Purification and partial characterization of mitochondrial ribosomes from a HeLa cell line resistant to chloramphenicol

Techniques are described for the purification of mitochondrial ribosomes from two lines of HeLa cells, S(,3) and 296-1, one of which (296-1) is resistant to chloramphenicol in vivo. The in vitro protein synthetic activity of purified mitochondrial ribosomes from both strains are compared for their sensitivity to a variety of drugs including chloramphenicol;For the preparation of mitochondrial ribosomes from either cell line, crude mitochondria are washed several times in buffer containing 0.002M EDTA, and mitoplasts are prepared by treatment with digitonin at a digitonin to mitochondrial protein ratio of 0.15. The mitoplasts are lysed by the addition of Brij-58 and Nonidet P-40 at a final concentration of 0.5% each (w/v) and mitochondrial ribosomes are collected by pelleting through a 1M sucrose pad (105,000 x G(,ave) for 16 hours);HeLa cell mitochondrial ribosomes prepared in this manner are free of contaminating cytoplasmic ribosomes as judged by sucrose gradient centrifugation and by extraction and analysis of the ribosomal RNA in polyacrylamide gels;The preparations are active in both poly(U) and poly(C,U)-directed polypeptide synthesis when supplemented with tRNA and supernatant factors from E. coli. However, mitochondrial supernatant factors (S-100) from rat, rabbit and HeLa cells were not able to replace those from E. coli. Despite repeated attempts to subfractionate these S-100 preparations, a potent inhibitor of in vitro polypeptide synthesis remained. At least one of these preparations was shown to contain significant RNase activity;Mitochondrial ribosomes from these cell lines differ in their sensitivity to chloramphenicol. When assayed for poly(C,U)-directed polypeptide synthesis, HeLa S(,3) mitochondrial ribosomes are inhibited 50% by 200 (mu)g/mL chloramphenicol. Mitochondrial ribosomes from HeLa 296-1 are unaffected at concentrations up to 400 (mu)g/mL. From these results, it is clear that for HeLa 296-1, the cytoplasmically inherited mutation to chloramphenicol resistance has affected a component of the mitochondrial ribosome. In vitro, polypeptide synthesis on HeLa 296-1 ribosomes is also resistant to inhibition by lincomycin at concentrations which were found to significantly inhibit HeLa S(,3) mitochondrial ribosomes. This is presently the only report of cross-resistance between these two drugs

IUCN, National Parks, and Protected Areas: Priorities for Action

In order to ensure that the full range of species and natural ecosystems continue to form part of the human habitat, national parks and other types of protected areas must be better designed and managed than is usually the case at present. While IUCN has a long history of involvement in protected areas, often in cooperation with UNESCO, the World Wildlife Fund, and UNEP, the World National Parks Congress (held in Bali, Indonesia, during 11-22 October 1982) marked a major turning-point in promoting protected areas as part of the social and economic development process. In this approach, the Congress provided ways and means for the philosophy of the World Conservation Strategy (1980) to be put into action on the groun

Mesurol (methiocarb) residues in grapes and wine and their possible effects on fermentation and wine quality

Wines were made from Pinot Noir and White Riesling grapes which had received field application rates of 2 and 4 lb./acre of the experimental bird repellent Mesurol (methiocarb or 3, 5 dimethyl- 4(methylthio)phenylmethylcarbamate). In addition, Pinot Noir must and press juice and White Riesling press juice were fortified with 15 ppm Mesurol (active ingredient) and vinified. Mesurol and its sulfoxide and sulfone metabolites were determined by gas chromstographic analysis as mesylate derivatives following fractionation on Florisil. Residues were also oxidized to and expressed as total sulfone. Mesurol residues, methiocarb and total sulfone, in field treated grapes sprayed with 4 lb./acre Mesurol exceeded the 15 ppm temporary tolerance established by the Environmental Protection Agency. Methiocarb residues in pomace, calculated on a dry weight basis, obtained from grapes harvested one day after the final application of Mesurol were in excess of the 75 ppm EPA temporary tolerance. Approximately 40 to 51% of the methiocarb residues measured in field, treated grapes remained associated with the pomace after pressing. Finished Pinot Noir and White Riesling wine, prior to bottling, contained in the order of 3-8 ppm representing 34 to 49% and. 13%, respectively, of the methiocarb residues originally present in grapes. The fate of Mesurol sulfide, sulfoxide and sulfone residues during the processing of Mesurol fortified White Reisling press juice was investigated. Settling of press juice was found to reduce methiocarb residues. Oxidation during vinification was minimal. Mesurol sulfide, sulfoxide, and sulfone residues in finished wine remained unchanged after 12 months of storage at cellar temperatures. Trace amounts of Mesurol sulfide phenol were detected with Mass Spectrometry. Volclay bentonite (KWK) fining at 4 and 8 lb./1000 gal. and membrane filtration (0.45 μ) were not effective in reducing Mesurol residues in wine. Reference-difference tests were used to evaluate the effect of Mesurol on finished wine sensory qualities. The sensory properties of wines made from grapes harvested seven days after the last application of Mesurol and Mesurol Fortified must and juices were not affected. Also, the chemical composition of these wines was not altered. Lag time and fermentation rate of Mesurol Fortified. Pinot Noir must and juice processed into wine were not affected. The course of fermentation in the presence of Mesurol was also monitored by recording mass loss in model studies. A slight inhibition in fermentation rate was observed, at levels below 25 ppm while a stimulation was evident at concentrations of up to 500 ppm

