PubMed · 2374606
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B Furrer, U Candrian, P Wieland, J Lüthy. 1990-07-26. Improving PCR efficiency.. https://doi.org/10.1038/346324b0
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BACKGROUND: Localization of human regenerating gene (reg) mRNA and its product was investigated in normal and neoplastic human pancreas with the in situ hybridization method and immunohistochemical studies. METHODS: Both reg mRNA and reg protein were observed in acinar cells of the pancreas, but neither was found in ductal or islet cells. Immunoreactive reg was observed in an acinar cell carcinoma, a pancreatoblastoma, a solid and cystic tumor, and 5 of 20 duct cell carcinomas, but it was not found in 15 endocrine tumors and 2 microcystic adenomas. RESULTS: For comparison with reg, alpha-1-antitrypsin (AAT), lysozyme, chromogranin A (CMG), CA 19-9, carcinoembryonic antigen (CEA), cytokeratin, vimentin, and alpha-fetoprotein (AFP) were assessed in those tumors. An acinar cell carcinoma and a pancreatoblastoma had positive results for AAT, lysozyme, cytokeratin, and AFP but negative results for vimentin. An acinar cell carcinoma showed cells focally immunoreactive for CMG and CEA. A solid and cystic tumor had strongly positive results for AAT and vimentin and focally positive results for CMG and pancreatic hormones. Microcystic adenomas had abundant glycogen and strong immunoreactivity for cytokeratin. Ductal cell carcinomas showed cells focally positive for AAT, lysozyme, CMG, CA 19-9, and CEA. CONCLUSIONS: The localization of reg protein was not consist with that of any other proteins examined in the current study. Thus, reg protein was considered a useful marker for acinar cell differentiation; however, ectopic expression of reg also was observed in ductal cell carcinomas. In ductal cell carcinomas, expression of reg immunoreactivity was considered as one of phenotypic heterogeneity, as seen in AAT, lysozyme, and CMG immunoreactivity.
The D-galactose chemosensory receptor of Escherichia coli is a .32 kDa globular protein possessing two distinct structural domains, each organized in an alpha/beta folding motif. Helices I and X lie at adjacent approximately parallel positions on the surface of the N-terminal domain, near the hinge region. In order to analyze the relative thermal motions of these two helices, the present study utilizes a generalizable disulfide trapping approach: first, site-directed mutagenesis is used to place a pair of cysteine residues at locations of interest on the protein surface, then disulfide bond formation is used to trap intramolecular cysteine-cysteine collisions resulting from thermal motions. Specifically, four engineered di-cysteine receptors have been constructed, each possessing one cysteine at position 26 on helix I, and a second cysteine at varying positions on helix X. A fifth control receptor possesses one cysteine at position 26, and a second on the opposite surface of the molecule. These surface cysteine substitutions have little or no effect on the measurable receptor parameters as judged by ligand binding equilibria and kinetics, protein stability, and 19F nuclear magnetic resonance, indicating that the engineered receptors are useful probes of native backbone dynamics. Spatial and kinetic features of backbone motions have been investigated by measuring intramolecular disulfide formation rates for cysteine pairs in the fully liganded receptor. The resulting rates decrease monotonically with increasing distance between cysteines in the crystal structure, while no disulfide formation is observed for the control pair unless the molecule is unfolded. The minimum translational amplitudes of the observed backbone motions range from 4.5 to 15.2 A, and the minimum rotational amplitudes are as large as 35 degrees. For each motion the rate of intramolecular sulfhydryl-sulfhydryl collision has been estimated from the measured rate of disulfide formation: the 4.5 and 15.2 A translations yield approximately 10(4) and approximately 10 collisions s-1 molecule-1, respectively. These collision rates, which are faster than ligand dissociation, likely underestimate the actual motional frequencies since only an undetermined fraction of the total motions yield collisions. The simplest plausible trajectory capable of producing such collisions is a rate-limiting translation of one or both helices along their long axes, coupled with minor helix rotations. When sugar is removed from the receptor, a substantial increase in backbone dynamics is observed, indicating the presence of new long-range backbone trajectories. Overall, the results suggest that internal motions in proteins may have larger amplitudes than previously observed.
The effect of the treatment of HeLa cells with a tumor-promoting phorbol ester, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of the genes for the calpain family has been examined. Among the mRNAs for the calpain family, only the mRNA for the large subunit of human m-calpain (calpain mL) was specifically induced by treatment of cells with TPA, suggesting its specific function in response to cellular stimuli. The effect of TPA on the expression of the calpain mL gene was further examined using fusion genes containing the promoter/enhancer region of the calpain mL gene fused upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene, showing that the promoter/enhancer sequence of the calpain mL gene contains a cis-acting element which responds to TPA and activates transcription of the downstream sequence.