scholarly journals Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius

2021 ◽  
Vol 12 ◽  
Author(s):  
Areum Lee ◽  
Eunji Bae ◽  
Jihee Park ◽  
Kyoung-Hwa Choi ◽  
Jaeho Cha
Keyword(s):  
Death Rate ◽  
Deletion Mutant ◽  
Glycogen Content ◽  
Glycogen Metabolism ◽  
Sulfolobus Acidocaldarius ◽  
Growth Phases ◽  
Wild Type ◽  
Promoter Assay ◽  
Branching Points

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC–gaa–glgX and amyA–glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC–gaa–glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

The Critical Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPalpha Mutations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2213-2213
Author(s):  
J. Pulikkan ◽  
A. Peer Zada ◽  
M. Geletu ◽  
V. Dengler ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Wild Type ◽  
Rt Pcr ◽  
Promoter Assay ◽  
Type C ◽  
Acute Myeloid

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is a myeloid specific transcription factor that coordinates cellular differentiation and cell cycle arrest. Loss of C/EBPα expression or function in leukemic blasts contributes to a block in myeloid cell differentiation. C/EBPα is mutated in around 9% of acute myeloid leukemia (AML). The mutations reported in C/EBPα are frame shift mutations and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. The role of peptidyl-prolyl cis/trans isomerase, Pin1 in tumorogenesis and its overexpression in many cancers led us to investigate its role in acute myeloid leukemia with C/EBPα mutation. Here we show that Pin1 is upregulated in patients with acute myeloid leukemia by affymetrix analysis. By quantitative Real-Time RT-PCR analysis, we show C/EBPα-p30 could induce Pin1 transcription, while the wild type C/EBPα downregulates Pin1 expression. Luciferase promoter assay for the Pin1 promoter shows that wild type C/EBPα is able to block Pin1 promoter activity. Mean while, C/EBPα-p30 couldn’t block Pin1 promotor activity. By silencing Pin1 by RNA Interference as well as with inhibitor against Pin1 (PiB) we could show myeloid differentiation in human CD34+ cord blood cells as well as in Kasumi-6 cells as assessed by FACS analysis with granulocytic markers. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We report that Pin1 increases the transcriptional activity of the oncogene c-jun. We also show that c-jun blocks the DNA binding and transactivation of C/EBPα protein as assessed by gel shift assay and promoter assay respectively. We have previously shown that c-jun expression is high in AML patients with C/EBPα mutation and c-jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that inhibition of Pin1 by the inhibitor PiB downregulates c-jun mRNA expression. In conclusion, inhibition of Pin1 leads to granulocytic differentiation. Our results show Pin1 as a novel target in treating AML patients with C/EBPα mutation.

scholarly journals Role of Alpha/Beta Interferons in the Attenuation and Immunogenicity of Recombinant Bovine Respiratory Syncytial Viruses Lacking NS Proteins

2003 ◽  
Vol 77 (15) ◽  
pp. 8426-8439 ◽  
Author(s):  
Jean-Francois Valarcher ◽  
Julie Furze ◽  
Sara Wyld ◽  
Roy Cook ◽  
Karl-Klaus Conzelmann ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Deletion Mutant ◽  
Wild Type ◽  
Antibody Titers ◽  
Ifn Γ ◽  
Respiratory Syncytial Viruses ◽  
Alpha Beta ◽  
Syncytial Virus ◽  
Prior Infection

ABSTRACT Alpha/beta interferons (IFN-α/β) are not only a powerful first line of defense against pathogens but also have potent immunomodulatory activities. Many viruses have developed mechanisms of subverting the IFN system to enhance their virulence. Previous studies have demonstrated that the nonstructural (NS) genes of bovine respiratory syncytial virus (BRSV) counteract the antiviral effects of IFN-α/β. Here we demonstrate that, in contrast to wild-type BRSVs, recombinant BRSVs (rBRSVs) lacking the NS proteins, and those lacking NS2 in particular, are strong inducers of IFN-α/β in bovine nasal fibroblasts and bronchoalveolar macrophages. Furthermore, whereas the NS deletion mutants replicated to wild-type rBRSV levels in cells lacking a functional IFN-α/β system, their replication was severely attenuated in IFN-competent cells and in young calves. These results suggest that the NS proteins block the induction of IFN-α/β gene expression and thereby increase the virulence of BRSV. Despite their poor replication in the respiratory tract of young calves, prior infection with virus lacking either the NS1 or the NS2 protein induced serum antibodies and protection against challenge with virulent BRSV. The greater level of protection induced by the NS2, than by the NS1, deletion mutant, was associated with higher BRSV-specific antibody titers and greater priming of BRSV-specific, IFN-γ-producing CD4+ T cells. Since there were no detectable differences in the ability of these mutants to replicate in the bovine respiratory tract, the greater immunogenicity of the NS2 deletion mutant may be associated with the greater ability of this virus to induce IFN-α/β.

