Gestational and tissue-specific regulation of C1C-2 chloride channel expression

1996 ◽  
Vol 271 (5) ◽  
pp. L829-L837 ◽  
Author(s):  
C. B. Murray ◽  
S. Chu ◽  
P. L. Zeitlin
Keyword(s):  
Protein Expression ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Chloride Secretion ◽  
The Body ◽  
Late Gestation ◽  
Ribonuclease Protection Assay ◽  
Mrna Transcripts ◽  
Specific Regulation ◽  
Ribonuclease Protection

Chloride channels supply critical functions in epithelial cells throughout the body. Although function of the volume- and voltage-gated C1C-2 is uncertain, its wide tissue distribution of mRNA suggests C1C-2 has important housekeeping functions. This study's objective was to identify the extent of not only C1C-2 mRNA expression but also protein expression as a measure of the capacity for C1C-2 chloride secretion in epithelial tissues. Using quantitative ribonuclease protection assay, we found that C1C-2 mRNA transcripts were abundant in fetal and postnatal brain, fetal kidney, liver, intestine, and lung. In contrast to brain, C1C-2 mRNA transcripts were downregulated during late gestation in lung, kidney, and intestine. The lung expressed the least C1C-2 mRNA. Immunoblotting demonstrated similar tissue- and gestation-dependent variations in C1C-2 protein expression. To determine if there is a correlation between the sites of C1C-2 protein expression and cystic fibrosis transmembrane conductance regulator (CFTR), another epithelial chloride channel, a polyclonal COOH-terminal C1C-2 antibody and an anti-R domain CFTR anti-body were used. C1C-2 and CFTR were expressed in different sites in lung and kidney.

ClC-5: ontogeny of an alternative chloride channel in respiratory epithelia

2002 ◽  
Vol 282 (3) ◽  
pp. L501-L507 ◽  
Author(s):  
Rebecca D. Edmonds ◽  
Ian V. Silva ◽  
William B. Guggino ◽  
Robert B. Butler ◽  
Pamela L. Zeitlin ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Lung Disease ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Chloride Transport ◽  
Premature Death ◽  
Fetal Lung ◽  
Chloride Secretion ◽  
Normal Lung ◽  
Ribonuclease Protection Assay

Chloride transport is critical to many functions of the lung. Molecular defects in the best-known chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), lead to impaired function of airway defensins, hydration of airway surface fluid, and mucociliary clearance leading to chronic lung disease, and premature death, but do not cause defects in lung development. We examined the expression of one member of the ClC family of volume- and voltage-regulated channels using the ribonuclease protection assay and Western blot analysis in rats. ClC-5 mRNA and protein are most strongly expressed in the fetal lung, and expression is maintained although downregulated postnatally. In addition, using immunocytochemistry, we find that ClC-5 is predominantly expressed along the luminal surface of the airway epithelium, suggesting that ClC-5 may participate in lung chloride secretion. Identifying candidate genes for critical ion transport functions is essential for understanding normal lung morphogenesis and the pathophysiology of several lung diseases. In addition, the manipulation of non-CFTR chloride channels may provide a viable approach for treating cystic fibrosis lung disease.

scholarly journals Size ofAT(n)Insertions in Promoter Region ModulatesGmhsp17.6-LmRNA Transcript Levels

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Renata Fuganti ◽  
Maria de Fátima Pires da Silva Machado ◽  
Valéria Stefania Lopes ◽  
João Flávio Veloso Silva ◽  
Carlos Alberto Arrabal Arias ◽  
...  
Keyword(s):  
Promoter Region ◽  
Perfect Match ◽  
Ribonuclease Protection Assay ◽  
Coding Region ◽  
Root Knot Nematode ◽  
Gene Coding ◽  
Mrna Transcripts ◽  
Soybean Genotypes ◽  
Ribonuclease Protection ◽  
Resistant Genotypes

