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

2011 ◽  
Vol 22 (1) ◽  
pp. 141-152 ◽  
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.

Inhibition of LH-stimulated cyclic AMP formation in pre-ovulatory rat granulosa cells by follicular fluid

1980 ◽  
Vol 95 (1) ◽  
pp. 84-89 ◽  
Author(s):  
Knut Nordenström ◽  
Anita Sjögren ◽  
Lars Hamberger
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Phosphodiesterase Inhibitor ◽  
Inhibitory Effect ◽  
Female Rats ◽  
Bicarbonate Buffer ◽  
Ovulatory Follicles

Abstract. Immature female rats were injected sc with a single dose of PMSG to induce growth and maturation of ovarian follicles. In the morning of prooestrus the rats were given a single ip injection of LH (10 μg/rat) or 0.154 m NaCl, 2 h prior to sacrifice. Granulosa cells were isolated from the pre-ovulatory follicles and incubated in Krebs bicarbonate buffer, for 1 h with or without in vitro addition of various test substances. Following incubation the amounts of cAMP in tissue plus medium were determined. It was found that the isolated granulosa cells exposed to LH in vivo responded to the addition of LH in vitro with a production of high amounts of cAMP, i.e. these cells were not refractory to LH stimulation and in fact responded better than granulosa cells isolated from ovaries not exposed to LH in vivo. The addition to the incubation medium of follicular fluid (FFl) obtained from pre-ovulatory follicles decreased the effect of LH in vitro when added at a final concentration of 1% and completely abolished it at a concentration of 3%. Removal of steroids from the FFl did not influence the inhibitory effect and the addition of a phosphodiesterase inhibitor (IBMX) in vitro did not alter the results in principle. These results point to the existence of a factor in the FF1 which interacts with the sensitivity of the isolated preovulatory granulosa cells to repeated exposures to LH. Characterization of this factor is subject to further investigations.

scholarly journals Identification and characterization of T reg–like cells in zebrafish

2017 ◽  
Vol 214 (12) ◽  
pp. 3519-3530 ◽  
Author(s):  
Melissa Kasheta ◽  
Corrie A. Painter ◽  
Finola E. Moore ◽  
Riadh Lobbardi ◽  
Alysia Bryll ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Live Cell Imaging ◽  
Genetic Ablation ◽  
Functional Studies ◽  
Normal Functions ◽  
Inflammatory Phenotype ◽  
Identification And Characterization

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.

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

2010 ◽  
Vol 78 (6) ◽  
pp. 2370-2376 ◽  
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.

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

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.

scholarly journals Cocoa (Theobroma cacao L.) Seed Proteins’ Anti-Obesity Potential through Lipase Inhibition Using In Silico, In Vitro and In Vivo Models

Foods ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1359
Author(s):  
Luis Jorge Coronado-Cáceres ◽  
Griselda Rabadán-Chávez ◽  
Luis Mojica ◽  
Blanca Hernández-Ledesma ◽  
Lucía Quevedo-Corona ◽  
...  
Keyword(s):  
In Silico ◽  
Seed Proteins ◽  
Inhibitory Effect ◽  
Mean Value ◽  
Intragastric Administration ◽  
Degree Of Hydrolysis ◽  
In Vivo Models ◽  
Hydrolysis Of

The aim of this study was to determine the pancreatic lipase (PL) inhibitory effect of cocoa protein (CP) hydrolysates (CPH) using in silico and in vitro approaches, and an in vivo high-fat diet (HF) obese rat model. The results showed better theoretical affinity on PL for cocoa peptides EEQR, GGER, QTGVQ, and VSTDVNIE released from vicilin and albumins (−6.5, −6.3, −6.2, and −6.1 kcal/mol, respectively). Absorption, distribution, metabolism, and excretion (ADMET) prediction showed the human intestinal absorption (HIA) capacity of orlistat and eight cocoa peptides, demonstrating that they presented a low probability of toxicity with values lower than 0.6, while the orlistat has a high probability of hepatotoxicity with a mean value of 0.9. CPH (degree of hydrolysis of 55%) inhibited PL with an IC50 (concentration needed to inhibit 50% of enzyme activity) value of 1.38 mg/mL. The intragastric administration of 150 mg CP/kg/day to rats increased total lipids and triglycerides excretion in feces, ranging from 11% to 15% compared to the HF-diet. The HF + CP-diet also significantly decreased (p < 0.05) the apparent rate of fat absorption compared with the HF group. These results suggest that CP has anti-obesity potential by inhibiting PL, thus helping to prevent the development of non-communicable diseases.

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

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.

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

2009 ◽  
Vol 75 (16) ◽  
pp. 5290-5299 ◽  
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.

Identification and characterization of variant alleles of human acetyltransferase NAT1 with defective function using p-aminosalicylate as an in-vivo and in-vitro probe

1998 ◽  
Vol 8 (1) ◽  
pp. 55-66 ◽  
Author(s):  
Nicola C. Hughes ◽  
Susan A. Janezic ◽  
Karina L. McQueen ◽  
Michael A.S. Jewett ◽  
Trisha Castranio ◽  
...  
Keyword(s):  
Variant Alleles ◽  
Identification And Characterization
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