Post-VX exposure treatment of rats with engineered phosphotriesterases

AbstractThe biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M−1 min−1) towards VX and a preferential hydrolysis of the more toxic P(−) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg−1 i.v.: PTE-2 dosed at 1.3 mg kg−1 i.v. (PTE-2.1) and 2.6 mg kg−1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg−1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.

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Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes

Microbiology ◽

10.1099/mic.0.27066-0 ◽

2004 ◽

Vol 150 (7) ◽

pp. 2257-2266 ◽

Cited By ~ 68

Author(s):

Helmuth Adelsberger ◽

Christian Hertel ◽

Erich Glawischnig ◽

Vladimir V. Zverlov ◽

Wolfgang H. Schwarz

Keyword(s):

Extracellular Enzymes ◽

Enzyme System ◽

Thermophilic Bacterium ◽

Recombinant Enzymes ◽

Type Mode ◽

Complete Set ◽

First Time ◽

Hydrolysis Of

Four extracellular enzymes of the thermophilic bacterium Clostridium stercorarium are involved in the depolymerization of de-esterified arabinoxylan: Xyn11A, Xyn10C, Bxl3B, and Arf51B. They were identified in a collection of eight clones producing enzymes hydrolysing xylan (xynA, xynB, xynC), β-xyloside (bxlA, bxlB, bglZ) and α-arabinofuranoside (arfA, arfB). The modular enzymes Xyn11A and Xyn10C represent the major xylanases in the culture supernatant of C. stercorarium. Both hydrolyse arabinoxylan in an endo-type mode, but differ in the pattern of the oligosaccharides produced. Of the glycosidases, Bxl3B degrades xylobiose and xylooligosaccharides to xylose, and Arf51B is able to release arabinose residues from de-esterified arabinoxylan and from the oligosaccharides generated. The other glycosidases either did not attack or only marginally attacked these oligosaccharides. Significantly more xylanase and xylosidase activity was produced during growth on xylose and xylan. This is believed to be the first time that, in a single thermophilic micro-organism, the complete set of enzymes (as well as the respective genes) to completely hydrolyse de-esterified arabinoxylan to its monomeric sugar constituents, xylose and arabinose, has been identified and the enzymes produced in vivo. The active enzyme system was reconstituted in vitro from recombinant enzymes.

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Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

Circulation Research ◽

10.1161/res.109.suppl_1.ap283 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Allen M Andres ◽

Chengqun Huang ◽

Eric P Ratliff ◽

Genaro Hernandez ◽

Pamela Lee ◽

...

Keyword(s):

Ex Vivo ◽

Wild Type ◽

Simulated Ischemia ◽

Selective Targeting ◽

Cardioprotective Effects ◽

Mitochondrial Turnover ◽

First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

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Essential roles for Pot1b in HSC self-renewal and survival

Blood ◽

10.1182/blood-2011-06-361527 ◽

2011 ◽

Vol 118 (23) ◽

pp. 6068-6077 ◽

Cited By ~ 22

Author(s):

Yang Wang ◽

Mei-Feng Shen ◽

Sandy Chang

Keyword(s):

Progenitor Cell ◽

Essential Role ◽

Dyskeratosis Congenita ◽

Telomere Maintenance ◽

Wild Type ◽

Telomere Shortening ◽

Damage Response ◽

First Time

Abstract Maintenance of mammalian telomeres requires both the enzyme telomerase and shelterin, which protect telomeres from inappropriately activating DNA damage response checkpoints. Dyskeratosis congenita is an inherited BM failure syndrome disorder because of defects in telomere maintenance. We have previously shown that deletion of the shelterin component Pot1b in the setting of telomerase haploinsufficiency results in rapid telomere shortening and fatal BM failure in mice, eliciting phenotypes that strongly resemble human syskeratosis congenita. However, it was unclear why BM failure occurred in the setting of Pot1b deletion. In this study, we show that Pot1b plays an essential role in HSC survival. Deletion of Pot1b results in increased apoptosis, leading to severe depletion of the HSC reserve. BM from Pot1bΔ/Δ mice cannot compete with BM from wild-type mice to provide multilineage reconstitution, indicating that there is an intrinsic requirement for Pot1b the maintenance of HSC function in vivo. Elimination of the p53-dependent apoptotic function increased HSC survival and significantly extended the lifespan of Pot1b-null mice deficient in telomerase function. Our results document for the first time the essential role of a component of the shelterin complex in the maintenance of HSC and progenitor cell survival.

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Epoxide hydrolase 3 (Ephx3) gene disruption reduces ceramide linoleate epoxide hydrolysis and impairs skin barrier function

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016570 ◽

2020 ◽

pp. jbc.RA120.016570

Author(s):

Matthew L. Edin ◽

Haruto Yamanashi ◽

William E. Boeglin ◽

Joan P. Graves ◽

Laura M. DeGraff ◽

...

