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111.
Natochin M  Barren B  Artemyev NO 《Biochemistry》2006,45(20):6488-6494
Mutations counterpart to dominant negative RasSer17Asn in the alpha-subunits of heterotrimeric G-proteins are known to also produce dominant negative effects. The mechanism of these mutations remains poorly understood. Here, we examined the effects and mechanism of the Ser43Cys and Ser43Asn mutants of transducin-like chimeric Gtalpha* in the visual signaling system. Our analysis showed that both mutants have reduced affinity for GDP and are likely to exist in an empty-or partially occupied-pocket state. S43C and S43N retained the ability to interact with Gtbetagamma and, as heterotrimeric proteins, bind to photoexcited rhodopsin (R*). The interaction with R* is unproductive as the mutants failed to bind GTPgammaS and become activated. S43C and S43N inhibited R*-dependent activation of Gtalpha* and Gtalpha, apparently by blocking R*. Finally, both Gtalpha* mutants lacked interaction with the gamma-subunit of PDE6, an effector protein in phototransduction. These results indicate that the S43C and S43N mutants of Gtalpha* are dominant negative inhibitors that bind and block the activated receptor in a mechanism that parallels that of RasSer17Asn. Dominant negative mutants of Gtalpha sequestering R*, such as S43C and S43N, may become useful instruments in probing the mechanisms of visual dysfunctions caused by abnormal phototransduction signaling.  相似文献   
112.
Methods development in chromatographic purification processes is a complex operation and has traditionally relied on trial and error approaches. The availability of a large number of commercial media, choice of different modes of chromatography, and diverse operating conditions contribute to the challenging task of accelerating methods development. In this paper, we describe a novel microtiter-plate based screening method to identify the appropriate sequence of chromatographic steps that result in high purities of bioproducts from their respective culture broths. Protein mixtures containing the bioproduct were loaded on aliquots of different chromatographic media in microtiter plates. Serial step elution of the proteins, in concert with bioproduct-specific assays, resulted in the identification of "active fractions" containing the bioproduct. The identification of a successful chromatographic step was based on the purity of the active fractions, which were then pooled and used as starting material for screening the next chromatographic dimension. This procedure was repeated across subsequent dimensions until single band purities of the protein were obtained. The sequence of chromatographic steps and the corresponding operating conditions identified from the screen were validated under scaled-up conditions. Various modes of chromatography including hydrophobic interaction, ion exchange (cation and anion exchange) and hydrophobic charge-induction chromatography (HCIC), and different operating conditions (pH, salt concentration and type, etc.) were employed in the screen. This approach was employed to determine the sequence of chromatographic steps for the purification of recombinant alpha-amylase from its cell-free culture broth. Recommendations from the screen resulted in single-band purity of the protein under scaled-up conditions. Similar results were observed for an scFv-beta-lactamase fusion protein. The use of a miniaturized screen enables the parallel screening of a wide variety of actual bioprocess media and conditions and represents a novel paradigm approach for the high-throughput process development of recombinant proteins.  相似文献   
113.
The anti-adhesive effects of cranberry have been attributed to both interactions of its components with the surface of bacterial cells and to inhibition of p-fimbriae expression. Previous reports also suggested that the presence of cranberry juice changed the Gram stain characteristics of Escherichia coli. Here, we show that the morphology of E. coli is changed when grown in the presence of juice or extract from Vaccinium macrocarpon (cranberry). Gene expression analysis indicates the down regulation of flagellar basal body rod and motor proteins. Consistent with this finding and previous reports, the SEM images indicate a decrease in the visible p-fimbriae. The iodine used in Gram-staining protocols was found to interact differently with the bacterial membrane when cells were cultured in spiked media. Slight alterations in the Gram stain protocol demonstrated that culturing in the presence of cranberry juice does not change the Gram stain characteristics contradicting other reports.  相似文献   
114.
N-Substituted porphyrins are well-known for the distortion they exhibit of the porphyrin plane through the sp(3) hybridization of one of the pyrrolenic units. They have served as model compounds in investigations of many biochemical processes. In this paper, we developed an efficient route to N-substituted porphyrins, and report the synthesis of a series of new N-substituted cobaltacarborane-porphyrins containing one or two cobaltabisdicarbollide anions linked by (CH(2)CH(2)O)(2) chains to either the core porphyrin nitrogens or to a meso-aminophenyl group. These conjugates show different degrees of distortion of the porphyrin macrocycle, which affect their spectroscopic and electrochemical properties. In particular, the core N-substituted conjugates show significant fluorescence quenching in comparison with the noncore substituted macrocycles. The X-ray structures of two targeted core N-cobaltacarborane porphyrin conjugates are presented. The electrochemical and spectroelectrochemical properties of these porphyrin conjugates were investigated; while the peripheral N-substituted cobaltacarboranylporphyrins undergo three reversible reductions and three reversible oxidations (two attributed to the porphyrin and one to the Co(III) cluster), the core N-substituted porphyrins exhibit complicated electrochemical behavior with coupled chemical reactions.  相似文献   
115.
116.
Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.  相似文献   
117.

