Characterization of genetically modified mice for phosphoglycerate mutase, a vitally-essential enzyme in glycolysis

Glycolytic metabolism is closely involved in physiological homeostasis and pathophysiological states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been reported to exert certain physiological role in vitro, whereas its impact on glucose metabolism in vivo remains unclear. Here, we report the characterization of Pgam1 knockout mice. We observed that homozygous knockout mice of Pgam1 were embryonic lethal. Although we previously reported that both PGAM-1 and -2 affect global glycolytic profile of cancers in vitro, in vivo glucose parameters were less affected both in the heterozygous knockout of Pgam1 and in Pgam2 transgenic mice. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before.

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Sigma 1 Receptor is Overexpressed in Hepatocellular Adenoma: Involvement of ERα and HNF1α

Cancers ◽

10.3390/cancers12082213 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2213

Author(s):

Laure Villemain ◽

Sylvie Prigent ◽

Aurélie Abou-Lovergne ◽

Laura Pelletier ◽

Magali Chiral ◽

...

Keyword(s):

Knockout Mice ◽

Hepatocellular Adenoma ◽

Integral Membrane Protein ◽

Hepatocyte Proliferation ◽

Sigma 1 Receptor ◽

Cellular Models ◽

Huh7 Cells ◽

The Impact

Sigma receptor 1 (SigR1) is an endoplasmic reticulum resident integral membrane protein whose functions remain unclear. Although the liver shows the highest expression of SigR1, its role in this organ is unknown. SigR1 is overexpressed in many cancers and its expression is correlated to hormonal status in hormone-dependent cancers. To better understand the role of SigR1 in hepatocytes we focused our work on the regulation of its expression in tumoral liver. In this context, hepatocellular adenomas, benign hepatic tumors associated with estrogen intake are of particular interest. The expression of SigR1 mRNA was assessed in hepatocellular adenoma (HCA) patients using qPCR. The impact of estrogen on the expression of SigR1 was studied in vivo (mice) and in vitro (HepG2 and Huh7 cells). The effect of HNF1α on the expression of SigR1 was studied in vivo by comparing wild type mice to HNF1 knockout mice. Estrogen enhanced SigR1 expression through its nuclear receptor ERα. HNF1α mutated HCA (H-HCA) significantly overexpressed SigR1 compared to all other HCA subtypes. HNF1 knockout mice showed an increase in SigR1 expression. Overexpressing SigR1 in cellular models increases proliferation rate and storage of lipid droplets, which phenocopies the H-HCA phenotype. SigR1 is involved in hepatocyte proliferation and steatosis and may play an important role in the control of the H-HCA phenotype.

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Transgenic overexpression of intraislet ghrelin does not affect insulin secretion or glucose metabolism in vivo

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00341.2011 ◽

2012 ◽

Vol 302 (4) ◽

pp. E403-E408 ◽

Cited By ~ 8

Author(s):

Mika Bando ◽

Hiroshi Iwakura ◽

Hiroyuki Ariyasu ◽

Hiroshi Hosoda ◽

Go Yamada ◽

...

Keyword(s):

Insulin Secretion ◽

Glucose Metabolism ◽

Medium Chain Triglyceride ◽

Physiological Role ◽

Standard Diet ◽

Entry Site ◽

Insulin Secretion In Vivo

Whereas ghrelin is produced primarily in the stomach, a small amount of it is produced in pancreatic islets. Although exogenous administration of ghrelin suppresses insulin secretion in vitro or in vivo, the role of intraislet ghrelin in the regulation of insulin secretion in vivo remains unclear. To understand the physiological role of intraislet ghrelin in insulin secretion and glucose metabolism, we developed a transgenic (Tg) mouse model, rat insulin II promoter ghrelin-internal ribosomal entry site-ghrelin O-acyl transferase (RIP-GG) Tg mice, in which mouse ghrelin cDNA and ghrelin O-acyltransferase are overexpressed under the control of the rat insulin II promoter. Although pancreatic desacyl ghrelin levels were elevated in RIP-GG Tg mice, pancreatic ghrelin levels were not altered in animals on a standard diet. However, when Tg mice were fed a medium-chain triglyceride-rich diet (MCTD), pancreatic ghrelin levels were elevated to ∼16 times that seen in control animals. It seems likely that the gastric ghrelin cells possess specific machinery to provide the octanoyl acid necessary for ghrelin acylation but that this machinery is absent from pancreatic β-cells. Despite the overexpression of ghrelin, plasma ghrelin levels in the portal veins of RIP-GG Tg mice were unchanged from control levels. Glucose tolerance, insulin secretion, and islet architecture in RIP-GG Tg mice were not significantly different even when the mice were fed a MCTD. These results indicate that intraislet ghrelin does not play a major role in the regulation of insulin secretion in vivo.

