scholarly journals Characterization of Bacteroides fragilis Hemolysins and Regulation and Synergistic Interactions of HlyA and HlyB

2006 ◽  
Vol 74 (4) ◽  
pp. 2304-2316 ◽  
Author(s):  
Kirstin P. Robertson ◽  
C. Jeffrey Smith ◽  
Andrea M. Gough ◽  
Edson R. Rocha
Keyword(s):  
Hemolytic Activity ◽  
Blot Hybridization ◽  
Bacteroides Fragilis ◽  
Blood Agar ◽  
Northern Blot Hybridization ◽  
Rich Medium ◽  
Independent Manner ◽  
Hemolytic Assay ◽  
Growth Deficiency

ABSTRACT This study describes the presence of 10 hemolysin orthologs in the genome of the opportunistic human anaerobic pathogen Bacteroides fragilis, which is currently classified as a nonhemolytic bacterium. The hemolysins were designated HlyA through HlyI plus HlyIII. All cloned hemolysin genes were able to confer hemolytic activity to a nonhemolytic Escherichia coli strain on blood agar plates. Interestingly, HlyH was found to be present in the genome of the B. fragilis NCTC9343 strain but absent in strains 638R, YCH46, and Bacteroides thetaiotaomicron VPI-5482. The hemolysins HlyA, HlyB, and HlyIII were selected for further characterization. HlyA, HlyB, and HlyIII were cytolytic to erythrocytes on liquid hemolytic assay. When hlyA and hlyB were expressed together in a nonhemolytic E. coli strain, the strain showed enhanced hemolytic activity on blood agar plates. Further analysis revealed that HlyA and HlyB have synergistic hemolytic activity as detected by the liquid hemolytic assay. In addition, the two-component hemolysins HlyA and HlyB form a protein-protein complex in vivo as determined by bacterial two-hybrid system assay. The hlyB and hlyA genes are organized in an operon that is coordinately regulated by iron and oxygen. Northern blot hybridization analysis revealed that hlyBA were expressed as a bicistronic mRNA induced approximately 2.5-fold under low-iron conditions and repressed in iron-rich medium. The normal iron-regulated expression of hlyBA mRNA was lost in the furA mutant strain. In contrast, the hlyA gene was also expressed as a single mRNA in iron-rich medium, but its expression was reduced approximately threefold under low-iron conditions in a Fur-independent manner. This suggests that hlyA alone is regulated by an unidentified iron-dependent regulator. Moreover, the expression levels of hlyBA and hlyA were reduced about threefold following oxygen exposure and treatment with hydrogen peroxide. Taken together, these results suggest that iron and oxidative stress have an effect on the control of hlyBA and hlyA transcriptional levels. A hlyBA mutant was constructed, and its hemolytic activity was greatly diminished compared to those of the hlyIII mutant and parent strains. In addition, the hlyBA mutant had a significant modification in colony morphology and growth deficiency compared to the parent strain. The implications of these findings for the pathophysiology of B. fragilis in extraintestinal infections and competition in ecological systems for this organism are discussed.

scholarly journals Deletion of the anion exchanger Slc26a4 (pendrin) decreases apical Cl−/HCO3− exchanger activity and impairs bicarbonate secretion in kidney collecting duct

2010 ◽  
Vol 299 (1) ◽  
pp. C33-C41 ◽  
Author(s):  
Hassane Amlal ◽  
Snezana Petrovic ◽  
Jie Xu ◽  
Zhaohui Wang ◽  
Xuming Sun ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Ph Monitoring ◽  
Collecting Duct ◽  
Blot Hybridization ◽  
Elevated Serum ◽  
Northern Blot Hybridization ◽  
Ammonium Excretion ◽  
Bicarbonate Secretion ◽  
Immunofluorescence Labeling