During earlier experiments, an SSR molecular marker (176 Soy HSP) showing high correlation (70%) with resistance/susceptibility to javanese root-knot nematodeMeloidogyne javanicawas identified in soybean. After being sequenced, results indicated that the SSR 176 Soy HSP marker was inserted in the promoter region ofGmhsp17.6-Lgene. It was also detected in this region that resistant genotypes presented insertions betweenAT(31)andAT(33)in size and susceptible genotypes,AT(9).Gmhsp17.6-Lgene coding region presented a perfect match in amino acid sequence in all soybean genotypes. A ribonuclease protection assay showed thatGmhsp17.6-Lgene mRNA transcripts were present in all genotypes. A real-time relative quantification (qPCR) indicated in the resistant individuals higher mRNA transcripts levels, which presented in the sequencing moreAT(n)insertions. These results suggest that the number ofAT(n)insertions inside this promoter region could modulate up or down gene levels. Those findings can lead to the possibility of manipulating, between some limits, the mRNA transcripts levels using different sizes ofAT(n)insertions.

RNA expression as a prognostic tool in idiopathic nephrotic syndrome

2007 ◽  
Vol 148 (23) ◽  
pp. 1067-1075
Author(s):  
Krisztina Fischer ◽  
Orsolya Galamb ◽  
Béla Molnár ◽  
Zsolt Tulassay ◽  
András Szabó
Keyword(s):  
Nephrotic Syndrome ◽  
Northern Blot ◽  
Idiopathic Nephrotic Syndrome ◽  
Minimal Change ◽  
Ribonuclease Protection Assay ◽  
Rna Expression ◽  
Rt Pcr ◽  
Protection Assay ◽  
Ribonuclease Protection

A gyermekkori nephrosis 90%-a idiopathiás nephrosis szindróma. Az idetartozó három kórkép, a minimal change betegség, a mesangialis proliferatio és a focalis sclerosis hasonló klinikai képpel jelentkező, eltérő prognózisú és terápiás válaszú betegség. Dolgozatunk célja az idiopathiás nephrosis szindrómába tartozó kórképek kialakulásával, progressziójával összefüggő genetikai ismeretek, génexpressziós változások áttekintése és funkcionális csoportosítása. A génexpressziós változások meghatározásának eszközeként, dolgozatunk röviden összefoglalja a northern blot, a ribonuclease protection assay, az in situ RNS-hibridizáció, a kvantitatív RT-PCR és a microarray módszerek lényegét. Az eddig elvégzett vizsgálatok a DNS-szintézis és repair gének, növekedési faktorok, extracelluláris mátrix, extracelluláris ligandreceptorok, extracelluláris jelátvitel zavarai mellett kiemelik a metabolikus és transzporter gének, illetve az immunszabályozó gének molekuláris eltéréseit, amelyek összefüggésben vannak az idiopathiás nephrosis szindróma eddig megismert molekuláris hátterével. A chiptechnológia fejlődésével és elterjedésével ezek a markerek és a hagyományos vizsgálati módszerek párhuzamos alkalmazása rutindiagnosztikai szempontból is fontossá válhat.

scholarly journals The Role of Serum Chloride in Acute and Chronic Heart Failure: A Narrative Review

2021 ◽  
Vol 11 (2) ◽  
pp. 87-98
Author(s):  
Frederick Berro Rivera ◽  
Pia Alfonso ◽  
Jem Marie Golbin ◽  
Kevin Lo ◽  
Edgar Lerma ◽  
...  
Keyword(s):  
Heart Failure ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Prognostic Value ◽  
Diuretic Therapy ◽  
Juxtaglomerular Apparatus ◽  
Sodium Chloride Cotransporter ◽  
Serum Chloride ◽  
And Function

