A Mechano-Activated Cell Reporter System as a Proxy for Flow-Dependent Endothelial Atheroprotection

The vascular endothelium plays a critical role in the health and disease of the cardiovascular system. Importantly, biomechanical stimuli generated by blood flow and sensed by the endothelium constitute important local inputs that are translated into transcriptional programs and functional endothelial phenotypes. Pulsatile, laminar flow, characteristic of regions in the vasculature that are resistant to atherosclerosis, evokes an atheroprotective endothelial phenotype. This atheroprotective phenotype is integrated by the transcription factor Kruppel-like factor-2 (KLF2), and therefore the expression of KLF2 can be used as a proxy for endothelial atheroprotection. Here, we report the generation and characterization of a cellular KLF2 reporter system, based on green fluorescence protein (GFP) expression driven by the human KLF2 promoter. This reporter is induced selectively by an atheroprotective shear stress waveform in human endothelial cells, is regulated by endogenous signaling events, and is activated by the pharmacological inducer of KLF2, simvastatin, in a dose-dependent manner. This reporter system can now be used to probe KLF2 signaling and for the discovery of a novel chemical-biological space capable of acting as the “pharmacomimetics of atheroprotective flow” on the vascular endothelium.

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The P-element-induced silencing effect of KP transposons is dose dependent in Drosophila melanogaster

Genome ◽

10.1139/g11-023 ◽

2011 ◽

Vol 54 (9) ◽

pp. 752-762 ◽

Cited By ~ 7

Author(s):

Alireza Sameny ◽

John Locke

Keyword(s):

Drosophila Melanogaster ◽

Critical Role ◽

Mutagenic Effect ◽

P Element ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

P Elements ◽

Single Well ◽

Dose Dependent

Transposable elements are found in the genomes of all eukaryotes and play a critical role in altering gene expression and genome organization. In Drosophila melanogaster, transposable P elements are responsible for the phenomenon of hybrid dysgenesis. KP elements, a deletion-derivative of the complete P element, can suppress this mutagenic effect. KP elements can also silence the expression of certain other P-element-mediated transgenes in a process called P-element-dependent silencing (PDS), which is thought to involve the recruitment of heterochromatin proteins. To explore the mechanism of this silencing, we have mobilized KP elements to create a series of strains that contain single, well-defined KP insertions that show PDS. To understand the quantitative role of KP elements in PDS, these single inserts were combined in a series of crosses to obtain genotypes with zero, one, or two KP elements, from which we could examine the effect of KP gene dose. The extent of PDS in these genotypes was shown to be dose dependent in a logarithmic rather than linear fashion. A logarithmic dose dependency is consistent with the KP products interacting with heterochromatic proteins in a concentration-dependent manner such that two molecules are needed to induce gene silencing.

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Microbial trend analysis for common dynamic trend, group comparison and classification in longitudinal microbiome study

10.1101/2020.01.30.926824 ◽

2020 ◽

Author(s):

Chan Wang ◽

Jiyuan Hu ◽

Martin J. Blaser ◽

Huilin Li

Keyword(s):

Trend Analysis ◽

Critical Role ◽

Human Microbiome ◽

Real Data ◽

Microbial Dynamics ◽

Individual Subject ◽

Microbial Profiling ◽

Health And Disease ◽

Microbiome Data

AbstractMotivationThe human microbiome is inherently dynamic and its dynamic nature plays a critical role in maintaining health and driving disease. With an increasing number of longitudinal microbiome studies, scientists are eager to learn the comprehensive characterization of microbial dynamics and their implications to the health and disease-related phenotypes. However, due to the challenging structure of longitudinal microbiome data, few analytic methods are available to characterize the microbial dynamics over time.ResultsWe propose a microbial trend analysis (MTA) framework for the high-dimensional and phylogenetically-based longitudinal microbiome data. In particular, MTA can perform three tasks: 1) capture the common microbial dynamic trends for a group of subjects on the community level and identify the dominant taxa; 2) examine whether or not the microbial overall dynamic trends are significantly different in groups; 3) classify an individual subject based on its longitudinal microbial profiling. Our extensive simulations demonstrate that the proposed MTA framework is robust and powerful in hypothesis testing, taxon identification, and subject classification. Our real data analyses further illustrate the utility of MTA through a longitudinal study in mice.ConclusionsThe proposed MTA framework is an attractive and effective tool in investigating dynamic microbial pattern from longitudinal microbiome studies.

