scholarly journals Function of the CysD domain of the gel-forming MUC2 mucin

2011 ◽  
Vol 436 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Daniel Ambort ◽  
Sjoerd van der Post ◽  
Malin E. V. Johansson ◽  
Jenny MacKenzie ◽  
Elisabeth Thomsson ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Tandem Repeats ◽  
Gel Filtration ◽  
Middle Part ◽  
Domain Of Unknown Function ◽  
Cross Links ◽  
Native Gel ◽  
The Individual ◽  
Covalent Nature

The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.

scholarly journals Involvement of Very Short DNA Tandem Repeats and the Influence of the RAD52 Gene on the Occurrence of Deletions in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 549-557 ◽  
Author(s):  
Anne J Welcker ◽  
Jacky de Montigny ◽  
Serge Potier ◽  
Jean-Luc Souciet
Keyword(s):  
Fusion Proteins ◽  
Tandem Repeats ◽  
Chromosomal Rearrangements ◽  
Reversion Rate ◽  
Wild Type ◽  
Recombination Mechanism ◽  
Nonhomologous Recombination ◽  
Deletion Rate ◽  
Dna Tandem Repeats

Abstract Chromosomal rearrangements, such as deletions, duplications, or Ty transposition, are rare events. We devised a method to select for such events as Ura+ revertants of a particular ura2 mutant. Among 133 Ura+ revertants, 14 were identified as the result of a deletion in URA2. Of seven classes of deletions, six had very short regions of identity at their junctions (from 7 to 13 bp long). This strongly suggests a nonhomologous recombination mechanism for the formation of these deletions. The total Ura+ reversion rate was increased 4.2-fold in a rad52Δ strain compared to the wild type, and the deletion rate was significantly increased. All the deletions selected in the rad52Δ context had microhomologies at their junctions. We propose two mechanisms to explain the occurrence of these deletions and discuss the role of microhomology stretches in the formation of fusion proteins.

scholarly journals Kinetical Study, Thermo-Mechanical Characteristics and Recyclability of Epoxidized Camelina Oil Cured with Antagonist Structure (Aliphatic/Aromatic) or Functionality (Acid/Amine) Hardeners

Polymers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 2503
Author(s):  
Chiara Di Mauro ◽  
Aratz Genua ◽  
Alice Mija
Keyword(s):  
Disulfide Bonds ◽  
Mechanical Characteristics ◽  
Dicarboxylic Acids ◽  
Thermomechanical Properties ◽  
Chemical Recycling ◽  
Camelina Oil ◽  
Ft Ir ◽  
Tan Δ ◽  
The Individual ◽  
First Time

In an attempt to prepare sustainable epoxy thermosets, this study introduces for the first time the idea to use antagonist structures (aromatic/aliphatic) or functionalities (acid/amine) as hardeners to produce reprocessable resins based on epoxidized camelina oil (ECMO). Two kinds of mixtures were tested: one combines aromatic/aliphatic dicarboxylic acids: 2,2′-dithiodibenzoic acid (DTBA) and 3,3′-dithiodipropionic acid (DTDA); another is the combination of two aromatic structures with acid/amine functionality: DTBA and 4-aminophenyl disulfide (4-AFD). DSC and FT-IR analyses were used as methods to analyze the curing reaction of ECMO with the hardeners. It was found that the thermosets obtained with the dual crosslinked mechanism needed reduced curing temperatures and reprocessing protocols compared to the individual crosslinked thermosets. Thanks to the contribution of disulfide bonds in the network topology, the obtained thermosets showed recycling ability. The final thermomechanical properties of the virgin and mechanical reprocessed materials were analyzed by DMA and TGA. The obtained thermosets range from elastomeric to rigid materials. As an example, the ECMO/DTBA704-AFD30 virgin or reprocessed thermosets have tan δ values reaching 82–83 °C. The study also investigates the chemical recycling and the solvent resistance of these vitrimer-like materials.

