Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping

Mocharla, Hanna; Mocharla, Raman; Me, Hodes

doi:10.1016/0378-1119(90)90235-j

Cited by 80 publications

(35 citation statements)

References 11 publications

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“…The cells were incubated with the first antibody (mouse monoclonal antibody specific for the ␣-SM actin isoform, DAKO A/S) diluted 1:100 in PBS for 1 hour and rinsed with PBS for 30 RT-PCR was performed as described previously. 12 In brief, aliquots of RNA (1 g The primers targeted to SM22␣ and matrix Gla genes were based on rat SM22␣ and matrix Gla cDNA sequences. 13 The primers used for PCR for osteopontin, 14 cathepsin D, 15 ACE, 16 transforming growth factor (TGF)-␤1, 17 platelet-derived growth factor (PDGF) A-chain, 18 and basic fibroblast growth factor (bFGF) 19 were as previously described and are shown in Table I (which appears online at http://atvb.ahajournal.org).…”

Section: Immunofluorescence For ␣-Sm Actinmentioning

confidence: 99%

Phenotypic Modulation by Fibronectin Enhances the Angiotensin II–Generating System in Cultured Vascular Smooth Muscle Cells

Fukuda

Satoh

et al. 2000

ATVB

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Abstract-We previously demonstrated that homogeneous cultures of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats produce angiotensin II (Ang II) in response to increases in the levels of angiotensinogen, cathepsin D, and angiotensin-converting enzyme (ACE). The change of VSMCs from the contractile to the synthetic phenotype increased the amount of synthetic organelles, resulting in the production of proteases and growth factors. To evaluate the contribution of the synthetic phenotype to the generation of Ang II, we examined the effect of fibronectin (FN), which reportedly induces the synthetic phenotype, on the Ang II-generating system in VSMCs. Cultured VSMCs from Wistar-Kyoto rats were incubated with an active fragment of FN, Arg-Gly-Asp-Ser, for 24, 48, or 72 hours after synchronization of the cell cycle with 0.2% calf serum for 48 hours. Immunofluorescence and protein levels of ␣-smooth muscle (SM) actin and expression of SM22␣ mRNA, apparent in the contractile phenotype, were suppressed by FN, whereas expression of matrix Gla mRNA and osteopontin mRNA and protein, apparent in the synthetic phenotype, was increased. FN (1 to 1000 g/mL) dose-dependently increased DNA synthesis in the VSMCs, which was inhibited by the Ang II type 1 receptor antagonist CV-11974. Ang II-like immunoreactivity as determined by radioimmunoassay was significantly increased in conditioned medium from the VSMCs. In addition, mRNA for the Ang II-generating proteases cathepsin D and ACE was increased by FN. Expression of transforming growth factor-␤1, platelet-derived growth factor A-chain, and basic fibroblast growth factor mRNAs was also increased by FN. These results indicate that the changes accompanying the alteration to the synthetic phenotype in homogeneous cultures of VSMCs increase expression of proteases such as cathepsin D and ACE, which then produce Ang II, and that these changes increase expression of growth factors that then induce growth of VSMCs.

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Section: Immunofluorescence For ␣-Sm Actinmentioning

confidence: 99%

Phenotypic Modulation by Fibronectin Enhances the Angiotensin II–Generating System in Cultured Vascular Smooth Muscle Cells

Fukuda

Satoh

et al. 2000

ATVB

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“…In order to reveal the number of AMY1 genes in some of the discussed families that show quantitative variation at the protein level, we used the PCR technique. Mocharla et al (1990) described a PCR that makes isozyme genotyping possible, using gene transcripts of the AMY1 and AMY2 genes, which differ from one another in length because of an insertion in intron S of the AMY1 gene. We have modified this technique by using genomic DNA instead of mRNA, and by using different isozyme-specific primers.…”

Section: Haplotypes Encoding Amy1mentioning

confidence: 99%

“…7). The differences in length in the amplified region of the AMY1 and AMY2 genes is, as in the method of Mocharla et al (1990), caused by an A-rich stretch that is present in AMY1 (nucleotide -233/-212, see Groot et al 1988), but absent in AMY2. As the human haploid genome contains two pancreatic amylase genes (Groot et al 1988(Groot et al , 1989Gumucio et al 1988) and a variable number of salivary amylase genes (Groot et al 1989), the 582-bp fragment can act as an internal standard.…”

Section: Haplotypes Encoding Amy1mentioning

confidence: 99%

Variation in gene copy number and polymorphism of the human salivary amylase isoenzyme system in Caucasians

et al. 1992

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Summary. The polymorphic patterns of human salivary amylase of a large number of individuals of Caucasian origin were determined by using isoelectric focusing and polyacrylamide gel electrophoresis. Nine different salivary amylase protein variants were found; three of them are recorded for the first time and their heredity is shown. Some of the variants are encoded by haplotypes expressing three allozymes. Most variants display low frequencies. Analysis of the relative intensities of variant-specific isozyme bands, combined with segregation analysis, show that extensive quantitative variation is present in the population. The numbers of salivary amylase genes in some families showing quantitative variation at the protein level have been estimated by the polymerase chain reaction. We present evidence that quantitative variations in amylase protein patterns do not always reflect variations in gene copy number but that other mechanisms are also involved.

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“…RT-PCR analyses of rat VEGF, Flt-1, and PECAM-1 mRNAs, and 18S ribosomal RNA (rRNA) as an internal control were performed with 1 µg of total RNA, as described previously (10). The amplification conditions were 30 cycles of denaturing at 94 ºC for 1 min, primer annealing at 60 ºC for 1 min, and extension at 72 ºC for 1 min in a DNA thermal cycler (Perkin-Elmer-Cetus, Norwalk, USA).…”

Section: Reverse Transcription-polymerase Chain Reaction (Rt-pcr) Anamentioning

confidence: 99%

Development of Angiogenic Cell and Gene Therapy by Transplantation of Umbilical Cord Blood with Vascular Endothelial Growth Factor Gene

et al. 2004

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Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping

Cited by 80 publications

References 11 publications

Phenotypic Modulation by Fibronectin Enhances the Angiotensin II–Generating System in Cultured Vascular Smooth Muscle Cells

Phenotypic Modulation by Fibronectin Enhances the Angiotensin II–Generating System in Cultured Vascular Smooth Muscle Cells

Variation in gene copy number and polymorphism of the human salivary amylase isoenzyme system in Caucasians

Development of Angiogenic Cell and Gene Therapy by Transplantation of Umbilical Cord Blood with Vascular Endothelial Growth Factor Gene

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