Synbindin Downregulation Participates in Slit Diaphragm Dysfunction

2021 ◽  
pp. 1-10
Author(s):  
Veniamin Ivanov ◽  
Yoshiyasu Fukusumi ◽  
Ying Zhang ◽  
Hidenori Yasuda ◽  
Meiko Kitazawa ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Transmembrane Protein ◽  
Immunohistochemical Analysis ◽  
Subtractive Hybridization ◽  
Minimal Change Nephrotic Syndrome ◽  
Slit Diaphragm ◽  
Hybridization Technique ◽  
Filamentous Actin ◽  
Puromycin Aminonucleoside ◽  
Diaphragm Dysfunction

<b><i>Introduction:</i></b> Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of synbindin in glomeruli was downregulated in puromycin aminonucleoside (PAN) nephropathy, a mimic of minimal-change nephrotic syndrome. <b><i>Methods:</i></b> The expression of synbindin in podocytes was analyzed in normal rats and 2 types of rat nephrotic models, anti-nephrin antibody-induced nephropathy, a pure slit diaphragm injury model, and PAN nephropathy, by immunohistochemical analysis and RT-PCR techniques. To elucidate the function of synbindin, a gene silencing study with human cultured podocytes was performed. <b><i>Results:</i></b> Synbindin was mainly expressed at the slit diaphragm area of glomerular epithelial cells (podocytes). In both nephrotic models, decreased mRNA expression and the altered staining of synbindin were already detected at the early phase when proteinuria and the altered staining of nephrin, a key molecule of slit diaphragm, were not detected yet. Synbindin staining was clearly reduced when severe proteinuria was observed. When the cultured podocytes were treated with siRNA for synbindin, the cell changed to a round shape, and filamentous actin structure was clearly altered. The expression of ephrin-B1, a transmembrane protein at slit diaphragm, was clearly lowered, and synaptic vesicle-associated protein 2B (SV2B) was upregulated in the synbindin knockdown cells. <b><i>Conclusion:</i></b> Synbindin participates in maintaining foot processes and slit diaphragm as a downstream molecule of SV2B-mediated vesicle transport. Synbindin downregulation participates in slit diaphragm dysfunction. Synbindin can be an early marker to detect podocyte injury.

scholarly journals CD9 Is Associated with Leukemia Inhibitory Factor-mediated Maintenance of Embryonic Stem Cells

2002 ◽  
Vol 13 (4) ◽  
pp. 1274-1281 ◽  
Author(s):  
Masahiro Oka ◽  
Kenichi Tagoku ◽  
Thomas L. Russell ◽  
Yuka Nakano ◽  
Takashi Hamazaki ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Leukemia Inhibitory Factor ◽  
Transmembrane Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Immunohistochemical Analysis ◽  
Inhibitory Factor ◽  
Cdna Subtraction ◽  
Differentiated Cells ◽  
Undifferentiated State

Mouse embryonic stem (ES) cells can proliferate indefinitely in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), or differentiate into all three germ layers upon removal of this factor. To determine cellular factors associated with self-renewal of undifferentiated ES cells, we used polymerase chain reaction-assisted cDNA subtraction to screen genes that are expressed in undifferentiated ES cells and down-regulated after incubating these cells in a differentiation medium without LIF for 48 h. The mRNA expression of a tetraspanin transmembrane protein, CD9, was high in undifferentiated ES cells and decreased shortly after cell differentiation. An immunohistochemical analysis confirmed that plasma membrane-associated CD9 was expressed in undifferentiated ES cells but low in the differentiated cells. Addition of LIF to differentiating ES cells reinduced mRNA expression of CD9, and CD9 expression was accompanied with a reappearance of undifferentiated ES cells. Furthermore, activation of STAT3 induced the expression of CD9, indicating the LIF/STAT3 pathway is critical for maintaining CD9 expression. Finally, addition of anti-CD9 antibody blocked ES cell colony formation and reduced cell viability. These results indicate that CD9 may play a role in LIF-mediated maintenance of undifferentiated ES cells.

scholarly journals Matrix GLA protein modulates branching morphogenesis in fetal rat lung