scholarly journals Genetic Reconstruction and Forensic Analysis of Chinese Shandong and Yunnan Han Populations by Co-Analyzing Y Chromosomal STRs and SNPs

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 743
Author(s):  
Caiyong Yin ◽  
Kaiyuan Su ◽  
Ziwei He ◽  
Dian Zhai ◽  
Kejian Guo ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Forensic Analysis ◽  
Copy Number Variations ◽  
Null Alleles ◽  
Nucleotide Polymorphisms ◽  
Evolutionary Pathway ◽  
Individual Level ◽  
Population Comparisons ◽  
Heated Debate ◽  
The Individual

Y chromosomal short tandem repeats (Y-STRs) have been widely harnessed for forensic applications, such as pedigree source searching from public security databases and male identification from male–female mixed samples. For various populations, databases composed of Y-STR haplotypes have been built to provide investigating leads for solving difficult or cold cases. Recently, the supplementary application of Y chromosomal haplogroup-determining single-nucleotide polymorphisms (SNPs) for forensic purposes was under heated debate. This study provides Y-STR haplotypes for 27 markers typed by the Yfiler™ Plus kit and Y-SNP haplogroups defined by 24 loci within the Y-SNP Pedigree Tagging System for Shandong Han (n = 305) and Yunnan Han (n = 565) populations. The genetic backgrounds of these two populations were explicitly characterized by the analysis of molecular variance (AMOVA) and multi-dimensional scaling (MDS) plots based on 27 Y-STRs. Then, population comparisons were conducted by observing Y-SNP allelic frequencies and Y-SNP haplogroups distribution, estimating forensic parameters, and depicting distribution spectrums of Y-STR alleles in sub-haplogroups. The Y-STR variants, including null alleles, intermedia alleles, and copy number variations (CNVs), were co-listed, and a strong correlation between Y-STR allele variants (“DYS518~.2” alleles) and the Y-SNP haplogroup QR-M45 was observed. A network was reconstructed to illustrate the evolutionary pathway and to figure out the ancestral mutation event. Also, a phylogenetic tree on the individual level was constructed to observe the relevance of the Y-STR haplotypes to the Y-SNP haplogroups. This study provides the evidence that basic genetic backgrounds, which were revealed by both Y-STR and Y-SNP loci, would be useful for uncovering detailed population differences and, more importantly, demonstrates the contributing role of Y-SNPs in population differentiation and male pedigree discrimination.

Galectin-13/placental protein 13: redox-active disulfides as switches for regulating structure, function and cellular distribution

Glycobiology ◽  
2019 ◽  
Vol 30 (2) ◽  
pp. 120-129 ◽  
Author(s):  
Tong Yang ◽  
Yuan Yao ◽  
Xing Wang ◽  
Yuying Li ◽  
Yunlong Si ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
Gel Filtration ◽  
Wild Type ◽  
High Concentration ◽  
Redox Active ◽  
Placental Protein ◽  
The Relationship ◽  
Low Expression

Abstract Galectin-13 (Gal-13) plays numerous roles in regulating the relationship between maternal and fetal tissues. Low expression levels or mutations of the lectin can result in pre-eclampsia. The previous crystal structure and gel filtration data show that Gal-13 dimerizes via formation of two disulfide bonds formed by Cys136 and Cys138. In the present study, we mutated them to serine (C136S, C138S and C136S/C138S), crystalized the variants and solved their crystal structures. All variants crystallized as monomers. In the C136S structure, Cys138 formed a disulfide bond with Cys19, indicating that Cys19 is important for regulation of reversible disulfide bond formation in this lectin. Hemagglutination assays demonstrated that all variants are inactive at inducing erythrocyte agglutination, even though gel filtration profiles indicate that C136S and C138S could still form dimers, suggesting that these dimers do not exhibit the same activity as wild-type (WT) Gal-13. In HeLa cells, the three variants were found to be distributed the same as with WT Gal-13. However, a Gal-13 variant (delT221) truncated at T221 could not be transported into the nucleus, possibly explaining why women having this variant get pre-eclampsia. Considering the normally high concentration of glutathione in cells, WT Gal-13 should exist mostly as a monomer in cytoplasm, consistent with the monomeric variant C136S/C138S, which has a similar ability to interact with HOXA1 as WT Gal-13.

