bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glycerol-Mediated Repression of Glucose Metabolism and Glycerol Kinase as the Sole Route of Glycerol Catabolism in the Haloarchaeon
Haloferax volcanii
Authors: Sherwood Katherine E. , Cano David José , Maupin-Furlow Julie A. 
Published: May 2009

ABSTRACT

Although glycerol is the primary carbon source available to halophilic heterotrophic communities, little is known regarding haloarchaeal glycerol metabolism. In this study, a gene encoding a glycerol kinase homolog (
glpK
; HVO_1541) was deleted from the genome of the haloarchaeon
Haloferax volcanii
by a markerless knockout strategy. The
glpK
mutant, KS4, readily grew on yeast extract-peptone complex medium and glucose minimal medium but was incapable of growth on glycerol. Glycerol kinase activity was dependent on the
glpK
gene and readily detected in cells grown on glucose and/or glycerol, with the activity level higher in medium supplemented with glycerol (with or without glucose) than in medium with glucose alone. An analysis of carbon utilization revealed that glycerol suppressed the metabolism of glucose in both the parent H26 and
glpK
mutant strains, with catabolite repression more pronounced in the glycerol kinase mutant. Transcripts specific for
glpK
and an upstream gene,
gpdA
, encoding a homolog of glycerol-3-phosphate dehydrogenase subunit A, were upregulated (8- and 74-fold, respectively) in the presence of glycerol and glucose compared to those in the presence of glucose alone. Furthermore,
glpK
was transcriptionally linked to the
gpdC
gene of the putative glycerol-3-phosphate dehydrogenase operon (
gpdABC
), based on the findings of reverse transcriptase PCR analysis. The results presented here provide genetic and biochemical evidence that glycerol metabolism proceeds through a glycerol kinase encoded by
glpK
and suggest that a glycerol-3-phosphate dehydrogenase encoded by the upstream
gpdABC
operon is also involved in this pathway. Furthermore, our findings reveal a unique example of glycerol-induced repression of glucose metabolism in
H. volcanii
.
Autotrophic Carbon Dioxide Assimilation in
Thermoproteales
Revisited
Authors: Ramos-Vera W. Hugo , Berg Ivan A. , Fuchs Georg 
Published: May 2009

ABSTRACT

For
Crenarchaea
, two new autotrophic carbon fixation cycles were recently described.
Sulfolobales
use the 3-hydroxypropionate/4-hydroxybutyrate cycle, with acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme.
Ignicoccus hospitalis
(
Desulfurococcales
) uses the dicarboxylate/4-hydroxybutyrate cycle, with pyruvate synthase and phosphoenolpyruvate carboxylase being responsible for CO
2
fixation. In the two cycles, acetyl-CoA and two inorganic carbons are transformed to succinyl-CoA by different routes, whereas the regeneration of acetyl-CoA from succinyl-CoA proceeds via the same route.
Thermoproteales
would be an exception to this unifying concept, since for
Thermoproteus neutrophilus
, the reductive citric acid cycle was proposed as a carbon fixation mechanism. Here, evidence is presented for the operation of the dicarboxylate/4-hydroxybutyrate cycle in this archaeon. All required enzyme activities were detected in large amounts. The key enzymes of the cycle were strongly upregulated under autotrophic growth conditions, indicating their involvement in autotrophic CO
2
fixation. The corresponding genes were identified in the genome.
14
C-labeled 4-hydroxybutyrate was incorporated into the central building blocks in accordance with the key position of this compound in the cycle. Moreover, the results of previous
13
C-labeling studies, which could be reconciled with a reductive citric acid cycle only when some assumptions were made, were perfectly in line with the new proposal. We conclude that the dicarboxylate/4-hydroxybutyrate cycle is operating in CO
2
fixation in the strict anaerobic
Thermoproteales
as well as in
Desulfurococcales
.
Characterization of an Alcohol Dehydrogenase from the Cyanobacterium
Synechocystis
sp. Strain PCC 6803 That Responds to Environmental Stress Conditions via the Hik34-Rre1 Two-Component System
Authors: Vidal Rebeca , López-Maury Luis , Guerrero Miguel G. , Florencio Francisco J. 
Published: May 2009

