Urine Proteomics Profiling and Functional Characterization of Knee Osteoarthritis Using iTRAQ Technology

2019 ◽  
Vol 51 (11) ◽  
pp. 735-740 ◽  
Author(s):  
Ke Xiao ◽  
Lingjia Yu ◽  
Lisi Zhu ◽  
Zhihong Wu ◽  
Xisheng Weng ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Joint Function ◽  
Urine Protein ◽  
Western Blots ◽  
Urine Proteomics ◽  
Protein Levels ◽  
Mass Spectrometry Technology ◽  
Proteome Profile ◽  
Insight Into

AbstractOsteoarthritis (OA) is a degenerative chronic disease affecting the whole joint structures. With the increment in life expectancy and aging population, OA has become one of the largest socioeconomic burdens, associated with pain and loss of joint function. However, early laboratory tests of OA are still lacking. Therefore, new diagnostic tests for this disease are urgently needed. In this study, to gain an insight into the pathogenesis and the potential biomarkers of OA, we implemented a comparative urine proteomics study on OA patients and health people using iTRAQ-based mass spectrometry technology. Western blotting was used to validate the relative changes in urine protein levels for four of the identified proteins. We constructed a comprehensive urine proteome profile of the OA patients and identified 102 proteins differently changed in abundance. Forty-six proteins were upregulated and 56 proteins were significantly downregulated in OA patients. Furthermore, the proteins, COL-4, MMP9, adiponectin, and BBOX1 were validated through Western blots, which can serve as valuable candidate biomarkers and help to illustrate the pathogenesis of OA. These findings may provide clues for promising biomarkers for the early diagnosis and also offer a theoretical basis for the early treatment of OA.

scholarly journals Codon Optimization of Insect Odorant Receptor Genes May Increase Their Stable Expression for Functional Characterization in HEK293 Cells

2021 ◽  
Vol 15 ◽  
Author(s):  
Rebecca E. Roberts ◽  
Jothi Kumar Yuvaraj ◽  
Martin N. Andersson
Keyword(s):  
Bark Beetles ◽  
Codon Optimization ◽  
Odorant Receptor ◽  
Functional Characterization ◽  
Stable Expression ◽  
Hek293 Cells ◽  
Western Blots ◽  
Protein Levels ◽  
Hek Cells

Insect odorant receptor (OR) genes are routinely expressed in Human Embryonic Kidney (HEK) 293 cells for functional characterization (“de-orphanization”) using transient or stable expression. However, progress in this research field has been hampered because some insect ORs are not functional in this system, which may be due to insufficient protein levels. We investigated whether codon optimization of insect OR sequences for expression in human cells could facilitate their functional characterization in HEK293 cells with stable and inducible expression. We tested the olfactory receptor co-receptor (Orco) proteins from the bark beetles Ips typographus (“Ityp”) and Dendroctonus ponderosae (“Dpon”), and six ItypORs previously characterized in Xenopus laevis oocytes and/or HEK cells. Western blot analysis indicated that codon optimization yielded increased cellular protein levels for seven of the eight receptors. Our experimental assays demonstrated that codon optimization enabled functional characterization of two ORs (ItypOR25 and ItypOR29) which are unresponsive when expressed from wildtype (non-codon optimized) genes. Similar to previous Xenopus oocyte recordings, ItypOR25 responded primarily to the host/conifer monoterpene (+)-3-carene. ItypOR29 responded primarily to (+)-isopinochamphone and similar ketones produced by fungal symbionts and trees. Codon optimization also resulted in significantly increased responses in ItypOR49 to its pheromone ligand (R)-(−)-ipsdienol, and improved responses to the Orco agonist VUAA1 in ItypOrco. However, codon optimization did not result in functional expression of DponOrco, ItypOR23, ItypOR27, and ItypOR28 despite higher protein levels as indicated by Western blots. We conclude that codon optimization may enable or improve the functional characterization of insect ORs in HEK cells, although this method is not sufficient for all ORs that are not functionally expressed from wildtype genes.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

scholarly journals Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA.

1988 ◽  
Vol 8 (4) ◽  
pp. 1518-1524 ◽  
Author(s):  
D Soldati ◽  
D Schümperli
Keyword(s):  
Sea Urchin ◽  
Functional Characterization ◽  
Rnase H ◽  
Primer Extension ◽  
Small Nuclear Rna ◽  
Nuclear Rna ◽  
Processing Signal ◽  
Insight Into

Oligonucleotides derived from the spacer element of the histone RNA 3' processing signal were used to characterize mouse U7 small nuclear RNA (snRNA), i.e., the snRNA component active in 3' processing of histone pre-mRNA. Under RNase H conditions, such oligonucleotides inhibited the processing reaction, indicating the formation of a DNA-RNA hybrid with a functional ribonucleoprotein component. Moreover, these oligonucleotides hybridized to a single nuclear RNA species of approximately 65 nucleotides. The sequence of this RNA was determined by primer extension experiments and was found to bear several structural similarities with sea urchin U7 snRNA. The comparison of mouse and sea urchin U7 snRNA structures yields some further insight into the mechanism of histone RNA 3' processing.

