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61.
Nani Van Gerven Parveen Goyal Guy Vandenbussche Maia De Kerpel Wim Jonckheere Henri De Greve Han Remaut 《Molecular microbiology》2014,91(5):1022-1035
Curli are functional amyloids expressed as fibres on the surface of Enterobacteriaceae. Contrary to the protein misfolding events associated with pathogenic amyloidosis, curli are the result of a dedicated biosynthetic pathway. A specialized transporter in the outer membrane, CsgG, operates in conjunction with the two accessory proteins CsgE and CsgF to secrete curlin subunits to the extracellular surface, where they nucleate into cross‐beta strand fibres. Here we investigate the substrate tolerance of the CsgG transporter and the capability of heterologous sequences to be built into curli fibres. Non‐native polypeptides ranging up to at least 260 residues were exported when fused to the curli subunit CsgA. Secretion efficiency depended on the folding properties of the passenger sequences, with substrates exceeding an approximately 2 nm transverse diameter blocking passage through the transport channel. Secretion of smaller passengers was compatible with prior DsbA‐mediated disulphide bridge formation in the fusion partner, indicating that CsgG is capable of translocating non‐linear polypeptide stretches. Using fusions we further demonstrate the exported or secreted heterologous passenger proteins can attain their native, active fold, establishing curli biogenesis pathway as a platform for the secretion and surface display of small heterologous proteins. 相似文献
62.
Sarcoplasmic reticulum (SR) Ca2+ release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca2+ channel and the intra-SR Ca2+ buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca2+ regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca2+ dependence of open probability (Po) over the physiologically relevant range (0.05-1 mM Ca2+) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca2+ sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca2+ sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca2+ regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca2+ regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca2+ buffer. 相似文献
63.
Maria Pajunen Eini Poussu Hilkka Turakainen Harri Savilahti 《Methods (San Diego, Calif.)》2009,49(3):255
High-precision mapping of regions involved in protein–protein interfaces of interacting protein partners is an essential component on a path to understand various cellular functions. Transposon-based systems, particularly those involving in vitro reactions, offer exhaustive insertion mutant libraries and high-throughput platforms for many types of genetic analyses. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision that is based on transposition-assisted construction, sampling, and analysis of a comprehensive insertion mutant library. The methodology integrates random pentapeptide mutagenesis of proteins, yeast two-hybrid screening, and high-resolution genetic footprinting. This straightforward strategy is general, and it provides a rapid and easy means to identify critical contact regions in proteins without the requirement of prior structural knowledge. 相似文献
64.
Deficiencies in Cu, Se, and Zn impair one or more biochemical functions, and excess are associated with toxicity. Baseline
studies on the Ghanaian population are scanty. The study was undertaken to determine whether significant rural/urban differences
in the serum levels of Cu, Se, and Zn did exist. Forty males/60 females from rural and 50 males/50 females from urban Ghanaian
communities were sampled. Serum Cu, Se, and Zn were determined using flame atomic absorption spectrometry. Cu level for rural
and urban subjects was 997 ± 333 and 979 ± 290 μg/L, respectively (p = 0.68). However, Cu levels were significantly higher in the rural females (1,063 ± 367 μg/L) than the rural males (898 ± 249 μg/L;
p = 0.0085). Se levels for rural/urban subjects were 97 ± 36 and 87 ± 31 μg/L, respectively (p = 0.03). Zn levels in the rural/urban subjects were 312 ± 218 and 150 ± 102 μg/L, respectively (p = 0.002). Additionally, Zn was significantly higher in rural females (428 ± 204 μg/L) than the urban females (166 ± 103 μg/L;
p = 0.0002). Finally, Zn was significantly higher in rural females (428 ± 204 μg/L) than males (172 ± 116 μg/L; p = 0.0028). In conclusion, Cu, Se, and Zn were higher in the rural group compared to the urban group, and the generally low
Zn levels were confirmed in another cohort follow-up study. 相似文献
65.
66.