Keyword(s):

Epoxide Hydrolase ◽

Skin Barrier ◽

Skin Barrier Function ◽

Potential Contribution ◽

Wild Type ◽

Lipoxygenase Pathway ◽

Physiological Relevance ◽

Hydrolysis Of

The mammalian epoxide hydrolase EPHX3 is known from in vitro experiments to efficiently hydrolyze the linoleate epoxides 9,10-epoxyoctadecamonoenoic acid (EpOME) and epoxyalcohol 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoate to corresponding diols and triols, respectively. Herein we examined the physiological relevance of EPHX3 to hydrolysis of both substrates in vivo. Ephx3-/- mice show no deficiency in EpOME-derived plasma diols, discounting a role for EPHX3 in their formation, whereas epoxyalcohol-derived triols esterified in acylceramides of the epidermal 12R-lipoxygenase pathway are reduced. Although the Ephx3-/- pups appear normal, measurements of trans-epidermal water loss detected a modest and statistically significant increase compared to the wild-type or heterozygote mice, reflecting a skin barrier impairment that was not evident in the knockouts of mouse microsomal epoxide hydrolase (EPHX1/mEH) or soluble epoxide hydrolase (EPHX2/sEH). This barrier phenotype in the Ephx3-/- pups was associated with a significant decrease in the covalently bound ceramides in the epidermis (40% reduction, p<0.05), indicating a corresponding structural impairment in the integrity of the water barrier. Quantitative LC-MS analysis of the esterified linoleate-derived triols in the murine epidermis revealed a marked and isomer-specific reduction (~85%) in the Ephx3-/- epidermis of the major trihydroxy isomer 9R,10S,13R-trihydroxy-11E-octadecenoate. We conclude EPHX3 (and not EPHX1 or EPHX2) catalyzes hydrolysis of the 12R-LOX/eLOX3-derived epoxyalcohol esterified in acylceramide, and may function to control flux through the alternative and crucial route of metabolism via the dehydrogenation pathway of SDR9C7. Importantly, our findings also identify a functional role for EPHX3 in transformation of a naturally esterified epoxide substrate, pointing to its potential contribution in other tissues.

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A large panel of chicken cells are invaded in vivo by Salmonella Typhimurium even when depleted of all known invasion factors

Open Biology ◽

10.1098/rsob.210117 ◽

2021 ◽

Vol 11 (11) ◽

Author(s):

S. M. Roche ◽

S. Holbert ◽

Y. Le Vern ◽

C. Rossignol ◽

A. Rossignol ◽

...

Keyword(s):

Gall Bladder ◽

Cell Types ◽

Human Infection ◽

Cell Tropism ◽

Wild Type ◽

Phagocytic Cells ◽

Bladder Cells ◽

First Time

Poultry are the main source of human infection by Salmonella . As infected poultry are asymptomatic, identifying infected poultry farms is difficult, thus controlling animal infections is of primary importance. As cell tropism is known to govern disease, our aim was therefore to identify infected host–cell types in the organs of chicks known to be involved in Salmonella infection and investigate the role of the three known invasion factors in this process (T3SS-1, Rck and PagN). Chicks were inoculated with wild-type or isogenic fluorescent Salmonella Typhimurium mutants via the intracoelomic route. Our results show that liver, spleen, gall bladder and aortic vessels could be foci of infection, and that phagocytic and non-phagocytic cells, including immune, epithelial and endothelial cells, are invaded in vivo in each organ. Moreover, a mutant defective for the T3SS-1, Rck and PagN remained able to colonize organs like the wild-type strain and invaded non-phagocytic cells in each organ studied. As the infection of the gall bladder had not previously been described in chicks, invasion of gall bladder cells was confirmed by immunohistochemistry and infection was shown to last several weeks after inoculation. Altogether, for the first time these findings provide insights into cell tropism of Salmonella in relevant organs involved in Salmonella infection in chicks and also demonstrate that the known invasion factors are not required for entry into these cell types.

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Neurons expressing pathological Tau protein trigger dramatic changes in microglial morphology and dynamics

10.1101/731901 ◽

2019 ◽

Author(s):

Rahma Hassan-Abdi ◽

Alexandre Brenet ◽

Mohamed Bennis ◽

Constantin Yanicostas ◽

Nadia Soussi-Yanicostas

Keyword(s):

Morphological Changes ◽

Pathological Process ◽

Wild Type ◽

Zebrafish Model ◽

Genetic Ablation ◽

Microglial Morphology ◽

First Time ◽

The Brain ◽

Heterogeneous Class

AbstractMicroglial cells, the resident macrophages of the brain, are important players in the pathological process of numerous neurodegenerative disorders, including tauopathies, a heterogeneous class of diseases characterized by intraneuronal Tau aggregates. However, microglia response in Tau pathologies remains poorly understood. Here we exploit a genetic zebrafish model of tauopathy, combined with live microglia imaging, to investigate the behaviour of microglia in vivo in the disease context. Results show that while microglia were almost immobile and displayed long and highly dynamic branches in a wild-type context, in presence of diseased neurons cells became highly mobile and displayed morphological changes, with highly mobile cell bodies together with fewer and shorter processes. We also imaged, for the first time to our knowledge, the phagocytosis of apoptotic tauopathic neurons by microglia in vivo and observed that microglia engulfed about as twice materials as in controls. Finally, genetic ablation of microglia in zebrafish tauopathy model significantly increased Tau hyperphosphorylation, suggesting that microglia provide neuroprotection to diseased neurons. Our findings demonstrate for the first time the dynamics of microglia in contact with tauopathic neurons in vivo and open perspectives for the real-time study of microglia in many neuronal diseases.

