Viscoelastic Properties of a Mixed Culture Biofilm from Rheometer Creep Analysis

The mechanical properties of mixed culture biofilms were determined by creep analysis using an AR1000 rotating disk rheometer. The biofilms were grown directly on the rheometer disks which were rotated in a chemostat for 12 d. The resulting biofilms were heterogeneous and ranged from 35?μm to 50?μm in thickness. The creep curves were all viscoelastic in nature. The close agreement between stress and strain ratio of a sample tested at 0.1 and 0.5 Pa suggested that the biofilms were tested in the linear viscoelastic range and supported the use of linear viscoelastic theory in the development of a constitutive law. The experimental data was fit to a 4-element Burger spring and dashpot model. The shear modulus (G) ranged from 0.2 to 24 Pa and the viscous coefficient (η) from 10 to 3000 Pa. These values were in the same range as those previously estimated from fluid shear deformation of biofilms in flow cells. A viscoelastic biofilm model will help to predict shear related biofilm phenomena such as elevated pressure drop, detachment, and the flow of biofilms over solid surfaces.     

Functional Characterization of a Glucosyltransferase Specific to Flavonoid 7-O-Glucosides from Withania somnifera

Flavonoids are a large class of phenylpropanoid-derived secondary metabolites, which are usually glycosylated by UDP-glycosyltransferases with one or more sugar groups. Here, we report the cloning and biochemical characterization of a flavonoid glycosyltransferase gene from Withania somnifera (WsGT), which is an important medicinal plant used in Ayurvedic formulations. Using PCR primers, designed for a highly conserved region of previously reported glycosyltransferases, we were able to isolate the corresponding fragment of the WsGT gene. Rapid amplification of cDNA ends (RACE) was then employed to isolate full-length cDNA, which had an open reading frame of 1,371 bp that encode for 456 amino acids. Phylogenetic analysis indicated that WsGT was similar to that of family 1 GT-B glycosyltransferase. Biochemical analysis revealed that WsGT interacts with UDP-glucose and was capable of regiospecifically glycosylating flavonoid-7-ols, such as apigenin, naringenin, luteolin, diadzein and genistein. Expression profiling studies showed that WsGT was highly expressed in young and mature leaves of W. somnifera. Furthermore, exposure to salicylic acid enhanced the expression of WsGT in the leaves and heat shock treatment resulted in decreased expression of WsGT after an initial increase. This may suggest the role of WsGT in response to abiotic/biotic stresses.     

in Silico mutagenesis and docking studies of active site residues suggest altered substrate specificity and possible physiological role of Cinnamoyl CoA Reductase 1 (Ll-CCRH1)

Cinnamoyl CoA reductase (CCR) carries out the first committed step in monolignol biosynthesis and acts as a first regulatory point in lignin formation. CCR shows multiple substrate specificity towards various cinnamoyl CoA esters. Here, in Silico mutagenesis studies of active site residues of Ll-CCRH1 were carried out. Homology modeling based modeled 3D structure of Ll-CCRH1 was used as template for in Silico mutant preparations. Docking simulations of Ll-CCRH1 mutants with CoA esters by AutoDock Vina tools showed altered substrate specificity as compared to wild type. The study evidences that conformational changes, and change in geometry or architecture of active site pocket occurred following mutations. The altered substrate specificity for active site mutants suggests the possible physiological role of CCR either in lignin formation or in defense system in plants.

Abbreviations

Ll-CCRH1 - Leucaena leucocephala cinnamoyl CoA reductase 1, OPLS - Optimized Potentials for Liquid Simulations, RMSD - Root Mean Square Deviation.     

Exploration of pyridine containing heteroaryl analogs of biaryl ureas as DGAT1 inhibitors

The diacylglycerol acyltransferase enzyme, DGAT1, presents itself as a potential target for obesity as this enzyme is dedicated to the final committed step in triglyceride biosynthesis. Biphenyl ureas, exemplified by compound 4, have been reported to be potent hDGAT1 inhibitors. We have synthesized and evaluated 2-pyridyl and 3-pyridyl containing biaryl ureas as hDGAT1 inhibitors. Our aim was to incorporate a heteroaryl scaffold within these molecules thereby improving the cLogP profile and making these compounds more drug-like. Compounds within this series exhibited potent hDGAT1 inhibition when evaluated using an in vitro enzymatic assay. Selected compounds were also subjected to an oral fat tolerance test in mice where the percent triglyceride reduction versus a vehicle control was evaluated. Of the studied heteroaryl analogs compound 44 exhibited an in vitro IC(50) of 17nM and a plasma triglyceride reduction of 79% along with a 12-fold improvement in solubility over the biphenyl urea compound 4.     

Taxonomy of five species of cyrtophorids (Protozoa: Ciliophora) including consideration of the phylogeny of two new genera

Cyrtophorids are a specialized group of ciliated protozoa with multitudinous morphotypes. In the present work, the morphology and infraciliature of two new and three rarely known species, including two new genera of cyrtophorid ciliates, Heterohartmannula fangi gen. et sp. nov. , Aporthotrochilia pulex (Deroux, 1976) gen. et comb. nov. , Trochilia alveolata sp. nov. , Trochochilodon flavus Deroux, 1976, and Hypocoma acinetarum Collin, 1907, are described. Heterohartmannula gen. nov. is mainly characterized by a combination of features: two circumoral kineties obliquely arranged, podite not surrounded by somatic kineties, and no distinct gap between left and right ciliary field. Aporthotrochilia gen. nov. is diagnosed mainly by: podite present, oral ciliature reduced to two fragments, several kinety fragments positioned on the right posterior of frontoventral kineties and several terminal fragments. Phylogenetic analyses based on the small subunit rRNA (SSU rRNA) gene sequences support the establishment of two new genera and indicate that Heterohartmannula is most closely related to Hartmannula, and Aporthotrochilia is basal to the Cyrtophoria‐Chonotrichia clade. Trochilia alveolata sp. nov. differs from its congeners mainly by having a conspicuous alveolar layer. In addition, detailed live and infraciliature data of Hypocoma acinetarum and Trochochilodon flavus are supplied. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 1–17.     

Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.     

Biodiversity of stream insects in the Malaysian Peninsula: spatial patterns and environmental constraints

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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.     

Use of Dactylopius coccus Costa (Hemiptera: Coccoidea) Pigments in the Detection of Larvae and Pupae of Plutella xylostella (L.) (Lepidoptera: Yponomeutidae)

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