Biomedical subjects
Publications and source records attributed to J R Emanuel.
We transferred murine NIH 3T3 metaphase chromosomes into monkey CV-1 cells to investigate the different ouabain sensitivities of rodent and primate cells. In 16 ouabain-resistant transferents, the mouse Na,K-ATPase alpha 1 subunit gene was detected, suggesting that structural differences between the rodent and primate alpha 1 subunits determine the different ouabain sensitivities.
We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.
We have isolated a cDNA clone for the rat brain Na,K-ATPase alpha subunit. A lambda gt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, lambda rb5, containing a 1200-base-pair cDNA insert expressed a beta-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the alpha subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of lambda rb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney greater than brain greater than liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to lambda rb5 cDNA were amplified approximately 40-fold. Analysis of the lambda rb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase alpha-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the lambda rb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase alpha subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.
A reproducible and sensitive assay for melanotropic agents is described employing mouse melanoma cells in culture and measuring tyrosinase activity in terms of production of tritiated water from L-(ring-3,5-3H)-tyrosine. Molar concentrations of peptides inducing one-half maximal stimulation of tyrosinase activity were: beta-MSH, 1 +/- 2 x 10(-9); alpha-MSH and Beta h-LPH, 1 +/- 2 x 10(-8); ACTHp, 1 +/- 2 x 10(-7). Beta p 9-18-MSH and melanotropin potentiating factor, beta s 88-91-LPH exhibited no activity at concentrations as high as 10(-5)M.
During meiosis and sporulation in Saccharomyces cerevisiae, the recessive genetic marker for cycloheximide resistance, believed to be due to an altered ribosomal protein (C. S. McLaughlin, p. 815-827, in M. Nomura et al., ed., Ribosomes, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), is expressed as early as meiosis II. Ribosomal ribonucleic acid synthesis peaks near the time that cycloheximide resistance begins to appear. Less than 25% of the 17S and 25S ribonucleic acid of the vegetative cells persists in spores, but pulse-labeling studies indicate that greater than 90% of the stable ribonucleic acid made after 6 h survives in spores. These results indicate that the haploid daughter genomes begin to function near the time of meiosis II.
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OBJECTIVE: To evaluate the sensitivity and specificity of the carcinoembryonic antigen (CEA) immunoassay and Ki-ras genotyping as adjuncts to the cytologic diagnosis of pancreatic fine needle aspirates (FNAs). STUDY DESIGN: A retrospective study of 30 patients with pancreatic masses evaluated with CEA immunoassay and gel or hybridization analysis of allele-specific polymerase chain reaction for mutant Ki-ras (codons 12 and 13). DNA was isolated from fixed, paraffin-embedded samples. Diagnoses were correlated with cytologic evaluations and patient outcome. RESULTS: Diagnoses included 17 pancreatic carcinomas, 3 other malignancies and 10 benign lesions. Sixty-five percent of all FNAs had mutated Ki-ras, and 42% of samples with altered Ki-ras had multiple mutations. Replicate FNA samplings in five of six patients had concordant genotypes. Sensitivities for diagnosis were as follows: cytology alone, 76%; CEA alone, 82%; Ki-ras alone, 82%; cytology plus CEA, 100%; cytology plus Ki-ras, 94%. Although specificities for Ki-ras (30%) and CEA (50%) individually were low, elevated CEA level and mutated Ki-ras in a sample with negative cytology strongly indicated false negative cytology. CONCLUSION: The addition of either or both the CEA assay and Ki-ras mutation analysis enhances the sensitivity of the cytologic diagnosis of pancreatic carcinoma by FNA.