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Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0050159 ◽

1990 ◽

Vol 5 (2) ◽

pp. 159-166 ◽

Cited By ~ 6

Author(s):

N. G. N. Milton ◽

E. W. Hillhouse ◽

S. A. Nicholson ◽

C. H. Self ◽

A. M. McGregor

Keyword(s):

Monoclonal Antibodies ◽

Enzyme Linked Immunosorbent Assay ◽

Dependent Manner ◽

Corticotrophin Releasing Factor ◽

Tissue Extracts ◽

Rat Pituitary ◽

Hypothalamic Tissue ◽

Dose Dependent

ABSTRACT Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine γ-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB 003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7·1 and 6·8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.

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Isolation and Characterization of a Lectin-like Protein (SBLP) from the Dried Roots of Scutellaria baicalensis (Lamiaceae)

Natural Product Communications ◽

10.1177/1934578x1801301232 ◽

2018 ◽

Vol 13 (12) ◽

pp. 1934578X1801301

Author(s):

Huiqin Wang ◽

Guanzhen Gao ◽

Lijing Ke ◽

Jianwu Zhou ◽

Pingfan Rao

Keyword(s):

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Scutellaria Baicalensis ◽

Dependent Manner ◽

Scutellaria Baicalensis Georgi ◽

Isolation And Characterization ◽

Pas Staining ◽

Ph Range ◽

Dose Dependent

A novel lectin-like protein with MW 63.2 kDa, designated as SBLP, has been isolated and characterized from the dried roots of Scutellaria baicalensis Georgi (Lamiaceae). SBLP was purified by ammonium sulfate precipitation and anion exchange chromatography. It is a glycoprotein according to a PAS staining assay and consisting of protein (86.0%) and sugar (14.0%). Its N-terminal amino acid sequence was determined as GSAVGFLY by Edman degradation. SBLP showed hemagglutinating activity against human and rooster erythrocytes, which were stable below 60°C and in the pH range of 4 −10. Furthermore, SBLP was found to be stimulated by Ca2+, Na+, Ba2+, Zn2+ ions, which suggested it was a metal-dependent lectin. SBLP inhibited the growth of Fusarium oxysporum f.sp. lycopersici and Alternaria eichhorniae in the a dose-dependent manner, and suppressed the proliferation of HepG2 tumor cells with an IC50 of 1.00 μM. This is the first report of a lectin from Radix Scutellariae.

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Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.01382-07 ◽

2007 ◽

Vol 73 (17) ◽

pp. 5411-5420 ◽

Cited By ~ 22

Author(s):

Yu-Sin Jang ◽

Young Ryul Jung ◽

Sang Yup Lee ◽

Ji Mahn Kim ◽

Jeong Wook Lee ◽

...

Keyword(s):

Succinic Acid ◽

Shuttle Vector ◽

Green Fluorescence Protein ◽

Expression Vectors ◽

Rumen Bacteria ◽

Gene Encoding ◽

Shuttle Vectors ◽

Genes Encoding ◽

Fluorescence Protein

ABSTRACT Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 106 and 7.1 × 106 transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

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Characterization of the role of CaMKI-like kinase (CKLiK) in human granulocyte function

Blood ◽

10.1182/blood-2004-09-3755 ◽

2005 ◽

Vol 106 (3) ◽

pp. 1076-1083 ◽

Cited By ~ 29

Author(s):

Sandra Verploegen ◽

Laurien Ulfman ◽

Hanneke W. M. van Deutekom ◽

Corneli van Aalst ◽

Henk Honing ◽

...

