Cysteine immobilisation on the polyethylene terephthalate surfaces and its effect on the haemocompatibility

Abstract Nitric oxide (NO) is an important signalling molecule involved in haemostasis. NO, present as endogenous S-nitrosothiols, is released by cysteine through a transnitrosation reaction. To exploit this mechanism, cysteine was immobilised onto the different carboxylated polyethylene terephthalate (PET) surfaces using 1-step EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) crosslinking mechanism. Immobilised cysteine concentration and NO release were dependent on the surface carboxyl density. Stability studies showed that the immobilised cysteine concentration and NO release reduced within 6 h. Immobilisation of cysteine derivatives eliminated the possibility of formation of polycysteine and its electrostatic interaction with the carboxylated PET. The immobilised cysteine concentration did not recover after DTT treatment, eliminating the possibility of disulphide bond formation. Further, cysteine was immobilised using a 2-step EDC crosslinking mechanism. Although the cysteine concentration reduced during stability studies, it recovered upon DTT treatment, indicating that cysteine forms amide bonds with the carboxylated PET and the observed decrease in cysteine concentration is probably due to the formation of disulphide bonds. The haemocompatibility of the cysteine immobilised PET surfaces showed similar results compared to the carboxylated PET. The loss of thiol groups due to the disulphide bond restricts the transnitrosation reaction. Hence, these materials can be used primarily in short-term applications.

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Exploring the Intracrine Functions of VEGF-A

Biomolecules ◽

10.3390/biom11010128 ◽

2021 ◽

Vol 11 (1) ◽

pp. 128

Author(s):

Sophie Wiszniak ◽

Quenten Schwarz

Keyword(s):

Cell Growth ◽

Molecular Mechanisms ◽

Direct Action ◽

Cancer Therapeutics ◽

Cell Types ◽

Vascular Endothelial ◽

Signalling Molecule ◽

Endothelial Growth Factor ◽

Important Signalling Molecule ◽

Vegf Signalling

Vascular endothelial growth factor A (VEGF-A or VEGF) is a highly conserved secreted signalling protein best known for its roles in vascular development and angiogenesis. Many non-endothelial roles for VEGF are now established, with the discovery that VEGF and its receptors VEGFR1 and VEGFR2 are expressed in many non-vascular cell-types, as well as various cancers. In addition to secreted VEGF binding to its receptors in the extracellular space at the cell membrane (i.e., in a paracrine or autocrine mode), intracellularly localised VEGF is emerging as an important signalling molecule regulating cell growth, survival, and metabolism. This intracellular mode of signalling has been termed “intracrine”, and refers to the direct action of a signalling molecule within the cell without being secreted. In this review, we describe examples of intracrine VEGF signalling in regulating cell growth, differentiation and survival, both in normal cell homeostasis and development, as well as in cancer. We further discuss emerging evidence for the molecular mechanisms underpinning VEGF intracrine function, as well as the implications this intracellular mode of VEGF signalling may have for use and design of anti-VEGF cancer therapeutics.

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Mononuclear Ni(II) and dinuclear Cd(II) complexes of 4-phenyl-2H-1,2,4-triazole-3-thione and Mn(II) catalyzed disulphide bond formation in 3,3′-dithio bis (4-phenyl-1,2,4-triazole): Syntheses, structural characterization, thermal analysis and DFT calculation

Inorganica Chimica Acta ◽

10.1016/j.ica.2014.06.037 ◽

2014 ◽

Vol 421 ◽

pp. 519-530 ◽

Cited By ~ 8

Author(s):

R.K. Dani ◽

M.K. Bharty ◽

S. Paswan ◽

Sanjay Singh ◽

N.K. Singh

Keyword(s):

Thermal Analysis ◽

Structural Characterization ◽

Dft Calculation ◽

Bond Formation ◽

Disulphide Bond ◽

Disulphide Bond Formation

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Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation

Molecular Microbiology ◽

10.1111/j.1365-2958.1995.mmi_17061035.x ◽

1995 ◽

Vol 17 (6) ◽

pp. 1035-1044 ◽

Cited By ~ 49

Author(s):

Kim R. Hardie ◽

Angela Schulze ◽

Michael W. Parker ◽

J. Thomas Buckley

Keyword(s):

Bond Formation ◽

Disulphide Bond ◽

Disulphide Bond Formation ◽

Vibrio Spp

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Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF)

Biochemical Journal ◽

10.1042/bj2560213 ◽

1988 ◽

Vol 256 (1) ◽

pp. 213-218 ◽

Cited By ~ 22

Author(s):

P Wingfield ◽

R Benedict ◽

G Turcatti ◽

B Allet ◽

J J Mermod ◽

...

Keyword(s):

Coli Strain ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Wild Type ◽

Disulphide Bonds ◽

Disulphide Bond Formation ◽

Stimulating Factor ◽

Factor G

Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.