Role of the A+ Helix in Heparin Binding to Protein C Inhibitor

1996 ◽  
Vol 75 (05) ◽  
pp. 760-766 ◽  
Author(s):  
Marc G L M Elisen ◽  
Machiel H H Maseland ◽  
Frank C Church ◽  
Bonno N Bouma ◽  
Joost C M Meijers
Keyword(s):  
Protein C ◽  
Electrostatic Interactions ◽  
Deletion Mutant ◽  
Activated Protein C ◽  
Structural Features ◽  
Heparin Binding ◽  
Wild Type ◽  
Protein C Inhibitor ◽  
Positively Charged

SummaryInteractions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A+ helix, is located at the N-terminus (residues 1-11), whereas the second region, the H helix, is positioned between residues 264 and 280 of PCI. Competition experiments with synthetic peptides based on the sequence of these regions demonstrated that the H helix has the highest affinity for heparin. In contrast to previous observations we found that the A+ helix peptide competed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide.Recombinant PCI was also used to investigate the role of the A+ helix in heparin binding. Full-length (wild-type) rPCI as well as an A+ helix deletion mutant of PCI (rPCI-Δ2-l 1) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A+ helix deletion mutant showed a decreased affinity for heparin in inhibition reactions with activated protein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A+ helix peptide competed with rPCI-Δ2-11 for binding to heparin. This indicated that the interaction between PCI and heparin is fairly non-specific and that the interaction is primarily based on electrostatic interactions.In summary, our data suggest that the H helix of PCI is the main heparin binding region of PCI, but the A+ helix increases the overall affinity for the PCI-heparin interaction by contributing a second positively charged region to the surface of PCI.

scholarly journals The Enterococcus faecalis fsrB Gene, a Key Component of the fsr Quorum-Sensing System, Is Associated with Virulence in the Rabbit Endophthalmitis Model

2002 ◽  
Vol 70 (8) ◽  
pp. 4678-4681 ◽  
Author(s):  
Eleftherios Mylonakis ◽  
Michael Engelbert ◽  
Xiang Qin ◽  
Costi D. Sifri ◽  
Barbara E. Murray ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Enterococcus Faecalis ◽  
Deletion Mutant ◽  
Wild Type ◽  
Sensing System ◽  
Quorum Sensing System

ABSTRACT We used a rabbit endophthalmitis model to explore the role of fsrB, a gene required for the function of the fsr quorum-sensing system of Enterococcus faecalis, in pathogenicity. A nonpolar deletion mutant of fsrB had significantly reduced virulence compared to wild type. Complementation of mutation restored virulence. These data corroborate the role of fsrB in E. faecalis pathogenesis and suggest that the rabbit endophthalmitis model can be used to study the in vivo role of quorum sensing.

Significant Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPα Mutations.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 55-55 ◽  
Author(s):  
John Anto Pulikkan ◽  
Viola Dengler ◽  
Abdul A. Peer Zada ◽  
Mulu Gelutu ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mrna Expression ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Wild Type ◽  
Promoter Assay ◽  
Type C ◽  
Acute Myeloid

Abstract Transcription factor CCAAT enhancer binding protein α (C/EBPα) is crucial for the differentiation of granulocytes. Experimental data from animal models as well as patient samples suggest that loss of function or expression of C/EBPα provides a platform on which acute myeloid leukemia (AML) develops. C/EBPα is mutated in around 9% of acute myeloid leukemia. The mutations reported in C/EBPα are frame shift mutations at N-terminal domain and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. Peptidyl-prolyl cis/trans isomerase, Pin1 binds to and isomerizes the peptidyl-prolyl bond in specific phosphorylated Ser/Thr-Pro motifs. A growing number of studies show that Pin1 is overexpressed in many cancers and has significant role in tumorigenesis. In the present study we investigated the role of Pin1 in acute myeloid leukemia with C/EBPα mutation. Here we report C/EBPα-p30 could induce Pin1 transcription as assessed by quantitative Real-Time RT-PCR analysis. Affymetrix mRNA expression analysis show that Pin1 is upregulated in patients with acute myeloid leukemia. Silencing of Pin1 could overcome the dominant negative action of the C/EBPα-p30 over the C/EBPα-p42 transactivation capacity as analyzed by promoter assay. By silencing Pin1 with inhibitor against Pin1 (PiB), we could show myeloid differentiation in Kasumi-6 cells by FACS analysis with granulocytic specific markers. Western blot analysis shows that Pin1 inhibition by PiB could upregulate wild type C/EBPα protein level. Luciferase promoter assay for the Pin1 promoter shows that C/EBPα-p30 induces Pin1 promoter activity in association with E2F1. Mean while, wild type C/EBPα interferes with transactivation of the Pin1 promoter and downregulates Pin1 mRNA expression. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We have previously shown that c-Jun expression is high in AML patients with C/EBPα mutation and c-Jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that overexpression of Pin1 induces c-Jun mRNA expression, while inhibition of Pin1 by the inhibitor PiB downregulates c-Jun mRNA expression. We show that c-Jun blocks the DNA binding and transactivation of wild type C/EBPα protein as assessed by gel shift assay and promoter assay respectively. In conclusion, inhibition of Pin1 leads to granulocytic differentiation of human myeloid cells. Our findings suggest inhibition of Pin1 as a novel strategy in treating AML patients with C/EBPα mutation.

