1093 Identification and Characterization of Colonic-Exosomes: micro-RNA Mediated Horizontal Gene Transfer Within the Colonic Epithelia In Vitro and In Vivo

Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg–deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg–like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.

Download Full-text

Identification and Characterization of Two Bordetella avium Gene Products Required for Hemagglutination

Infection and Immunity ◽

10.1128/iai.00140-10 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2370-2376 ◽

Cited By ~ 14

Author(s):

Louise M. Temple ◽

David M. Miyamoto ◽

Manju Mehta ◽

Christian M. Capitini ◽

Stephen Von Stetina ◽

...

Keyword(s):

Whooping Cough ◽

Bioinformatic Analysis ◽

Tracheal Ring ◽

Tracheal Cell ◽

Bordetella Avium ◽

Identification And Characterization

ABSTRACT Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

Download Full-text

A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0665 ◽

2011 ◽

Vol 22 (1) ◽

pp. 141-152 ◽

Cited By ~ 65

Author(s):

Xiao-Wei Chen ◽

Dara Leto ◽

Tingting Xiong ◽

Genggeng Yu ◽

Alan Cheng ◽

...

Keyword(s):

Glucose Transporter ◽

Critical Role ◽

Inhibitory Effect ◽

Small Gtpase ◽

Regulatory Subunit ◽

Hydrolysis Of ◽

Identification And Characterization

Insulin stimulates glucose transport in muscle and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation.

Download Full-text

Computational Insight into the Effect of Natural Compounds on the Destabilization of Preformed Amyloid-β(1-40) Fibrils

10.20944/preprints201805.0380.v1 ◽

2018 ◽

Author(s):

Francesco Tavanti ◽

Alfonso Pedone ◽

Maria Cristina Menziani

Keyword(s):

Therapeutic Strategy ◽

Therapeutic Potential ◽

Structure Stability ◽

Insoluble Form ◽

The Brain ◽

Identification And Characterization ◽

Insight Into

One of the principal hallmarks of Alzheimer’s disease (AD) is related to the aggregation of amyloid-β fibrils in an insoluble form in the brain, also known as amyloidosis. Therefore, a prominent therapeutic strategy against AD consists either in blocking the amyloid aggregation and/or destroying the already formed aggregates. Natural products have shown significant therapeutic potential as amyloid inhibitors from in vitro studies as well as in vivo animal tests. In this study, the interaction of five natural biophenols (curcumin, dopamine, (-)-Epigallocatechin-3-gallate, Quercetin, and Rosmarinic acid) with the amyloid-β(1-40) fibrils has been studied through computational simulations. The results allowed the identification and characterization of the different binding modalities of each compounds and their consequences on fibril dynamics and aggregation. It emerges that the lateral aggregation of the fibrils is strongly influenced by the intercalation of the ligands, which modulate the double-layered structure stability.

Download Full-text

Identification and Characterization of Metabolites of Irinotecan in-vivo and in-vitro matrixes by HPLC/LC-MS

Research in Pharmacy and Health Sciences ◽

10.32463/rphs.2016.v02i03.42 ◽

2016 ◽

Vol 2 (3) ◽

pp. 211-218

Author(s):

Nidhi Srivastava ◽

Vishal Dubey ◽

Madhumita Sengar ◽

Rastogi Sameer

Keyword(s):

Method Development ◽

Biological Fluids ◽

Parent Compound ◽

Enzymatic Reaction ◽

Liver Microsomes ◽

Metabolite Identification ◽

Identification And Characterization

In the present study metabolite identification and characterization has done by using HPLC and LC-MS. During method development various mobile phases have tried for identification of metabolites. The matrixes selected for in- vivo study were urine because nearly all the metabolites of irinotecan were obtained in it. The extraction mixtures have selected to retain maximum amount of analyte with less effort. During experiment four extraction solvents were used in six different concentrations out of which TBME suit our method. In-vitro study done by Human Liver microsomes by using Phosphate buffer (pH 7.4) and NADPH as co-factors for initiation of enzymatic reaction. Irinotecan is a prodrug that is converted in the liver to an active metabolite, SN-38. It is eliminate in Bile and Faeces and thus its dose reduced in Hepatic Failure. Irinotecan act by inhibiting Topoisomerase-1.It is the enzyme which nicks, introduces negative supercoils and reseals the DNA strand. Conventionally, drug metabolite identification in the past has usually been based on the comparison of ultraviolet (UV) spectral data and high-performance liquid chromatography (HPLC) retention times of isolated ‘unknown’ metabolites with those of synthesised standards. Such a method of detecting and characterising drug metabolites is an uncertain, time-consuming and expensive process, as well as affording very limited structural information. Furthermore, Phase I metabolism of a drug candidate often results in only minor structural modification of the parent compound; these minor changes can make it particularly difficult to determine suitable chromatographic conditions to effect HPLC separation of metabolites. This study describes contemporary approach to identification and characterization of xenobiotic metabolites in complex biological fluids derived from drug metabolism studies.

Download Full-text

Identification and Characterization of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase from Bacillus megaterium

Applied and Environmental Microbiology ◽

10.1128/aem.00621-09 ◽

2009 ◽

Vol 75 (16) ◽

pp. 5290-5299 ◽

Cited By ~ 24

Author(s):

Hui-Ju Chen ◽

Shih-Chuan Pan ◽

Gwo-Chyuan Shaw

Keyword(s):

Bacillus Megaterium ◽

Surface Proteins ◽

In Vitro Degradation ◽

Transmission Electron ◽

Almost All ◽

Identification And Characterization ◽

Striking Contrast

ABSTRACT A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.

Download Full-text