The anion exchanger Pendrin, which is encoded by SLC26A4 (human)/Slc26a4 (mouse) gene, is localized on the apical membrane of non-acid-secreting intercalated (IC) cells in the kidney cortical collecting duct (CCD). To examine its role in the mediation of bicarbonate secretion in vivo and the apical Cl−/HCO3− exchanger in the kidney CCD, mice with genetic deletion of pendrin were generated. The mutant mice show the complete absence of pendrin expression in their kidneys as assessed by Northern blot hybridization, Western blot, and immunofluorescence labeling. Pendrin knockout (KO) mice display significantly acidic urine at baseline [pH 5.20 in KO vs. 6.01 in wild type (WT); P < 0.0001] along with elevated serum HCO3− concentration (27.4 vs. 24 meq/l in KO vs. WT, respectively; P < 0.02), consistent with decreased bicarbonate secretion in vivo. The urine chloride excretion was comparable in WT and KO mice. For functional studies, CCDs were microperfused and IC cells were identified by their ability to trap the pH fluorescent dye BCECF. The apical Cl−/HCO3− exchanger activity in B-IC and non-A, non-B-IC cells, as assessed by intracellular pH monitoring, was significantly reduced in pendrin-null mice. The basolateral Cl−/HCO3− exchanger activity in A-IC cells and in non-A, non-B-IC cells, was not different in pendrin KO mice relative to WT animals. Urine NH4+ (ammonium) excretion increased significantly, consistent with increased trapping of NH3 in the collecting duct in pendrin KO mice. We conclude that Slc26a4 (pendrin) deletion impairs the secretion of bicarbonate in vivo and reduces apical Cl−/HCO3− exchanger activity in B-IC and non-A, non-B-IC cells in CCD. Additional apical Cl−/HCO3− exchanger(s) is (are) present in the CCD.

scholarly journals Thioredoxin Reductase Is Essential for Thiol/Disulfide Redox Control and Oxidative Stress Survival of the Anaerobe Bacteroides fragilis

2007 ◽  
Vol 189 (22) ◽  
pp. 8015-8023 ◽  
Author(s):  
Edson R. Rocha ◽  
Arthur O. Tzianabos ◽  
C. Jeffrey Smith
Keyword(s):  
Parent Strain ◽  
Culture Media ◽  
Blot Hybridization ◽  
Thioredoxin Reductase ◽  
Bacteroides Fragilis ◽  
Redox System ◽  
Opportunistic Pathogen ◽  
Abscess Formation ◽  
Infection Model ◽  
Northern Blot Hybridization

ABSTRACT Results of this study showed that the anaerobic, opportunistic pathogen Bacteroides fragilis lacks the glutathione/glutaredoxin redox system and possesses an extensive number of putative thioredoxin (Trx) orthologs. Analysis of the genome sequence revealed six Trx orthologs and an absence of genes required for synthesis of glutathione and glutaredoxins. In addition, it was shown that the thioredoxin reductase (TrxB)/Trx system is the major or sole redox system for thiol/disulfide cellular homeostasis in this anaerobic bacterium. Expression of the B. fragilis trxB gene was induced following treatment with diamide or H2O2 or exposure to oxygen. This inducible trxB expression was OxyR independent. Northern blot hybridization analysis showed that the trxB mRNA was cotranscribed with lolA as a bicistronic transcript or was present as a monocistronic transcript that was also highly induced under the same conditions. The role of LolA, a prokaryotic periplasmic lipoprotein-specific molecular chaperone in the thiol/disulfide redox system, is unknown. A trxB deletion mutant was more sensitive to the effects of diamide and oxygen than the parent strain. In addition, the trxB mutant was unable to grow in culture media without addition of a reductant. Furthermore, the trxB mutant was not able to induce intraabdominal abscess formation in a mouse model, whereas the parent strain was. Taken together, these data strongly suggest that TrxB/Trx is the major, if not the sole, thiol/disulfide redox system in this anaerobe required for survival and abscess formation in a peritoneal cavity infection model.

scholarly journals Glucose represses transcription of Saccharomyces cerevisiae nuclear genes that encode mitochondrial components.

