scholarly journals Isolated domains of recombinant human apo-metallothionein 1A are folded at neutral pH: a denaturant and heat-induced unfolding study using ESI-MS

2018 ◽  
Vol 38 (4) ◽  
Author(s):  
Gordon W. Irvine ◽  
Natalie Korkola ◽  
Martin J. Stillman
Keyword(s):  
Critical Role ◽  
Cysteine Residues ◽  
Small Molecular Weight ◽  
Esi Ms ◽  
Neutral Ph ◽  
Induced Folding ◽  
Starting Point ◽  
High Metal ◽  
High Concentrations ◽  
Product Species

Metallothioneins (MTs) are characterized by their high metal loading capacity, small molecular weight, and abundant cysteine residues. It has long been thought that metal-free, or apo-MT peptides were unstructured and only adopted as a distinct conformation upon forming the metal clusters, described as metal-induced folding. More recent studies have suggested that the presence of a globular, yet loosely defined structure actually exists that can be disrupted or unfolded. Residue modification and ion-mobility ESI (IM-ESI)-MS have been used to examine this unusual unfolding process. The structure of apo-MT plays a critical role as the starting point in the flexible metalation pathways that can accommodate numerous soft metals. ESI-MS measurements of the product species formed following the cysteine alkylation of the isolated domain fragments of recombinant human apo-MT 1A with n-ethylmaleimide (NEM) were used in the present study to monitor the denaturant- and heat-induced unfolding at physiological pH. The results indicate that these apo-MT fragments adopt distinct structures at neutral pH that react co-operatively with NEM when folded and non-cooperatively when heated or exposed to high concentrations of the denaturant guanidinium chloride (GdmCl). From these studies, we can conclude that at neutral pH, the domain fragments are folded into globular structures where some of the free cysteine residues are buried within the core and are stabilized by hydrogen bonds. Metalation therefore, must take place from the folded conformation.

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

2019 ◽  
Vol 22 (5) ◽  
pp. 346-354
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Large Scale ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Reporter System ◽  
E Coli ◽  
Starting Point ◽  
High Concentrations ◽  
Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

scholarly journals The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis

2007 ◽  
Vol 81 (10) ◽  
pp. 5212-5224 ◽  
Author(s):  
Michael Mach ◽  
Karolina Osinski ◽  
Barbara Kropff ◽  
Ursula Schloetzer-Schrehardt ◽  
Magdalena Krzyzaniak ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Critical Role ◽  
Point Mutations ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Type I ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Assembly Compartment

ABSTRACT Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.

Night-time chemistry of biomass burning plumes in urban areas: A dual mobile chamber study

2021 ◽  
Author(s):  
Spiro Jorga ◽  
Kalliopi Florou ◽  
Christos Kaltsonoudis ◽  
John Kodros ◽  
Christina Vasilakopoulou ◽  
...  
Keyword(s):  
Biomass Burning ◽  
Urban Areas ◽  
Mass Spectra ◽  
Prescribed Burning ◽  
Organic Aerosol ◽  
Photochemical Activity ◽  
Organic Nitrate ◽  
Night Time ◽  
Starting Point ◽  
High Concentrations

<p>Biomass burning including residential heating, agricultural fires, prescribed burning, and wildfires is a major source of gaseous and particulate pollutants in the atmosphere. Although, important changes in the size distributions and the chemical composition of the biomass burning aerosol during daytime chemistry have been observed, the corresponding changes at nighttime or in winter where photochemistry is slow, have received relatively little attention. In this study, we tested the hypothesis that nightime chemistry in biomass burning plumes can be rapid in urban areas using a dual smog chamber system.</p><p> </p><p>Ambient urban air during winter nighttime periods with high concentrations of ambient biomass burning organic aerosol is used as the starting point. Ozone was added in the perturbed chamber to simulate mixing with background air (and subsequent NO<sub>3</sub> production and aging) while the second chamber was used as a reference. Following the injection of ozone rapid organic aerosol (OA) formation was observed in all experiments leading to increases of the OA concentration by 20-70%. The oxygen to carbon ratio of the OA increased by 50% on average and the mass spectra of the produced OA was quite similar to that of the oxidized OA mass spectra reported during winter in urban areas. Good correlation was also observed with the produced mass spectra from nocturnal aging of laboratory biomass burning emissions showing the strong contribution of biomass burning emissions in the SOA formation during cold nights with high biomass burning activities. Concentrations of NO<sub>3</sub> radicals as high as 25 ppt were measured in the perturbed chamber with an accompanying production of 0.2-1.2 μg m<sup>-3</sup> of organic nitrate. These results strongly indicate that the OA in biomass burning plumes can evolve rapidly even during wintertime periods with low photochemical activity.</p>

scholarly journals Some Inequalities for LR-$$\left({h}_{1}, {h}_{2}\right)$$-Convex Interval-Valued Functions by Means of Pseudo Order Relation