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MR imaging of model drug distribution in simulated vitreous

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2015-0059 ◽

2015 ◽

Vol 1 (1) ◽

pp. 236-239 ◽

Cited By ~ 2

Author(s):

Sandra Stein ◽

Christian Simroth-Loch ◽

Sönke Langner ◽

Stefan Hadlich ◽

Oliver Stachs ◽

...

Keyword(s):

Molecular Weight ◽

Ex Vivo ◽

Drug Distribution ◽

Injection Technique ◽

Innovative Therapy ◽

Age Related ◽

The Impact

AbstractThe in vitro and in vivo characterization of intravitreal injections plays an important role in developing innovative therapy approaches. Using the established vitreous model (VM) and eye movement system (EyeMoS) the distribution of contrast agents with different molecular weight was studied in vitro. The impact of the simulated age-related vitreal liquefaction (VL) on drug distribution in VM was examined either with injection through the gel phase or through the liquid phase. For comparison the distribution was studied ex vivo in the porcine vitreous. The studies were performed in a magnetic resonance (MR) scanner. As expected, with increasing molecular weight the diffusion velocity and the visual distribution of the injected substances decreased. Similar drug distribution was observed in VM and in porcine eye. VL causes enhanced convective flow and faster distribution in VM. Confirming the importance of the injection technique in progress of VL, injection through gelatinous phase caused faster distribution into peripheral regions of the VM than following injection through liquefied phase. VM and MR scanner in combination present a new approach for the in vitro characterization of drug release and distribution of intravitreal dosage forms.

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Purification and characterization of a mannose-containing disaccharide obtained from human pregnancy urine. A new immunoregulatory saccharide.

Journal of Experimental Medicine ◽

10.1084/jem.160.6.1672 ◽

1984 ◽

Vol 160 (6) ◽

pp. 1672-1685 ◽

Cited By ~ 17

Author(s):

A V Muchmore ◽

J M Decker ◽

R M Blaese ◽

B Nilsson

Keyword(s):

Lymphocyte Culture ◽

Membrane Receptors ◽

Cell Wall Polysaccharide ◽

Human Pregnancy ◽

Isolation And Characterization ◽

Pregnancy Urine ◽

The Impact

Endogenous mammalian lectin-like sugar-binding molecules have been previously described that have immunoregulatory properties. Further, the addition of defined simple saccharides to lymphocyte cultures has been shown to inhibit a variety of in vitro lymphocyte functions, presumably because these sugars are able to compete with the binding of endogenous lectins to critical membrane receptors. In this report, we describe the isolation and characterization of a D-mannose-containing disaccharide in human pregnancy urine that inhibits the proliferative response of human T lymphocytes. The inhibitory disaccharide was purified to homogeneity by sequential steps including affinity chromatography on immobilized concanavalin A and molecular sizing on Sephadex G-75 and then Fractogel 40S columns, with final purification on high-performance thin-layer chromatography. By mass spectrometry of the purified material as its permethylated derivative, the deduced structure of this compound was alpha-D-Manp 1-6-D-Man. To confirm that this disaccharide was in fact immunosuppressive, an identical disaccharide was prepared by sequential digestion of yeast cell wall polysaccharide. The urinary and yeast disaccharides had identical immunosuppressive properties. It has been previously reported that D-mannose is inhibitory for antigen-specific proliferative assays in the range of 10-50 mM. The purified alpha-D-Manp 1-6-D-Man disaccharide was inhibitory at 100-fold-lower concentrations. Further, while D-mannose inhibits T cell proliferation when added at anytime up to 24 h before harvest of a 6-d lymphocyte culture, alpha-D-Manp 1-6-D-Man disaccharide was inhibitory only if added at the initiation of culture and had no inhibitory effect if added just 24 h later. These data support the concept that simple sugar compounds can exhibit marked immunoregulatory activity in vitro. The impact of these molecules on the regulation of immune responses in vivo is unknown, as is their precise mechanism of action, but structural and chemical identification should now permit a detailed analysis of these issues.

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Different SUMO Paralogs Determine the Fate of WT and Mutant CFTRs: Biogenesis vs. Degradation

10.1101/340851 ◽

2018 ◽

Author(s):

Xiaoyan Gong ◽

Yong Liao ◽

Annette Ahner ◽

Mads Breum Larsen ◽

Xiaohui Wang ◽

...