1984 ◽  
Vol 4 (5) ◽  
pp. 939-946 ◽  
Author(s):  
E Szekely ◽  
D L Montgomery
Keyword(s):  
Blot Hybridization ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Northern Blot Hybridization ◽  
Rich Medium ◽  
Genes Encoding ◽  
Atpase Subunits ◽  
Steady State Levels ◽  
Alpha Beta ◽  
Kinetics Of

By Northern blot hybridization analysis, we demonstrated that the steady-state levels of mRNAs specifying the alpha subunit of ATPase, the beta subunit of ATPase, and the ATP/ADP translocator are all reduced in cells grown in glucose-rich medium. The extent to which glucose represses the levels of alpha, beta, and translocator mRNAs varies from strain to strain, from 2.5- to 7-fold. Furthermore, by hybridization experiments with an excess of DNA, we showed that glucose represses the rates of synthesis of these mRNAs. The kinetics of repression and depression of transcription were also studied. Finally, a mutant was characterized which appears to be defective in depression of transcription of the genes encoding the alpha and beta ATPase subunits as well as the ATP/ADP translocator.

scholarly journals Isolation of Bacteroides fragilis Mutants with In Vivo Growth Defects by Using Tn4400′, a Modified Tn4400 Transposition System, and a New Screening Method

2000 ◽  
Vol 68 (1) ◽  
pp. 415-419 ◽  
Author(s):  
Yixin P. Tang ◽  
Michael H. Malamy
Keyword(s):  
Tissue Culture ◽  
Screening Method ◽  
Bacteroides Fragilis ◽  
Normal Growth ◽  
Growth Characteristics ◽  
Rich Medium ◽  
Growth Defects ◽  
Mutant Strains ◽  
In Vivo Growth

ABSTRACT A modified version of the Bacteroides fragilistransposon Tn4400, designated Tn4400′, enabling rapid isolation and analysis of B. fragilis mutants has been constructed. To identify potential virulence factors, Tn4400′-generated mutants were screened by a new method; this resulted in the isolation of 21 mutant strains with impaired growth characteristics on tissue culture monolayers but normal growth in rich medium anaerobically.

scholarly journals Improving Crustacean Aquaculture Production Efficiencies through Development of Monosex Populations Using Endocrine and Molecular Manipulations

2010 ◽  
Author(s):  
Abigail Elizur ◽  
Amir Sagi ◽  
Gideon Hulata ◽  
Clive Jones ◽  
Wayne Knibb
Keyword(s):  
Cdna Library ◽  
Developmental Stages ◽  
Blot Hybridization ◽  
Repeated Administration ◽  
Sex Reversal ◽  
Northern Blot Hybridization ◽  
Female Population ◽  
Original Proposal ◽  
Aquaculture Production