ABSTRACT

The slr1192 (
adhA
) gene from
Synechocystis
sp. strain PCC 6803 encodes a member of the medium-chain alcohol dehydrogenase/reductase family. The gene product AdhA exhibits NADP-dependent alcohol dehydrogenase activity, acting on a broad variety of aromatic and aliphatic primary alcohols and aldehydes but not on secondary alcohols or ketones. It exhibits superior catalytic efficiency for aldehyde reduction compared to that for alcohol oxidation. The enzyme is a cytosolic protein present in photoautotrophically grown
Synechocystis
cells. The expression of AdhA is enhanced upon the exposure of cells to different environmental stresses, although it is not essential for survival even under such stress conditions. The induction of the expression of the
adhA
gene is dependent on the Hik34-Rre1 two-component system, as it is severely impaired in mutant strains lacking either the histidine kinase Hik34 or the response regulator Rre1. In vitro DNA-protein interaction analysis reveals that the response regulator Rre1 binds specifically to the promoter region of the
adhA
gene.
The C-Terminal Domain of AcrA Is Essential for the Assembly and Function of the Multidrug Efflux Pump AcrAB-TolC
Authors: Ge Qiang , Yamada Yoichi , Zgurskaya Helen 
Published: May 2009

ABSTRACTPeriplasmic membrane fusion proteins (MFPs) are essential components of multidrug efflux pumps and type I protein secretion systems of gram-negative bacteria. Located in the periplasm, MFPs function by creating a physical link between inner membrane transporters and outer membrane channels. The most conserved sequence of MFPs is located in their distal C-terminal domain. However, neither the structure nor the function of this domain is known. In this study, we investigated the structural and functional role of the C-terminal domain ofEscherichia coliAcrA, a periplasmic component of the multidrug efflux pump AcrAB-TolC. Using trypsin proteolysis, we identified the proteolytically labile sites in the C-terminal domain (amino acid residues 315 to 397) of AcrA in vitro. We next used these sites as a map to evaluate the structural integrity of this domain of AcrA inside the periplasm. We found that the C-terminal domain of AcrA is protected from trypsin when the tripartite efflux pump AcrAB-TolC is assembled. In contrast, this domain remains proteolytically labile in cells producing only one of the AcrB or TolC components of the complex. Site-directed mutagenesis of 12 highly conserved amino acid residues of the C-terminal domain of AcrA showed that a single G363C substitution dramatically impairs the multidrug efflux activity of AcrAB-TolC. The G363C mutant interacts with both AcrB and TolC but fails to properly assemble into a functional complex. We conclude that the C-terminal domain of AcrA plays an important role in the assembly and function of AcrAB-TolC efflux pump.
Regulation of Gene Expression by PrrA in
Rhodobacter sphaeroides
2.4.1: Role of Polyamines and DNA Topology
Authors: Eraso Jesus M. , Kaplan Samuel 
Published: May 2009

ABSTRACT

In the present study, we show in vitro binding of PrrA, a global regulator in
Rhodobacter sphaeroides
2.4.1, to the PrrA site 2, within the RSP3361 locus. Specific binding, as shown by competition experiments, requires the phosphorylation of PrrA. The binding affinity of PrrA for site 2 was found to increase 4- to 10-fold when spermidine was added to the binding reaction. The presence of extracellular concentrations of spermidine in growing cultures of
R. sphaeroides
gave rise to a twofold increase in the expression of the photosynthesis genes
pucB
and
pufB
, as well as the RSP3361 gene, under aerobic growth conditions, as shown by the use of
lacZ
transcriptional fusions, and led to the production of light-harvesting spectral complexes. In addition, we show that negative supercoiling positively regulates the expression of the RSP3361 gene, as well as
pucB
. We show the importance of supercoiling through an evaluation of the regulation of gene expression in situ by supercoiling, in the case of the former gene, as well as using the DNA gyrase inhibitor novobiocin. We propose that polyamines and DNA supercoiling act synergistically to regulate expression of the RSP3361 gene, partly by affecting the affinity of PrrA binding to the PrrA site 2 within the RSP3361 gene.
Half-Site DNA Sequence and Spacing Length Contributions to PrrA Binding to PrrA Site 2 of RSP3361 in
Rhodobacter sphaeroides
2.4.1
Authors: Eraso Jesus M. , Kaplan Samuel 
Published: May 2009