Keyword(s):

Respiratory Burst ◽

Critical Role ◽

Neutrophil Migration ◽

Platelet Activating Factor ◽

Dependent Manner ◽

Functional Responses ◽

Effector Functions ◽

C Terminus ◽

Human Granulocytes ◽

Dose Dependent

AbstractActivation of granulocyte effector functions, such as induction of the respiratory burst and migration, are regulated by a variety of relatively ill-defined signaling pathways. Recently, we identified a novel Ca2+/calmodulin-dependent kinase I-like kinase, CKLiK, which exhibits restricted mRNA expression to human granulocytes. Using a novel antibody generated against the C-terminus of CKLiK, CKLiK was detected in CD34+-derived neutrophils and eosinophils, as well as in mature peripheral blood granulocytes. Activation of human granulocytes by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF), but not the phorbol ester PMA (phorbol 12-myristate-13-acetate), resulted in induction of CKLiK activity, in parallel with a rise of intracellular Ca2+ [Ca2+]i. To study the functionality of CKLiK in human granulocytes, a cell-permeable CKLiK peptide inhibitor (CKLiK297-321) was generated which was able to inhibit kinase activity in a dose-dependent manner. The effect of this peptide was studied on specific granulocyte effector functions such as phagocytosis, respiratory burst, migration, and adhesion. Phagocytosis of Aspergillus fumigatus particles was reduced in the presence of CKLiK297-321 and fMLP-induced reactive oxygen species (ROS) production was potently inhibited by CKLiK297-321 in a dose-dependent manner. Furthermore, fMLP-induced neutrophil migration on albumin-coated surfaces was perturbed, as well as β2-integrin-mediated adhesion. These findings suggest a critical role for CKLiK in modulating chemoattractant-induced functional responses in human granulocytes.

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Gene and cDNA cloning and characterization of the mouse V3/V1b pituitary vasopressin receptor

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0220251 ◽

1999 ◽

Vol 22 (3) ◽

pp. 251-260 ◽

Cited By ~ 33

Author(s):

MA Ventura ◽

P Rene ◽

Y de Keyzer ◽

X Bertagna ◽

E Clauser

Keyword(s):

Inositol Phosphate ◽

Rank Order ◽

Single Gene ◽

Dependent Manner ◽

Binding Studies ◽

V1b Receptor ◽

Competition Binding ◽

Receptor Cdna ◽

Dose Dependent

The gene of the mouse V3/V1b receptor was identified by homology cloning. One of the genomic clones contained the entire coding sequence. The cDNA presented high identity with rat (92%) and human (84%) sequences. Southern blot analysis indicated the existence of a single gene. Tissue distribution was studied by RT-PCR. The major site of expression was the pituitary. A faint signal was also present in hypothalamus, brain, adrenal, pancreas and colon. The mouse corticotroph cell line, AtT20, did not express the transcript. In order to confirm the identity of the sequence, the V3/V1b receptor cDNA was cloned and stably expressed in CHO-AA8 Tet-Off cells under the control of tetracycline. When transfected cells were treated with arginine vasopressin (AVP), inositol phosphate production increased in a dose-dependent manner, indicating that the V3/V1b receptor couples to phospholipase C. Moreover, AVP did not stimulate cAMP production. Binding studies with [3H]AVP indicated that the affinity of the mouse V3/V1b receptor (Kd=0.5 nM) is similar to that reported for rat and human receptors. The rank order of potency established in competition binding experiments with different analogues was representative of a V3/V1b profile, distinct from V1a and V2. However, significant differences were found between human and mouse receptors tested in parallel. Thus the pharmacology of V3/V1b receptors can not be transposed among different species.

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Actin-dependent Lamellipodia Formation and Microtubule-dependent Tail Retraction Control-directed Cell Migration

Molecular Biology of the Cell ◽

10.1091/mbc.11.9.2999 ◽

2000 ◽

Vol 11 (9) ◽

pp. 2999-3012 ◽

Cited By ~ 141

Author(s):

Christoph Ballestrem ◽

Bernhard Wehrle-Haller ◽

Boris Hinz ◽

Beat A. Imhof

Keyword(s):

