Adult human circulating CD34−Lin−CD45−CD133− cells can differentiate into hematopoietic and endothelial cells

Abstract A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34−Lin−CD45−CD133− cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34−Lin−CD45−CD133− cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34−Lin−CD45−CD133− cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34−Lin−CD45−CD133− cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34−Lin−CD45−CD133− cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

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Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽

10.1099/mic.0.28753-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2129-2135 ◽

Cited By ~ 17

Author(s):

Taku Oshima ◽

Francis Biville

Keyword(s):

Functional Characterization ◽

Anaerobic Growth ◽

Functional Identification ◽

New Members ◽

E Coli ◽

Inner Membrane Protein ◽

Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Microorganisms ◽

10.3390/microorganisms9051107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1107

Author(s):

Wonho Choi ◽

Yoshihiro Yamaguchi ◽

Ji-Young Park ◽

Sang-Hyun Park ◽

Hyeok-Won Lee ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Physiological Function ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

In Vitro Assays ◽

Cell Growth Inhibition ◽

The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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Omics profiling identifies MAPK/ERK pathway as a gatekeeper of nephron progenitor metabolism

10.1101/2021.09.27.461969 ◽

2021 ◽

Author(s):

Hyuk Nam Kwon ◽

Kristen Kurtzeborn ◽

Xing Jin ◽

Bruno Reversade ◽

Sunghyouk Park ◽

...

Keyword(s):

Functional Characterization ◽

Mitogen Activated Protein Kinase ◽

Metabolic Effects ◽

Erk Pathway ◽

Double Knockout ◽

Nephron Progenitor ◽

Progenitor Maintenance

Nephron endowment is defined by fetal kidney growth and it critically dictates renal health in adults. Despite the advances in understanding the molecular regulation of nephron progenitor maintenance, propagation, and differentiation, the causes for low congenital nephron count and contribution of basic metabolism to nephron progenitor regulation remain poorly studied. Here we have analyzed the metabolic effects that depend on and are triggered by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which is an essential intracellular cascade required for nephron progenitor maintenance. Our combined approach utilizing LC/MS-based metabolomics and transcriptional profiling of MAPK/ERK-deficient cells identified 18 out of total 46 metabolites (38 untargeted and 8 targeted) that were down-regulated. These represent glycolysis, gluconeogenesis, pentose phosphate, glycine, and proline pathways among others. We focused our functional characterization of identified metabolites on pyruvate and proline. Use of in vitro kidney cultures revealed dosage-specific functions for pyruvate in not only controlling ureteric bud branching but also determining progenitor and differentiated (tip-trunk) cell identities. Our in vivo characterization of Pycr1/2 double knockout kidneys revealed functional requirement for proline metabolism in nephron progenitor maintenance. In summary, our results demonstrate that MAPK/ERK cascade regulates energy and amino acid metabolism in developing kidney where these metabolic pathways specifically regulate progenitor preservation.

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Metformin-stimulated AMPK-α1 promotes microvascular repair in acute lung injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00173.2013 ◽

2013 ◽

Vol 305 (11) ◽

pp. L844-L855 ◽

Cited By ~ 47

Author(s):

Ming-Yuan Jian ◽

Mikhail F. Alexeyev ◽

Paul E. Wolkowicz ◽

Jaroslaw W. Zmijewski ◽

Judy R. Creighton

Keyword(s):

