Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This article represents the first evidence that the renal secretion of the commonly used drug, digoxin, is mediated by P-glycoprotein. In this study, it was demonstrated that digoxin is a substrate of P-glycoprotein, and the mechanism of a clinically important drug interaction, such as digoxin-quinidine, was elucidated. Human P-glycoprotein was expressed on the apical membrane of the porcine kidney epithelial cell line, LLC-PK1 by transfecting with human MDR1 cDNA. The expression and function of P-glycoprotein were confirmed by Southern and Western blotting, RNase protection assay, immunostaining and transporting activity for vinblastine. The transepithelial transport of [3H]digoxin was measured across the cell monolayers grown on microporous polycarbonate membrane filters. The transfectant cells exhibited markedly greater basal-to-apical transport and less apical-to-basal transport than the host cells, and the former was 8-fold greater than the latter. The augmented transepithelial transport resulted from the increased efflux from cells to apical side. This oriented transport was inhibited by the presence of 20 microM vinblastine, quinidine or verapamil. The rate of efflux to the apical side was 2-fold greater than that to the basal side. Quinidine inhibited the efflux to the apical side but did not affect transport into the basal side. These findings demonstrate that digoxin is transported by human P-glycoprotein, which is a previously undiscovered drug transport system in the kidney other than organic cation and anion transport systems, and suggest a molecular mechanism for the renal tubular secretion of digoxin as well as clinically important digoxin-quinidine interaction via P-glycoprotein.
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PMID:Transport of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell line (LLC-PK1). 135 20

Agarose-encapsulated nuclear matrix preparations of the lower eukaryote Physarum polycephalum and the mammalian renal epithelial LLC-PK1 cell line were analyzed after various experimental protocols with respect to the protein composition. The effect of the mode of deproteinization (2 M NaCl, 0.25 M ammonium sulfate or 25 mM lithium diiodosalicylate), presence of 2-mercaptoethanol, Ca2+, Cu2+, chelating agents, the sequence of protein extraction and nuclease digestion, the use of RNase, the temperature at which the experimental manipulations were performed and the use of hypotonic or isotonic conditions was investigated. No significant differences in the final nuclear matrix composition could be observed, regardless of the experimental procedure applied. In Physarum, the major nuclear matrix proteins range over 12-70 kDa with prominent bands at 24, 31, 37 and 45 kDa; the proteins of the matrix in LLC-PK1 cells extend predominantly over 40-80 kDa. Furthermore, no essential differences in the protein composition could be observed when type I and type II nuclear matrices from the highly differentiated LLC-PK1 cell line were compared. The same was found for analogous matrix preparations of Physarum. Therefore, in both systems a distinction between type I/II matrix is questionable. Immunoblotting of the matrix preparations with a variety of antibodies against intermediate filament proteins and with antinuclear autoantibodies revealed the presence of intermediate filament proteins as components of the nuclear matrix. We conclude that the nuclear matrix represents a much more stable and reproducible structure than has been proposed so far, largely independent of changes in the preparation protocol.
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PMID:Nuclear matrix of the lower eukaryote Physarum polycephalum and the mammalian epithelial LLC-PK1 cell line. A comprehensive investigation of different preparation procedures. 768 Mar 12

Myosin diversity in the human epithelial cell line Caco-2BBe, the porcine epithelial cell line LLC-PK1 (CL-4), human peripheral blood leukocytes, and human liver was analyzed. PCR amplification yielded 8-11 putative myosins (depending on the cDNA source) representing six distinct myosin classes. Analysis of clones obtained by hybridization screening demonstrated that the original PCR products correspond to bona fide myosins, based on the presence of sequences highly conserved in other myosins. RNase protection analysis confirmed mRNA expression of 11 myosins in Caco-2BBe cells. Immunoblot analysis showed that at least 6 myosin immunogens are expressed in Caco-2BBe cells. The results reveal the existence of at least 11 unconventional human myosin genes, most of which are expressed in an overlapping fashion in different cell types. The abundance of myosins suggests that the myosin I vs. myosin II paradigm is inadequate to explain actin-based cellular motility.
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PMID:Identification and overlapping expression of multiple unconventional myosin genes in vertebrate cell types. 797 38