Background Most of Australian prawn aquaculture production is based on P. monodon. However, the Australian industry is under intense competition from lower priced overseas imports. The availability of all-female monosex populations, by virtue of their large size and associated premium prize, will offer competitive advantage to the industry which desperately needs to counteract competitors within this market. As for the redclaw production in Israel, although it is at its infancy, the growers realized that the production of males is extremely advantageous and that such management strategy will change the economic assumptions and performances of this aquaculture to attract many more growers. Original objectives (as in original proposal) Investigating the sex inheritance mechanism in the tiger prawn. Identification of genes expressed uniquely in the androgenic gland (AG) of prawns and crayfish. The above genes and/or their products will be used to localize the AG in the prawn and manipulate the AG activity in both species. Production of monosex populations through AG manipulation. In the prawn, production of all-female populations and in the crayfish, all-male populations. Achievements In the crayfish, the AG cDNA library was further screened and a third AG specific transcript, designated Cq-AG3, had been identified. Simultaneously the two AG specific genes, which were previously identified, were further characterized. Tissue specificity of one of those genes, termed Cq-AG2, was demonstrated by northern blot hybridization and RNA in-situ hybridization. Bioinformatics prediction, which suggested a 42 amino acid long signal anchor at the N-terminus of the deduced Cq-AG2, was confirmed by immunolocalization of a recombinant protein. Cq-IAG's functionality was demonstrated by dsRNA in-vivo injections to intersex crayfish. Cq-IAGsilencing induced dramatic sex-related alterations, including male feature feminization, reduced sperm production, extensive testicular apoptosis, induction of the vitellogeningene expression and accumulation of yolk proteins in the ovaries. In the prawn, the AG was identified and a cDNA library was created. The putative P. monodonAG hormone encoding gene (Pm-IAG) was identified, isolated and characterized for time of expression and histological localization. Implantation of the AG into prawn post larvae (PL) and juveniles resulted in phenotypic transformation which included the appearance of appendix masculina and enlarged petasma. The transformation however did not result in sex change or the creation of neo males thus the population genetics stage to be executed with Prof. Hulata did not materialized. Repeated AG implantation is currently being trialed. Major conclusions and Implications, both scientific and agricultural Cq-IAG's involvement in male sexual differentiation had been demonstrated and it is strongly suggested that this gene encodes an AG hormone in this crayfish. A thorough screening of the AG cDNA library shows Cq-IAG is the prominent transcript within the library. However, the identification of two additional transcripts hints that Cq-IAG is not the only gene mediating the AG effects. The successful gene silencing of Cq-IAG, if performed at earlier developmental stages, might accomplish full and functional sex reversal which will enable the production of all-male crayfish populations. Pm-IAG is likely to play a similar role in prawns. It is possible that repeated administration of the AG into prawn will lead to the desired full sex reversal, so that WZ neo males, crossed with WZ females can result in WW females, which will form the basis for monosex all-female population.  

Glucose represses transcription of Saccharomyces cerevisiae nuclear genes that encode mitochondrial components

1984 ◽  
Vol 4 (5) ◽  
pp. 939-946
Author(s):  
E Szekely ◽  
D L Montgomery
Keyword(s):  
Blot Hybridization ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Northern Blot Hybridization ◽  
Rich Medium ◽  
Genes Encoding ◽  
Atpase Subunits ◽  
Steady State Levels ◽  
Alpha Beta ◽  
Kinetics Of

By Northern blot hybridization analysis, we demonstrated that the steady-state levels of mRNAs specifying the alpha subunit of ATPase, the beta subunit of ATPase, and the ATP/ADP translocator are all reduced in cells grown in glucose-rich medium. The extent to which glucose represses the levels of alpha, beta, and translocator mRNAs varies from strain to strain, from 2.5- to 7-fold. Furthermore, by hybridization experiments with an excess of DNA, we showed that glucose represses the rates of synthesis of these mRNAs. The kinetics of repression and depression of transcription were also studied. Finally, a mutant was characterized which appears to be defective in depression of transcription of the genes encoding the alpha and beta ATPase subunits as well as the ATP/ADP translocator.

Expression and potential role of Fsrg1, a murine bromodomain-containing homologue of the Drosophila gene female sterile homeotic

1998 ◽  
Vol 111 (23) ◽  
pp. 3541-3550 ◽  
Author(s):  
K. Rhee ◽  
M. Brunori ◽  
V. Besset ◽  
R. Trousdale ◽  
D.J. Wolgemuth
Keyword(s):  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Blot Hybridization ◽  
Immunoblot Analysis ◽  
Hybridization Analysis ◽  
Relative Molecular Mass ◽  
Northern Blot Hybridization ◽  
Specific Substrate ◽  
Drosophila Gene