Endothelial Cells ◽

Acute Lung Injury ◽

Lung Injury ◽

Ex Vivo ◽

Endothelial Permeability ◽

Beneficial Effects ◽

Isolated Lung

Acute lung injury secondary to sepsis is a leading cause of mortality in sepsis-related death. Present therapies are not effective in reversing endothelial cell dysfunction, which plays a key role in increased vascular permeability and compromised lung function. AMP-activated protein kinase (AMPK) is a molecular sensor important for detection and mediation of cellular adaptations to vascular disruptive stimuli. In this study, we sought to determine the role of AMPK in resolving increased endothelial permeability in the sepsis-injured lung. AMPK function was determined in vivo using a rat model of endotoxin-induced lung injury, ex vivo using the isolated lung, and in vitro using cultured rat pulmonary microvascular endothelial cells (PMVECs). AMPK stimulation using N1-(α-d-ribofuranosyl)-5-aminoimidizole-4-carboxamide or metformin decreased the LPS-induced increase in permeability, as determined by filtration coefficient ( Kf) measurements, and resolved edema as indicated by decreased wet-to-dry ratios. The role of AMPK in the endothelial response to LPS was determined by shRNA designed to decrease expression of the AMPK-α1 isoform in capillary endothelial cells. Permeability, wounding, and barrier resistance assays using PMVECs identified AMPK-α1 as the molecule responsible for the beneficial effects of AMPK in the lung. Our findings provide novel evidence for AMPK-α1 as a vascular repair mechanism important in the pulmonary response to sepsis and identify a role for metformin treatment in the management of capillary injury.

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Abstract 23: Microrna302-367 Sphingosine 1 Phosphate Receptor 1 Pathway Prevents Tumor Growth via Restricting Angiogenesis and Enhancing Vascular Stability

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.23 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Jinjiang Pi ◽

Ting Tao ◽

Tao Zhuang ◽

Huimin Sun ◽

Xiaoli Chen ◽

...

Keyword(s):

Endothelial Cells ◽

Tumor Growth ◽

Ex Vivo ◽

Downstream Target ◽

Sprouting Angiogenesis ◽

Pathological Angiogenesis ◽

Vascular Stability ◽

Sphingosine 1 Phosphate

Angiogenic hypersprouting and leaky immature vessels of pathological angiogenesis are essential for tumor growth. MicroRNAs have unique therapeutic advantages by targeting multiple pathways of tumor-associated angiogenesis, but the function of individual miRNAs in angiogenesis and tumors has not yet been fully evaluated. Here, we show that miR302-367 elevation in endothelial cells reduces retina sprouting angiogenesis and promotes vascular stability in vivo, ex vivo and in vitro. Erk1/2 are identified as direct targets of miR302-367, and down-regulation of Erk1/2 upon miR302-367 elevation in endothelial cells increases the expression of Klf2 and in turn S1pr1 and its downstream target VE-cadherin, suppressing angiogenesis and improving vascular stability. Conversely, both pharmacological blockade and genetic deletion of S1pr1 in endothelial cells reverse the anti-angiogenic and vascular stabilizing effect of miR302-367 in mice. Pathological angiogenesis in tumors shares features of developmental angiogenesis, and endothelial specific elevation of miR302-367 reduces tumor growth by restricting sprout angiogenesis and decreasing vascular permeability via the same Erk1/2-Klf2-S1pr1 pathways. In conclusion, miR302-367 regulation of an Erk1/2-Klf2-S1pr1 pathway in the endothelium advances our understanding of angiogenesis, meanwhile also provides opportunities for therapeutic intervention of tumor growth.

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Molecular and functional characterization of somatostatin-type signalling in a deuterostome invertebrate

Open Biology ◽

10.1098/rsob.200172 ◽

2020 ◽

Vol 10 (9) ◽

pp. 200172

Author(s):

Ya Zhang ◽

Luis Alfonso Yañez Guerra ◽

Michaela Egertová ◽

Cleidiane G. Zampronio ◽

Alexandra M. Jones ◽

...

Keyword(s):

Functional Characterization ◽

The Body ◽

Pharmacological Characterization ◽

Cardiac Stomach ◽

Physiological Processes ◽

Pharmacological Tests ◽

The Central Nervous System

Somatostatin (SS) and allatostatin-C (ASTC) are structurally and evolutionarily related neuropeptides that act as inhibitory regulators of physiological processes in mammals and insects, respectively. Here, we report the first molecular and functional characterization of SS/ASTC-type signalling in a deuterostome invertebrate—the starfish Asterias rubens (phylum Echinodermata). Two SS/ASTC-type precursors were identified in A. rubens (ArSSP1 and ArSSP2) and the structures of neuropeptides derived from these proteins (ArSS1 and ArSS2) were analysed using mass spectrometry. Pharmacological characterization of three cloned A. rubens SS/ASTC-type receptors (ArSSR1–3) revealed that ArSS2, but not ArSS1, acts as a ligand for all three receptors. Analysis of ArSS2 expression in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed stained cells/fibres in the central nervous system, the digestive system (e.g. cardiac stomach) and the body wall and its appendages (e.g. tube feet). Furthermore, in vitro pharmacological tests revealed that ArSS2 causes dose-dependent relaxation of tube foot and cardiac stomach preparations, while injection of ArSS2 in vivo causes partial eversion of the cardiac stomach. Our findings provide new insights into the molecular evolution of SS/ASTC-type signalling in the animal kingdom and reveal an ancient role of SS-type neuropeptides as inhibitory regulators of muscle contractility.