We recently cloned a novel rabbit gene that encodes a 725-amino acid protein (Kcn1) (Y. Yao, A. S. Segal, P. Welling, X. Zhang, C. M. McNicholas, D. Engel, E. L. Boulpaep, and G. Desir. Proc. Natl. Acad. Sci. USA 92: 11711-11715, 1995). Kcn1 RNA injected in Xenopus oocytes leads to the expression of potassium channels that are specifically activated by guanosine 3',5'-cyclic monophosphate (cGMP). Northern blot and ribonuclease (RNase) protection analysis show that Kcn1 is differentially expressed in kidney, aorta, brain, and heart. The purpose of present study is to determine the structure of Kcn1 gene, analyze the promoter region, and identify cis-regulatory elements responsible for transcription. We find that the coding region of Kcn is intronless. The major transcription initiation site was identified by primer extension. Sequence analysis of the 5'-flanking region indicates that, although the gene lacks a typical TATA box, it does have a TATA-box-like region (-TAT-). Using luciferase reporter constructs transfected in the porcine kidney cell line (LLC-PK1), the promoter region and a 5' enhancer element were identified by deletion analysis. Phorbol esters (12-O-tetradecanoylphorbol-13-acetate) and forskolin stimulated Kcn1 gene expression 2.5- and 3.5-fold, respectively. In conclusion, we have identified the region of the novel potassium channel gene, Kcn1, that contains the promoter, a 5' enhancer, and several cis-regulatory elements and shown that gene transcription is stimulated by cAMP and phorbol esters.
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PMID:Genomic structure and regulation of Kcn1, a cGMP-gated potassium channel. 876 Feb 41

Expression of aquaporin-2 (AQP2) is exclusively limited to kidney collecting duct cells, and this strictly limited expression could be mediated by transcription of the gene. We first examined AQP2 mRNA expression in many cultured epithelial cells derived from kidney. Northern blot using OK, LLC-PK1, Madin-Darby canine kidney, and outer medullary collecting duct (OMCD) cells and primary culture of inner medullary collecting duct (IMCD) cells did not reveal any significant signal. A more sensitive method, ribonuclease protection assay, could detect a faint signal in OMCD cells when they were bathed in a hypertonic medium. Reverse-transcribed polymerase chain reaction applied to primary culture of IMCD cells showed a rapid dissipation of AQP2 mRNA within 4 days after culture. A reporter gene assay performed in the 1st day of primary culture of IMCD cells showed that the 5' region up to -2.9 kb worked as a promoter. Deletion experiments showed that at least two regions, from -434 to -364 and from -153 to -84, contain negatively acting cis-elements. When connected to a heterologous promoter, these regions repressed the activity in an orientation-dependent manner. These results suggest that transcription of AQP2 gene is strictly regulated and its ability is rapidly depressed in culture condition. This cell differentiation-specific expression of the gene may be, at least in part, mediated by the repressors present in its 5'-flanking region.
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PMID:Repressive regulation of the aquaporin-2 gene. 889 15

The presence of advanced glycation end products (AGEs) formed because of hyperglycemia in diabetic patients has been strongly linked to the development of diabetic complications and disturbances in cellular function. In this report, we describe the isolation and identification of novel AGE-binding proteins from diabetic rat kidneys. The proteins were purified by cation exchange and AGE-modified bovine serum albumin (AGE-BSA) affinity chromatography. NH2-terminal and internal sequencing identified the proteins as the NH2-terminal domains of ezrin, radixin, and moesin (ERM proteins). Using BIAcore biosensor analysis, human N-ezrin-(1-324) bound to immobilized AGE-BSA with a KD of 5.3 +/- 2.1 x 10 -7 m, whereas full-length ezrin-(1-586) and C-ezrin-(323-586) did not bind. Other glycated proteins such as AGE-RNase, N in -carboxymethyllysine (CML)-BSA, and glycated human serum albumin isolated from hyperglycemic diabetic sera competed with the immobilized AGE-BSA for binding to N-ezrin, but non-glycated BSA and RNase did not. Thus N-ezrin binds to AGEs in a glycation- and concentration-dependent manner. Phosphorylated ezrin plays a crucial role in cell shape changes, cell attachment, and cell adhesion. The effect of AGE-BSA on ezrin function was studied in a tubulogenesis model in which LLC-PK1 cell tubule formation is dependent on phosphorylated ezrin. Addition of AGE-BSA completely inhibited the ability of the cells to produce tubules. Furthermore, in vitro tyrosine phosphorylation of N-ezrin and ezrin was also inhibited by AGE-BSA. These proteins represent a novel family of intracellular binding molecules for glycated proteins and provide a potential new target for therapeutic intervention in the prevention or treatment of diabetic complications.
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PMID:The amino-terminal domains of the ezrin, radixin, and moesin (ERM) proteins bind advanced glycation end products, an interaction that may play a role in the development of diabetic complications. 1273 2