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Conversion of TSH Heterodimer to a Single Polypeptide Chain Increases Bioactivity and Longevity

Endocrinology ◽

10.1210/en.2011-1856 ◽

2012 ◽

Vol 153 (2) ◽

pp. 954-960 ◽

Cited By ~ 8

Author(s):

Naiel Azzam ◽

Rinat Bar-Shalom ◽

Fuad Fares

Keyword(s):

Binding Affinity ◽

Half Life ◽

Chinese Hamster ◽

Β Subunit ◽

Wild Type ◽

Single Chain ◽

Glycoprotein Hormone ◽

Α Subunit

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and β genes with [TSHβ- carboxyl-terminal peptide (CTP)-α] or without (TSHβ-α) the CTP of human chorionic gonadotropin β-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHβ-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T4 in circulation after 2 and 4 h of treatment. However, the level of T4 after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHβ-α and TSHβ-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHβ-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

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Twitchin kinase inhibits muscle activity

Molecular Biology of the Cell ◽

10.1091/mbc.e16-10-0707 ◽

2017 ◽

Vol 28 (12) ◽

pp. 1591-1600 ◽

Cited By ~ 4

Author(s):

Yohei Matsunaga ◽

Hyundoo Hwang ◽

Barbara Franke ◽

Rhys Williams ◽

McKenna Penley ◽

...

Keyword(s):

Muscle Activity ◽

Kinase Activity ◽

Kinase Domain ◽

Wild Type ◽

Competitive Fitness ◽

Binding Partners ◽

Fibronectin Domains ◽

First Time

Muscle sarcomeres contain giant polypeptides composed of multiple immunoglobulin and fibronectin domains and one or two protein kinase domains. Although binding partners for a number of this family’s kinase domains have been identified, the catalytic necessity of these kinase domains remains unknown. In addition, various members of this kinase family are suspected pseudokinases with no or little activity. Here we address catalytic necessity for the first time, using the prototypic invertebrate representative twitchin (UNC-22) from Caenorhabditis elegans. In in vitro experiments, change of a conserved lysine (K) that is involved in ATP coordination to alanine (A) resulted in elimination of kinase activity without affecting the overall structure of the kinase domain. The same mutation, unc-22(sf21), was generated in the endogenous twitchin gene. The unc-22(sf21) worms have well-organized sarcomeres. However, unc-22(sf21) mutants move faster than wild-type worms and, by optogenetic experiments, contract more. Wild-type nematodes exhibited greater competitive fitness than unc-22(sf21) mutants. Thus the catalytic activity of twitchin kinase has a role in vivo, where it inhibits muscle activity and is likely maintained by selection.

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Mapping actin surfaces required for functional interactions in vivo.

The Journal of Cell Biology ◽

10.1083/jcb.126.2.423 ◽

1994 ◽

Vol 126 (2) ◽

pp. 423-432 ◽

Cited By ~ 44

Author(s):

D A Holtzman ◽

K F Wertman ◽

D G Drubin

Keyword(s):

Binding Protein ◽

Null Alleles ◽

Actin Binding ◽

Null Mutation ◽

Wild Type ◽

Actin Binding Protein ◽

Functional Interactions ◽

Type Gene ◽

First Time

An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed. This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein. 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein. Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene. Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction. Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin.

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PGI synthase overexpression protects against bleomycin-induced mortality and is associated with increased Nqo 1 expression

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00224.2010 ◽

2011 ◽

Vol 301 (4) ◽

pp. L615-L622 ◽

Cited By ~ 14

Author(s):

Weisong Zhou ◽

Dustin R. Dowell ◽

Mark W. Geraci ◽

Timothy S. Blackwell ◽

Robert D. Collins ◽

...

Keyword(s):

Airway Epithelium ◽

Death Rate ◽

Treatment Strategies ◽

Oxygen Species ◽

In Vitro Experiments ◽

Wild Type ◽

Potential Therapeutic Target ◽

First Time

The mortality rate for acute lung injury (ALI) is reported to be between 35–40%, and there are very few treatment strategies that improve the death rate from this condition. Previous studies have suggested that signaling through the prostaglandin (PG) I2 receptor may protect against bleomycin-induced ALI in mice. We found that mice that overexpress PGI synthase (PGIS) in the airway epithelium were significantly protected against bleomycin-induced mortality and had reduced parenchymal consolidation, apoptosis of lung tissue, and generation of F2-isoprostanes compared with littermate wild-type controls. In addition, we show for the first time in both in vivo and in vitro experiments that PGI2 induced the expression of NADP (H): quinoneoxidoreductase 1 (Nqo 1), an enzyme that prevents the generation of reactive oxygen species. PGI2 induction of Nqo 1 provides a possible novel mechanism by which this prostanoid protects against bleomycin-induced mortality and identifies a potential therapeutic target for human ALI.

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