Changes in climate and in browsing pressure are expected to alter the abundance of tundra shrubs thereby influencing the composition and species richness of plant communities. We investigated the associations between browsing, tundra shrub canopies and their understory vegetation by utilizing a long‐term (10–13 seasons) experiment controlling reindeer and ptarmigan herbivory in the subarctic forest tundra ecotone in northwestern Fennoscandia. In this area, there has also been a consistent increase in the yearly thermal sum and precipitation during the study period. The cover of shrubs increased 2.8–7.8 fold in exclosures and these contrasted with browsed control areas creating a sharp gradient of canopy cover of tundra shrubs across a variety of vegetation types. Browsing exclusions caused significant shifts in more productive vegetation types, whereas little or no shift occurred in low‐productive tundra communities. The increased deciduous shrub cover was associated with significant losses of understory plant species and shifts in functional composition, the latter being clearest in the most productive plant community types. The total cover of understory vegetation decreased along with increasing shrub cover, while the cover of litter showed the opposite response. The cover of cryptogams decreased along with increasing shrub cover, while the cover of forbs was favoured by a shrub cover. Increasing shrub cover decreased species richness of understory vegetation, which was mainly due to the decrease in the cryptogam species. The effects were consistent across different types of forest tundra vegetation indicating that shrub increase may have broad impacts on arctic vegetation diversity. Deciduous shrub cover is strongly regulated by reindeer browsing pressure and altered browsing pressure may result in a profound shrub expansion over the next one or two decades. Results suggest that the impact of an increase in shrubs on tundra plant richness is strong and browsing pressure effectively counteracts the effects of climate warming‐driven shrub expansion and hence maintains species richness. 相似文献
67.
A. Dorn M. Müller H. -G. Bernstein A. Pajunen M. Järvinen 《The Histochemical journal》1985,17(6):731-733
Summary Ornithine decarboxylase was localized immunohistochemically in paraffin sections of prostate glands of the rat using Sternberger's PAP technique. Two types of immunoreactive cells were detected in the gland epithelium: strongly positive ones and weakly positive ones. Unreactive cells were also present. The functional role of the enzyme in the secretory process of the glands is discussed in the light of these findings. 相似文献
68.
69.
Jaepyeong Cha Roger R. Nani Michael P. Luciano Gabriel Kline Aline Broch Kihoon Kim Jung-Man Namgoong Rhushikesh A. Kulkarni Jordan L. Meier Peter Kim Martin J. Schnermann 《Bioorganic & medicinal chemistry letters》2018,28(16):2741-2745
Surgical methods guided by exogenous fluorescent markers have the potential to define tissue types in real time. Small molecule dyes with efficient and selective renal clearance could enable visualization of the ureter during surgical procedures involving the abdomen and pelvis. These studies report the design and synthesis of a water soluble, net neutral C4′-O-alkyl heptamethine cyanine, Ureter-Label (UL)-766, with excellent properties for ureter visualization. This compound is accessed through a concise synthetic sequence involving an N- to O-transposition reaction that provides other inaccessible C4′-O-alkyl heptamethine cyanines. Unlike molecules containing a C4′-O-aryl substituent, which have also been used for ureter visualization, UL-766 is not reactive towards glutathione and the cellular proteome. In addition, rat models of abdominal surgery reveal that UL-766 undergoes efficient and nearly exclusive renal clearance in vivo. In total, this molecule represents a promising candidate for visualizing the ureter during a variety of surgical interventions. 相似文献
70.
Bovine enterotoxigenic Escherichia coli (ETEC) carrying F17a fimbriae attach to the intestinal epithelium by means of the F17a-G adhesin. Since filamentous bacteriophages
can be employed for the display of foreign peptides, we tested the applicability of this system to F17a-G. The receptor-binding
domain of the F17a-G adhesin was expressed on bacteriophage M13, as an amino-terminal fusion with the phage protein pIII.
This domain retained its N-acetyl-beta-d-glucosamine binding activity. The phage presenting the fimbrial receptor-binding domain elicited an IgG response against
F17a-G after intraperitoneal immunisation of mice. 相似文献