Background

Human infection with Bwamba virus (BWAV) and the closely related Pongola virus (PGAV), as well as Nyando virus (NDV), are important causes of febrile illness in Africa. However, despite seroprevalence studies that indicate high rates of infection in many countries, these viruses remain relatively unknown and unstudied. In addition, a number of unclassified bunyaviruses have been isolated over the years often with uncertain relationships to human disease.

Methodology/Principal Findings

In order to better understand the genetic and evolutionary relationships among orthobunyaviruses associated with human disease, we have sequenced the complete genomes for all 3 segments of multiple strains of BWAV (n = 2), PGAV (n = 2) and NDV (n = 4), as well as the previously unclassified Mojuí dos Campos (MDCV) and Kaeng Khoi viruses (KKV). Based on phylogenetic analysis, we show that these viruses populate 2 distinct branches, one made up of BWAV and PGAV and the other composed of NDV, MDCV and KKV. Interestingly, the NDV strains analyzed form two distinct clades which differed by >10% on the amino acid level across all protein products. In addition, the assignment of two bat-associated bunyaviruses into the NDV group, which is clearly associated with mosquito-borne infection, led us to analyze the ability of these different viruses to grow in bat (RE05 and Tb 1 Lu) and mosquito (C6/36) cell lines, and indeed all the viruses tested were capable of efficient growth in these cell types.

Conclusions/Significance

On the basis of our analyses, it is proposed to reclassify the NDV strains ERET147 and YM176-66 as a new virus species. Further, our analysis definitively identifies the previously unclassified bunyaviruses MDCV and KKV as distinct species within the NDV group and suggests that these viruses may have a broader host range than is currently appreciated.  相似文献   
118.
119.
A high utility integrated map of the pig genome   总被引:2,自引:1,他引:1  

Background

The domestic pig is being increasingly exploited as a system for modeling human disease. It also has substantial economic importance for meat-based protein production. Physical clone maps have underpinned large-scale genomic sequencing and enabled focused cloning efforts for many genomes. Comparative genetic maps indicate that there is more structural similarity between pig and human than, for example, mouse and human, and we have used this close relationship between human and pig as a way of facilitating map construction.

Results

Here we report the construction of the most highly continuous bacterial artificial chromosome (BAC) map of any mammalian genome, for the pig (Sus scrofa domestica) genome. The map provides a template for the generation and assembly of high-quality anchored sequence across the genome. The physical map integrates previous landmark maps with restriction fingerprints and BAC end sequences from over 260,000 BACs derived from 4 BAC libraries and takes advantage of alignments to the human genome to improve the continuity and local ordering of the clone contigs. We estimate that over 98% of the euchromatin of the 18 pig autosomes and the X chromosome along with localized coverage on Y is represented in 172 contigs, with chromosome 13 (218 Mb) represented by a single contig. The map is accessible through pre-Ensembl, where links to marker and sequence data can be found.

Conclusion

The map will enable immediate electronic positional cloning of genes, benefiting the pig research community and further facilitating use of the pig as an alternative animal model for human disease. The clone map and BAC end sequence data can also help to support the assembly of maps and genome sequences of other artiodactyls.  相似文献   
120.
Epidemiological evidence suggests that long term treatment with hydroxymethylglutaryl-CoA reductase inhibitors, or statins, decreases the risk for developing Alzheimer disease (AD). However, statin-mediated AD protection cannot be fully explained by reduction of cholesterol levels. In addition to their cholesterol lowering effects, statins have pleiotropic actions and act to lower the concentrations of isoprenoid intermediates, such as geranylgeranyl pyrophosphate and farnesyl pyrophosphate. The Rho and Rab family small G-proteins require addition of these isoprenyl moieties at their C termini for normal GTPase function. In neuroblastoma cell lines, treatment with statins inhibits the membrane localization of Rho and Rab proteins at statin doses as low as 200 nm, without affecting cellular cholesterol levels. In addition, we show for the first time that at low, physiologically relevant, doses statins preferentially inhibit the isoprenylation of a subset of GTPases. The amyloid precursor protein (APP) is proteolytically cleaved to generate beta-amyloid (Abeta), which is the major component of senile plaques found in AD. We show that inhibition of protein isoprenylation by statins causes the accumulation of APP within the cell through inhibition of Rab family proteins involved in vesicular trafficking. Moreover, inhibition of Rho family protein function reduces levels of APP C-terminal fragments due to enhanced lysosomal dependent degradation. Statin inhibition of protein isoprenylation results in decreased Abeta secretion. In summary, we show that statins selectively inhibit GTPase isoprenylation at clinically relevant doses, leading to reduced Abeta production in an isoprenoid-dependent manner. These studies provide insight into the mechanisms by which statins may reduce AD pathogenesis.  相似文献   
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