We have isolated a cDNA which is a murine homologue of the Drosophila gene female sterile homeotic (fsh). This homologue, which we have designated Fsrg1*, contains two bromodomains and an ET motif characteristic of the Fsh sub-class of bromodomain-containing proteins. Northern blot hybridization analysis of adult tissues revealed that Fsrg1 was expressed at low levels rather ubiquitously, but most abundantly in the testis and ovary. Polyclonal antibodies raised against an Fsrg1 fusion protein were used to characterize the Fsrg1 gene product in tissues. Constructs were also generated in which the Fsrg1 cDNA was tagged with epitopes for hemaglutinin and used in transfection experiments. Immunoblot analysis revealed that the Fsrg1 protein migrates with a relative molecular mass of approximately 110 kDa, although the cDNA sequence would predict a protein of approximately 88 kDa. The migration at approximately 110 kDa was observed for both in vivo protein and protein produced in cultured cells. The Fsrg1 protein was localized to the nucleus when expressed in cultured cells, consistent with the presence of a nuclear localization signal motif in the Fsrg1 sequence. No kinase activity was detected for this nuclear protein as assessed in either autokinase or specific substrate assays. In situ hybridization analysis revealed strikingly high expression of Fsrg1 in granulosa cells of growing follicles in the adult ovary and suggested its possible involvement in folliculogenesis. Additional clues to its potential function were provided by the demonstration of its high level of expression in epithelia of tissues which undergo hormonally-modulated remodeling.

scholarly journals Overexpression of eCLCA1 in Small Airways of Horses with Recurrent Airway Obstruction

2005 ◽  
Vol 53 (8) ◽  
pp. 1011-1021 ◽  
Author(s):  
Friederike Anton ◽  
Ina Leverkoehne ◽  
Lars Mundhenk ◽  
Wallace B. Thoreson ◽  
Achim D. Gruber
Keyword(s):  
Airway Obstruction ◽  
Blot Hybridization ◽  
Hek293 Cells ◽  
Sweat Glands ◽  
Northern Blot Hybridization ◽  
Small Airways ◽  
Recurrent Airway Obstruction ◽  
Nucleotide Polymorphisms ◽  
Coding Region

The human hCLCA1 and murine mCLCA3 (chloride channels, calcium-activated) have recently been identified as promising therapeutic targets in asthma. Recurrent airway obstruction in horses is an important animal model of human asthma. Here, we have cloned and characterized the first equine CLCA family member, eCLCA1. The 913 amino acids eCLCA1 polypeptide forms a 120-kDa transmembrane glycoprotein that is processed to an 80-kDa protein in vivo. Three single nucleotide polymorphisms were detected in the eCLCA1 coding region in 14 horses, resulting in two amino acid changes (485H/R and 490V/L). However, no functional differences were recorded between the channel properties of the two variants in transfected HEK293 cells. The eCLCA1 protein was detected immunohistochemically in mucin-producing cells in the respiratory and intestinal tracts, cutaneous sweat glands, and renal mucous glands. Strong overexpression of eCLCA1 was observed in the airways of horses with recurrent airway obstruction using Northern blot hybridization, Western blotting, immunohistochemistry, and real-time quantitative RT-PCR. The results suggest that spontaneous or experimental recurrent airway obstruction in horses may serve as a model to study the role of CLCA homologs in chronic airway disease with overproduction of mucins.

Evaluation of the In Vivo Acute Toxicity and In Vitro Hemolytic and Immunomodulatory Activities of the Moringa oleifera Flower Trypsin Inhibitor (MoFTI)

2020 ◽  
Vol 27 ◽  
Author(s):  
Leydianne Leite de Siqueira Patriota ◽  
Dayane Kelly Dias do Nascimento Santos ◽  
Bárbara Rafaela da Silva Barros ◽  
Lethícia Maria de Souza Aguiar ◽  
Yasmym Araújo Silva ◽  
...  
Keyword(s):  
Acute Toxicity ◽  
Trypsin Inhibitor ◽  
Hemolytic Activity ◽  
Moringa Oleifera ◽  
Biological Activities ◽  
Hematological Parameters ◽  
Behavioral Alterations ◽  
Female Mice