Keyword(s):

Physiological Role ◽

Array Analysis ◽

Specific Effects ◽

Ubiquitin Conjugation ◽

Proteosomal Degradation ◽

The Common ◽

Protein Array Analysis ◽

The Impact

ABSTRACTA pathway for CFTR degradation is initiated by Hsp27 which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogs form poly-chains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, PIAS4, which increased WT and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR three-fold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation 3-fold and stabilized mature WT CFTR at the PM. PIAS4 knockdown reduced WT and F508del CFTR expression by 40-50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1in vivoand reduced its conjugation to SUMO-2/3. These SUMO paralog specific effects of PIAS4 were reproducedin vitrousing purified F508del NBD1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ~50%, and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogs determine the fates of WT and mutant CFTRs, and they suggest that a paralog switch during biogenesis can direct these proteins to different outcomes: biogenesis vs. degradation.

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Could β-Lactam Antibiotics Block Humoral Immunity?

Frontiers in Immunology ◽

10.3389/fimmu.2021.680146 ◽

2021 ◽

Vol 12 ◽

Author(s):

Cléa Melenotte ◽

Pierre Pontarotti ◽

Lucile Pinault ◽

Jean-Louis Mège ◽

Christian Devaux ◽

...

Keyword(s):

Immune Response ◽

Immune Cell ◽

Opportunistic Infections ◽

Severe Combined Immunodeficiency ◽

Physiological Role ◽

Zinc Ion ◽

Variable Regions ◽

The Impact

It has been reported that treatment with β-lactam antibiotics induces leukopenia and candidemia, worsens the clinical response to anticancer immunotherapy and decreases immune response to vaccination. β-lactamases can cleave β-lactam antibiotics by blocking their activity. Two distincts superfamilies of β-lactamases are described, the serine β-lactamases and the zinc ion dependent metallo-β-lactamases. In human, 18 metallo-β-lactamases encoding genes (hMBLs) have been identified. While the physiological role of most of them remains unknown, it is well established that the SNM1A, B and C proteins are involved in DNA repair. The SNM1C/Artemis protein is precisely associated in the V(D)J segments rearrangement, that leads to immunoglobulin (Ig) and T-cell receptor variable regions, which have a crucial role in the immune response. Thus in humans, SNM1C/Artemis mutation is associated with severe combined immunodeficiency characterized by hypogammaglobulinemia deficient cellular immunity and opportunistic infections. While catalytic site of hMBLs and especially that of the SNM1 family is highly conserved, in vitro studies showed that some β-lactam antibiotics, and precisely third generation of cephalosporin and ampicillin, inhibit the metallo-β-lactamase proteins SNM1A & B and the SNM1C/Artemis protein complex. By analogy, the question arises as to whether β-lactam antibiotics can block the SNM1C/Artemis protein in humans inducing transient immunodeficiency. We reviewed here the literature data supporting this hypothesis based on in silico, in vitro and in vivo evidences. Understanding the impact of β-lactam antibiotics on the immune cell will offer new therapeutic clues and new clinical approaches in oncology, immunology, and infectious diseases.

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Different SUMO paralogues determine the fate of wild-type and mutant CFTRs: biogenesis versus degradation

Molecular Biology of the Cell ◽

10.1091/mbc.e18-04-0252 ◽

2019 ◽

Vol 30 (1) ◽

pp. 4-16 ◽

Cited By ~ 3

Author(s):

Xiaoyan Gong ◽

Yong Liao ◽

Annette Ahner ◽

Mads Breum Larsen ◽

Xiaohui Wang ◽

...

Keyword(s):

Physiological Role ◽

Transmembrane Conductance Regulator ◽

Wild Type ◽

Transmembrane Conductance ◽

Specific Effects ◽

Ubiquitin Conjugation ◽

Protein Array Analysis ◽

The Impact

A pathway for cystic fibrosis transmembrane conductance regulator (CFTR) degradation is initiated by Hsp27, which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogues form polychains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, protein inhibitor of activated STAT 4 (PIAS4), which increased wild-type (WT) and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR threefold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation threefold and stabilized mature WT CFTR at the plasma membrance. PIAS4 knockdown reduced WT and F508del CFTR expression by 40–50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralogue-specific effects of PIAS4 were reproduced in vitro using purified F508del nucleotide-binding domain 1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ∼50% and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogues determine the fates of WT and mutant CFTRs, and they suggest that a paralogue switch during biogenesis can direct these proteins to different outcomes: biogenesis versus degradation.

