. The optimized microspheres showed drug entrapment efficiency of 74.73%, particle size 707.26μm and bioadhesion 71.68% in simulated gastric fluid (pH 1.2) after 8h with floating lag time 40s. The average size of all the dried microspheres ranged from 608.24 to 720.80μm. The drug entrapment efficiency of microspheres ranged from 41.67% to 87.58% and bioadhesion ranged from 62% to 86%. Accelerated stability study was performed on optimized formulation as per ICH guidelines and no significant change was found in drug content on storage.     

Large‐scale survey and database of high affinity ligands for peptide recognition modules

Many proteins involved in signal transduction contain peptide recognition modules (PRMs) that recognize short linear motifs (SLiMs) within their interaction partners. Here, we used large‐scale peptide‐phage display methods to derive optimal ligands for 163 unique PRMs representing 79 distinct structural families. We combined the new data with previous data that we collected for the large SH3, PDZ, and WW domain families to assemble a database containing 7,984 unique peptide ligands for 500 PRMs representing 82 structural families. For 74 PRMs, we acquired enough new data to map the specificity profiles in detail and derived position weight matrices and binding specificity logos based on multiple peptide ligands. These analyses showed that optimal peptide ligands resembled peptides observed in existing structures of PRM‐ligand complexes, indicating that a large majority of the phage‐derived peptides are likely to target natural peptide‐binding sites and could thus act as inhibitors of natural protein–protein interactions. The complete dataset has been assembled in an online database (http://www.prm‐db.org) that will enable many structural, functional, and biological studies of PRMs and SLiMs.