The Redox Potential of the β-93-Cysteine Thiol Group in Human Hemoglobin Estimated from In Vitro Oxidant Challenge Experiments

Glutathionyl hemoglobin is a minor form of hemoglobin with intriguing properties. The measurement of the redox potential of its reactive β-93-Cysteine is useful to improve understanding of the response of erythrocytes to transient and chronic conditions of oxidative stress, where the level of glutathionyl hemoglobin is increased. An independent literature experiment describes the recovery of human erythrocytes exposed to an oxidant burst by measuring glutathione, glutathione disulfide and glutathionyl hemoglobin in a two-hour period. This article calculates a value for the redox potential E0 of the β-93-Cysteine, considering the erythrocyte as a closed system at equilibrium described by the Nernst equation and using the measurements of the literature experiment. The obtained value of E0 of −121 mV at pH 7.4 places hemoglobin as the most oxidizing thiol of the erythrocyte. By using as synthetic indicators of the concentrations the electrochemical potentials of the two main redox pairs in the erythrocytes, those of glutathione–glutathione disulfide and of glutathionyl–hemoglobin, the mechanism of the recovery phase can be hypothesized. Hemoglobin acts as the redox buffer that scavenges oxidized glutathione in the oxidative phase and releases it in the recovery phase, by acting as the substrate of the NAD(P)H-cofactored enzymes.

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Kangaroo IGF-II is structurally and functionally similar to the human [Ser29]-IGF-II variant

Journal of Endocrinology ◽

10.1677/joe.0.1610445 ◽

1999 ◽

Vol 161 (3) ◽

pp. 445-453 ◽

Cited By ~ 7

Author(s):

CA Yandell ◽

GL Francis ◽

JF Wheldrake ◽

Z Upton

Keyword(s):

Amino Acid ◽

Human Serum ◽

Cdna Sequence ◽

Mammalian Species ◽

In Vitro Assays ◽

Igf Binding Proteins ◽

Western Grey Kangaroo ◽

Minor Form ◽

A Minor

Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser29]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein. The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser29]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cation-independent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser29]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.

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In vivo Measurement of the Volume of Distribution of Water in Cerebral Grey Matter: Effects on the Calculation of Regional Cerebral Blood Flow

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1992.39 ◽

1992 ◽

Vol 12 (2) ◽

pp. 291-295 ◽

Cited By ~ 11

Author(s):

A. A. Lammertsma ◽

A. J. Martin ◽

K. J. Friston ◽

T. Jones

Keyword(s):

Volume Of Distribution ◽

Regional Cerebral Blood ◽

Generating Functional ◽

In Vivo Measurement ◽

A Value ◽

Cerebral Grey Matter ◽

Washout Phase ◽

A Minor

The present study was undertaken to determine the apparent value for the volume of distribution of water to be used in the dynamic/integral technique for generating functional CBF images. A value of 0.86 resulted in only a minor loss of accuracy compared to the more accurate (but time-consuming) dynamic only analysis, which incorporated the regionally fitted estimates of the volume of distribution of water. In contrast to the traditionally used in vitro value of 0.95, the value of 0.86 allows for the inclusion of a significant part of the washout phase in the integral analysis, thereby producing statistically improved CBF images.

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Isolation of Bacillus subtilis mutants altered in expression of a gene transcribed in vitro by a minor form of RNA polymerase (E-sigma 37).

Journal of Bacteriology ◽

10.1128/jb.161.2.515-522.1985 ◽

1985 ◽

Vol 161 (2) ◽

pp. 515-522 ◽

Cited By ~ 7

Author(s):

C L Truitt ◽

G L Ray ◽

J E Trempy ◽

Z Da-Jian ◽

W G Haldenwang

Keyword(s):

Bacillus Subtilis ◽

Rna Polymerase ◽

Minor Form ◽

A Minor

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Generation Of Factor XII-Dependent Plasminogen Activator Activity In Human Plasma Measured With A Fluorogenic Substrate

10.1055/s-0038-1652135 ◽

1981 ◽

Author(s):

G Dooijewaard ◽

C Kluft

Keyword(s):

