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941.
Moreland D. Gibbs Rosalind A. Reeves David Mandelman Qingli Mi Jun Lee Peter L. Bergquist 《Extremophiles : life under extreme conditions》2009,13(5):817-826
Thermus aquaticus DNA polymerase (Taq polymerase) made the polymerase chain reaction feasible and led to a paradigm shift in genomic analysis. Other Thermus polymerases were reported to have comparable performance in PCR and there was an analysis of their properties in the 1990s.
We re-evaluated our earlier phylogeny of Thermus species on the basis of 16S rDNA sequences and concluded that the genus could be divided into eight clades. We examined 22
representative isolates and isolated their DNA polymerase I genes. The eight most diverse polymerase genes were selected to
represent the eight clades and cloned into an expression vector coding for a His-tag. Six of the eight polymerases were expressed
so that there was sufficient protein for purification. The proteins were purified to homogeneity and examination of the biochemical
characteristics showed that although they were competent to perform PCR, none was as thermostable as commercially available
Taq polymerase; all had similar error-frequencies to Taq polymerase and all showed the expected 5′–3′ exonuclease activity. We conclude that the initial selection of T. aquaticus for DNA polymerase purification was a far-reaching and fortuitous choice but simple mutagenesis procedures on other Thermus-derived polymerases should provide comparable thermostability for the PCR reaction. 相似文献
942.
Stojałowski S Myśków B Milczarski P Masojć P 《Cellular & molecular biology letters》2009,14(2):190-198
Four F2 mapping populations derived from crosses between rye inbred lines DS2×RXL10, 541×Ot1-3, S120×S76 and 544×Ot0-20 were used
to develop a consensus map of chromosome 6R. Thirteen marker loci that were polymorphic in more than one mapping population
constituted the basis for the alignment of the four maps using the JoinMap v. 3.0 software package. The consensus map consists
of 104 molecular marker loci including RFLPs, RAPDs, AFLPs, SSRs, ISSRs, SCARs, STSs and isozymes. The average distance between
the marker loci is 1.3 cM, and the total map length is 135.5 cM. This consensus map may be used as a source of molecular markers
for the rapid development of new maps of chromosome 6R in any mapping population. 相似文献
943.
Olga Wesołowska Andrzej B. Hendrich Barbara Łania-Pietrzak Jerzy Wiśniewski Joseph Molnar Imre Ocsovszki Krystyna Michalak 《Cellular & molecular biology letters》2009,14(2):199-221
The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant
phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane
perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and
flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1
fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity
of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry
and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones
and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their
potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible
for inhibition of MRP1 by the flavonoids. 相似文献
944.
945.
Bong-Hyun Jun Mi Suk Noh Homan Kang Woo-Jae Chung Min-Soo Kim Myung-Haing Cho Dae Hong Jeong Yoon-Sik Lee 《Analytical biochemistry》2009,391(1):24-286
This article presents a prototype of a surface-enhanced Raman spectroscopy (SERS)-encoded magnetic bead of 8 μm diameter. The core part of the bead is composed of a magnetic nanoparticle (NP)-embedded sulfonated polystyrene bead. The outer part of the bead is embedded with Ag NPs on which labeling molecules generating specific SERS bands are adsorbed. A silica shell is fabricated for further bioconjugation and protection of SERS signaling. Benzenethiol, 4-mercaptotoluene, 2-naphthalenethiol, and 4-aminothiophenol are used as labeling molecules. The magnetic SERS beads are used as substrates for protein sensing and screening with easy handling. As a model application, streptavidin-bound magnetic SERS beads are used to illustrate selective separation in a flow cytometry system, and the screened beads are spectrally recognized by Raman spectroscopy. The proposed magnetic SERS beads are likely to be used as a versatile solid support for protein sensing and screening in multiple assay technology. 相似文献
946.
Marian Adamkov L’udovít Lauko Július Raj?áni Soňa Bálentová Silvia Rybárová Du?an Mi?tuna Dagmar Statelová 《Biologia》2009,64(4):840-844
We examined the expression of potential tumor marker survivin by immunohistochemical staining using antisurvivin antibody
(DAKO, Clone 12C4) in a panel of 25 malignant melanomas. In each section, we assessed the percentage of positively stained
tumor cells, the intensity of staining and its subcellular localization. Survivin was present in 23 out of 25 cases (92%).
Nuclear staining was found in 2 of these 23 cases (8.7%) only, while cytoplasmic staining only was seen in 3 of them (13%).
The combined nuclear as well as cytoplasmic localization of survivin was demonstrated in 18 out of 23 cases (78.3%). In 2
cases revealing nuclear staining only, the worse histological features were more pronounced than in 3 cases with cytoplasmic
staining only. Our results suggest that nuclear positivity of survivin may correlate with the degree of malignancy. In addition,
we conclude that overexpression of survivin involved in the pathogenesis of melanoma represents an important diagnostic marker. 相似文献
947.
948.
The effect is described of selenium supplemented in an inorganic and organic form on the innate immune response of goats.
Though the phagocytic activity (as a marker of the immune function) was found to be lower in organic-Se-treated group than
in control (54.5 ± 4.32 vs. 60.2 ± 9.15 %), it did not generally exhibit any significant differences; similarly, no differences were found in the phagocytic
index. The production of reactive oxygen species (ROS) was determined using the luminol-enhanced chemiluminescence (CL) (estimated
as peak CL, integral CL and a peak time after addition of calcium ionophore A23187, opsonised zymosan (OZP) and phorbol-12-myristate-13-acetate
as effectors. A significant ROS increase reflected in integral CL and a peak time was found in the inorganic-Se-treated group
when OZP was used as activator; other parameters did not exhibit significant changes. The supplementation of Se in inorganic
form can thus be seen to influence positively the innate immune system of kids. 相似文献
949.
950.
Jung‐Il Chae Janghwan Kim Sun‐Mi Woo Hyo‐Won Han Young Keun Cho Keon‐Bong Oh Ki‐Hoan Nam Yong‐Kook Kang 《Proteomics》2009,9(5):1128-1141
The ability to generate neural lineages from human embryonic stem cells (hESCs) in a controlled manner would further investigation of human neurogenesis and development of potential cell therapeutic applications to treat neurological diseases; however, generating such neural stem cells (NSCs) remains a challenge. In an attempt to characterize the cellular mechanisms involved in hESC differentiation into neuroprogenitor cells, we performed 2‐DE using protein extracts from hESC‐derived embryoid bodies (EBs) and neuroectodermal spheres (NESs) bearing neuroprogenitors. Of 47 differentially expressed protein spots, 28 nonredundant spots were shown to be upregulated in the NESs; these protein spots included neurogenesis‐related proteins (TAF1, SEPT2, NPH3, and CRABP), as expected. Interestingly, 6 of these 28 protein spots were cytoskeleton‐associated proteins (CSAP) such as Fascin‐1, Cofilin‐1, and Stathmin‐1. Western‐blot analyses confirmed the increased levels of these proteins in the NESs. Furthermore, immunostaining analysis showed that both Fascin‐1 and Stathmin‐1 were preferentially expressed in the inner rims of neural rosettes, which are characteristic features of neuroprogenitors in culture. We also confirmed prominent expression of Fascin‐1 in (sub‐)ventricular zone in E15.5 mouse fetal brain. Our results suggest that, in addition to the induction of those genes involved in neural development, hESC differentiation into the NES is associated with a marked reorganization of the cellular cytoskeleton. 相似文献