IS6110-Based PCR Methods for Detection of Mycobacterium tuberculosis Kent et al. (3) detected Mycobacterium tuberculosis by a nested PCR test that amplified a 181-bp target located in insertion sequence IS6110, but they also encountered many false-positive results when the test was applied to isolates of mycobacteria that do not belong to the M. tuberculosis complex. Because the investigators attributed the false positives to homology between IS6110 and genomic DNA of the other mycobacterial species, they warned that false-positive PCR results could be a problem with some IS6110-based methods for detection of M. tuberculosis complex. Our recent experience in using IS6110-based amplifications for detection of M. tuberculosis complex is informative on this issue. We selected two such amplifications that others had developed, with respective 123-bp (2) and 317-bp (1, 4) targets, and after slightly modifying the procedures incorporated them into a dual-PCR clinical protocol that we applied to mycobacterial colonies grown on Löwenstein-Jensen or Middlebrook 7H11 medium. The modifications that we made included use of dUTP (rather than dTTP) in both reaction mixes and use of a cycling program that was the same for both amplfications (30 cycles; cycle 1: 94ЊC for 5 min, 68ЊC for 45 s, 72ЊC for 90 s; cycles 2 through 30: 94ЊC for 45 s, 68ЊC for 45 s, 72ЊC for 90 s). The two amplifications were performed in separate tubes in a single thermal-cycler run, and the two amplified products were then electrophoresed in separate lanes of the same ethidium bromide-stained 2% agarose gel. In initial validation studies and in subsequent routine clinical examinations, we used this dual-PCR procedure in blindly testing 116 mycobacterial isolates, all of which were also identified to the species (or complex) level by the Mycobacteriology Laboratory of the New Jersey State Department of Health on the basis of their colony characteristics, chemical reactions, patterns shown on highpressure liquid chromatography, and DNA-rRNA hybridizations. The results produced by the dual-PCR procedure and the final identifications by the state laboratory were in total agreement for all 116 isolates: they were positive for M. tuberculosis complex in 46 cases and negative in the remaining 70. Identifications made by the state laboratory on the 70 isolates negative for M. tuberculosis complex were as follows: M. avium-M. intracellulare complex, 50; M. xenopi, 7; M. gordonae, 7; M. fortuitum, 5; and M. kansasii, 1. (In the entire series of 116 cultures, there was a single specimen for which the initial PCR results were discrepant, i.e., the 123-bp target was amplified but the 317-bp target was not. An advantage of the dual-PCR approach was demonstrated in th is case, in that duplicate repeats with the two sets of applicable primer pairs produced amplification of both targets, thus identifying the culture as positive for M. tuberculosis complex. This diagnosis was later confirmed by the state laboratory.) Kent et al.'s call for careful validation of any IS61...
Smooth-muscle neoplasms are rarely located in the spleen. They have been previously reported in five cases of children with human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS). Two cases of children with HIV infection/AIDS with autopsy and surgical pathology evidence of multiple smooth-muscle neoplasms with splenic involvement are presented. DNA was extracted from histology slides in both cases for analysis for Epstein Barr (EB) virus. In both cases, the presence of EB virus was confirmed. This paper documents two additional cases of the unusual phenomenon of splenic involvement by smooth-muscle neoplasms in the setting of AIDS in childhood and further supports the role of EB virus in the development of these neoplasms.
Asparaginase synthesis by Vibrio succinogenes is induced by ammonium ions. Synthesis occurs throughout exponential phase, and in early stationary phase asparaginase accounts for about 5% of the total soluble protein. The organism grows best when fumarate is provided as the terminal electron acceptor of the formate-oxidizing cytochrome system. Yeast extract or enzyme-hydrolyzed proteins are effective nutrient sources. In an ammonium formate-sodium fumarate medium, where maximum growth and asparaginase synthesis occurs, the total enzyme yield (international units per liter of culture) is about one-tenth that obtainable with a good asparaginase-producing strain of Escherichia coli. The energetic inefficiency of V. succinogenes appears to cause a low yield of cells and therefore low total enzyme yield. However, the levels of asparaginase accumulated within cells raise questions about the organism's protein synthesizing system.
Puromycin aminonucleoside selectively inhibits the synthesis of ribosomal RNA in human lung fibroblasts transformed by the oncogenic virus SV40. The mechanism of this inhibition was studied utilizing nuclei and nucleoli isolated from cells treated for 18 hours with 100 micrograms/ml of this compound. It was established that for a limited period of time nuclei and nucleoli isolated from the fibroblasts continue synthesis of RNA of size classes seen in intact cells, and that the inhibitory effect of aminonucleoside persists after isolation of these organelles. The inhibition was shown to be directed primarily to the activity of RNA polymerase I. Studies of the mechanism of this inhibition have indicated that the decreased rate of the polymerase reaction is not due to the impairment of the template function of nucleolar chromatin, and that unbound, as well as chromatin-bound, RNA polymerase I is present in both control and treated nucleoli. Analysis of the size distribution of the products of cell-free RNA synthesis showed that aminonucleoside pretreatment results in marked reduction in the synthesis of preribosomal 45S RNA, abnormal accumulation of 32S RNA, and reduced formation of mature ribosomal RNA species in the in vitro system. The data suggest that the inhibitory effect of aminonucleoside on ribosomal synthesis is due in part to a lower rate of transcription by RNA polymerase I of preribosomal RNA, and in part to its impaired maturation.
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