ABSTRACT

The consensus DNA binding sequence for PrrA, a global regulator in
Rhodobacter sphaeroides
2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of
lacZ
transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using
lacZ
transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.
An Overlap between the Control of Programmed Cell Death in
Bacillus anthracis
and Sporulation
Authors: Chandramohan Lakshmi , Ahn Jong-Sam , Weaver Keith E. , Bayles Kenneth W. 
Published: May 2009

ABSTRACT

The
Staphylococcus aureus cid
and
lrg
operons have been shown to control cell death and lysis in a manner thought to be analogous to programmed cell death (apoptosis) in eukaryotic organisms. Although orthologous operons are present in a wide variety of bacterial species, members of the
Bacillus cereus
group are unique in that they have a total of four
cid
-
/lrg
-like operons. Two of these operons are similar to the
S. aureus cid
and
lrg
operons, while the other two (designated
clhAB
1
and
clhAB
2
) are unique to this group. In the present study, the functions and regulation of these loci were examined. Interestingly, the
Bacillus anthracis lrgAB
mutant displayed decreased stationary-phase survival, whereas the
clhAB
2
mutant exhibited increased stationary-phase survival compared to the parental and complementation strains. However, neither mutation had a dramatic effect on murein hydrolase activity or autolysis. Furthermore, a quantitative analysis of the sporulation efficiency revealed that both mutants formed fewer spores than did the parental strain. Similar to
S. aureus
,
B. anthracis lrgAB
transcription was shown to be induced by gramicidin and CCCP, agents known to dissipate the proton motive force, in a
lytSR
-dependent manner. Northern blot analyses also demonstrated a positive role for
lytSR
in the
clhAB
2
transcription. Taken together, the results of the present study demonstrate that
B. anthracis lrgAB
and
clhAB
2
play important roles in the control of cell death and lysis and reveal a previously unrecognized role of this system in sporulation.
The Iron-Hydrogenase of
Thermotoga maritima
Utilizes Ferredoxin and NADH Synergistically: a New Perspective on Anaerobic Hydrogen Production
Authors: Schut Gerrit J. , Adams Michael W. W. 
Published: May 2009

ABSTRACT

The hyperthermophilic and anaerobic bacterium
Thermotoga maritima
ferments a wide variety of carbohydrates, producing acetate, CO
2
, and H
2
. Glucose is degraded through a classical Embden-Meyerhof pathway, and both NADH and reduced ferredoxin are generated. The oxidation of these electron carriers must be coupled to H
2
production, but the mechanism by which this occurs is unknown. The trimeric [FeFe]-type hydrogenase that was previously purified from
T. maritima
does not use either reduced ferredoxin or NADH as a sole electron donor. This problem has now been resolved by the demonstration that this hydrogenase requires the presence of both electron carriers for catalysis of H
2
production. The enzyme oxidizes NADH and ferredoxin simultaneously in an approximately 1:1 ratio and in a synergistic fashion to produce H
2
. It is proposed that the enzyme represents a new class of bifurcating [FeFe] hydrogenase in which the exergonic oxidation of ferredoxin (midpoint potential, −453 mV) is used to drive the unfavorable oxidation of NADH (
E
0
′ = −320 mV) to produce H
2
(
E
0
′ = −420 mV). From genome sequence analysis, it is now clear that there are two major types of [FeFe] hydrogenases: the trimeric bifurcating enzyme and the more well-studied monomeric ferredoxin-dependent [FeFe] hydrogenase. Almost one-third of the known H
2
-producing anaerobes appear to contain homologs of the trimeric bifurcating enzyme, although many of them also harbor one or more homologs of the simpler ferredoxin-dependent hydrogenase. The discovery of the bifurcating hydrogenase gives a new perspective on our understanding of the bioenergetics and mechanism of H
2
production and of anaerobic metabolism in general.
Loss of the SMRT/NCoR2 Corepressor Correlates with JAG2 Overexpression in Multiple Myeloma
Authors: Ghoshal Pushpankur , Nganga Alain J. , Moran-Giuati Joseph , Szafranek Angela , Johnson Timothy R. , Bigelow Ashley J. , Houde Christiane M. , Avet-Loiseau Herve , Smiraglia Dominic J. , Ersing Noreen , Chanan-Khan Asher A. , Coignet Lionel J. 
Published: May 2009

