Localization of angiotensin peptide-forming enzymes of 3T3-F442A adipocytes

1993 ◽  
Vol 264 (6) ◽  
pp. C1570-C1576 ◽  
Author(s):  
J. A. Saye ◽  
N. V. Ragsdale ◽  
R. M. Carey ◽  
M. J. Peach
Keyword(s):  
Northern Blot Analysis ◽  
Cell System ◽  
Chain Reaction ◽  
Peptide Formation ◽  
Renin Mrna ◽  
Sensitive Polymerase Chain Reaction ◽  
The Media ◽  
Angiotensin Peptide ◽  
Polymerase Chain ◽  
Hydrolysis Of

We have demonstrated that angiotensinogen is synthesized by 3T3-F442A cells and is hydrolyzed to angiotensins I and II (ANG I and II) by this model adipocyte system. This study was designed to determine whether ANG I is generated by renin or some other enzyme and where the formation of ANG I and/or II occurs in 3T3-F442A cells. Renin mRNA was not detected by Northern blot analysis of poly(A)(+)-selected RNA from cultures of fully differentiated adipocytes nor by the more sensitive polymerase chain reaction, implying that renin is not synthesized in this model adipocyte system. Hydrolysis of angiotensinogen to ANG I and II was demonstrated to be associated with the cell but not the media. Inhibitors, including EDTA, aimed at inactivating enzymes belonging to the serine, acid, or aspartyl proteases, and metalloproteases were ineffective in preventing the formation of either ANG I or II. Therefore the model adipocyte 3T3-F442A cell system forms ANG I and II in the absence of renin and angiotensin-converting enzyme. The unidentified enzymes responsible for peptide formation are associated with the cell itself.

Molecular Biology for the Clinician: Understanding Current Methods

2006 ◽  
Vol 42 (5) ◽  
pp. 326-335 ◽  
Author(s):  
Heather L. Chandler ◽  
Carmen M.H. Colitz
Keyword(s):  
Molecular Biology ◽  
Blot Analysis ◽  
Deoxyribonucleic Acid ◽  
Northern Blot Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Biochemical Assays ◽  
Basic Understanding ◽  
Polymerase Chain ◽  
Pcr Microarray

The basics of molecular biology involve the replication of deoxyribonucleic acid and its transcription and translation into proteins. Biochemical assays such as the Southern blot analysis, polymerase chain reaction (PCR), Northern blot analysis, reverse-transcriptase PCR, microarray technology, Western blot analysis, immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry utilize various aspects of molecular biology. To understand these assays requires some basic understanding of the principles of molecular biology. This paper provides basic information on the methodology and techniques used in these assays.

Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel

1997 ◽  
Vol 272 (4) ◽  
pp. C1335-C1344 ◽  
Author(s):  
C. Ding ◽  
E. D. Potter ◽  
W. Qiu ◽  
S. L. Coon ◽  
M. A. Levine ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Pineal Gland ◽  
Northern Blot ◽  
Blot Analysis ◽  
Cyclic Nucleotide ◽  
Northern Blot Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.

scholarly journals Novel members of the cdc2-related kinase family in Drosophila: cdk4/6, cdk5, PFTAIRE, and PITSLRE kinase.

1996 ◽  
Vol 7 (11) ◽  
pp. 1759-1769 ◽  
Author(s):  
K Sauer ◽  
K Weigmann ◽  
S Sigrist ◽  
C F Lehner
Keyword(s):  
Polymerase Chain Reaction ◽  
Northern Blot Analysis ◽  
Kinase Domain ◽  
Early Embryogenesis ◽  
Cellular Functions ◽  
Chain Reaction ◽  
Sequence Comparisons ◽  
Polymerase Chain ◽  
Two Hybrid

In addition to the previously identified Drosophila cdc2 and cdc2c genes, we have identified four additional cdc2-related genes with low stringency and polymerase chain reaction approaches. Sequence comparisons suggest that the four putative kinases represent the Drosophila homologues of vertebrate cdk4/6, cdk5, PCTAIRE, and PITSLRE kinases. Although the similarity between human and Drosophila homologues is extensive in the case of cdk5, PCTAIRE, and PITSLRE kinases (78%, 58%, and 65% identity in the kinase domain), only limited conservation is observed for Drosophila cdk4/6 (47% identity). However, like vertebrate cdk4 and cdk6, Drosophila cdk4/6 binds also to a D-type cyclin according to the results of two-hybrid experiments in yeast. Northern blot analysis indicated that the four Drosophila kinases are expressed throughout embryogenesis. Expression in early embryogenesis appeared to be ubiquitous according to in situ hybridization. Abundant expression already at the start of embryogenesis and long before neuron differentiation was also observed in the case of cdk5 protein, which has been described as predominantly neuron specific in mice. Sequence conservation and expression pattern, therefore, suggest that all of these kinases perform important cellular functions.

scholarly journals Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription.

