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231.
Therese S. Høiem Maria K. Andersen Marta Martin-Lorenzo Rémi Longuespée Britt S.R. Claes Anna Nordborg Frédéric Dewez Benjamin Balluff Marco Giampà Animesh Sharma Lars Hagen Ron M.A. Heeren Tone F. Bathen Guro F. Giskeødegård Sebastian Krossa May-Britt Tessem 《Proteomics》2022,22(10):2100223
MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H2O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 μg/mm2. Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types. 相似文献
232.
Richard Wetherbee Vanessa Rossetto Marcelino Joana F. Costa Brenna Grant Simon Crawford Ross F. Waller Robert A. Andersen Drew Berry Geoffrey I. McFadden Heroen Verbruggen 《Journal of phycology》2019,55(6):1210-1225
Prasinophytes (Chlorophyta) are a diverse, paraphyletic group of planktonic microalgae for which benthic species are largely unknown. Here, we report a sand‐dwelling, marine prasinophyte with several novel features observed in clonal cultures established from numerous locations around Australia. The new genus and species, which we name Microrhizoidea pickettheapsiorum (Mamiellophyceae), alternates between a benthic palmelloid colony, where cell division occurs, and a planktonic flagellate. Flagellates are short lived, settle and quickly resorb their flagella, the basal bodies then nucleate novel tubular appendages, termed “microrhizoids”, that lack an axoneme and function to anchor benthic cells to the substratum. To our knowledge, microrhizoids have not been observed in any other green alga or protist, are slightly smaller in diameter than flagella, generally contain nine microtubules, are long (3–5 times the length of flagella) and are not encased in scales. Following settlement, cell divisions result in a loose, palmelloid colony, each cell connected to the substratum by two microrhizoids. Flagellates are round to bean‐shaped with two long, slightly uneven flagella. Both benthic cells and flagellates, along with their flagella, are encased in thin scales. Phylogenies based on the complete chloroplast genome of Microrhizoidea show that it is clearly a member of the Mamiellophyceae, most closely related to Dolichomastix tenuilepsis. More taxon‐rich phylogenetic analyses of the 18S rRNA gene, including metabarcodes from the Tara Oceans and Ocean Sampling Day projects, confidently show the distinctive nature of Microrhizoidea, and that the described biodiversity of the Mamiellophyceae is a fraction of its real biodiversity. The discovery of a largely benthic prasinophyte changes our perspective on this group of algae and, along with the observation of other potential benthic lineages in environmental sequences, illustrates that benthic habitats can be a rich ground for algal biodiscovery. 相似文献
233.
Andersen Julie Høgsgaard Tjørnhøj-Thomsen Tine Reventlow Susanne Davidsen Annette Sofie 《Culture, medicine and psychiatry》2019,43(3):425-441
Culture, Medicine, and Psychiatry - An increasing number of young adults in Denmark experience difficulties in completing their education and holding down a job. Many of these young adults have... 相似文献
234.
Over the last 20 years, studies of Caenorhabditis elegans natural diversity have demonstrated the power of quantitative genetic approaches to reveal the evolutionary, ecological, and genetic factors that shape traits. These studies complement the use of the laboratory-adapted strain N2 and enable additional discoveries not possible using only one genetic background. In this chapter, we describe how to perform quantitative genetic studies in Caenorhabditis, with an emphasis on C. elegans. These approaches use correlations between genotype and phenotype across populations of genetically diverse individuals to discover the genetic causes of phenotypic variation. We present methods that use linkage, near-isogenic lines, association, and bulk-segregant mapping, and we describe the advantages and disadvantages of each approach. The power of C. elegans quantitative genetic mapping is best shown in the ability to connect phenotypic differences to specific genes and variants. We will present methods to narrow genomic regions to candidate genes and then tests to identify the gene or variant involved in a quantitative trait. The same features that make C. elegans a preeminent experimental model animal contribute to its exceptional value as a tool to understand natural phenotypic variation. 相似文献
235.
236.
Peptides corresponding to the entire sequence of the alpha-subunit of the human glycoprotein hormones were synthesized by using standard solid-phase procedures. Purified peptides were incubated in the presence of alpha- and beta-subunits of bovine lutropin, and subunit recombination was monitored by difference spectroscopy, reverse-phase high-pressure liquid chromatography, and gel filtration chromatography. Although the binding of alpha-peptides to either subunit could not be detected by these techniques, it was possible to demonstrate that some peptides could inhibit the recombination of alpha- and beta-subunits. Specifically, alpha-peptide 33-58 allowed only 0-11% of subunit recombination in 24 h (38-56% after 48 h), while alpha-peptide 51-65 allowed 10-60% of subunits to recombine in 24 h (65-94% in 48 h). Peptides 1-15, 11-27, 22-39, 61-78, and 73-92 of the alpha-subunit could not inhibit subunit recombination at any time or at any concentration tested. The data suggest that at least a portion of the alpha-subunit contact site has been identified, and results are discussed in terms of protein structure assessment tools. 相似文献
237.
Lisby M Olesen JR Skouboe C Krogh BO Straub T Boege F Velmurugan S Martensen PM Andersen AH Jayaram M Westergaard O Knudsen BR 《The Journal of biological chemistry》2001,276(23):20220-20227
All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme. 相似文献
238.
