scholarly journals Glutamate-64, a newly identified residue of the functionally conserved electron-sharing network contributes to catalysis and structural integrity of glutathione transferases

2007 ◽  
Vol 402 (2) ◽  
pp. 339-348 ◽  
Author(s):  
Pakorn Winayanuwattikun ◽  
Albert J. Ketterman
Keyword(s):  
Structural Integrity ◽  
Glutathione Transferase ◽  
Product Formation ◽  
Glutathione Transferases ◽  
Nucleophilic Attack ◽  
Anopheles Dirus ◽  
Catalytic Function ◽  
Equivalent Residue ◽  
Vitro Analysis

In Anopheles dirus glutathione transferase D3-3, position 64 is occupied by a functionally conserved glutamate residue, which interacts directly with the γ-glutamate moiety of GSH (glutathione) as part of an electron-sharing network present in all soluble GSTs (glutathione transferases). Primary sequence alignment of all GST classes suggests that Glu64 is one of a few residues that is functionally conserved in the GST superfamily. Available crystal structures as well as consideration of the property of the equivalent residue at position 64, acidic or polar, suggest that the GST electron-sharing motif can be divided into two types. Electrostatic interaction between the GSH glutamyl and carboxylic Glu64, as well as with Arg66 and Asp100, was observed to extend the electron-sharing motif identified previously. Glu64 contributes to the catalytic function of this motif and the ‘base-assisted deprotonation’ that are essential for GSH ionization during catalysis. Moreover, this residue also appears to affect multiple steps in the enzyme catalytic strategy, including binding of GSH, nucleophilic attack by thiolate at the electrophilic centre and product formation, probably through active-site packing effects. Replacement with non-functionally-conserved amino acids alters initial packing or folding by favouring aggregation during heterologous expression. Thermodynamic and reactivation in vitro analysis indicated that Glu64 also contributes to the initial folding pathway and overall structural stability. Therefore Glu64 also appears to impact upon catalysis through roles in both initial folding and structural maintenance.

scholarly journals The soluble glutathione transferase superfamily: Role of Mu class in Triclabendazole sulphoxide challenge in Fasciola hepatica

2020 ◽  
Author(s):  
Rebekah B. Stuart ◽  
Suzanne Zwaanswijk ◽  
Neil D. MacKintosh ◽  
Boontarikaan Witikornkul ◽  
Mark Prescott ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Fasciola Hepatica ◽  
Affinity Purification ◽  
Economic Loss ◽  
Glutathione Transferases ◽  
Parasitic Helminths ◽  
Food Borne ◽  
Production Industry

AbstractFasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock production industry and is re-emerging as a food borne disease of humans. In the absence of vaccines the commonly used method of treatment control is by anthelmintics; with only Triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and detoxification by flukes might contribute to the mechanism. However, there is limited Phase I capacity in adult parasitic helminths and the major Phase II detoxification system in adults is the soluble Glutathione transferases (GST) superfamily. Previous global proteomic studies have shown that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-Sulphoxide (TCBZ-SO), the likely active metabolite, challenge during in vitro culture ex-host. We have extended this finding by using a sub-proteomic lead approach to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated a FhGST-Mu29 and FhGST-Mu26 response following affinity purification using both GSH and S-hexyl GSH affinity resins. Furthermore, a low affinity Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a novel low affinity mu class GST. Low affinity GST isoforms within the GST-ome was not limited to FhGST-Mu5 with second likely low affinity sigma class GST (FhGST-S2) uncovered through genome analysis. This study represents the most complete Fasciola GST-ome generated to date and has supported the sub proteomic analysis on individual adult flukes.

Dengue protease activity: the structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target

2011 ◽  
Vol 31 (5) ◽  
pp. 399-409 ◽  
Author(s):  
Wai Y. Phong ◽  
Nicole J. Moreland ◽  
Siew P. Lim ◽  
Daying Wen ◽  
Prasad N. Paradkar ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Protease Activity ◽  
Structural Integrity ◽  
Glutathione Transferase ◽  
Human Monoclonal Antibody ◽  
Site Formation ◽  
Single Chain ◽  
Ns3 Protease