scholarly journals Protein disulfide isomerase activity is released by activated platelets

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2226-2228 ◽  
Author(s):  
K Chen ◽  
Y Lin ◽  
TC Detwiler
Keyword(s):  
Disulfide Bonds ◽  
Protein Disulfide Isomerase ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
Activated Platelets ◽  
Protein Disulfide ◽  
Supernatant Solution ◽  
Disulfide Isomerase Activity

Abstract The release of protein disulfide isomerase by activated platelets was hypothesized on the basis of reported intermolecular and intramolecular thiol-disulfide exchange and disulfide reduction involving released thrombospondin in the supernatant solution of activated platelets (Danishefsky, Alexander, Detwiler: Biochemistry, 23:4984, 1984; Speziale, Detwiler: J Biol Chem, 265:17859, 1990; Speziale, Detwiler: Arch Biochem Biophys 286:546, 1991). Protein disulfide isomerase activity, measured by catalysis of the renaturation of ribonuclease inactivated by randomization of disulfide bonds, was detected in the supernatant solution after platelet activation. The activity was inhibited by peptides known to inhibit protein disulfide isomerase; the peptides also inhibited formation of disulfide-linked thrombospondin- thrombin complexes. The reaction catalyzed by the supernatant solution showed a pH dependence distinct from that of the uncatalyzed reaction. The activity was excluded by a 50-Kd dialysis membrane, and it was eluted in the void volume of a gel-filtration column, indicating that it was associated with a macromolecule. The activity was not removed by centrifugation at 100,000 g for 150 minutes indicating that it was not associated with membrane microvesicles. Possible functions for the release of protein disulfide isomerase by activated platelets are discussed.

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

Abstract The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

Concentration of collagen cross-links in human dentin bears no relation to the individual age

2002 ◽  
Vol 116 (6) ◽  
pp. 340-343 ◽  
Author(s):  
Y. Açil ◽  
I. Springer ◽  
J. Prasse ◽  
J. Hedderich ◽  
S. Jepsen
Keyword(s):  
Human Dentin ◽  
Cross Links ◽  
The Individual

Non-homologous end-joining partners in a helical dance: structural studies of XLF–XRCC4 interactions

2011 ◽  
Vol 39 (5) ◽  
pp. 1387-1392 ◽  
Author(s):  
Qian Wu ◽  
Takashi Ochi ◽  
Dijana Matak-Vinkovic ◽  
Carol V. Robinson ◽  
Dimitri Y. Chirgadze ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Coiled Coil ◽  
Helical Structure ◽  
Complementation Group ◽  
Helical Axis ◽  
Structural Studies ◽  
End Joining ◽  
Individual Crystal ◽  
Non Homologous End Joining ◽  
The Individual

XRCC4 (X-ray cross-complementation group 4) and XLF (XRCC4-like factor) are two essential interacting proteins in the human NHEJ (non-homologous end-joining) pathway that repairs DNA DSBs (double-strand breaks). The individual crystal structures show that the dimeric proteins are homologues with protomers containing head domains and helical coiled-coil tails related by approximate two-fold symmetry. Biochemical, mutagenesis, biophysical and structural studies have identified the regions of interaction between the two proteins and suggested models for the XLF–XRCC4 complex. An 8.5 Å (1 Å=0.1 nm) resolution crystal structure of XLF–XRCC4 solved by molecular replacement, together with gel filtration and nano-ESI (nano-electrospray ionization)–MS results, demonstrates that XLF and XRCC4 dimers interact through their head domains and form an alternating left-handed helical structure with polypeptide coiled coils and pseudo-dyads of individual XLF and XRCC4 dimers at right angles to the helical axis.