Phylogenetic analysis of fungal aquaporins provides insight into their possible role in water transport of mycorrhizal associations

Botany ◽  
2013 ◽  
Vol 91 (8) ◽  
pp. 495-504 ◽  
Author(s):  
Hao Xu ◽  
Janice E.K. Cooke ◽  
Janusz J. Zwiazek
Keyword(s):  
Water Transport ◽  
Mycorrhizal Fungi ◽  
Functional Characterization ◽  
Arbuscular Mycorrhizal ◽  
Gene Families ◽  
Fungal Species ◽  
Comparative Genomic ◽  
Mycorrhizal Associations ◽  
Insight Into

In mycorrhizal associations, water transport properties of the fungal hyphae may have a profound effect on water transport of the host plant. The importance of aquaporins, water-transporting members of the major intrinsic protein (MIP) family, in facilitating water transport has been widely acknowledged and extensively studied in plants. However, until recently, relatively little was known about the structure, function, and regulation of fungal MIPs. The rapid increase in the number of sequenced fungal genomes, including Laccaria bicolor and other mycorrhizal fungi, has enabled functional and comparative genomic investigations to delineate the role that fungal MIPs play in mycorrhizal-facilitated plant water transport. Phylogenic analysis of 229 fungal MIPs from 88 species revealed that MIPs of mycorrhizal fungal species fall into four clusters delineated by functionally characterized fungal MIPs: the orthodox aquaporins, the aquaglyceroporins, the facultative fungal aquaporins, and the X intrinsic proteins. This comparative genomics analysis, together with in silico structural characterization of predicted MIPs and recently published functional characterization of MIPs from a small number of ectomycorrhizal and arbuscular mycorrhizal species, provide new insight into MIP gene families of mycorrhizal fungi and possible roles for fungal aquaporins in water relations of mycorrhizal plant–fungus symbioses.

scholarly journals Discovery and characterization of Hv1-type proton channels in reef-building corals.

2021 ◽  
Author(s):  
Gisela Rangel-Tescas ◽  
Cecilia Cervantes ◽  
Miguel A Cervantes-Rocha ◽  
Esteban Suarez-Delgado ◽  
Anastazia T Banaszak ◽  
...  
Keyword(s):  
Voltage Dependence ◽  
Ph Regulation ◽  
Functional Characterization ◽  
Gene Encoding ◽  
Proton Channels ◽  
Gating Model ◽  
Mechanistic Insight ◽  
Voltage Dependent ◽  
Insight Into

Voltage-dependent proton-permeable channels are membrane proteins mediating a number of important physiological functions. Here we report the presence of a gene encoding for Hv1 voltage-dependent, proton-permeable channels in two species of reef-building corals. We performed a characterization of their biophysical properties and found that these channels are fast-activating and modulated by the pH gradient in a manner that makes them interesting models for studying these processes more easily. We have also developed an allosteric gating model that provides mechanistic insight into the modulation of voltage-dependence by protons. This work also represents the first functional characterization of any ion channel in scleractinian corals. We discuss the implications of the presence of these channels in the membranes of coral cells in the calcification and pH regulation processes and possible consequences of ocean acidification related to the function of these channels.

scholarly journals Functional characterization of tlmH in Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insight into tallysomycinbiosynthesis and affording a novel bleomycin analog

2010 ◽  
Vol 6 (2) ◽  
pp. 349-356 ◽  
Author(s):  
Meifeng Tao ◽  
Liyan Wang ◽  
Evelyn Wendt-Pienkowski ◽  
Ningning Zhang ◽  
Dong Yang ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Insight Into

scholarly journals Genome-wide identification and characterization of the ALOG gene family in Petunia

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Feng Chen ◽  
Qin Zhou ◽  
Lan Wu ◽  
Fei Li ◽  
Baojun Liu ◽  
...  
Keyword(s):  
Gene Family ◽  
Expression Patterns ◽  
Functional Characterization ◽  
Reproductive Organs ◽  
Genome Wide ◽  
Domain Of Unknown Function ◽  
Intron Structure ◽  
Identification And Characterization ◽  
Insight Into

Abstract Background The ALOG (Arabidopsis LSH1 and Oryza G1) family of proteins, namely DUF640 (domain of unknown function 640) domain proteins, were found in land plants. Functional characterization of a few ALOG members in model plants such as Arabidopsis and rice suggested they play important regulatory roles in plant development. The information about its evolution, however, is largely limited, and there was no any report on the ALOG genes in Petunia, an important ornamental species. Results The ALOG genes were identified in four species of Petunia including P. axillaris, P. inflata, P. integrifolia, and P. exserta based on the genome and/or transcriptome databases, which were further confirmed by cloning from P. hybrida ‘W115’ (Mitchel diploid), a popular laboratorial petunia line susceptible to genetic transformation. Phylogenetic analysis indicated that Petunia ALOG genes (named as LSHs according to their closest Arabidopsis homologs) were grouped into four clades, which can be further divided into eight groups, and similar exon-intron structure and motifs are reflected in the same group. The PhLSH genes of hybrid petunia ‘W115’ were mainly derived from P. axillaris. The qPCR analysis revealed distinct spatial expression patterns among them suggesting potentially functional diversification. Moreover, over-expressing PhLSH7a and PhLSH7b in Arabidopsis uncovered their functions in the development of both vegetative and reproductive organs. Conclusions Petunia genome includes 11 ALOG genes that can be divided into eight distinct groups, and they also show different expression patterns. Among these genes, PhLSH7b and PhLSH7a play significant roles in plant growth and development, especially in fruit development. Our results provide new insight into the evolution of ALOG gene family and have laid a good foundation for the study of petunia LSH gene in the future.

scholarly journals New Insight into Phaeodactylum tricornutum Fatty Acid Metabolism. Cloning and Functional Characterization of Plastidial and Microsomal Δ12-Fatty Acid Desaturases

2003 ◽  
Vol 131 (4) ◽  
pp. 1648-1660 ◽  
Author(s):  
Frédéric Domergue ◽  
Patricia Spiekermann ◽  
Jens Lerchl ◽  
Christoph Beckmann ◽  
Oliver Kilian ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Metabolism ◽  
Phaeodactylum Tricornutum ◽  
Acid Metabolism ◽  
Functional Characterization ◽  
Fatty Acid Desaturases ◽  
Insight Into
Sign in / Sign up

Export Citation Format

Share Document