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Muhammad Bilal Khan ◽  
Muhammad Aslam Noor ◽  
Khalida Inayat Noor ◽  
Kottakkaran Sooppy Nisar ◽  
Khadiga Ahmed Ismail ◽  
...  
Keyword(s):  
Applied Mathematics ◽  
Critical Role ◽  
Order Relation ◽  
Strong Relationship ◽  
New Class ◽  
Starting Point ◽  
Interval Valued Function ◽  
Fejér Inequality ◽  
Definition Of ◽  
Interval Valued

AbstractIn both theoretical and applied mathematics fields, integral inequalities play a critical role. Due to the behavior of the definition of convexity, both concepts convexity and integral inequality depend on each other. Therefore, the relationship between convexity and symmetry is strong. Whichever one we work on, we introduced the new class of generalized convex function is known as LR-$$\left({h}_{1}, {h}_{2}\right)$$ h 1 , h 2 -convex interval-valued function (LR-$$\left({h}_{1}, {h}_{2}\right)$$ h 1 , h 2 -IVF) by means of pseudo order relation. Then, we established its strong relationship between Hermite–Hadamard inequality (HH-inequality)) and their variant forms. Besides, we derive the Hermite–Hadamard–Fejér inequality (HH–Fejér inequality)) for LR-$$\left({h}_{1}, {h}_{2}\right)$$ h 1 , h 2 -convex interval-valued functions. Several exceptional cases are also obtained which can be viewed as its applications of this new concept of convexity. Useful examples are given that verify the validity of the theory established in this research. This paper’s concepts and techniques may be the starting point for further research in this field.

SUSTAINABLE DESIGN IN PROJECT DELIVERY: A DISCUSSION ON CURRENT AND FUTURE TRENDS

2016 ◽  
Vol 11 (2) ◽  
pp. 39-56 ◽  
Author(s):  
Karno Widjaja
Keyword(s):  
Construction Projects ◽  
Sustainable Design ◽  
Current System ◽  
Critical Role ◽  
Project Delivery ◽  
Starting Point ◽  
New Construction ◽  
Communication And Coordination ◽  
The Individual ◽  
Sustainability Principles

INTRODUCTION Sustainability principles have become an integral part of the design and construction process for many new construction projects. The selection of the project delivery method (PDM) is extremely important in the effective execution of the project, and plays a critical role in establishing communication and coordination between the key entities: owner, architect, and contractor. The goal of this paper is to hopefully serve as a starting point for further discussion to improve on the AEC industry's current integration of sustainability principles in PDMs. The first step consists of an assessment of current project delivery systems from a sustainable design perspective. This is followed by a determination of the current limitations, and examination of the various disruptions in the industry. Various literature sources are analyzed to form a framework to discuss improvements and optimization strategies beyond the current system. Thereafter, proposed solutions are introduced at both stakeholders, as well as PDM scales. In this paper, the focus for the conducted analysis and proposed methodologies is predicated on new construction projects instead of retrofits due to the resources available. However, the principles can similarly be applied to retrofit scenarios as well, depending on the specific requirements of the individual project at play.

Can alcohol retain the reproductive and genetic potential of sperm nuclei? Chromosome analysis of mouse spermatozoa stored in alcohol

Zygote ◽  
1998 ◽  
Vol 6 (3) ◽  
pp. 233-238 ◽  
Author(s):  
Hiroyuki Tateno ◽  
Teruhiko Wakayama ◽  
W. Steven Ward ◽  
R. Yanagimachi
Keyword(s):  
Primary Structure ◽  
Reproductive Potential ◽  
Chromosome Analysis ◽  
Sperm Chromatin ◽  
Small Molecular Weight ◽  
Simple Method ◽  
Sperm Nuclei ◽  
High Concentrations ◽  
Dna Strand ◽  
Normal Offspring

Alcohol is known to preserve genomic DNA and the primary structure of sperm protamines. To determine whether alcohol can retain the genetic and reproductive potential of mammalian sperm nuclei, mature mouse spermatozoa were stored in 70% ethanol or propanol for up to 2 months before injection into oocytes. Live offspring were obtained after injection of spermatozoa stored in 70% ethanol for 1 day at -20 °C. About 20% of the spermatozoa stored under this condition had normal chromosomes. The remaining 80% of spermatozoa and all the spermatozoa stored in 70% ethanol for 2 months had structurally aberrant chromosomes, and none could support the development of normal embryos. High concentrations of alcohol do not alter the primary structure of either DNA or small-molecular-weight protamines. However, alcohol may modify protamine—protamine or protamine—DNA interactions in a manner that results in the induction of DNA strand breaks during sperm chromatin decondensation within the oocyte. The limited success in obtaining normal offspring with ethanol-stored spermatozoa is encouraging. It may be possible to overcome these problems and develop a simple method for preserving mammalian spermatozoa without freezing.

scholarly journals Inherited dysfunctional platelet P2Y12 receptor mutations associated with bleeding disorders

