PubMed · 7938595
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I Endo. 1994. [Genome analysis system].. https://pubmed.ncbi.nlm.nih.gov/7938595/
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A rapid, sensitive, semiautomated method to measure the activity of mast cell-derived tryptase has been developed. The assay is based on the tryptase-mediated hydrolysis of vasoactive intestinal peptide that was modified to include an N-terminal dansyl reporter group. Tryptase cleaves vasoactive intestinal peptide (VIP) producing two major and two minor products. Full-length VIP was separated from the proteolysis products by reverse-phase (C18) chromatography. The amount of each product as well as the amount of full-length substrate remaining was determined using an in-line fluorescence detector. As little as 0.5 pmol of peptide could be detected. Predominant cleavages were after Arg-14 and Lys-20 with minor cleavages after Lys-15 and Lys-21. The hydrolysis of VIP at Arg-14 was slightly affected by the presence of the dansyl group at the N-terminus. While the Km value was unaffected, the kcat value decreased, yielding a kcat/Km ratio that was eightfold lower. The addition of calcium chloride to the reaction mixture resulted in a slight decrease in the kcat/Km ratio. Cleavage of dansyl-VIP by tryptase was completely inhibited by DesPro2-Arg15-Aprotinin.
A fast method is reported for the precise and accurate quantification of cytokine mRNA, based on a quantitative polymerase chain reaction (PCR) assay. Post-PCR detection of the amplification products is achieved using an automated electrochemiluminescent (ECL) detection system. The target is amplified using a biotinylated forward and a tris(2,2'-bipyridine)ruthenium (II) (TBR)-labeled reverse primer. The amplification products are then captured on streptavidin coated paramagnetic beads and quantified by measuring the ECL signal of the TBR label. The results obtained are reproducible and accurate over a wide range (3 orders of magnitude) of concentrations. Quantitative results can be obtained using a standard curve which is generated with a synthetic external standard. This technique was applied to quantify tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin-2(IL-2) mRNA levels in human T cells transfected with the corresponding genes.
After more than four decades of research into automation of the process of screening Papanicolaou (Pap) smears, attempts to develop commercially viable automated screening machines have increased in recent years. These developments have been made possible in part because of the improving price-to-performance ratios in computers and other electronics. Although the Pap smear has been responsible for a very significant decrease in mortality of cervical cancer over the past 40 years, concern has arisen over false-negative cases, with their effects on patients, and the associated legal liability, particularly in the United States. In addition, shortages of cytotechnologists, which have been exacerbated by new regulations limiting the number of slides that may be examined per day, have caused concern about handling the workload, which will probably increase as more individuals gain access to preventive health care. Automated screening machines can potentially allow detection of abnormal cases that are missed with conventional screening, although they may substantially increase the cost of Pap smears. The use of automated screening machines represents a change in the way cervical cytology specimens are processed, and with some machines, a significant change in the operation of the cytology laboratory. Current methods for processing and evaluating Pap smears have not changed significantly for the past four decades. This review discusses some of the principles of operation and practical aspects of automated screening machines.