Human Plasma ◽

Plasminogen Activator ◽

Minor Role ◽

Factor Xii ◽

Dextran Sulphate ◽

Fluorogenic Substrate ◽

A Value ◽

Substrate Activation ◽

Factor Xiia ◽

A Minor

A rapid fluorometric assay for measurement of amidolytic activity in human plasma was developed, using the plasminogen activator sensitive synthetic substrate t-BOC-L-valyl--glycyl-L-arginine-β-naphthylamide. The plasma is diluted in a reaction cuvet containing 0.050 M Tris HC1 buffer (pH 8.0) and 150 μM substrate. Activation of plasminogen proactivator(s) is initiated at 37°C by the addition of 10 μg dextran sulphate (MW 500,000)/ml. The concentration of β-naphthyl- amide released is recorded fluorometrically as a function of time. The slope of this curve at any time t is proportional to the concentration of activator. Thus, in a single assay, the entire time-dependent profile of activation and subsequent inhibition is monitored; this provides 1. a value for an optimum plasminogen activator content in the plasma, and 2. the time it takes to reach the optimum. The plot of optimum activator content against μl of plasma added is linear for dilutions more than 100-fold, suggesting that under these conditions the optimum content approaches the content of proactivator(s) originally present.The activator content measured predominantly consists of contributions of a factor XII-dependent process since 1. without dextran sulphate or with plasmas deficient in factor XII or prekallikrein no activity could be generated, and 2. plots of optimum activator content against dextran sulphate concentration show sigmoidal-shaped saturation curves as found previously for the kallikrein generation in human plasma. Contributions of factor XIIa and kallikrein only partly account for the content measured and studies with plasmas deficient in factor XI point to a minor role for this factor, if any. Further identification of the activator (s) involved is in progress.

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1250. Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1434 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S643-S643

Author(s):

Maria F Mojica ◽

Christopher Bethel ◽

Emilia Caselli ◽

Magdalena A Taracila ◽

Fabio Prati ◽

...

Keyword(s):

Active Site ◽

Inhibitory Activity ◽

Covalent Bond ◽

Thiol Group ◽

Viable Cell ◽

Free Thiol ◽

Transition State Analogs ◽

Cell Counts ◽

Free Thiol Group

Abstract Background Catalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs- form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn2+ ions and displace the nucleophilic OH-. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL. Methods Exploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBL-producing strains. Results Kinetic assays revealed that IC50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log10-fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL- and MBL-producing E. coli (Table 1). Table 1 Conclusion Addition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases. Disclosures Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

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In-Vitro Evaluation of the Antioxidant and Anti-Inflammatory Activity of Volatile Compounds and Minerals in Five Different Onion Varieties

Separations ◽

10.3390/separations8050057 ◽

2021 ◽

Vol 8 (5) ◽

pp. 57

Author(s):

Rokayya Sami ◽

Abeer Elhakem ◽

Mona Alharbi ◽

Manal Almatrafi ◽

Nada Benajiba ◽

...

Keyword(s):

Volatile Compounds ◽

Dimethyl Sulfide ◽

Antioxidant Activities ◽

Dry Weight ◽

No Production ◽

Oxidative Stresses ◽

Anti Inflammatory ◽

Red Variety ◽

A Minor

Onions contain high antioxidants compounds that fight inflammation against many diseases. The purpose was to investigate some selected bioactive activities of onion varieties (Yellow, Red, Green, Leek, and Baby). Antioxidant assays and anti-inflammatory activities such as NO production with the addition of some bioactive components were determined and analyzed by using a spectrophotometer. Gas chromatography and mass spectrometry (GC–MS) was used for the volatile compounds, while an Atomic absorption spectrometer was used for mineral determinations. Red variety achieved the highest antioxidant activities. The total flavonoids were between (12.56 and 353.53 mg Quercetin/gin dry weight) (dw) and the total phenol was (8.75–25.73 mg/g dw). Leek, Yellow and Green extracts achieved highly anti-inflammatory values (3.71–4.01 μg/mL) followed by Red and Baby extracts, respectively. The highest contents of sodium, potassium, zinc, and calcium were established for Red onions. Furfuraldehyde, 5-Methyl-2-furfuraldehyde, 2-Methyl-2-pentenal, and 1-Propanethiol were the most predominant, followed by a minor abundance of the other compounds such as Dimethyl sulfide, Methyl allyl disulfide, Methyl-trans-propenyl-disulfide, and Methyl propyl disulfide. The results recommend that these varieties could act as sources of essential antioxidants and anti-inflammatories to decrease inflammation and oxidative stresses, especially red onions that recorded high activities.