Background: Protease inhibitors have been isolated from plants and present several biological activities, including immunomod-ulatory action. Objective: This work aimed to evaluate a Moringa oleifera flower trypsin inhibitor (MoFTI) for acute toxicity in mice, hemolytic activity on mice erythrocytes and immunomodulatory effects on mice splenocytes. Methods: The acute toxicity was evaluated using Swiss female mice that received a single dose of the vehicle control or MoFTI (300 mg/kg, i.p.). Behavioral alterations were observed 15–240 min after administration, and survival, weight gain, and water and food consumption were analyzed daily. Organ weights and hematological parameters were analyzed after 14 days. Hemolytic activity of MoFTI was tested using Swiss female mice erythrocytes. Splenocytes obtained from BALB/c mice were cultured in the absence or presence of MoFTI for the evaluation of cell viability and proliferation. Mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) levels were also determined. Furthermore, the culture supernatants were analyzed for the presence of cytokines and nitric oxide (NO). Results: MoFTI did not cause death or any adverse effects on the mice except for abdominal contortions at 15–30 min after administration. MoFTI did not exhibit a significant hemolytic effect. In addition, MoFTI did not induce apoptosis or necrosis in splenocytes and had no effect on cell proliferation. Increases in cytosolic and mitochondrial ROS release, as well as ΔΨm reduction, were observed in MoFTI-treated cells. MoFTI was observed to induce TNF-α, IFN-γ, IL-6, IL-10, and NO release. Conclusion: These results contribute to the ongoing evaluation of the antitumor potential of MoFTI and its effects on other immunological targets.

scholarly journals Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jingjing Yang ◽  
Yulu Zhou ◽  
Shuduo Xie ◽  
Ji Wang ◽  
Zhaoqing Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Treatment ◽  
Morphological Changes ◽  
Tumor Model ◽  
Independent Manner ◽  
Regulated Cell Death ◽  
Anti Cancer ◽  
Cancer Agent ◽  
Approved Drugs

Abstract Background Ferroptosis is a newly defined form of regulated cell death characterized by the iron-dependent accumulation of lipid peroxidation and is involved in various pathophysiological conditions, including cancer. Targeting ferroptosis is considered to be a novel anti-cancer strategy. The identification of FDA-approved drugs as ferroptosis inducers is proposed to be a new promising approach for cancer treatment. Despite a growing body of evidence indicating the potential efficacy of the anti-diabetic metformin as an anti-cancer agent, the exact mechanism underlying this efficacy has not yet been fully elucidated. Methods The UFMylation of SLC7A11 is detected by immunoprecipitation and the expression of UFM1 and SLC7A11 in tumor tissues was detected by immunohistochemical staining. The level of ferroptosis is determined by the level of free iron, total/lipid Ros and GSH in the cells and the morphological changes of mitochondria are observed by transmission electron microscope. The mechanism in vivo was verified by in situ implantation tumor model in nude mice. Results Metformin induces ferroptosis in an AMPK-independent manner to suppress tumor growth. Mechanistically, we demonstrate that metformin increases the intracellular Fe2+ and lipid ROS levels. Specifically, metformin reduces the protein stability of SLC7A11, which is a critical ferroptosis regulator, by inhibiting its UFMylation process. Furthermore, metformin combined with sulfasalazine, the system xc− inhibitor, can work in a synergistic manner to induce ferroptosis and inhibit the proliferation of breast cancer cells. Conclusions This study is the first to demonstrate that the ability of metformin to induce ferroptosis may be a novel mechanism underlying its anti-cancer effect. In addition, we identified SLC7A11 as a new UFMylation substrate and found that targeting the UFM1/SLC7A11 pathway could be a promising cancer treatment strategy.

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