scholarly journals Establishment and characterization of Neu1-knockout zebrafish and its abnormal clinical phenotypes

2020 ◽  
Author(s):  
Keiji Okada ◽  
Ryo Takase ◽  
Yurie Hamaoka ◽  
Akinobu Honda ◽  
Asami Ikeda ◽  
...  
Keyword(s):  
Animal Model ◽  
Plasma Membrane ◽  
Sialic Acid ◽  
Danio Rerio ◽  
Cancer Metastasis ◽  
Expression Patterns ◽  
Wild Type ◽  
Fish Embryo ◽  
Neu1 Sialidase

Mammalian sialidase Neu1 is involved in various physiological functions, including cell adhesion, differentiation, cancer metastasis, and diabetes through lysosomal catabolism and desialylation of glycoproteins at the plasma membrane. Various animal models have been established to further explore the functions of vertebrate Neu1. The present study focused on zebrafish (Danio rerio) belonging to Cypriniformes as an experimental animal model with neu1 gene deficiency. The results revealed that the zebrafish Neu1 desialyzed both a2-3 and a2-6 sialic acid linkages from oligosaccharides and glycoproteins at pH 4.5, and it is highly conserved with other fish species and mammalian Neu1. Further, Neu1-knockout zebrafish (Neu1-KO) was established through CRISPR/Cas9 genome editing. Neu1-KO fish exhibited slight abnormal embryogenesis with the accumulation of pleural effusion; however, no embryonic lethality was observed. Although Neu1-KO fish were able to be maintained as homozygous, they showed smaller body length and weight than the wild type (WT) fish, and muscle atrophy and curvature of the vertebra were observed in adult Neu1-KO fish (8 months). The expression patterns of myod and myog transcription factors regulating muscle differentiation varied between Neu1-KO and WT fish embryo. Expression of lysosomal-related genes, including ctsa,lamp1a, and tfeb were upregulated in adult Neu1-KO muscle as compared to WT. Furthermore, the expression pattern of genes involved in bone remodeling (runx2a, runx2b, and mmp9) was decreased in Neu1-KO fish. These phenotypes were quite similar to those of Neu1-KO mice and human sialidosis patients, indicating the effectiveness of the established Neu1-KO zebrafish for the study of vertebrate Neu1 sialidase.

scholarly journals Characterization of Sialic Acid-Binding Immunoglobulin-Type Lectins in Fish Reveals Teleost-Specific Structures and Expression Patterns

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 836
Author(s):  
Kim F. Bornhöfft ◽  
Joan Martorell Ribera ◽  
Torsten Viergutz ◽  
Marzia T. Venuto ◽  
Ulrike Gimsa ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Sander Lucioperca ◽  
Handling Stress ◽  
Sialic Acid Binding ◽  
Specific Manner ◽  
Genes Encoding ◽  
Myelin Associated Glycoprotein

The cellular glycocalyx of vertebrates is frequently decorated with sialic acid residues. These sialylated structures are recognized by sialic acid-binding immunoglobulin-type lectins (Siglecs) of immune cells, which modulate their responsiveness. Fifteen Siglecs are known to be expressed in humans, but only four Siglecs are regularly present in fish: Siglec1, CD22, myelin-associated glycoprotein (MAG), and Siglec15. While several studies have dealt with the physiological roles of these four Siglecs in mammals, little is known about Siglecs in fish. In the present manuscript, the expression landscapes of these Siglecs were determined in the two salmonid species Oncorhynchus mykiss and Coregonus maraena and in the percid fish Sander lucioperca. This gene-expression profiling revealed that the expression of MAG is not restricted to neuronal cells but is detectable in all analyzed blood cells, including erythrocytes. The teleostean MAG contains the inhibitory motif ITIM; therefore, an additional immunomodulatory function of MAG is likely to be present in fish. Besides MAG, Siglec1, CD22, and Siglec15 were also expressed in all analyzed blood cell populations. Interestingly, the expression profiles of genes encoding Siglecs and particular associated enzymes changed in a gene- and tissue-specific manner when Coregonus maraena was exposed to handling stress. Thus, the obtained data indicate once more that stress directly affects immune-associated processes.