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Characterization of the SARS-CoV-2 coronavirus X4-like accessory protein

Egyptian Journal of Medical Human Genetics ◽

10.1186/s43042-021-00160-1 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Olanrewaju Ayodeji Durojaye ◽

Nkwachukwu Oziamara Okoro ◽

Arome Solomon Odiba

Keyword(s):

Rapid Development ◽

Protein A ◽

The Novel ◽

Quality Model ◽

A Value ◽

Model Protein ◽

Novel Coronavirus ◽

Global Threat

Abstract Background The novel coronavirus SARS-CoV-2 is currently a global threat to health and economies. Therapeutics and vaccines are in rapid development; however, none of these therapeutics are considered as absolute cure, and the potential to mutate makes it necessary to find therapeutics that target a highly conserved regions of the viral structure. Results In this study, we characterized an essential but poorly understood coronavirus accessory X4 protein, a core and stable component of the SARS-CoV family. Sequence analysis shows a conserved ~ 90% identity between the SARS-CoV-2 and previously characterized X4 protein in the database. QMEAN Z score of the model protein shows a value of around 0.5, within the acceptable range 0–1. A MolProbity score of 2.96 was obtained for the model protein and indicates a good quality model. The model has Ramachandran values of φ = − 57o and ψ = − 47o for α-helices and values of φ = − 130o and ψ = + 140o for twisted sheets. Conclusions The protein data obtained from this study provides robust information for further in vitro and in vivo experiment, targeted at devising therapeutics against the virus. Phylogenetic analysis further supports previous evidence that the SARS-CoV-2 is positioned with the SL-CoVZC45, BtRs-BetaCoV/YN2018B and the RS4231 Bat SARS-like corona viruses.

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Species pattern of phosphatidylinositol from lung surfactant and a comparison of the species pattern of phosphatidylinositol and phosphatidylglycerol synthesized de novo in lung microsomal fractions

Biochemical Journal ◽

10.1042/bj2540067 ◽

1988 ◽

Vol 254 (1) ◽

pp. 67-71 ◽

Cited By ~ 9

Author(s):

B Rüstow ◽

Y Nakagawa ◽

H Rabe ◽

K Waku ◽

D Kunze

Keyword(s):

Lung Function ◽

Molecular Species ◽

De Novo ◽

Lung Surfactant ◽

Minor Component ◽

Main Species ◽

Surfactant Phospholipids ◽

Species Pattern ◽

A Minor

1. Phosphatidylinositol (PI) is a minor component of lung surfactant which may be able to replace the functionally important phosphatidylglycerol (PG) [Beppu, Clements & Goerke (1983) J. Appl. Physiol. 55, 496-502] without disturbing lung function. The dipalmitoyl species is one of the main species for both PI (14.4%) and PG (16.9%). Besides the C16:0--C16:0 species, the C16:0--C18:0, C16:0--C18:1, C16:0--C18:2 and C18:0--C18:1 species showed comparable proportions in the PG and PI fractions. These similarities of the species patterns and the acidic character of both phospholipids could explain why surfactant PG may be replaced by PI. 2. PI and PG were radiolabelled by incubation of microsomal fractions with [14C]glycerol 3-phosphate (Gro3P). For 11 out of 14 molecular species of PI and PG we measured comparable proportions of radioactivity. The radioactivity of these 11 species accounted together for more than 80% of the total. The addition of inositol to the incubation system decreased the incorporation in vitro of Gro3P into PG and CDP-DG (diacylglycerol) of lung microsomes (microsomal fractions), but did not change the distribution of radioactivity among the molecular species of PG. These results supported the idea that both acidic surfactant phospholipids may be synthesized de novo from a common CDP-DG pool in lung microsomes.

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The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

Journal of Virology ◽

10.1128/jvi.76.3.1015-1024.2002 ◽

2002 ◽

Vol 76 (3) ◽

pp. 1015-1024 ◽

Cited By ~ 36

Author(s):

Barbara Müller ◽

Tilo Patschinsky ◽

Hans-Georg Kräusslich

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Metabolic Labeling ◽

Immunodeficiency Virus ◽

Infected Cells ◽

A Minor ◽

Hiv 1

ABSTRACT The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho- 32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.

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