2004 ◽  
Vol 286 (6) ◽  
pp. L1179-L1187 ◽  
Author(s):  
Kirk A. Gilbert ◽  
Stephen R. Rannels
Keyword(s):  
Mrna Expression ◽  
Time Course ◽  
Immunohistochemical Analysis ◽  
Fetal Lung ◽  
Branching Morphogenesis ◽  
Matrix Gla Protein ◽  
Antibody Treatment ◽  
Developmentally Regulated

The regulation of matrix γ-carboxyglutamic acid protein (MGP) expression during the process of lung branching morphogenesis and development was investigated. MGP mRNA expression was determined over an embryonic and postnatal time course and shown to be developmentally regulated. Immunohistochemical analysis revealed increased staining for MGP in peripheral mesenchyme surrounding distal epithelial tubules. Fetal lung explants were used as an in vitro growth model to examine expression and regulation of MGP during branching morphogenesis. MGP mRNA expression over the culture interval mimicked the in vivo time course. Explants cultured in the presence of antibodies against MGP showed gross dilation and reduced terminal lung bud counts, accompanied by changes in MGP, sonic hedgehog, and patched mRNA expression. Similarly, antifibronectin antibody treatment resulted in explant dilation and reduced MGP expression, providing evidence for an interaction with MGP and fibronectin. Conversely, intraluminal microinjection of anti-MGP antibodies had no effect either on explant growth or MGP expression, supporting the hypothesis that MGP exerts its effects through the mesenchyme. Taken together, the results suggest that MGP plays a role in lung growth and development, likely via temporally and spatially specific interactions with other branching morphogenesis-related proteins to influence growth processes.

scholarly journals Isolation and Expression Analysis of Novel Silicon Absorption Gene from Roots of Mangrove(Rhizophora apiculata) viaSuppression Subtractive Hybridization

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Mahbod Sahebi ◽  
Mohamed M. Hanafi ◽  
Siti Nor Akmar Abdullah ◽  
Mohd Y. Rafii ◽  
Parisa Azizi ◽  
...  
Keyword(s):  
Essential Element ◽  
Subtractive Hybridization ◽  
Hybridization Technique ◽  
Plant Tolerance ◽  
Rt Pcr ◽  
Abiotic And Biotic Stresses ◽  
Biotic Stresses ◽  
Mangrove Plants ◽  
Rhizophora Apiculata ◽  
Abundant Element

Silicon (Si) is the second most abundant element in soil after oxygen. It is not an essential element for plant growth and formation but plays an important role in increasing plant tolerance towards different kinds of abiotic and biotic stresses. The molecular mechanism of Si absorption and accumulation may differ between plants, such as monocotyledons and dicotyledons. Silicon absorption and accumulation in mangrove plants are affected indirectly by some proteins rich in serine and proline amino acids. The expression level of the genes responsible for Si absorption varies in different parts of plants. In this study, Si is mainly observed in the epidermal roots’ cell walls of mangrove plants compared to other parts. The present work was carried out to discover further information on Si stress responsive genes inRhizophora apiculata, using the suppression subtractive hybridization technique. To construct the cDNA library, two-month-old seedlings were exposed to 0.5, 1, and 1.5 mM SiO2for 15 hrs and for 1 to 6 days resulting in a total of 360 high quality ESTs gained. Further examination by RT-PCR and real-time qRT-PCR showed the expression of a candidate gene ofserine-rich protein.

scholarly journals Personalized approach to assessing mRNA expression of histidine-rich glycoprotein and immunohistochemical markers in diseases of the breast

2019 ◽  
Vol 484 (5) ◽  
pp. 624-628 ◽  
Author(s):  
A. I. Autenshlyus ◽  
A. V. Golovanova ◽  
A. A. Studenikina ◽  
I. I. Brusentsov ◽  
A. V. Proskura ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Epithelial Mesenchymal Transition ◽  
Immunohistochemical Analysis ◽  
Breast Diseases ◽  
Biopsy Material ◽  
Mesenchymal Transition ◽  
Benign Breast Diseases ◽  
Immunohistochemical Markers ◽  
Atypical Cells ◽  
Personalized Approach

Biopsy material of patients with malignant and benign breast diseases was examined. HRG mRNA expression was detected in 70% of cases in biopsy material obtained from patients with nonspecific invasive carcinoma and in 66.7% of cases in biopsy material of patients with benign breast diseases. Immunohistochemical analysis revealed expression of collagen II, the beta-1 integrin, and E-cadherin – markers of epithelial–mesenchymal transition. The use of RT-qPCR combined with immunohistochemical study made it possible to identify atypical cells, which can be regarded as precancerous changes, in individual patients.