Clinical guidelines include diuretics for the treatment of heart failure (HF), not to decrease mortality but to decrease symptoms and hospitalizations. More attention has been paid to the worse outcomes, including mortality, associated with continual diuretic therapy due to hypochloremia. Studies have revealed a pivotal role for serum chloride in the pathophysiology of HF and is now a target of treatment to decrease mortality. The prognostic value of serum chloride in HF has been the subject of much attention. Mechanistically, the macula densa, a region in the renal juxtaglomerular apparatus, relies on chloride levels to sense salt and volume status. The recent discovery of with-no-lysine (K) (WNK) protein kinase as an intracellular chloride sensor sheds light on the possible reason of diuretic resistance in HF. The action of chloride on WNKs results in the upregulation of the sodium-potassium-chloride cotransporter and sodium-chloride cotransporter receptors, which could lead to increased electrolyte and fluid reabsorption. Genetic studies have revealed that a variant of a voltage-sensitive chloride channel (CLCNKA) gene leads to almost a 50% decrease in current amplitude and function of the renal chloride channel. This variant increases the risk of HF. Several trials exploring the prognostic value of chloride in both acute and chronic HF have shown mostly positive results, some even suggesting a stronger role than sodium. However, so far, interventional trials exploring serum chloride as a therapeutic target have been largely inconclusive. This study is a review of the pathophysiologic effects of hypochloremia in HF, the genetics of chloride channels, and clinical trials that are underway to investigate novel approaches to HF management.

Effects of maternal periconceptional undernutrition in sheep on offspring glucose–insulin axis function into adulthood

2020 ◽  
pp. 1-7
Author(s):  
Mark H. Oliver ◽  
Frank H. Bloomfield ◽  
Amita Bansal ◽  
Hui Hui Phua ◽  
Eric B. Thorstensen ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Glucose Tolerance ◽  
Protein Expression ◽  
Insulin Signalling ◽  
Area Under The Curve ◽  
Late Gestation ◽  
Maternal Undernutrition ◽  
Kinase C ◽  
First Phase Insulin Secretion ◽  
Protein Kinase C Zeta

Abstract Maternal periconceptional undernutrition (PCUN) affected fetal pancreatic maturation in late gestation lambs and impaired glucose tolerance in 10-month-old sheep. To examine the importance of the timing of maternal undernutrition around conception, a further cohort was born to PCUN ewes [undernourished for 61 d before conception (PreC), 30 d after conception (PostC), or 61 d before until 30 d after conception (PrePostC)], or normally fed ewes (Control) (n = 15–20/group). We compared glucose tolerance, insulin secretion, and sensitivity at 36 months of age. We also examined protein expression of insulin signalling proteins in muscle from these animals and in muscle from a fetal cohort (132 d of gestation; n = 7–10/group). Adult PostC and PrePostC sheep had higher glucose area under the curve than Controls (P = 0.07 and P = 0.02, respectively), whereas PreC sheep were similar to Controls (P = 0.97). PostC and PrePostC had reduced first-phase insulin secretion compared with Control (P = 0.03 and P = 0.02, respectively). PreC was similar to Control (P = 0.12). Skeletal muscle SLC2A4 protein expression in PostC and PrePostC was increased 19%–58% in fetuses (P = 0.004), but decreased 39%–43% in adult sheep (P = 0.003) compared with Controls. Consistent with this, protein kinase C zeta (PKCζ) protein expression tended to be increased in fetal (P = 0.09) and reduced in adult (P = 0.07) offspring of all PCUN ewes compared with Controls. Maternal PCUN alters several aspects of offspring glucose homeostasis into adulthood. These findings suggest that maternal periconceptional nutrition has a lasting impact on metabolic homeostasis of the offspring.

scholarly journals Upregulation of Cytokines Is Detected in the Placentas of Cattle Infected with Neospora caninum and Is More Marked Early in Gestation When Fetal Death Is Observed