Cell Migration ◽

Cell Body ◽

Actin Polymerization ◽

Green Fluorescence Protein ◽

Time Lapse ◽

Dependent Manner ◽

Directed Cell Migration ◽

Cell Front ◽

Fluorescence Protein

Migrating cells are polarized with a protrusive lamella at the cell front followed by the main cell body and a retractable tail at the rear of the cell. The lamella terminates in ruffling lamellipodia that face the direction of migration. Although the role of actin in the formation of lamellipodia is well established, it remains unclear to what degree microtubules contribute to this process. Herein, we have studied the contribution of microtubules to cell motility by time-lapse video microscopy on green flourescence protein-actin- and tubulin-green fluorescence protein–transfected melanoma cells. Treatment of cells with either the microtubule-disrupting agent nocodazole or with the stabilizing agent taxol showed decreased ruffling and lamellipodium formation. However, this was not due to an intrinsic inability to form ruffles and lamellipodia because both were restored by stimulation of cells with phorbol 12-myristate 13-acetate in a Rac-dependent manner, and by stem cell factor in melanoblasts expressing the receptor tyrosine kinase c-kit. Although ruffling and lamellipodia were formed without microtubules, the microtubular network was needed for advancement of the cell body and the subsequent retraction of the tail. In conclusion, we demonstrate that the formation of lamellipodia can occur via actin polymerization independently of microtubules, but that microtubules are required for cell migration, tail retraction, and modulation of cell adhesion.

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Characterization of adenosine receptor(s) involved in adenosine-induced bronchoconstriction in an allergic mouse model

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00353.2002 ◽

2003 ◽

Vol 284 (6) ◽

pp. L1012-L1019 ◽

Cited By ~ 33

Author(s):

Ming Fan ◽

Weixi Qin ◽

S. Jamal Mustafa

Keyword(s):

Mouse Model ◽

Adenosine Receptor ◽

Receptor Subtypes ◽

Dependent Manner ◽

Transcript Levels ◽

Allergen Sensitization ◽

Selective Agonist ◽

Dose Dependent ◽

Allergic Mouse

We recently reported that adenosine caused bronchoconstriction and enhanced airway inflammation in an allergic mouse model. In this study, we further report the characterization of the subtype of adenosine receptor(s) involved in bronchoconstriction. 5′-( N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine agonist, elicited bronchoconstriction in a dose-dependent manner. Little effects of N 6-cyclopentyladenosine (A1-selective agonist) and 2- p-(2-carboxyethyl)phenethylamino-5′- N-ethylcarboxamidoadenosine (A2A-selective agonist) compared with NECA were observed in this model. 2-Chloro- N 6-(3-iodobenzyl)-9-[5-(methylcarbamoyl)-β-d-ribofuranosyl]adenosine, an A3-selective receptor agonist, produced a dose-dependent bronchoconstrictor response, which was blocked by selective A3 antagonist 2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS1523). However, MRS1523 only partially inhibited NECA-induced bronchoconstriction. Neither selective A1 nor A2A antagonists affected NECA-induced bronchoconstriction. Enprofylline, a relatively selective A2B receptor antagonist, blocked partly NECA-induced bronchoconstriction. Furthermore, a combination of enprofylline and MRS1523 completely abolished NECA-induced bronchoconstrictor response. Using RT-PCR, we found that all four adenosine receptor subtypes are expressed in control lungs. Allergen sensitization and challenge significantly increased transcript levels of the A2B and A3receptors, whereas the A1 receptor message decreased. No change in transcript levels of A2A receptors was observed after allergen sensitization and challenge. These findings suggest that A2B and A3 adenosine receptors play an important role in adenosine-induced bronchoconstriction in our allergic mouse model. Finally, whether the airway effects of the receptor agonists/antagonists are direct or indirect needs further investigations.

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Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/506363 ◽

2010 ◽

Vol 2010 ◽

pp. 1-11 ◽

Cited By ~ 5

Author(s):

Yi-Shin Pan ◽

Hung-Ju Wei ◽

Chung-Chieh Chang ◽

Chung-Hung Lin ◽

Ting-Shyang Wei ◽

...

Keyword(s):

Insect Cell ◽

Matrix Protein ◽

Green Fluorescence Protein ◽

Structural Rearrangement ◽

Jurkat Cells ◽

Proton Channel ◽

Receptor Interaction ◽

Transmission Electron ◽

Fluorescence Protein

We have constructed virus-like particles (VLPs) harboring hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1) ,and proton channel protein (M2) using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP) was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

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