Flaviviral NS3 serine proteases require the NS2B cofactor region (cNS2B) to be active. Recent crystal structures of WNV (West Nile virus) protease in complex with inhibitors revealed that cNS2B participates in the formation of the protease active site. No crystal structures of ternary complexes are currently available for DENV (dengue virus) to validate the role of cNS2B in active site formation. In the present study, a GST (glutathione transferase) fusion protein of DENV-2 cNS2B49–95 was used as a bait to pull down DENV-2 protease domain (NS3pro). The affinity of NS3pro for cNS2B was strong (equilibrium-binding constant <200 nM) and the heterodimeric complex displayed a catalytic efficiency similar to that of single-chain DENV-2 cNS2B/NS3pro. Various truncations and mutations in the cNS2B sequence showed that conformational integrity of the entire 47 amino acids is critical for protease activity. Furthermore, DENV-2 NS3 protease can be pulled down and transactivated by cNS2B cofactors from DENV-1, -3, -4 and WNV, suggesting that mechanisms for activation are conserved across the flavivirus genus. To validate NS2B as a potential target in allosteric inhibitor development, a cNS2B-specific human monoclonal antibody (3F10) was utilized. 3F10 disrupted the interaction between cNS2B and NS3 in vitro and reduced DENV viral replication in HEK (human embryonic kidney)-293 cells. This provides proof-of-concept for developing assays to find inhibitors that block the interaction between NS2B and NS3 during viral translation.

The native structure of the eukaryotic ribosome

1983 ◽  
Vol 41 ◽  
pp. 432-433
Author(s):  
R.A. Milligan ◽  
P.N.T. Unwin
Keyword(s):  
Ribosomal Proteins ◽  
Crystal Form ◽  
Native Structure ◽  
X Ray Diffraction ◽  
Eukaryotic Ribosome ◽  
Vitro Analysis ◽  
In Vitro Analysis ◽  
Resolution Structure ◽  
Stable Unit

A detailed understanding of the mechanism of protein synthesis will ultimately depend on knowledge of the native structure of the ribosome. Towards this end we have investigated the low resolution structure of the eukaryotic ribosome embedded in frozen buffer, making use of a system in which the ribosomes crystallize naturally.The ribosomes in the cells of early chicken embryos form crystalline arrays when the embryos are cooled at 4°C. We have developed methods to isolate the stable unit of these arrays, the ribosome tetramer, and have determined conditions for the growth of two-dimensional crystals in vitro, Analysis of the proteins in the crystals by 2-D gel electrophoresis demonstrates the presence of all ribosomal proteins normally found in polysomes. There are in addition, four proteins which may facilitate crystallization. The crystals are built from two oppositely facing P4 layers and the predominant crystal form, accounting for >80% of the crystals, has the tetragonal space group P4212, X-ray diffraction of crystal pellets demonstrates that crystalline order extends to ~ 60Å.

Structural integrity after cold storage of human epidermal model in hypothermosol: A correlative ultrastructural and fluorescent assay

1994 ◽  
Vol 52 ◽  
pp. 270-271
Author(s):  
Henry H. Eichelberger ◽  
John G. Baust ◽  
Robert G. Van Buskirk
Keyword(s):  
Cold Storage ◽  
Structural Integrity ◽  
Culture Media ◽  
Cell Structure ◽  
Natural State ◽  
Sodium Cacodylate ◽  
Ruthenium Tetroxide ◽  
Cacodylate Buffer ◽  
Before And After

For research in cell differentiation and in vitro toxicology it is essential to provide a natural state of cell structure as a benchmark for interpreting results. Hypothermosol (Cryomedical Sciences, Rockville, MD) has proven useful in insuring the viability of synthetic human epidermis during cold-storage and in maintaining the epidermis’ ability to continue to differentiate following warming.Human epidermal equivalent, EpiDerm (MatTek Corporation, Ashland, MA) consisting of fully differentiated stratified human epidermal cells were grown on a microporous membrane. EpiDerm samples were fixed before and after cold-storage (4°C) for 5 days in Hypothermosol or skin culture media (MatTek Corporation) and allowed to recover for 7 days at 37°C. EpiDerm samples were fixed 1 hour in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2). A secondary fixation with 0.2% ruthenium tetroxide (Polysciences, Inc., Warrington, PA) in sodium cacodylate was carried out for 3 hours at 4°C. Other samples were similarly fixed, but with 1% Osmium tetroxide in place of ruthenium tetroxide. Samples were dehydrated through a graded acetone series, infiltrated with Spurrs resin (Polysciences Inc.) and polymerized at 70°C.