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A structure—function study of dihydrofolate reductase by protein engineering

Philosophical Transactions of the Royal Society of London. Series A, Mathematical and Physical Sciences ◽

10.1098/rsta.1986.0050 ◽

1986 ◽

Vol 317 (1540) ◽

pp. 405-413 ◽

Cited By ~ 6

Keyword(s):

Structure Function ◽

Dihydrofolate Reductase ◽

Conformational Changes ◽

Guanidine Hydrochloride ◽

Disulphide Bond ◽

E Coli ◽

Transition State Stabilization ◽

Disulphide Bond Formation ◽

High Resolution Structure ◽

Charge Interaction

Several mutants of the enzyme dihydrofolate reductase (DHFR) have been engineered by oligonucleotide-directed mutagenesis of the cloned E. coli gene. The mutations were designed to address specific questions about DHFR structure-function relations that arose from the analysis of the high-resolution structure. Mutations at the active site have revealed that the invariant residue aspartate-27 is involved in substrate protonation, and not in transition-state stabilization as previously thought. The 2.0 Å (1 Å = 10 -1 nm = 10 -10 m) refined structures of the Asn-27 and Ser-27 mutant enzymes reveal that the enhanced binding observed for the 2,4-diamino pteridine and pyrimidine inhibitors is probably not attributable to the charge interaction between Asp-27 and a protonated N-1 of the inhibitor. Substitution of a cysteine for a proline at position 39 places two sulphydryls within bonding distance, and under certain oxidation conditions they will quantitatively form a disulphide bond. The refined 2.0 Å structures of both reduced and oxidized forms of this mutant show that only minor conformational changes occur for disulphide bond formation. The crosslinked enzyme is significantly more conformationally stable to denaturants such as guanidine hydrochloride and urea.

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Current approaches to measure nitric oxide in plants

Journal of Experimental Botany ◽

10.1093/jxb/erz242 ◽

2019 ◽

Vol 70 (17) ◽

pp. 4333-4343 ◽

Cited By ~ 9

Author(s):

Abhaypratap Vishwakarma ◽

Aakanksha Wany ◽

Sonika Pandey ◽

Mallesham Bulle ◽

Aprajita Kumari ◽

...

Keyword(s):

Nitric Oxide ◽

Current Knowledge ◽

Reactive Intermediates ◽

Confounding Factors ◽

Biological Processes ◽

Cellular Redox ◽

Signalling Molecule ◽

Growth Development ◽

Scavenging Enzymes ◽

Important Signalling Molecule

AbstractNitric oxide (NO) is now established as an important signalling molecule in plants where it influences growth, development, and responses to stress. Despite extensive research, the most appropriate methods to measure and localize these signalling radicals are debated and still need investigation. Many confounding factors such as the presence of other reactive intermediates, scavenging enzymes, and compartmentation influence how accurately each can be measured. Further, these signalling radicals have short half-lives ranging from seconds to minutes based on the cellular redox condition. Hence, it is necessary to use sensitive and specific methods in order to understand the contribution of each signalling molecule to various biological processes. In this review, we summarize the current knowledge on NO measurement in plant samples, via various methods. We also discuss advantages, limitations, and wider applications of each method.

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Molecular and clinical spectrum of four pedigrees of TRAPS in Greece: results from a national referral center

Rheumatology ◽

10.1093/rheumatology/kez424 ◽

2019 ◽

Vol 59 (6) ◽

pp. 1241-1246 ◽

Cited By ~ 2

Author(s):

Adrianos Nezos ◽

Ourania D Argyropoulou ◽

Eleni Klinaki ◽

Nikolaos Marketos ◽

Panagiota Karagianni ◽

...

Keyword(s):

Clinical Phenotype ◽

Tumor Necrosis Factor Receptor ◽

Clinical Manifestations ◽

Pedigree Analysis ◽

Clinical Spectrum ◽

Mild Disease ◽

Disulphide Bonds ◽

Disulphide Bond Formation ◽

Tnfrsf1a Gene ◽

Necrosis Factor

Abstract Objective Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is a rare autosomal dominantly inherited autoinflammatory disease caused by mutations of the TNFRSF1A gene. To address the association between TNFRSF1A mutations and clinical phenotype, we analyzed four pedigrees of TRAPS patients. Methods Four Greek patients with TRAPS-like clinical features were screened for TNFRSF1A mutations by sequencing exons 2, 3 and 4. Following positive testing, twenty-two members of their families were also genetically and clinically screened. Results Twenty-six members of four unrelated Greek families were investigated. The C73Y (c.305G>A) mutation of the TNFRSF1A gene was identified in five patients, with two of the five carrying a concomitant R92Q variation. We also identified seven C73W (c.306C>G), two T50M (c.236C>T) and seven R92Q (c.362G>A) carriers. Symptoms varied and the C73Y, C73W and T50M mutations were associated with the most severe clinical manifestations. The R92Q phenotype ranged from asymptomatic to mild disease. Molecular modelling linked pathogenicity with aberrant TNFRSF1A disulphide bond formation. Conclusion In this first pedigree analysis of TRAPS in Greece, we identified the rare C73Y TNFRSF1A mutation. A wide clinical spectrum was observed with the C73Y, C73W and T50M mutations that affect TNFRSF1A disulphide bonds and are associated with worse symptoms.

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