Abstract 5183: Identification and characterization of wild type kinases driving prostate cancer metastasis

2015 ◽  
Author(s):  
Claire Faltermeier ◽  
Justin Drake ◽  
Peter Clark ◽  
Bryan Smith ◽  
Colleen Mathis ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Metastasis ◽  
Wild Type ◽  
Prostate Cancer Metastasis ◽  
Identification And Characterization

scholarly journals Eyespot-Assembly Mutants in Chlamydomonas reinhardtii

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 721-729 ◽  
Author(s):  
Mary Rose Lamb ◽  
Susan K Dutcher ◽  
Cathy K Worley ◽  
Carol L Dieckmann
Keyword(s):  
Plasma Membrane ◽  
Chlamydomonas Reinhardtii ◽  
Chloroplast Envelope ◽  
Ca Channel ◽  
Carotenoid Pigment ◽  
Wild Type ◽  
Ultrastructural Studies ◽  
Envelope Membranes ◽  
Synthetic Phenotype

Abstract Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca++-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.

scholarly journals STAT-1 Knockout Mice as a Model for Wild-Type Sudan Virus (SUDV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1388
Author(s):  
Olivier Escaffre ◽  
Terry L. Juelich ◽  
Natasha Neef ◽  
Shane Massey ◽  
Jeanon Smith ◽  
...  
Keyword(s):  
Animal Model ◽  
Democratic Republic Of Congo ◽  
White Blood Cells ◽  
Small Animal ◽  
Marburg Virus ◽  
Wild Type ◽  
Small Animal Model ◽  
Knock Out ◽  
Zaire Ebolavirus

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014–2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 101 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

Cleavage of Arpin by Calpain as a Potential Regulation Mechanism of the Arp2/3 Complex and Cell Motility

2018 ◽  
Author(s):  
Renee Potashner
Keyword(s):  
Plasma Membrane ◽  
Cell Motility ◽  
Cancer Metastasis ◽  
Competitive Inhibition ◽  
Cell Movement ◽  
Personal Communication ◽  
Cysteine Proteases ◽  
Regulation Mechanism ◽  
Wild Type ◽  
In The Wild

Cell movement is mediated by cycles of actin polymerisation. A novel protein, Arpin, was discovered that has been suggested to decrease cell motility through competitive inhibition of WASP family proteins, the activators of the complex driving actin polymerisation. In preliminary studies, Arpin was found to inhibit the Arp2/3 complex (Gautreau and Blanchoin, personal communication). Arpin contains sequence homology to the Arp2/3-binding site of WASP proteins. Calpains, a family of intracellular calcium-dependent cysteine proteases, can be found near the plasma membrane and the concentration of calcium ion required for activation is decreased when calpain is bound to the plasma membrane in the presence of phospholipids (Leloup et al. 2010). The common localization of calpain and Arpin, along with the known contributions calpain has in regulating other cell motility proteins, makes calpain a likely candidate for Arpin regulation. By transfecting calpain wild-type (pz/pz), knockdown (p/p) and lentivirus rescue mouse embryonic fibroblasts with a plasmid containing the Arpin gene (c15orf38), I hope to show the presence of differential cleavage of Arpin by calpain in the wild-type cells compared to the calpain knock-down cells through Western blot analysis. Understanding how Arpin is regulated will provide a basis for further research in cell motility, which has contributions to cancer metastasis and other diseases.  

scholarly journals The Plasma Membrane H+-ATPase from the Biotrophic Rust Fungus Uromyces fabae: Molecular Characterization of the Gene (PMA1) and Functional Expression of the Enzyme in Yeast

1998 ◽  
Vol 11 (6) ◽  
pp. 458-465 ◽  
Author(s):  
Christine Struck ◽  
Claudia Siebels ◽  
Oliver Rommel ◽  
Marcus Wernitz ◽  
Matthias Hahn
Keyword(s):  
Plasma Membrane ◽  
Rust Fungus ◽  
Single Gene ◽  
Functional Expression ◽  
Wild Type ◽  
Ph Optimum ◽  
Uromyces Fabae ◽  
Mutant Derivative ◽  
Regulatory Properties