scholarly journals Attenuation of the Activity of Caspofungin at High Concentrations against Candida albicans: Possible Role of Cell Wall Integrity and Calcineurin Pathways

2005 ◽  
Vol 49 (12) ◽  
pp. 5146-5148 ◽  
Author(s):  
Nathan P. Wiederhold ◽  
Dimitrios P. Kontoyiannis ◽  
Randall A. Prince ◽  
Russell E. Lewis
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Deletion Mutant ◽  
Cell Wall Integrity ◽  
Short Exposure ◽  
Wild Type ◽  
High Concentrations

ABSTRACT Caspofungin had diminished activity in vitro against Candida albicans at concentrations of 8 to 32 μg/ml. This phenomenon was markedly attenuated in a Δmkc1/Δmkc1 deletion mutant and by the addition of cyclosporine to the wild type. Short exposure to these caspofungin concentrations resulted in MKC1 up-regulation, suggesting roles of cell wall integrity and calcineurin pathways.

scholarly journals Role of Sigma E-Regulated Genes in Escherichia coli Uropathogenesis

2006 ◽  
Vol 74 (7) ◽  
pp. 4030-4038 ◽  
Author(s):  
Peter Redford ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Deletion Mutant ◽  
Cell Envelope ◽  
Infection Model ◽  
Wild Type ◽  
E Coli ◽  
Animal Infection ◽  
Strain Cft073 ◽  
Sigma E

ABSTRACT The sigma E regulon encodes proteins for maintenance and repair of the Escherichia coli cell envelope. Previously, we observed that an antirepressor of sigma E, DegS, is essential for uropathogenic E. coli virulence. Here we use a mouse urinary tract infection model to assay the virulence of mutants of E. coli genes described as sigma E dependent. Deletion mutants of candidate genes were made in the uropathogenic E. coli strain CFT073. Swiss Webster female mice were inoculated with a mixture of mutant and wild-type strains. Bladder and kidney homogenates were cultured 2 days after infection, and CFU of the wild type and mutant were compared. Eleven mutants were assayed, and two, CFT073 degP and CFT073 skp, showed significantly diminished survival compared to wild type. DegP is a chaperone and degradase active in the periplasm. Skp is also a periplasmic chaperone. The virulence of the skp deletion mutant could not be restored by complementation with skp. The virulence of the degP deletion mutant, in contrast, could be restored. However, complementation with a degP allele encoding a serine-to-alanine (S210A) mutation at the protease active site fails to restore virulence. Unlike degP mutants in other bacteria, the E. coli degP mutant is tolerant of oxidative stress. It disappears abruptly from bladder and kidney cultures between 6 and 12 hours after inoculation. A mutant of degQ, a close homolog of degP, was not attenuated in mice. This is the first report that the DegP degradase is an E. coli virulence factor in an animal infection model.

Role of glucocorticoid in the regulation of glycogen metabolism in skeletal muscle

1991 ◽  
Vol 260 (6) ◽  
pp. E927-E932 ◽  
Author(s):  
L. Coderre ◽  
A. K. Srivastava ◽  
J. L. Chiasson
Keyword(s):  
Skeletal Muscle ◽  
Muscle Contraction ◽  
Glycogen Phosphorylase ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Glycogen Metabolism ◽  
Activity Ratios ◽  
Resting Muscle ◽  
Glycogen Breakdown

