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Mohan D Rolston R Pal R Swalsky PA Sasatomi E Lee RE Finkelstein SD 《Human pathology》2004,35(4):482-487
The genetic diagnosis of Gaucher disease by molecular methods is complicated by the existence of a highly homologous transcribed pseudogene (96% identity) that is found in close proximity to the true gene on chromosome 1q21. In addition, the pseudogene sequence can mimic disease-causing mutations in the true gene. Selective polymerase chain reaction (PCR) amplification of the true gene can be accomplished in extracted DNA from fresh-frozen samples by designing oligonucleotide primers to hybridize to defined regions that are not present in the pseudogene. This standard molecular approach, which entails amplification of relatively long segments of intact DNA, is not feasible in archival, paraffin-embedded, solid-tissue specimens in which the negative effects of chemical fixation result in DNA strand scission and breakdown of nucleic acid. A novel approach, specifically created for use with archival, fixative-treated tissue specimens, was developed for detection and characterization of common mutations of Gaucher disease. Three separate robust PCR reactions were formulated, 2 for selective amplification of portions of only the true gene exons 2 and 9, with a third reaction targeting exon 10, wherein both the true and pseudogene were coamplified. In the latter, DNA sequencing was used to determine the presence of true and pseudogene allele content in addition to identification of base sequence alterations. This method, requiring a single, 4-microm-thick histologic section, was successfully applied to archival paraffin block tissue specimens that had been in storage for up to 75 years. It was capable of accurately genotyping common Gaucher disease mutations as well as discovering a novel mutation and genetic polymorphism. We recommend our approach when only fixative-treated tis sue is available for molecular genotyping. 相似文献
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Sass DA Chopra KB Finkelstein SD Schauer PR 《Archives of pathology & laboratory medicine》2004,128(2):214-217
In cases of obscure gastrointestinal bleeding, when a source for blood loss is not apparent from examination of the colon and upper gastrointestinal tract, the small bowel usually becomes the focus of investigation. A tumor with interesting pathologic features was found in a patient who presented with recurrent episodes of massive obscure gastrointestinal hemorrhage. This case highlights the importance of considering small intestinal tumors as the likely cause of obscure gastrointestinal hemorrhage in young patients and how a noninvasive test, eg, abdominal computed tomography scan, might obviate the need for more invasive testing. 相似文献
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Methods have been developed previously for rapid evaluation of compounds for antiviral activity in 96-well microplates, which include visual quantitation of antiviral activity based upon inhibition of virus-induced cytopathic effect (CPE) or by less subjective colorimetric or fluorometric means. In the present studies we compared a number of colorimetric (crystal violet, MTT [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide], and neutral red) and fluorometric (Alamar Blue, bisbenzimide [Hoechst 33258], fluorescein diacetate, and rhodamine 6G) methods to visual scoring of antiviral activity in influenza A virus infections in Madin Darby canine kidney (MDCK) cells. Toxicity determinations using these same methods were also made for anti-influenza inhibitors and other compounds known to inhibit cell proliferation. Against influenza A/Texas/36/91 (H1N1) and A/Sydney/05/97 (H3N2) viruses, visual scoring and dye or stain methods produced results that were not significantly different from each other in deriving 50% virus-inhibitory concentrations (EC(50) values) for six anti-influenza compounds (amantadine, rimantadine, ribavirin, RWJ-270201 [BCX-1812], oseltamivir carboxylate, and zanamivir), with the exception of Alamar Blue which quantified lower EC(50) values than expected. In uninfected replicating cells, the visual and Alamar Blue methods underestimated the 50% cytotoxic concentrations (IC(50) values) of ribavirin, 1-beta-D-arabinofuranosylcytosine, and 6-azauridine, but more accurately assessed the toxicities of amantadine, rimantadine, and cycloheximide. Visual scoring, coupled with the use of one of these dyes or stains except Alamar Blue, can be used to accurately and rapidly quantify the anti-influenza virus activities and toxicities of potential new influenza virus inhibitors. These methods should also be applicable to evaluating antiviral effects against other lytic virus infections. 相似文献
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Ruth Ludwig David S. Alberts Thomas P. Miller Sydney E. Salmon 《Cancer chemotherapy and pharmacology》1984,12(3):135-141
Summary A human tumor clonogenic assay (HTCA) has been used to evaluate standard and experimental anticancer drugs with respect to thrir inhibition of clonogenicity of both fresh human cancers and human tumor cell lines. By comparing the inhibitory effect on tumor colony-forming unit (TCFU) growth of 1-h and continuous drug exposures in the HTCA we were able to identify and separate schedule-dependent (e.g., bleomycin, vinblastine, and etoposide) and schedule-independent drugs (e.g., actinomycin D, adriamycin, basantrene, and cis-platinum). Vinblastine, bleomycin, and etoposide, which are known to have cell cycle-specific characteristics, caused exponential reduction in tumor colony formation when given by continuous exposure, whereas when given with a short exposure each of these drugs caused plateau-type dose-response curves. For comparison of the relative efficacy of the two dosing schedules, a ratio (1-h versus continuous exposures) was calculated of the drug concentrations which reduced growth of TCFU to 50% of the control values (ID50) for fresh human tumors and human tumor cell lines. For fresh tumors, ID50 ratios for adriamycin, actinomycin D, and bisantrene ranged between 2 and 60 (median 14), whereas the ID50 ratios for bleomycin, vinblastine, and etoposide ranged between 100 and 3,000 (median 600). The fact that actinomycin D, adriamycin, and bisantrene (a new anthracene-type drug) had similarly shaped dose-response curves and very low ID50 ratios suggests that the cytotoxicity of these compounds may not be schedule-dependent. On the other hand, the steep dose-survival curves we observed after continuous drug exposure and the high ID50 ratios of bleomycin, vinblastine, and etoposide suggest that these drugs may possess schedule-dependent cytotoxicity characteristics. Before final conclusions are drawn concerning a drug's schedule dependency it is essential to evaluate its in vitro stability and protein-binding characteristics. Finally, it must be emphasized that unlike the results obtained with 1-h exposure studies, the in vitro continuous exposure schedules have yet to be shown to be predictive of clinical response for any agent or tumor type. 相似文献
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Summary Esorubicin (4 deoxydoxorubicin) is a new analogue of the anthracycline, doxorubicin. This compound lacks the hydroxyl group at 4 position on the amino sugar of the anthracycline. Phase II study was designed to determine the clinical response rate and to define the qualitative and quantitative toxicities of esorubicin in patients with adenocarcinoma of the pancreas. Fifty-eight patients with inoperable adenocarcinoma of the pancreas were entered on the study, 47 were evaluable for response, and 57 were evaluable for toxicity. The dose of esorubicin was 30 mg/m2 for good risk patients and 25 mg/m2 for poor risk patients every 21 days and administered IV push through a side arm of a running IV. Diphenhydramine, 50 mg is administered IM prior to the administration of the drug to block local venous reaction. Subsequent doses of esorubicin were modified according to granulocyte and platelet nadirs and the drug was not administered until recovery of platelets (> 100,000/ul) and wbc (> 3000/ul). Three partial responses, 20 stable, and 31 with increased disease were observed. Forty-seven had severe granulocytopenia (< 250), and two patients had severe thrombocytopenia (< 25,000). One patient experienced a decrease in left ventricular ejection fraction with a total dose of 180 mg/m2. The dose of esorubicin in this study demonstrated that the drug has minimal activity in adenocarcinoma of the pancreas but the toxicity is tolerable. Search should continue for single agents with activity in this disease. 相似文献
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