Abstract
Multiple myeloma (MM) is a clonal B-cell neoplasm that accounts for 10% of all malignant hematologic neoplasms and that affects terminally differentiated B cells (i.e., plasma cells). It is now well recognized that the cytokine interleukin-6 (IL-6) is a major cytokine that promotes the proliferation of malignant plasma cells in MM. The IL-6 gene can be regulated by the NOTCH genes products. We have previously shown that the NOTCH ligand, JAG2, is overexpressed in MM. To investigate the mechanism(s) leading to JAG2 overexpression in MM, we assessed potential epigenetic modifications of the JAG2 promoter. We showed that the JAG2 promoter region is aberrantly acetylated in MM cell lines and patient samples. The acetylation state of histones is regulated by the recruitment of histone deacetylases (HDAC). HDACs are typically recruited to promoter regions through interaction with nuclear corepressors such as SMRT. SMRT levels were therefore investigated. Interestingly, MM cell lines and patient samples presented significantly reduced SMRT levels. The experiments suggest a correlation between constitutive acetylation of the JAG2 core promoter in the MM cell lines and reduced levels of the SMRT corepressor that recruits HDAC to promoter regions. Finally, SMRT function restoration induced JAG2 down-regulation as well as MM cell apoptosis. [Cancer Res 2009;69(10):4380–7]
Association between Transcriptional Activity, Local Chromatin Structure, and the Efficiencies of Both Subpathways of Nucleotide Excision Repair of Melphalan Adducts
Authors: Episkopou Hara , Kyrtopoulos Soterios A. , Sfikakis Petros P. , Fousteri Maria , Dimopoulos Meletios A. , Mullenders Leon H.F. , Souliotis Vassilis L. 
Published: May 2009

Abstract
The repair of melphalan-induced N-alkylpurine monoadducts and interstrand cross-links was examined in different repair backgrounds, focusing on four genes (β-actin, p53, N-ras, and δ-globin) with dissimilar transcription activities. Adducts were found to be substrates for both global genome repair (GGR) and transcription-coupled repair (TCR), with TCR being less efficient than GGR. In nucleotide excision repair–deficient cells, adducts accumulated to similar levels in all four genes. The repair efficiency in different gene loci varied in a qualitatively and quantitatively similar way in both GGR-deficient and TCR-deficient backgrounds and correlated with transcriptional activity and local chromatin condensation. No strand-specific repair was found in GGR+/TCR+ cells, implying that GGR dominated. Adducts were lost over two sharply demarcated phases: a rapid phase resulting in the removal within 1 hour of up to ∼80% of the adducts, and a subsequent phase with t1/2 ∼36 to 48 hours. Following pretreatment of cells with α-amanitin, the rate of transcription, the state of chromatin condensation, and the repair efficiencies (both TCR and GGR) of the transcribed β-actin, p53, and N-ras genes became similar to those of the nontranscribed δ-globin gene. In conclusion, a continuous, parallel variation of the state of transcription and local chromatin condensation, on one hand, and the rates of both GGR and TCR, on the other hand, have been shown. [Cancer Res 2009;69(10):4424–33]
Sunitinib Inhibits Tumor Growth and Synergizes with Cisplatin in Orthotopic Models of Cisplatin-Sensitive and Cisplatin-Resistant Human Testicular Germ Cell Tumors
Authors: Castillo-Ávila Wilmar , Piulats Josep Maria , Garcia del Muro Xavier , Vidal August , Condom Enric , Casanovas Oriol , Mora Josefina , Germà Josep Ramon , Capellà Gabriel , Villanueva Alberto , Viñals Francesc 
Published: May 2009

