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61.
Kozarov E Sweier D Shelburne C Progulske-Fox A Lopatin D 《Microbes and infection / Institut Pasteur》2006,8(3):687-693
This is the first study to analyze atheromatous plaques for the presence of bacterial DNA from ten species, including periodontal species and Chlamydia pneumoniae. We examined 129 samples of DNA extracted from atheromas from 29 individuals for the presence of bacterial 16S rDNA sequences from ten different species: Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans (A.a.), Tannerella forsythensis, Eikenella corrodens, Prevotella intermedia, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Treponema denticola and C. pneumoniae. All determinations were made using real-time quantitative polymerase chain reaction (PCR) methods employing SYBR Green. Species from the Bacteroides family were found in about 17% of the young but approximately 80% in elderly patients. Almost half of the samples contained DNA from A. a. and C. pneumoniae, although the proportion of the latter was minimal. S. aureus and S. epidermidis were found with the lowest frequency, 5 and 10%, respectively. S. mutans was found in approximately 20% of the samples. The proportions of each bacterial species were calculated relative to the total amount of prokaryotic DNA. The data support our previous findings of an association between periodontal organisms and vascular inflammation. We conclude that DNA from oral infectious agents is commonly found in atheromas from young but especially from elderly subjects, and that the contribution of C. pneumoniae to the inflammation may be minimal. 相似文献
62.
Domenica R. Massardo Grazia M. Borrelli Natale Di Fonzo Pietro Alifano Luigi Del Giudice 《Biotechnology letters》2002,24(14):1199-1202
An efficient method has been developed to improve preparation of phage particles by ammonium sulfate precipitation and to yield high quality DNA. The method, that has been used to screen plant DNA libraries constructed in vectors, is inexpensive, does not require purification of phage particles, and can be used from either plate stocks or liquid lysates. Up to 1100 g DNA was produced from 5 ml lysate obtained from agar plates. 相似文献
63.
We constructed two aptamers, each of which contains a 7-nt-long loop complementary to the anticodon loop of a suppressor tRNA. One of these aptamers can form a stable bimolecular complex with the suppressor tRNA in vitro and protects the 7 nt in the suppressor's anticodon loop from RNase S1. An Escherichia coli strain, carrying an amber mutation in the lac Z gene, produces beta-galactosidase only if the suppressor is present; the aptamer's coexpression in the cell inhibits the activity of the suppressor tRNA. Moreover, in E. coli extract, the aptamer partially inhibits the read-through of the stop codon on the part of the suppressor tRNA. These results point to a novel strategy that need not be limited to the suppressor tRNA. By constructing appropriate inducible aptamers, it may well be possible to effectively control translation in vivo. 相似文献
64.
In order to find new ways for the functionalization of the A and B rings of the steroid nucleus, the reaction of 5alpha-androst-2-en-17beta-ol 17-acetate (1), cholesta-2,4-diene (4) and cholesta-4,6-dien-3beta-ol 3-acetate (7) was examined using stoichiometric amounts of ruthenium tetraoxide to yield 1,2-cis diols and/or alpha-hydroxy ketones. The reaction of 5alpha-cholest-2-en-3-ol 3-acetate (9) with ruthenium tetraoxide was also carried out and afforded, apart from an alpha-hydroxy ketone, also a diketone and a seco-dicarboxylic acid. The structures of all new steroids, including stereochemical details, were deduced by analysis of spectral data. 相似文献
65.
Shiga toxin 1 (Stx1) catalyses the removal of a specific adenine from 28S rRNA within ribosomes (RNA-N-glycosylase activity) and the removal of multiple adenines from DNA (DNA-glycosylase activity). For the in vitro activity the toxin requires activation by trypsin, urea and DTT which releases the enzymatically active A1 fragment. We show that activated Stx1 acts on DNA as a heat-stable enzyme. Moreover, heat-treatment of the pro-enzyme at acidic pH turns it into an enzymatically active species which efficiently depurinates DNA. Although the effect of this treatment is centred on the enzyme and not on DNA, we found no evidence for covalent modification of the holotoxin. We suggest that high temperatures and acidic buffer induce unfolding of the holotoxin allowing the substrate to gain access to the active site. Possible practical applications (rapid assay for Stx1 detection, use of the toxin for DNA sequencing) are discussed. 相似文献
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69.
Jousson O Pretti C Di Bello D Cognetti-Varriale AM 《Diseases of aquatic organisms》2005,65(3):251-255
The main parasitic threat to freshwater fish is the ciliate Ichthyophthirius multifiliis. We developed a real-time PCR assay using SYBR Green intercalating fluorescent dye for rapid detection and quantification of I. multifiliis. This non-invasive assay was based on the quantification of I. multifiliis free-swimming stages from filtered water samples, and thus made it possible to preserve host individuals. An alignment of 18S rDNA sequences of I. multifiliis and related species of the ciliate order Hymenostomatida was used to design amplification primers specifically targeting the I. multifiliis 18S rDNA gene. Different standard curves consisting of 2-fold serial dilutions of DNA extracted from 20, 60, 100 and 1000 I. multifiliis cells were constructed. The assay was able to detect less than 0.5 cell equivalent and showed a strong linearity (R2 = 0.984). Water samples were collected from 2 tanks containing heavily infected and apparently uninfected Carassius auratus specimens and were used to test this technique. Positive signals were obtained from water samples collected from both tanks, with a deduced concentration ranging from 3 to 58 I. multifiliis cells l(-1). The assay can detect low concentrations of the parasite in water, presumably corresponding to an early phase of the disease. It may, thus, be a valuable tool in assisting in the monitoring and control of ichthyophthiriasis in aquaculture. 相似文献
70.
Michael B. Sherman Juan Trujillo Ian Leahy Dennis Razmus Robert DeHate Paul Lorcheim Mark A. Czarneski Domenica Zimmerman Je T’Aime M. Newton Andrew D. Haddow Scott C. Weaver 《Journal of structural biology》2013,181(3):223-233
A unique cryo-electron microscopy facility has been designed and constructed at the University of Texas Medical Branch (UTMB) to study the three-dimensional organization of viruses and bacteria classified as select agents at biological safety level (BSL)-3, and their interactions with host cells. A 200 keV high-end cryo-electron microscope was installed inside a BSL-3 containment laboratory and standard operating procedures were developed and implemented to ensure its safe and efficient operation. We also developed a new microscope decontamination protocol based on chlorine dioxide gas with a continuous flow system, which allowed us to expand the facility capabilities to study bacterial agents including spore-forming species. The new unified protocol does not require agent-specific treatment in contrast to the previously used heat decontamination. To optimize the use of the cryo-electron microscope and to improve safety conditions, it can be remotely controlled from a room outside of containment, or through a computer network world-wide. Automated data collection is provided by using JADAS (single particle imaging) and SerialEM (tomography). The facility has successfully operated for more than a year without an incident and was certified as a select agent facility by the Centers for Disease Control. 相似文献