With the use of the hindlimb perfusion technique, the effect of glucocorticoid on the regulation of glycogen metabolism was studied in rat skeletal muscle. Rats were adrenalectomized (ADX) or sham operated (controls) 14 days before the study. The ADX animals were treated with either saline or corticosterone, and the hindlimbs were perfused at rest or during muscle contraction with saline or epinephrine (10(-7) M). In the resting state, the glycogen content was 33.0 +/- 1.9 mumol/g in the controls, and the activity ratios of glycogen phosphorylase (GPase) and glycogen synthase (GSase) were 0.27 +/- 0.03 and 0.15 +/- 0.02, respectively. Epinephrine treatment increased GPase activity (0.78 +/- 0.03) and decreased GSase activity (0.05 +/- 0.01), which resulted in decreased glycogen content (25.7 +/- 0.9 mumol/g; P less than 0.01). Adrenalectomy induced a 35% reduction in glycogen content but had no effect on the activities of basal enzymes. Under these conditions, however, epinephrine had no effect on GPase activity, had a diminished effect on GSase activity (0.11 +/- 0.01), and did not induce further glycogen breakdown. Corticosterone replacement normalized muscle glycogen content in ADX rats as well as the response of the enzymes to epinephrine. Muscle contraction resulted in a decrease in glycogen content (8.9 +/- 1.3 mumol/g) and in GPase activity (0.14 +/- 0.02) and an increase in GSase activity (0.25 +/- 0.01); this was not affected by adrenalectomy nor by epinephrine. In conclusion, these data indicate that glucocorticoid is essential for the effects of epinephrine on GPase activation. on GSase inhibition, and consequently on glycogen breakdown in resting muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Neural control of glycogen content and its diurnal rhythm in mouse pineal cell.

1977 ◽  
Vol 232 (6) ◽  
pp. E584 ◽  
Author(s):  
T Kachi ◽  
T Ito
Keyword(s):  
Diurnal Rhythm ◽  
Neural Control ◽  
Glycogen Content ◽  
Sympathetic Innervation ◽  
Continuous Light ◽  
Glycogen Metabolism ◽  
Release Of Noradrenaline ◽  
Internal Mechanism ◽  
Dd Mice

In adult male dd mice, possible mechanisms regulating the glycogen content in the pineal cell were investigated by a semiquantitative histochemical method, with particular reference to the role of the sympathetic innervation. Reserpine, superior cervical ganglionectomy (SCGX), or decentralization of the ganglia (DC), as well as continuous light, prevented the nocturnal decrease in the glycogen content, causing a marked increase, and caused a gradual decrease in the size of the pineal cell. In the SCGX or DC group, the glycogen content reached a peak at 2 days and then decreased gradually. The nocturnal decrease was also prevented by propranolol. Noradrenaline caused a marked decrease in the glycogen content. These findings support the hypothesis that the glycogen metabolism and its diurnal rhythm in the pineal cell are regulated by the sympathetic nerve terminals innervating the pineal gland, presumably by the release of noradrenaline. In addition, the nature of the internal mechanism in the organism generating the pineal glycogen rhythm was examined. Light was considered to induce a phase shift in such a mechanism, but reserpine was not.

scholarly journals Altered proteome of a Burkholderia pseudomallei mutant defective in short-chain dehydrogenase affects cell adhesion, biofilm formation and heat stress tolerance

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8659 ◽  
Author(s):  
Onrapak Reamtong ◽  
Nitaya Indrawattana ◽  
Amporn Rungruengkitkun ◽  
Tipparat Thiangtrongjit ◽  
Taksaon Duangurai ◽  
...  
Keyword(s):  
Heat Stress ◽  
Biofilm Formation ◽  
Burkholderia Pseudomallei ◽  
Deletion Mutant ◽  
Stress Conditions ◽  
Host Cells ◽  
Short Chain ◽  
Wild Type ◽  
Short Chain Dehydrogenase

Burkholderia pseudomallei is a Gram-negative bacillus that causes melioidosis and is recognized as an important public health problem in southeast Asia and northeast Australia. The treatment of B. pseudomallei infection is hampered by resistance to a wide range of antimicrobial agents and no vaccine is currently available. At present, the underlying mechanisms of B. pseudomallei pathogenesis are poorly understood. In our previous study, we reported that a B. pseudomallei short-chain dehydrogenase (SDO; BPSS2242) mutant constructed by deletion mutagenesis showed reduced B. pseudomallei invasion and initial intracellular survival. This indicated that SDO is associated with the pathogenesis of melioidosis. In the present study, the role of B. pseudomallei SDO was further investigated using the SDO deletion mutant by a proteomic approach. The protein profiles of the SDO mutant and wild-type K96243 were investigated through gel-based proteomic analysis. Quantitative intensity analysis of three individual cultures of the B. pseudomallei SDO mutant revealed significant down-regulation of five protein spots compared with the wild-type. Q-TOF MS/MS identified the protein spots as a glutamate/aspartate ABC transporter, prolyl-tRNA synthetase, Hsp70 family protein, quinone oxidoreductase and a putative carboxypeptidase. Functional assays were performed to investigate the role of these differentially expressed proteins in adhesion to host cells, biofilm induction and survival under heat stress conditions. The SDO deletion mutant showed a decreased ability to adhere to host cells. Moreover, biofilm formation and the survival rate of bacteria under heat stress conditions were also reduced in the mutant strain. Our findings provide insight into the role of SDO in the survival and pathogenesis of B. pseudomallei at the molecular level, which may be applied to the prevention and control of B. pseudomallei infection.

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