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Functional and Genomic Characterization of Ligilactobacillus salivarius TUCO-L2 Isolated From Lama glama Milk: A Promising Immunobiotic Strain to Combat Infections

Frontiers in Microbiology ◽

10.3389/fmicb.2020.608752 ◽

2020 ◽

Vol 11 ◽

Author(s):

Sandra Quilodrán-Vega ◽

Leonardo Albarracin ◽

Flavia Mansilla ◽

Lorena Arce ◽

Binghui Zhou ◽

...

Keyword(s):

Epithelial Cells ◽

Functional Characterization ◽

Comparative Genomic ◽

Cytokines And Chemokines ◽

Isolation And Characterization ◽

Intestinal Pathogens ◽

Lama Glama

Potential probiotic or immunobiotic effects of lactic acid bacteria (LAB) isolated from the milk of the South American camelid llama (Lama glama) have not been reported in published studies. The aim of the present work was to isolate beneficial LAB from llama milk that can be used as potential probiotics active against bacterial pathogens. LAB strains were isolated from llama milk samples. In vitro functional characterization of the strains was performed by evaluating the resistance against gastrointestinal conditions and inhibition of the pathogen growth. Additionally, the adhesive and immunomodulatory properties of the strains were assessed. The functional studies were complemented with a comparative genomic evaluation and in vivo studies in mice. Ligilactobacillus salivarius TUCO-L2 showed enhanced probiotic/immunobiotic potential compared to that of other tested strains. The TUCO-L2 strain was resistant to pH and high bile salt concentrations and demonstrated antimicrobial activity against Gram-negative intestinal pathogens and adhesion to mucins and epithelial cells. L. salivarius TUCO-L2 modulated the innate immune response triggered by Toll-like receptor (TLR)-4 activation in intestinal epithelial cells. This effect involved differential regulation of the expression of inflammatory cytokines and chemokines mediated by the modulation of the negative regulators of the TLR signaling pathway. Moreover, the TUCO-L2 strain enhanced the resistance of mice to Salmonella infection. This is the first report on the isolation and characterization of a potential probiotic/immunobiotic strain from llama milk. The in vitro, in vivo, and in silico investigation performed in this study reveals several research directions that are needed to characterize the TUCO-L2 strain in detail to position this strain as a probiotic or immunobiotic that can be used against infections in humans or animals, including llama.

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Functional characterization of human Kindlin-2 core promoter identifies a key role of SP1 in Kindlin-2 transcriptional regulation

Cellular & Molecular Biology Letters ◽

10.2478/s11658-011-0028-6 ◽

2011 ◽

Vol 16 (4) ◽

Cited By ~ 1

Author(s):

Ammad Khan ◽

Takashi Shimokawa ◽

Staffan Strömblad ◽

Hongquan Zhang

Keyword(s):

Transcriptional Regulation ◽

Core Promoter ◽

Functional Characterization ◽

Ph Domain ◽

Interacting Protein ◽

Normal Tissues ◽

Functional Aspects

AbstractKindlin-2 is a recently identified FERM and PH domain containing integrin interacting protein. Kindlin-2 is ubiquitously expressed in normal tissues. So far, much effort has been spent exploring the functional aspects of Kindlin-2. However, the transcriptional regulation of Kindlin-2 has not yet been investigated. In this study we identified and functionally characterized the promoter of the human Kindlin-2 gene. We show that the core promoter of Kindlin-2 is a 39 base pair long GC rich fragment located −122/-83 upstream of the Kindlin-2 transcription start site. Functional characterization of this core promoter region by both in silico as well as in vitro/in vivo analysis shows that the transcription factor SP1 plays an important role in regulation of Kindlin-2 expression.

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