To study the molecular basis of biotrophic nutrient uptake by plant parasitic rust fungi, the gene (Uf-PMA1) encoding the plasma membrane H+-ATPase from Uromyces fabae was isolated. Uf-PMA1 exists probably as a single gene. However, two nearly identical sequences were identified; the similarity apparently is due to two Uf-PMA1 alleles in the dikaryotic hyphae. Multiple Uf-PMA1 transcripts were observed during early rust development, and reduced amounts of a single Uf-PMA1 mRNA were observed in haustoria and rust-infected leaves. This is in contrast to elevated enzyme activity in haustoria compared to germinated spores (C. Struck, M. Hahn, and K. Mendgen. Fungal Genet. Biol. 20:30–35, 1996). Unexpectedly, the PMA1-encoded rust protein is more similar to H+-ATPases from plants (55% identity) than from ascomycetous fungi (36% identity). When the rust PMA1 cDNA was expressed in Saccharomyces cerevisiae, both the wild-type enzyme and a mutant derivative (Δ76) deleted for the 76 C-terminal amino acids were able to support growth of a yeast strain lacking its own H+-ATPases. Compared to the wild-type, the Δ76 mutant enzyme displayed increased affinity to ATP, a higher vanadate sensitivity, and a more alkaline pH optimum. These results indicate that the C-terminal region of the rust enzyme exhibits auto-regulatory properties.

A new animal model for epithelial ion transport modeling (focusing on CFTR) - Characterization of pancreatic ductal fluid and bicarbonate secretion in wild type ferrets

Pancreatology ◽  
2017 ◽  
Vol 17 (3) ◽  
pp. S35
Author(s):  
Emese Tóth ◽  
József Maléth ◽  
Petra Pallagi ◽  
Viktória Venglovecz ◽  
Zoltán Rakonczay ◽  
...  
Keyword(s):  
Animal Model ◽  
Ion Transport ◽  
Transport Modeling ◽  
Bicarbonate Secretion ◽  
Wild Type ◽  
Epithelial Ion Transport ◽  
Ductal Fluid

scholarly journals Characterization of an Allele-Nonspecific Intragenic Suppressor in the Yeast Plasma Membrane H+-ATPase Gene (PMA1)

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 11-19
Author(s):  
Ana M Maldonado ◽  
Natalia de la Fuente ◽  
Francisco Portillo
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Dominant Negative ◽  
Wild Type ◽  
Dominant Lethal ◽  
Atpase Gene ◽  
Dominant Negative Allele ◽  
Biochemical Analyses ◽  
Dominant Lethality

Abstract We have analyzed the ability of A165V, V169I/D170N, and P536L mutations to suppress pma1 dominant lethal alleles and found that the P536L mutation is able to suppress the dominant lethality of the pma1-R271T, -D378N, -D378E, and -K474R mutant alleles. Genetic and biochemical analyses of site-directed mutants at Pro-536 suggest that this amino acid may not be essential for function but is important for biogenesis of the ATPase. Proteins encoded by dominant lethal pma1 alleles are retained in the endoplasmic reticulum, thus interfering with transport of wild-type Pma1. Immunofluorescence studies of yeast conditionally expressing revertant alleles show that the mutant enzymes are correctly located at the plasma membrane and do not disturb targeting of the wild-type enzyme. We propose that changes in Pro-536 may influence the folding of the protein encoded by a dominant negative allele so that it is no longer recognized and retained as a misfolded protein by the endoplasmic reticulum.

Electron Microscopic Detection and Characterization of Nonhuman Primate Enteric Viruses

1982 ◽  
Vol 40 ◽  
pp. 306-307
Author(s):  
G. C. Smith ◽  
R. L. Heberling ◽  
S. S. Kalter
Keyword(s):  
Electron Microscopy ◽  
Animal Model ◽  
Nonhuman Primate ◽  
Clinical Condition ◽  
Intestinal Tract ◽  
Enteric Viruses ◽  
Electron Microscopic ◽  
Microscopic Detection

A number of viral agents are recognized as and suspected of causing the clinical condition “gastroenteritis.” In our attempts to establish an animal model for studies of this entity, we have been examining the nonhuman primate to ascertain what viruses may be found in the intestinal tract of “normal” animals as well as animals with diarrhea. Several virus types including coronavirus, adenovirus, herpesvirus, and picornavirus (Table I) were detected in our colony; however, rotavirus, astrovirus, and calicivirus have not yet been observed. Fecal specimens were prepared for electron microscopy by procedures reported previously.

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