2008 ◽  
Vol 76 (6) ◽  
pp. 2352-2361 ◽  
Author(s):  
Anne Rosbottom ◽  
E. Helen Gibney ◽  
Catherine S. Guy ◽  
Anja Kipar ◽  
Robert F. Smith ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fetal Death ◽  
Neospora Caninum ◽  
Late Gestation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Cytokine Mrna ◽  
Early Gestation ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACT The protozoan parasite Neospora caninum causes fetal death after experimental infection of pregnant cattle in early gestation, but the fetus survives a similar infection in late gestation. An increase in Th1-type cytokines in the placenta in response to the presence of the parasite has been implicated as a contributory factor to fetal death due to immune-mediated pathological alterations. We measured, using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay, the levels of cytokines in the placentas of cattle experimentally infected with N. caninum in early and late gestation. After infection in early gestation, fetal death occurred, and the levels of mRNA of both Th1 and Th2 cytokines, including interleukin-2 (IL-2), gamma interferon (IFN-γ), IL-12p40, tumor necrosis factor alpha (TNF-α), IL-18, IL-10, and IL-4, were significantly (P < 0.01) increased by up to 1,000-fold. There was extensive placental necrosis and a corresponding infiltration of CD4+ T cells and macrophages. IFN-γ protein expression was also highly increased, and a modest increase in transforming growth factor β was detected. A much smaller increase in the same cytokines and IFN-γ protein expression, with minimal placental necrosis and inflammatory infiltration, occurred after N. caninum infection in late gestation when the fetuses survived. Comparison of cytokine mRNA levels in separated maternal and fetal placental tissue that showed maternal tissue was the major source of all cytokine mRNA except for IL-10 and TNF-α, which were similar in both maternal and fetal tissues. These results suggest that the magnitude of the cytokine response correlates with but is not necessarily the cause of fetal death and demonstrate that a polarized Th1 response was not evident in the placentas of N. caninum-infected cattle.

scholarly journals mtCLIC/CLIC4, an Organellular Chloride Channel Protein, Is Increased by DNA Damage and Participates in the Apoptotic Response to p53

2002 ◽  
Vol 22 (11) ◽  
pp. 3610-3620 ◽  
Author(s):  
Ester Fernández-Salas ◽  
Kwang S. Suh ◽  
Vladislav V. Speransky ◽  
Wendy L. Bowers ◽  
Joshua M. Levy ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Mitochondrial Membrane ◽  
Cellular Response ◽  
Mitochondrial Protein ◽  
Inner Mitochondrial Membrane ◽  
Necrosis Factor Alpha ◽  
Channel Protein ◽  
Factor Alpha

ABSTRACT mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.

Abstract 294: Expression of Matrix Metalloproteases during the Differentiation of Porcine Adopse-Drived Mesenchymal Stem Cells ro Endothelial Cells

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Sami G Almalki ◽  
Velidi Rao ◽  
Divya Pankajakshan ◽  
Devendra K Agrawal
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Enzyme Activity ◽  
Protein Expression ◽  
Western Blot ◽  
Gelatin Zymography ◽  
Matrix Metalloproteases ◽  
Positive Staining ◽  
Mrna Transcripts

Rationale Adipose-derived mesenchymal stem cells (ADMSCs) are multipotent cells that have the potential to differentiate into different cell linages, and represent promising tools in various clinical applications. However, the molecular mechanisms that control the ability of ADMSCs to remodel 3-dimensional extracellular matrix (ECM) barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of ADMSCs to endothelial cells (ECs) in vitro . Methods MSCs were isolated from porcine abdominal adipose tissue, and characterized by positive staining for MSC markers, CD44, CD73, CD90, and negative staining for CD11b, CD34 and CD45. The plasticity of MSCs was detected by bi-lineage differentiation to osteocytes, and adipocytes. The mRNA transcripts for different MMPs and TIMPs and protein expression of EC markers were analyzed by RT-PCR and immunostaining. The enzyme activity and protein expression were also analyzed by gelatin zymography, ELISA, and Western blot. Results The differentiation of ADMSCs to ECs was confirmed by the positive staining and mRNA expression of the endothelial markers. The mRNA transcripts for MMP-2 and membrane type 1 MMP (MT1-MMP) was significantly increased by 2.5 and 2.0 fold, respectively, during the differentiation of MSCs into ECs. Western blot and ELISA showed an elevated MT1-MMP and MMP-2 expression. The enzyme activity of MMP-2 was also observed by gelatin zymography. Conclusion We demonstrated that porcine ADMSCs have the ability to differentiate into ECs, and this process involves the up-regulation of MMP-2 and MT1-MMP. The increase in the expression of MMP-2 and MT1-MMP may, at least partially, facilitate the change in morphology of MSCs by degrading the ECM barriers. These findings may provide a potential mechanism for the role of MMP2 and MT1-MMP in the differentiation of ADMSCs into ECs.

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