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Modulation of Staphylococcus aureus Response to Antimicrobials by the Candida albicans Quorum Sensing Molecule Farnesol

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01573-17 ◽

2017 ◽

Vol 61 (12) ◽

Cited By ~ 27

Author(s):

Eric F. Kong ◽

Christina Tsui ◽

Sona Kucharíková ◽

Patrick Van Dijck ◽

Mary Ann Jabra-Rizk

Keyword(s):

Staphylococcus Aureus ◽

Candida Albicans ◽

Quorum Sensing ◽

Physiological Role ◽

Efflux Pumps ◽

Content Type ◽

The Impact ◽

Quorum Sensing Molecule

ABSTRACT In microbial biofilms, microorganisms utilize secreted signaling chemical molecules to coordinate their collective behavior. Farnesol is a quorum sensing molecule secreted by the fungal species Candida albicans and shown to play a central physiological role during fungal biofilm growth. Our pervious in vitro and in vivo studies characterized an intricate interaction between C. albicans and the bacterial pathogen Staphylococcus aureus, as these species coexist in biofilm. In this study, we aimed to investigate the impact of farnesol on S. aureus survival, biofilm formation, and response to antimicrobials. The results demonstrated that in the presence of exogenously supplemented farnesol or farnesol secreted by C. albicans in biofilm, S. aureus exhibited significantly enhanced tolerance to antimicrobials. By using gene expression studies, S. aureus mutant strains, and chemical inhibitors, the mechanism for the enhanced tolerance was attributed to upregulation of drug efflux pumps. Importantly, we showed that sequential exposure of S. aureus to farnesol generated a phenotype of high resistance to antimicrobials. Based on the presence of intracellular reactive oxygen species upon farnesol exposure, we hypothesize that antimicrobial tolerance in S. aureus may be mediated by farnesol-induced oxidative stress triggering the upregulation of efflux pumps, as part of a general stress response system. Hence, in mixed biofilms, C. albicans may influence the pathogenicity of S. aureus through acquisition of a drug-tolerant phenotype, with important therapeutic implications. Understanding interspecies signaling in polymicrobial biofilms and the specific drug resistance responses to secreted molecules may lead to the identification of novel targets for drug development.

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Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein

mBio ◽

10.1128/mbio.00310-18 ◽

2018 ◽

Vol 9 (2) ◽

Cited By ~ 10

Author(s):

Johannes G. Rebelein ◽

Chi Chung Lee ◽

Megan Newcomb ◽

Yilin Hu ◽

Markus W. Ribbe

Keyword(s):

Fine Tuning ◽

Ambient Conditions ◽

Protein Scaffold ◽

Reducing Activity ◽

Co Reduction ◽

The Impact ◽

Protein Environment

ABSTRACTThe Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase inAzotobacter vinelandiiand compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase.IMPORTANCEThis is the first report on thein vivogeneration andin vitrocharacterization of an M-cluster-containing V-nitrogenase hybrid. The “normalization” of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe proteins) housing the same cofactor (M-cluster) allows for an effective assessment of the impact of the protein environment on the CO reactivity of nitrogenase. The results of this study provide a first look into the “weighted” contributions of protein environment and cofactor properties to the overall activity of CO reduction; more importantly, they establish a useful platform for further investigation of the structural elements attributing to the CO-reducing activity of nitrogenase.

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Zinc Supplementation Modulates NETs Release and Neutrophils’ Degranulation

Nutrients ◽

10.3390/nu13010051 ◽

2020 ◽

Vol 13 (1) ◽

pp. 51

Author(s):

Weronika Kuźmicka ◽

Aneta Manda-Handzlik ◽

Adrianna Cieloch ◽

Agnieszka Mroczek ◽

Urszula Demkow ◽

...

Keyword(s):

Zinc Supplementation ◽

Physiological Role ◽

Zinc Level ◽

Entire Body ◽

Isolated Cells ◽

Zinc Levels ◽

The Impact ◽

Deficient Mice

Zinc plays an important physiological role in the entire body, especially in the immune system. It is one of the most abundant microelements in our organism and an essential component of enzymes and antibacterial proteins. Zinc levels were reported to be correlated with the intensity of innate immunity responses, especially those triggered by neutrophils. However, as the results are fragmentary, the phenomenon is still not fully understood and requires further research. In this study, we aimed to perform a comprehensive assessment and study the impact of zinc on several basic neutrophils’ functions in various experimental setups. Human and murine neutrophils were preincubated in vitro with zinc, and then phagocytosis, oxidative burst, degranulation and release of neutrophil extracellular traps (NETs) were analyzed. Moreover, a murine model of zinc deficiency and zinc supplementation was introduced in the study and the functions of isolated cells were thoroughly studied. We showed that zinc inhibits NETs release as well as degranulation in both human and murine neutrophils. Our study revealed that zinc decreases NETs release by inhibiting citrullination of histone H3. On the other hand, studies performed in zinc-deficient mice demonstrated that low zinc levels result in increased release of NETs and enhanced neutrophils degranulation. Overall, it was shown that zinc affects neutrophils’ functions in vivo and in vitro. Proper zinc level is necessary to maintain efficient functioning of the innate immune response.

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