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871.
A genome-wide strategy for the identification of essential genes in Staphylococcus aureus 总被引:1,自引:0,他引:1
Forsyth RA Haselbeck RJ Ohlsen KL Yamamoto RT Xu H Trawick JD Wall D Wang L Brown-Driver V Froelich JM C KG King P McCarthy M Malone C Misiner B Robbins D Tan Z Zhu Zy ZY Carr G Mosca DA Zamudio C Foulkes JG Zyskind JW 《Molecular microbiology》2002,43(6):1387-1400
To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics. 相似文献
872.
A near infrared spectroscopic method was developed to determine drug content in a 20% (wt/wt) ibuprofen and spray-dried hydous
lactose blend. A blending profile was obtained after blending for 0.5, 1, 3, 5, 10, and 20 minutes. Stream sampling was used
to collect about 20 blend samples at each of the blending times from a laboratory scale V-blender. The samples collected were
used to develop a near infrared calibration model. The calibration model was then used to determine the drug content of unknown
samples from 2 validation blends. The validation blends were not included in the calibration model; they were used to evaluate
the effectiveness of the calibration model. A total of 45 samples from the 2 validation blends were predicted by the near
infrared calibration model and then analyzed by a validated UV spectrophotometric method. The root mean square error of prediction
for the first validation blend was 5.69 mg/g and 3.30 mg/g for the samples from the second blend. A paired t test at the 95%
confidence level did not indicate any differences between the drug content predicted by the near infrared spectroscopy (NIRS)
method and the validated UV method for the 2 blends. The results show that the NIRS method could be developed while the blending
profile is generated and used to thoroughly characterize a new formulation during development by analyzing a large number
of samples. The new formulation could be transferred to a manufacturing plant with an NIRS method to facilitate blend uniformity
analysis. 相似文献
873.
The electrophoresis of circular DNA, ranging in size from 4.4 kilobase pairs (kbp) to 220 kbp, was studied in agarose gels. Bacterial artificial chromosome (BAC) DNA was used as a source of large supercoiled and open circular (relaxed) forms. The open circles above approximately 50 kbp were trapped at the sample wells of 1% agarose gels during electrophoresis at 3 V/cm. Field inversion gel electrophoresis (FIGE) was used to relieve the trapping of the open circles in the gels. Using FIGE (30 s forward pulse time), open circles with sizes of 115 and 220 kbp required reverse pulse times of 3 and 6 s, respectively, to free the circles from open-ended gel fibers. A minimum in the gel velocity of the open circles was measured at approximately 20 kbp. Open circles below approximately 20 kbp migrated slower than the supercoiled forms, and above 20 kbp the order was reversed. These results indicate that when the size of the open circles exceeded the average pore size of a gel and it was forced to span multiple pores, the open circles gained a mobility advantage. Decreasing the ionic strength of the electrophoresis buffer significantly decreased the mobility of the smaller circles and slightly increased the mobility of the larger circles. 相似文献
874.
Navas CA 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2002,133(3):469-485
A well-defined macroecological pattern is the decline in biodiversity with altitude. However, this decline is taxa-specific. For example, amphibians are more diverse than squamates at extreme elevations in the tropical Andes, but this pattern is reversed at extreme elevations in the southern latitudes. Several ecophysiological and evolutionary factors may be related to this difference. At high-elevations in southern latitudes temperature differs dramatically among seasons and dry soils dominate, characteristics that appear to favor lizard physiological ecology. Tropical high altitudes, in contrast, are humid and offer abundant and diverse water resources. These characteristics allow for a richer anuran community but might complicate lizard egg development through temperature and oxygen constrains. Differences in strategies of thermal adaptation might also modulate diversity patterns. The thermal physiology of anurans is extremely labile so that behavioral and physiological performance is maintained despite an altitudinal decrease in field body temperature. Lizards, in contrast, exhibit a conservative thermal physiology and rely on behavioral thermoregulation to face cold and variable temperatures. Both, lizard behavioral strategies and anuran physiological adjustments seem equally efficient in allowing ecological success and diversification for both groups in the tropics up to approximately 3000 m. At higher elevations physiological thermal adaptation is required, and lizards are ecologically constrained, perhaps at various ontogenetic stages. Patterns of biodiversity along environmental clines can be better understood through a physiological approach, and can help to refine and propose hypotheses in evolutionary physiology. 相似文献
875.
Pieri Pde C Pereira DH Glina S Hallak J McElreavey K Moreira-Filho CA 《Genetic testing》2002,6(3):185-194
PCR-based screening of microdeletions in the azoospermic factor (AZF) on the Yq chromosome is an accepted means of identifying a common genetic cause of male infertility, responsible for 5-15% of cases associated with a low sperm count (=5 x 10(6) sptz/ml). Based on an extensive analysis of the literature, we have established a cost-effective preliminary PCR-based diagnostic screening test, with a set of six pairs of primers ("set-of-6") that have the capability of detecting up to 95% of the Y microdeletion cases already published. These primers are: sY84 in AZFa, sY114, sY129, sY143 in AZFb, and sY149, sY254 in AZFc. Initially, the set-of-6 was tested with 13 other pairs of primers covering the three AZF subregions. A sample of 114 infertile men was tested and 10 (8.8%) microdeletions were found, 3 of which were among the 26 (11.5%) idiopathic azoospermic men. These results showed that all detected microdeletions would be identified using the set-of-6 only. Another sample of 34 patients was subsequently tested using the set-of-6 and 3 (8.8%) microdeletions were found in this group. A comparison of our results with those reported in the literature showed similar microdeletion detection frequencies, demonstrating that the set-of-6 primers provides a reliable, simple and cost-effective way of detecting AZF deletions. 相似文献
876.