1991 ◽  
Vol 173 (3) ◽  
pp. 711-720 ◽  
Author(s):  
M S Schlissel ◽  
L M Corcoran ◽  
D Baltimore
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Gene Rearrangement ◽  
Gene Segment ◽  
Allelic Exclusion ◽  
Immunoglobulin Gene ◽  
Chain Reaction ◽  
Highly Sensitive ◽  
Sensitive Polymerase Chain Reaction ◽  
Polymerase Chain

Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (mu) protein in regulating gene rearrangement. Our analysis of pre-B cell cultures representing various stages of maturity revealed that transcription of each germline Ig locus precedes or is coincident with its rearrangement. Cell lines containing one functional rearranged H chain allele, however, continue to transcribe and to rearrange the allelic, unrearranged H chain locus. These cell lines appear to initiate but not terminate rearrangement events and therefore provide information about the requirements for activating rearrangement but not about allelic exclusion mechanisms.

scholarly journals Molecular diagnosis of Beckwith-Wiedemann Syndrome using quantitative methylation-sensitive polymerase chain reaction

2006 ◽  
Vol 8 (10) ◽  
pp. 628-634 ◽  
Author(s):  
Bradford Coffee ◽  
Kasinathan Muralidharan ◽  
William E Highsmith ◽  
Pablo Lapunzina ◽  
Stephen T Warren
Keyword(s):  
Polymerase Chain Reaction ◽  
Molecular Diagnosis ◽  
Chain Reaction ◽  
Sensitive Polymerase Chain Reaction ◽  
Polymerase Chain

scholarly journals Differential regulation of angiotensin-(1-12) in plasma and cardiac tissue in response to bilateral nephrectomy

2009 ◽  
Vol 296 (4) ◽  
pp. H1184-H1192 ◽  
Author(s):  
Carlos M. Ferrario ◽  
Jasmina Varagic ◽  
Javad Habibi ◽  
Sayaka Nagata ◽  
Johji Kato ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Tissue Expression ◽  
Ang Ii ◽  
Bilateral Nephrectomy ◽  
Functional Changes ◽  
Peptide Formation ◽  
Quantitative Immunofluorescence ◽  
Renin Mrna ◽  
Angiotensin Peptide ◽  
Wistar Kyoto

We examined the effects of 48 h bilateral nephrectomy on plasma and cardiac tissue expression of angiotensin-(1-12) [ANG-(1-12)], ANG I, and ANG II in adult Wistar-Kyoto rats to evaluate functional changes induced by removing renal renin. The goal was to expand the evidence of ANG-(1-12) being an alternate renin-independent, angiotensin-forming substrate. Nephrectomy yielded divergent effects on circulating and cardiac angiotensins. Significant decreases in plasma ANG-(1-12), ANG I, and ANG II levels postnephrectomy accompanied increases in cardiac ANG-(1-12), ANG I, and ANG II concentrations compared with controls. Plasma ANG-(1-12) decreased 34% following nephrectomy, which accompanied 78 and 66% decreases in plasma ANG I and ANG II, respectively ( P < 0.05 vs. controls). Contrastingly, cardiac ANG-(1-12) in anephric rats averaged 276 ± 24 fmol/mg compared with 144 ± 20 fmol/mg in controls ( P < 0.005). Cardiac ANG I and ANG II values were 300 ± 15 and 62 ± 7 fmol/mg, respectively, in anephric rats compared with 172 ± 8 fmol/mg for ANG I and 42 ± 4 fmol/mg for ANG II in controls ( P < 0.001). Quantitative immunofluorescence revealed significant increases in average grayscale density for cardiac tissue angiotensinogen, ANG I, ANG II, and AT1 receptors of WKY rats postnephrectomy. Faint staining of cardiac renin, unchanged by nephrectomy, was associated with an 80% decrease in cardiac renin mRNA. These changes were accompanied by significant increases in p47phox, Rac1, and Nox4 isoform expression. In conclusion, ANG-(1-12) may be a functional precursor for angiotensin peptide formation in the absence of circulating renin.

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