Ultrastructural and functional characterization of circulating hemocytes from the freshwater crayfish Astacus leptodactylus: Cell types and their role after in vivo artificial non-self challenge

Micron ◽  
2007 ◽  
Vol 38 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Piero Giulio Giulianini ◽  
Manuel Bierti ◽  
Simonetta Lorenzon ◽  
Silvia Battistella ◽  
Enrico Antonio Ferrero
Keyword(s):  
Functional Characterization ◽  
Cell Types ◽  
Freshwater Crayfish ◽  
Astacus Leptodactylus

scholarly journals Tyrosinases from crustaceans form hexamers

2003 ◽  
Vol 371 (2) ◽  
pp. 515-523 ◽  
Author(s):  
Elmar JAENICKE ◽  
Heinz DECKER
Keyword(s):  
Oxygen Carrier ◽  
Spiny Lobster ◽  
Physicochemical Characterization ◽  
Primary Immune Response ◽  
Freshwater Crayfish ◽  
Astacus Leptodactylus ◽  
Purification Protocol ◽  
Palinurus Elephas

Tyrosinases, which are widely distributed among animals, plants and fungi, are involved in many biologically essential functions, including pigmentation, sclerotization, primary immune response and host defence. In the present study, we present a structural and physicochemical characterization of two new tyrosinases from the crustaceans Palinurus elephas (European spiny lobster) and Astacus leptodactylus (freshwater crayfish). In vivo, the purified crustacean tyrosinases occur as hexamers composed of one subunit type with a molecular mass of approx. 71kDa. The tyrosinase hexamers appear to be similar to the haemocyanins, based on electron microscopy. Thus a careful purification protocol was developed to discriminate clearly between tyrosinases and the closely related haemocyanins. The physicochemical properties of haemocyanins and tyrosinases are different with respect to electronegativity and hydrophobicity. The hexameric nature of arthropod tyrosinases suggests that these proteins were the ideal predecessors from which to develop the oxygen-carrier protein haemocyanin, with its allosteric and co-operative properties, later on.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

scholarly journals Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

2013 ◽  
Vol 142-143 ◽  
pp. 447-457 ◽  
Author(s):  
Afonso C.D. Bainy ◽  
Akira Kubota ◽  
Jared V. Goldstone ◽  
Roger Lille-Langøy ◽  
Sibel I. Karchner ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Functional Characterization ◽  
Pregnane X Receptor ◽  
Full Length ◽  
Receptor Expression

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

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
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.

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

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.

scholarly journals Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Tracy M. Yamawaki ◽  
Daniel R. Lu ◽  
Daniel C. Ellwanger ◽  
Dev Bhatt ◽  
Paolo Manzanillo ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Immune Cell ◽  
Cell Types ◽  
Data Interpretation ◽  
Detection Sensitivity ◽  
Rna Seq ◽  
Cell Recovery

Abstract Background Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. Results Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. Conclusion Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

scholarly journals Functional Characterization of Interferon Regulation Element of Hepatitis B virus Genome In Vivo

2012 ◽  
Vol 23 (3) ◽  
pp. 278-285 ◽  
Author(s):  
Feng-Jun Liu ◽  
En-Qiang Chen ◽  
Qiao-Ling Zhou ◽  
Tao-You Zhou ◽  
Cong Liu ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Functional Characterization ◽  
Virus Genome ◽  
B Virus

Identification and functional characterization of legumain in amphioxus Branchiostoma belcheri

2009 ◽  
Vol 30 (3) ◽  
pp. 177-186 ◽  
Author(s):  
Lei Teng ◽  
Hiroshi Wada ◽  
Shicui Zhang
Keyword(s):  
Functional Characterization ◽  
Specific Substrate ◽  
Glycosylation Sites ◽  
Branchiostoma Belcheri ◽  
Hen’S Egg ◽  
Hind Gut ◽  
Catalytic Dyad ◽  
Pepstatin A

Legumain has been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates. The present study reports the first characterization of legumain cDNA from the protochordate Branchiostoma belcheri. The deduced 435-amino-acid-long protein is structurally characterized by the presence of a putative N-terminal signal peptide, a peptidase_C13 superfamily domain with the conserved Lys123-Gly124-Asp125 motif and catalytic dyad His153 and Cys195 and two potential Asn-glycosylation sites at Asn85 and Asn270. Phylogenetic analysis demonstrates that B. belcheri legumain forms an independent cluster together with ascidian legumain, and is positioned at the base of vertebrate legumains, suggesting that B. belcheri legumain gene may represent the archetype of vertebrate legumain genes. Both recombinant legumain expressed in yeast and endogenous legumain are able to be converted into active protein of ~37 kDa via a C-terminal autocleavage at acid pH values. The recombinant legumain efficiently degrades the legumain-specific substrate Z-Ala-Ala-Asn-MCA (benzyloxycarbonyl-L-alanyl-L-alanyl-L-asparagine-4-methylcoumaryl-7-amide) at optimum pH 5.5; and the enzymatic activity is inhibited potently by iodoacetamide and N-ethylmaleimide, partially by hen's-egg white cystatin, but not by E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane], PMSF and pepstatin A. In addition, legumain is expressed in vivo in a tissue-specific manner, with main expression in the hepatic caecum and hind-gut of B. belcheri. Altogether, these results suggest that B. belcheri legumain plays a role in the degradation of macromolecules in food.

scholarly journals Cloning and functional characterization of the human fractalkine receptor promoter regions

2002 ◽  
Vol 368 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Alexandre GARIN ◽  
Philippe PELLET ◽  
Philippe DETERRE ◽  
Patrice DEBRÉ ◽  
Christophe COMBADIÈRE
Keyword(s):  
Functional Characterization ◽  
Cell Types ◽  
Promoter Regions ◽  
Exon 2 ◽  
Reading Frame ◽  
Fractalkine Receptor ◽  
A Cell ◽  
Exon 4 ◽  
Coronary Events

We have previously shown that reduced expression of the fractalkine receptor, CX3CR1, is correlated with rapid HIV disease progression and with reduced susceptibility to acute coronary events. In order to elucidate the mechanisms underlying transcriptional regulation of CX3CR1 expression, we structurally and functionally characterized the CX3CR1 gene. It consists of four exons and three introns spanning over 18kb. Three transcripts are produced by splicing the three untranslated exons with exon 4, which contains the complete open reading frame. The transcript predominantly found in leucocytes corresponds to the splicing of exon 2 with exon 4. Transcripts corresponding to splicing of exons 1 and 4 are less abundant in leucocytes and splicing of exons 3 and 4 are rare longer transcripts. A constitutive promoter activity was found in the regions extending upstream from untranslated exons 1 and 2. Interestingly, exons 1 and 2 enhanced the activity of their respective promoters in a cell-specific manner. These data show that the CX3CR1 gene is controlled by three distinct promoter regions, which are regulated by their respective untranslated exons and that lead to the transcription of three mature messengers. This highly complex regulation may allow versatile and precise expression of CX3CR1 in various cell types.

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

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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