How do surfactants and DTT affect the size, dynamics, activity and growth of soluble lysozyme aggregates?

2008 ◽  
Vol 415 (2) ◽  
pp. 275-288 ◽  
Author(s):  
Satish Kumar ◽  
Vijay Kumar Ravi ◽  
Rajaram Swaminathan
Keyword(s):  
Disulfide Bonds ◽  
Molecular Mechanisms ◽  
Amyloid Fibrils ◽  
Gel Filtration ◽  
Segmental Motion ◽  
Initial Growth ◽  
Time Resolved ◽  
Force Microscopy ◽  
Intermolecular Disulfide Bonds ◽  
Soluble Aggregates

The early intermediates in the protein aggregation pathway, the elusive soluble aggregates, play a pivotal role in growth and maturation of ordered aggregates such as amyloid fibrils. Blocking the growth of soluble oligomers is an effective strategy to inhibit aggregation. To decipher the molecular mechanisms and develop better strategies to arrest aggregation, it is imperative to understand how the size, molecular dynamics, activity and growth kinetics of soluble aggregates are affected when aggregation is inhibited. With this objective, in the present study we have investigated the influence of additives such as SDS, CTAB (cetyltrimethylammonium bromide) and DTT (dithiothreitol) on the slow aggregation of HEWL (hen eggwhite lysozyme) at pH 12.2. For this purpose, techniques such as steady-state and time-resolved fluorescence anisotropy of covalently labelled dansyl probe, gel-filtration chromatography, estimation of free thiol groups, thioflavin T and ANS (8-anilinonaphthalene-1-sulfonic acid) fluorescence, CD and atomic-force microscopy were employed to monitor the soluble oligomers over a period spanning 30 days. The results of the present study reveal that: (i) the spontaneous formation of soluble aggregates is irreversible and abolishes activity; (ii) the initial growth of aggregates (0–24 h) is promoted by a gradual increase in the exposure of hydrophobic surfaces; (iii) subsequently intermolecular disulfide bonds are critical for the assembly and stability of aggregates; (iv) the tight molecular packing inside large aggregates which contributed to slow (∼5 ns) and restricted segmental motion of dansyl probe was clearly loosened up in the presence of additives, enabling fast (1–2 ns) and free motion (unlike DTT, the size of lysozyme complexes with surfactants, was large, due to a conglomeration of proteins and surfactants); (v) the aggregates show reduced helical content compared with native lysozyme, except in the presence of SDS; and (vi) DTT was more potent than SDS/CTAB in arresting the growth of aggregates.

Influence of diet on the development of antral gastrin-like immunoreactivity in the stomach of rats

1989 ◽  
Vol 120 (3) ◽  
pp. 374-378 ◽  
Author(s):  
Hiroyasu Okahata ◽  
Yoshikazu Nishi ◽  
Kotaro Muraki ◽  
Masaru Arai ◽  
Norio Kubo ◽  
...  
Keyword(s):  
Breast Milk ◽  
Gel Filtration ◽  
Essential Nutrients ◽  
Young Rats ◽  
Rich Food ◽  
Old Rats ◽  
The Individual ◽  
Milk Feeding

Abstract. The effects of individual food constituents on antral gastrin-like immunoreactivity concentrations were studied in young rats. Rats aged 7 to 20 days were given only rat breast milk and then weaned by various nutrients (regular laboratory chow, protein (ovalbumin)-, fat- or carbohydrate (starch)-rich food). Rats receiving rat breast milk only until 27 days of age were also studied. In rats on regular laboratory chow, antral gastrin-like immunoreactivity increased and reached adult levels on day 25. In rats on ovalbumin, fat-rich food or starch, it increased on day 23 but dropped thereafter. The increment by laboratory chow was higher than that by the individual nutrients. No increase was observed during milk feeding alone. Gel filtration of antral gastrin-like immunoreactivity from 25-day-old rats on laboratory chow or three essential nutrients showed the same results.

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