2016 ◽  
Vol 36 (04) ◽  
pp. 279-283 ◽  
Author(s):  
Anna Lecchi ◽  
Eti Femia ◽  
Silvia Paoletta ◽  
Arnaud Dupuis ◽  
Philippe Ohlmann ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Integrity ◽  
Present Report ◽  
Critical Role ◽  
Receptor Expression ◽  
Severe Bleeding ◽  
Modelling Studies ◽  
Agonist And Antagonist ◽  
High Concentrations

SummaryThe platelet adenosine 5’-diphosphate (ADP) receptor P2Y12 (P2Y12R) plays a critical role in platelet aggregation. The present report illustrates an update of dysfunctional platelet P2Y12R mutations diagnosed with congenital lifelong bleeding problems. Described patients with heterozygous or homozygous substitution in the P2Y12R gene and qualitative abnormalities of the platelet P2Y12R are summarized. Recently, a further dysfunctional variant of P2Y12R has been identified in two brothers who presented with a lifelong severe bleeding disorder. During in vitro aggregation studies, the patient´s platelets show a markedly reduced and rapid reversible ADP-promoted aggregation. A homozygous c.561T>A substitution that changes the codon for His187 to Gln (p.His187Gln) in the P2Y12R gene has been identified. This mutation causes no change in receptor expression but decreases the affinity of the ligand for the receptor, even at high concentrations. Structure modelling studies indicated that the p.His187Gln mutation, located in the fifth transmembrane spanning domain (TM5), impairs conformational changes of the receptor. Structural integrity of the TM5 region is necessary for agonist and antagonist binding and for correct receptor function.

Copper Resistance Mechanisms of Biomining Bacteria and Archaea Living under Extremely High Concentrations of Metals

2009 ◽  
Vol 71-73 ◽  
pp. 279-282 ◽  
Author(s):  
A. Orell ◽  
C.A. Navarro ◽  
Carlos A. Jerez
Keyword(s):  
Resistance Mechanisms ◽  
Metal Resistance ◽  
Copper Resistance ◽  
Inorganic Polyphosphate ◽  
Resistance Determinants ◽  
High Metal ◽  
Pi Complex ◽  
Cu Resistance ◽  
High Concentrations ◽  
Cu Tolerance

Extremophiles such as the acidophilic Sulfolobus metallicus (Archaea) and Acidithiobacillus ferrooxidans (Bacteria) can resist Cu (CuSO4) concentrations of 200 mM and 800 mM respectively. These microorganisms are important in biomining processes to extract copper and other metals. A. ferrooxidans grown at low Cu concentrations (5 mM) expressed genes coding for ATPases most likely involved in pumping the metal from the cytoplasm to the periplasm of the bacterium. At 100 mM Cu the previous systems were repressed and there was a great induction in the expression of efflux systems known to use the proton motive force energy to export the metal outside the cell. These Cu-resistance determinants from A. ferrooxidans were found to be functional since when expressed in Escherichia coli they conferred higher Cu tolerance to it. Novel Cu-resistance determinants for A. ferrooxidans were found and characterized. S. metallicus possessed at least 2 CopM metallochaperones and 2 CopA ATPases whose expressions were induced by Cu (5 to 50 mM). Furthermore, we previously reported that both microorganisms accumulate high levels of inorganic polyphosphate (PolyP) and that intracellular Cu concentration stimulates polyP hydrolysis. The resulting Pi would then be transported out of the cell as a metal-Pi complex to detoxify the cells. In addition, our results suggest that at high Cu concentrations polyP could also provide energy for the metal efflux. All the data suggest that both biomining microorganisms use different systems to respond to Cu depending on the extracellular concentrations of the metal and suggest that the presence of different additional systems to respond to Cu may explain the extremely high metal resistance of these extremophiles.

Isolation and characterization of a bacteriocin produced by Bacillus stearothermophilus strain NU-10

1976 ◽  
Vol 22 (12) ◽  
pp. 1743-1750 ◽  
Author(s):  
Ronald Yule ◽  
B. D. Barridge
Keyword(s):  
Molecular Weight ◽  
Stationary Phase ◽  
Bacillus Stearothermophilus ◽  
Proteolytic Enzymes ◽  
Small Molecular Weight ◽  
Optimal Production ◽  
Chemical Treatments ◽  
Neutral Ph ◽  
Isolation And Characterization

Broth-grown cultures of Bacillus stearothermophilus strain NU-10 produce a bacteriocin which exerts lethal activity on other strains of the bacterium. Optimal production occurs during late maximum stationary phase of growth, at neutral pH, and 55–65 °C. The bacteriocin can be substantially purified by a combination of precipitations, centrifugations, and gel filtrations. The thermocin is composed of protein and carbohydrate. It is partially destroyed by proteolytic enzymes but is resistant to DNase, RNase, and various chemical treatments. The bacteriocin has a small molecular weight and exhibits considerable thermostability.

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