scholarly journals In the non-insect-transmissible line of onion yellows phytoplasma (OY-NIM), the plasmid-encoded transmembrane protein ORF3 lacks the major promoter region

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 2058-2067 ◽  
Author(s):  
Yoshiko Ishii ◽  
Shigeyuki Kakizawa ◽  
Ayaka Hoshi ◽  
Kensaku Maejima ◽  
Satoshi Kagiwada ◽  
...  
Keyword(s):  
Promoter Region ◽  
Transmembrane Protein ◽  
Immunohistochemical Analysis ◽  
The Other ◽  
Insect Host ◽  
Gene Encoding ◽  
Phytopathogenic Bacterium ◽  
Orf3 Protein ◽  
Promoter Sequences

‘Candidatus Phytoplasma asteris’, onion yellows strain (OY), a mildly pathogenic line (OY-M), is a phytopathogenic bacterium transmitted by Macrosteles striifrons leafhoppers. OY-M contains two types of plasmids (EcOYM and pOYM), each of which possesses a gene encoding the putative transmembrane protein, ORF3. A non-insect-transmissible line of this phytoplasma (OY-NIM) has the corresponding plasmids (EcOYNIM and pOYNIM), but pOYNIM lacks orf3. Here we show that in OY-M, orf3 is transcribed from two putative promoters and that on EcOYNIM, one of the promoter sequences is mutated and the other deleted. We also show by immunohistochemical analysis that ORF3 is not expressed in OY-NIM-infected plants. Moreover, ORF3 protein seems to be preferentially expressed in OY-M-infected insects rather than in plants. We speculate that ORF3 may play a role in the interactions of OY with its insect host.

scholarly journals Proline-rich transmembrane protein 2 (PRRT2) regulates the actin cytoskeleton during synaptogenesis

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Elisa Savino ◽  
Romina Inès Cervigni ◽  
Miriana Povolo ◽  
Alessandra Stefanetti ◽  
Daniele Ferrante ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Neurotransmitter Release ◽  
Hippocampal Neurons ◽  
Neuronal Excitability ◽  
Transmembrane Protein ◽  
Neuronal Cell ◽  
Cell Aggregation ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Filamentous Actin

Abstract Mutations in proline-rich transmembrane protein 2 (PRRT2) have been recently identified as the leading cause of a clinically heterogeneous group of neurological disorders sharing a paroxysmal nature, including paroxysmal kinesigenic dyskinesia and benign familial infantile seizures. To date, studies aimed at understanding its physiological functions in neurons have mainly focused on its ability to regulate neurotransmitter release and neuronal excitability. Here, we show that PRRT2 expression in non-neuronal cell lines inhibits cell motility and focal adhesion turnover, increases cell aggregation propensity, and promotes the protrusion of filopodia, all processes impinging on the actin cytoskeleton. In primary hippocampal neurons, PRRT2 silencing affects the synaptic content of filamentous actin and perturbs actin dynamics. This is accompanied by defects in the density and maturation of dendritic spines. We identified cofilin, an actin-binding protein abundantly expressed at the synaptic level, as the ultimate effector of PRRT2. Indeed, PRRT2 silencing unbalances cofilin activity leading to the formation of cofilin-actin rods along neurites. The expression of a cofilin phospho-mimetic mutant (cof-S3E) is able to rescue PRRT2-dependent defects in synapse density, spine number and morphology, but not the alterations observed in neurotransmitter release. Our data support a novel function of PRRT2 in the regulation of the synaptic actin cytoskeleton and in the formation of synaptic contacts.

scholarly journals Using a Multiplex Nucleic Acid in situ Hybridization Technique to Determine HCN4 mRNA Expression in the Adult Rodent Brain

2019 ◽  
Vol 12 ◽  
Author(s):  
Julia Oyrer ◽  
Lauren E. Bleakley ◽  
Kay L. Richards ◽  
Snezana Maljevic ◽  
A. Marie Phillips ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Mrna Expression ◽  
Hybridization Technique ◽  
Rodent Brain
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