Patient-Centered Medicine and Prevention of Munchausen Syndrome by Proxy

Munchausen syndrome by proxy (MSbP) is known by many names and is considered the deadliest form of child abuse. Although the condition was named in 1976 and there is now a substantial body of scientific literature about this type of abuse, to date, patient‐centered approaches to early identification, intervention, and prevention have been absent from this literature. The purpose of this chapter is to recommend patient‐centered approaches to identifying MSbP in the clinical setting to facilitate prevention and early intervention. It also recommends patient‐centered practices that can be implemented to reduce the MSbP‐related morbidity and mortality contributed by the healthcare system. The evolving nomenclature and definition of MSbP abuse has been an obstacle to achieving scientific consensus on the topic. Yet, the body of scientific literature on the subject is large. This literature is reviewed to enumerate the healthcare system\u27s contribution to MSbP abuse. The Haddon matrix, a public health framework, is applied to MSbP abuse in order to guide the development of recommendations of patient‐centered approaches that should be implemented to reduce the healthcare system\u27s contribution to the morbidity and mortality that MSbP victims face

Spoil Type Influences Soil Genesis and Forest Development on an Appalachian Surface Coal Mine Ten Years After Placement

Surface mining for coal (or other mineral resources) is a major driver of land-use change around the world and especially in the Appalachian region of the United States. Intentional and well-informed reclamation of surface-mined land is critical for the restoration of healthy ecosystems on these disturbed sites. In Appalachia, the pre-mining land cover is predominately mixed hardwood forest, with rich species diversity. In recent years, Appalachian mine reforestation has become an issue of concern, prompting the development of the Forestry Reclamation Approach, a series of mine reforestation recommendations. One of these recommendations is to use the best available soil substitute; however, the characteristics of the “best” soil substitute have been an issue. This study was initiated to compare the suitability of several types of mine spoil common in the Appalachian region: brown sandstone (Brown), gray sandstone (Gray), mixed spoils (Mixed), and shale (Shale). Experimental plots were established in 2007 with each spoil type replicated three times. These plots were planted with a mix of native hardwood species. Ten years after plot construction and planting, tree growth and canopy cover were highest in Brown, followed by Shale, Mixed, and Gray. Soil conditions (particularly pH) in Brown and Shale were more favorable for native tree growth than Mixed or Gray, largely explaining these differences in tree growth and canopy cover. However, soil chemistry did not clearly explain differences in tree growth between Brown and Shale. These differences were more likely related to differences in near-surface soil temperature, which is related to soil color and available shade

Evaluating the Use of Tree Shelters for Direct Seeding of \u3cem\u3eCastanea\u3c/em\u3e on a Surface Mine in Appalachia

American chestnut (Castanea dentata), once a primary constituent of the eastern hardwood forest ecosystem, was nearly extirpated from the forest canopy by the accidental introduction of chestnut blight (Cryphonectria parasitica). An intensive breeding program has sought to breed blight resistance from Chinese chestnut into American chestnuts, while maintaining as much of the desirable American chestnut phenotypes as possible. Previous studies suggest that these blight resistant American chestnuts, termed “restoration chestnuts”, are capable of thriving on reclaimed surface mines. We direct seeded pure Chinese, pure American, and three backcross lines into brown sandstone minesoil on a mine site in Pike County, KY. To investigate the effects of tree sheltering on survival and growth, we installed tree shelters on half the plots, and left the rest of the plots unsheltered. Results indicated that shelters were highly effective at reducing initial mortality. In addition, while pure Chinese chestnut survival was highest, the three backcross lines have also survived well on this site. Our study demonstrates that American, Chinese, and backcrossed chestnuts can survive through five growing seasons on reclaimed surface mines with the use of tree shelters

Burden of respiratory viral infection in persons with human immunodeficiency virus

This study was conducted to determine the prevalence of respiratory viral infections (RVI) in persons living with HIV (PLH) admitted with a respiratory complaint using real-time reverse transcription polymerase chain reaction and primer-independent next-generation sequencing (NGS). Of 82 subjects, respiratory viruses were the most common pathogen identified in 27 (33%), followed by fungus and bacteria in 8 (10%) and 4 (5%) subjects, respectively. Among subjects with RVI, 11 (41%) required ICU admission and 16 (59%) required mechanical ventilation. The proportion of respiratory viruses identified, and the associated complicated hospital course highlights the significant role that RVIs play in the lung health of PLH