Generation of histocompatible tissues using nuclear transplantation 总被引:27,自引:0,他引:27
877.
Kumar V Carlson JE Ohgi KA Edwards TA Rose DW Escalante CR Rosenfeld MG Aggarwal AK 《Molecular cell》2002,9(4):857-869
The Lcd1p/Mec1p complex is crucial for normal S phase progression and for signaling DNA damage. We show that Lcd1p/Ddc2p and Mec1p in cell extracts bind to DNA ends. Although Lcd1p binds DNA independently of Mec1p, recruitment of Mec1p to DNA requires Lcd1p. DNA binding by Lcd1p is also independent of Rad9p, Rad17p, and Rad24p. Recombinant Lcd1p binds DNA, and this is impaired by Lcd1p mutations that abrogate its in vivo functions. Furthermore, Mec1p is recruited to cdc13-induced DNA damage and HO endonuclease-induced double-strand breaks in vivo. This requires Lcd1p, and recruitment of Lcd1p/Mec1p to cdc13-induced damage is abolished by Lcd1p mutations that abrogate its in vivo functions. Recruitment of Lcd1p to these lesions is independent of Mec1p and Rad9p/Rad24p. Thus, recruitment of Mec1p to DNA lesions by Lcd1p is crucial for the DNA damage response. 相似文献
878.
Population structure in the endangered Blanca Cacereña bovine breed demonstrated by RAPD analyses 总被引:1,自引:0,他引:1
Parejo JC Padilla JA Rabasco A Sansinforiano ME Martinez-Trancón M 《Genes & genetic systems》2002,77(1):51-58
RAPD analyses have been used to determine the genetic diversity and the population structure of the endangered Blanca Cacere?a bovine breed. Genetic variability was evaluated on the basis of 1048 loci produced by 71 primers. RAPD produced a number of polymorphic loci (30.44%), and it has been proved to be a useful method for evaluating polymorphisms in this breed. The dendrograms based on simililarity indexes and on Nei's genetic distances between 60 animals and the value of genetic differentiation among subpopulations (F(ST)) showed a clear population substructure defined by herds and a scarce genetic flow among herds. Analysis of molecular variance (AMOVA) showed that 32.4% of the total variance was due to differences among herds and confirmed the clustering found. The results of the present study allow us to plan more adequate mating in order to maintain the genetic diversity and to improve the efficiency of conservation for the Blanca Cacere?a bovine breed. 相似文献
879.
Cordova CA Siqueira IR Netto CA Yunes RA Volpato AM Cechinel Filho V Curi-Pedrosa R Creczynski-Pasa TB 《Redox report : communications in free radical research》2002,7(2):95-102
Calendula officinalis (marigold) has many pharmacological properties. It is used for the treatment of skin disorders, pain and also as a bactericide, antiseptic and anti-inflammatory. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to participate in the pathogenesis of various human diseases and may be involved in the conditions which C. officinalis is used to treat. The aim of this study was to investigate the relationship between the beneficial properties of this plant and its antioxidant action. The butanolic fraction (BF) was studied because it is non-cytotoxic and is rich in a variety of bioactive metabolites including flavonoids and terpenoids. Superoxide radicals (O(2)(*-)) and hydroxyl radicals (HO(*)) are observed in decreasing concentrations in the presence of increasing concentrations of BF with IC(50) values of 1.0 +/- 0.09 mg/ml and 0.5 +/- 0.02 mg/ml, respectively, suggesting a possible free radical scavenging effect. Lipid peroxidation in liver microsomes induced by Fe(2+)/ascorbate was 100% inhibited by 0.5 mg/ml of BF (IC(50) = 0.15 mg/ml). Its total reactive antioxidant potential (TRAP) (in microM Trolox equivalents) was 368.14 +/- 23.03 and its total antioxidant reactivity (TAR) was calculated to be 249.19 +/- 14.5 microM. The results obtained suggest that the butanolic fraction of C. officinalis possesses a significant free radical scavenging and antioxidant activity and that the proposed therapeutic efficacy of this plant could be due, in part, to these properties. 相似文献
880.
Fradera X De La Cruz X Silva CH Gelpí JL Luque FJ Orozco M 《Bioinformatics (Oxford, England)》2002,18(7):939-948
Classical molecular interaction potentials, in conjunction with other theoretical techniques, are used to analyze the dependence of the binding sites of representative proteins on the bound ligand. It is found that the ligand bound introduces in general small structural perturbations at the binding site of the protein. However, such small structural changes can lead to important alterations in the recognition pattern of the protein. The impact of these findings in docking procedures is discussed. 相似文献