Andersen JP Sorensen TL Povlsen K Vilsen B 《The Journal of biological chemistry》2001,276(26):23312-23321
The specific functional roles of various parts of the third transmembrane segment (M3) of the sarcoplasmic reticulum Ca(2+)-ATPase were examined by functionally characterizing a series of mutants with multiple or single substitutions of M3 residues. Steady-state and transient kinetic measurements, assisted by computer simulation of the time and Ca(2+) dependences of the phosphorylation level, were used to study the partial reaction steps of the enzyme cycle, including the binding and dissociation of Ca(2+) at the high affinity cytoplasmically facing sites. The mutation Lys-Leu-Asp-Glu(255) --> Glu-Ile-Glu-His resulted in a conspicuous increase in the rate of Ca(2+) dissociation as well as a displacement of the major conformational equilibria of the phosphoenzyme and dephosphoenzyme forms. The point mutant Phe(256) --> Ala also showed an increased rate of Ca(2+) dissociation, whereas a conspicuous decrease both in the rate of Ca(2+) dissociation and in the rate of Ca(2+) binding was found for the mutant Gly-Glu-Gln-Leu(260) --> Ile-His-Leu-Ile. These findings suggest that the NH(2)-terminal half of M3 is involved in control of the gateway to the Ca(2+) sites. The main effect of two mutations to the COOH-terminal half of M3, Ser-Lys-Val-Ile-Ser(265) --> Thr-Gly-Val-Ala-Val and Leu-Ile-Cys-Val-Ala-Val-Trp-Leu-Ile(274) --> Phe-Leu-Gly-Val-Ser-Phe-Phe-Ile-Leu, was a block of the dephosphorylation. 相似文献
239.
Dimeric fragment of the insulin receptor alpha-subunit binds insulin with full holoreceptor affinity
The insulin receptor (IR) is a dimeric receptor, and its activation is thought to involve cross-linking between monomers initiated by binding of a single insulin molecule to separate epitopes on each monomer. We have previously shown that a minimized insulin receptor consisting of the first three domains of the human IR fused to 16 amino acids from the C-terminal of the alpha-subunit was monomeric and bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Sch?ffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). To investigate the insulin binding properties of dimerized alpha-subunits, we have reintroduced the domains containing alpha-alpha disulfide bonds into this minireceptor. When inserting either the first fibronectin type III domain or the full-length sequence of exon 10, the receptor fragments were predominantly secreted as disulfide-linked dimers that both had nanomolar affinity for insulin, similar to the affinity found for the minireceptor. However, when both these domains were included we obtained a soluble dimeric receptor that bound insulin with 1000-fold higher affinity (4-8 pm) similar to what was obtained for the solubilized holoreceptor (14-24 pm). Moreover, dissociation of labeled insulin from this receptor was accelerated in the presence of unlabeled insulin, demonstrating another characteristic feature of the holoreceptor. This is the first direct demonstration showing that the alpha-subunit of IR contains all the epitopes required for binding insulin with full holoreceptor affinity. 相似文献
240.
Jude AR Providence LL Schmutzer SE Shobana S Greathouse DV Andersen OS Koeppe R 《Biochemistry》2001,40(5):1460-1472
To examine the structural and functional importance of backbone amide groups in ion channels for subunit folding, hydrogen bonding, ion solvation, and ion permeation, we replaced the peptide bond between Val(1) and Gly(2) in gramicidin A by an ester bond. The substitution is at the junction between the two channel subunits, where it removes an intramolecular hydrogen bond between the NH of Gly(2) and the C==O of Val(7) and perturbs an intermolecular hydrogen bond between the C==O of Val(1) in one subunit and the NH of Ala(5) in the other subunit. The substitution thus perturbs not only subunit folding but also dimer assembly, in addition to any effects on ion permeation. This backbone modification has large effects on channel function: It alters channel stability, as monitored by the channel forming ability and channel lifetime, and ion permeability, as monitored by changes in single-channel conductance and cation permeability ratios. In fact, the homodimeric channels, with two ester-containing subunits, have lifetimes so short that it becomes impossible to characterize them in any detail. The peptide --> ester substitution, however, does not affect the basic subunit fold because heterodimeric channels can form between a subunit with an ester bond and a native subunit. These heterodimeric channels, with only a single ester bond, are more easily characterized; the lone ester reduces the single-channel conductance about 4-fold and the lifetime about 200-fold as compared to the native homodimeric channels. The altered channel function results from a perturbation/disruption of the hydrogen bond network that stabilizes the backbone, as well as the membrane-spanning dimer, and that forms the lining of the ion-conducting pore. Molecular dynamics simulations show the expected destabilization of the modified heterodimeric or homodimeric channels, but the changes in backbone structure and dynamics are remarkably small. The ester bond is somewhat unstable, which precluded further structural characterization. The lability also led to a hydrolysis product that terminates with an alcohol and lacks formyl-Val. Symmetric channels formed by the hydrolyzed product again have short lifetimes, but the channels are distinctly different from those formed by the ester gramicidin A. Furthermore, well-behaved asymmetric channels form between the hydrolysis product and reference subunits that have either an L- or a D-residue at the formyl-NH-terminus. 相似文献