1163: Radial Dilation of Ureteral Balloons: A Comparative in-Vitro Analysis

2005 ◽  
Vol 173 (4S) ◽  
pp. 315-316
Author(s):  
Kari Hendlin ◽  
Brynn Lund ◽  
Manoj Monga
Keyword(s):  
Vitro Analysis ◽  
In Vitro Analysis

Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

1999 ◽  
Vol 81 (06) ◽  
pp. 951-956 ◽  
Author(s):  
J. Corral ◽  
R. González-Conejero ◽  
J. Rivera ◽  
F. Ortuño ◽  
P. Aparicio ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cell Types ◽  
Receptor Expression ◽  
Case Control Studies ◽  
Platelet Surface ◽  
Vitro Analysis ◽  
And Function ◽  
In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

An In Vitro Analysis of Sodium Hypochlorite Decontamination for the Reuse of Implant Healing Abutments

2020 ◽  
Author(s):  
Ahmad Almehmadi
Keyword(s):  
Sodium Hypochlorite ◽  
Bacterial Culture ◽  
Brain Heart Infusion ◽  
In Vitro Study ◽  
Vitro Study ◽  
Sem Images ◽  
Streptococcus Sanguis ◽  
Implant Dentistry ◽  
Vitro Analysis

Abstract The re-use of healing abutments (HAs) has become common practice in implant dentistry for economic concerns and the aim of this in-vitro study was to assess the effect of sodium hypochlorite (NaOCl) in decontamination of HAs. 122 HAs (Used and sterilized n=107; New n=15) were procured from 3 centers, of which 3 samples were discarded due to perforation in sterilization pouch.  For sterility assessment, the used HAs (n=80) were cultured in Brain Heart Infusion Broth (BHI) and Potato Dextrose Agar (PDA), bacterial isolates were identified in 7 samples. Also, 24 used HAs were stained with Phloxine B, photographed and compared to new HAs (n=5). Scanning electron microscope (SEM) assessed the differences between the two sets of HAs, following which the 7 contaminated HAs along with 24 used HAs from staining experiment (Total=31) were subsequently treated with sodium hypochlorite (NaOCl) and SEM images were observed. About 8.75% of HAs tested positive in bacterial culture; Streptococcus sanguis, Dermabacter hominis, Staphylococcus haemolyticus, and Aspergillus species were isolated. Phloxine B staining was positive for used and sterilized HAs when compared to controls. The SEM images revealed deposits in the used HAs and although treatment with NaOCl eliminated the contamination of cultured HAs, the SEM showed visible debris in the HA thread region. This in-vitro study concluded that SEM images showed debris in used HAs at screw-hole and thread regions even though they tested negative in bacterial culture. The treatment with NaOCl of used HAs showed no bacterial contamination but the debris was observed in SEM images. Future studies on the chemical composition, biological implications, and clinical influence is warranted before considering the reuse of HAs.

Hydrogels: A Versatile Drug Delivery Carrier Systems

1970 ◽  
Vol 5 (3) ◽  
pp. 1745-1756
Author(s):  
L H Baldaniya ◽  
Sarkhejiya N A
Keyword(s):  
Drug Delivery ◽  
Protein Delivery ◽  
Structural Integrity ◽  
Healing Process ◽  
Polymer Chains ◽  
Wound Healing Process ◽  
Aqueous Environment ◽  
Preparation Methods ◽  
Control Drug

Hydrogels are the material of choice for many applications in regenerative medicine due to their unique properties including biocompatibility, flexible methods of synthesis, range of constituents, and desirable physical characteristics. Hydrogel (also called Aquagel) is a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (contain ~99.9% water), natural or synthetic polymers. Hydrogel also possess a degree of flexibility very similar to natural tissue, due to its significant water content. It can serve as scaffolds that provide structural integrity to tissue constructs, control drug and protein delivery to tissues and cultures. Also serve as adhesives or barriers between tissue and material surfaces. The positive effect of hydrogels on wounds and enhanced wound healing process has been proven. Hydrogels provide a warm, moist environment for wound that makes it heal faster in addition to its useful mucoadhesive properties. Moreover, hydrogels can be used as carriers for liposomes containing variety of drugs, such as antimicrobial drugs. Hydrogels are water swollen polymer matrices, with a tendency to imbibe water when placed in aqueous environment. This ability to swell, under biological conditions, makes it an ideal material for use in drug delivery and immobilization of proteins, peptides, and other biological compounds. Hydrogels have been extensively investigated for use as constructs to engineer tissues in vitro. This review describes the properties, classification, preparation methods, applications, various monomer used in formulation and development of hydrogel products.

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