Abstract
Purpose: Germ cell tumors (GCT) of the testis are highly curable, but those patients who are refractory to cisplatin (CDDP)-based combination chemotherapy have a poor prognosis. Therefore, identifying new alternatives for treatment remains a priority. Several studies support an important role for angiogenesis in GCTs, suggesting that antiangiogenic treatment might be a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor inhibitor with antiangiogenic and antitumor activities. In the present study, we evaluated the effect of sunitinib, CDDP, or the combination of both drugs using an orthotopic model of human testicular GCT.
Experimental Design: Mice were implanted with four different testicular tumors: a yolk sac, two choriocarcinomas, and a CDDP-resistant choriocarcinoma variant induced in mice by continuous exposure to CDDP. Mice were treated with vehicle, CDDP, sunitinib, or the combination of both drugs and their effects on tumors were analyzed.
Results: We observed a significant inhibition in tumor growth accompanied by longer survival after sunitinib treatment. Combination therapy with CDDP significantly enhanced these effects. Sunitinib induced apoptosis, reduced tumor cell proliferation and tumor vasculature, and inhibited vascular endothelial growth factor receptor 1, 2, and 3 and platelet-derived growth factor receptor α phosphorylation without affecting phosphorylation of other tyrosine kinase receptors. More importantly, tumor growth inhibition induced by sunitinib was also observed in the induced CDDP-resistant choriocarcinoma model.
Conclusions: Taken together, these results suggest that sunitinib might be a new alternative for treatment of CDDP-refractory patients.
Single-Step Antigen Loading and Activation of Dendritic Cells by mRNA Electroporation for the Purpose of Therapeutic Vaccination in Melanoma Patients
Authors: Bonehill Aude , Van Nuffel An M.T. , Corthals Jurgen , Tuyaerts Sandra , Heirman Carlo , François Violaine , Colau Didier , van der Bruggen Pierre , Neyns Bart , Thielemans Kris 
Published: May 2009

Abstract
Purpose: A critical factor determining the effectiveness of currently used dendritic cell (DC)–based vaccines is the DC activation or maturation status. We have recently shown that the T-cell stimulatory capacity of DCs pulsed with tumor-antigen–derived peptides can be considerably increased by activating the DCs through electroporation with mRNA encoding CD40 ligand, CD70, and a constitutively active Toll-like receptor 4 (TriMix DCs). Here, we investigate whether TriMix DCs can be coelectroporated with whole tumor-antigen–encoding mRNA.
Experimental Design: The T-cell stimulatory capacity of TriMix DCs pulsed with the immunodominant MelanA-A2 peptide and that of TriMix DCs coelectroporated with MelanA mRNA were compared in vitro. TriMix DCs were also coelectroporated with mRNA encoding Mage-A3, Mage-C2, tyrosinase, or gp100. The capacity of these DCs to stimulate tumor-antigen–specific T cells in melanoma patients was investigated both in vitro before vaccination and after DC vaccination.
Results: Like peptide-pulsed TriMix DCs, TriMix DCs coelectroporated with MelanA mRNA are very potent in inducing MelanA-specific CD8+ T cells in vitro. These T cells have an activated phenotype, show cytolytic capacity, and produce inflammatory cytokines in response to specific stimulation. TriMix DCs coelectroporated with tyrosinase are able to stimulate tyrosinase-specific CD8+ T cells in vitro from the blood of nonvaccinated melanoma patients. Furthermore, TriMix DCs coelectroporated with Mage-A3, Mage-C2, or tyrosinase are able to induce antigen-specific CD8+ T cells through therapeutic DC vaccination.
Conclusions: TriMix DCs coelectroporated with whole tumor-antigen mRNA stimulate antigen-specific T cells in vitro and induce antigen-specific T-cell responses in melanoma patients through vaccination. Therefore, they represent a promising new approach for antitumor immunotherapy.
Increased Frequency and Suppression by Regulatory T Cells in Patients with Acute Myelogenous Leukemia
Authors: Szczepanski Miroslaw J. , Szajnik Marta , Czystowska Malgorzata , Mandapathil Magis , Strauss Laura , Welsh Ann , Foon Kenneth A. , Whiteside Theresa L. , Boyiadzis Michael 
Published: May 2009

AbstractPurpose: Regulatory CD4+CD25highFoxp3+ T cells (Treg) control peripheral immune tolerance. Patients with cancer, including those with hematologic malignancies, have elevated numbers of Treg in the peripheral circulation and in tumor tissues. However, mechanisms of suppression and clinical significance of Treg, especially in patients with acute myelogenous leukemia (AML), has not been well defined.Experimental Design: We prospectively evaluated the phenotype, function, and mechanisms of suppression used by Treg in newly diagnosed untreated AML patients. The relationship between the frequency of circulating Treg and the disease status as well as treatment outcome was also evaluated.Results: The percentage of circulating Treg was higher (P &lt; 0.0001) and their phenotype was distinct in AML patients relative to normal controls. Suppression mediated by Treg coincubated with proliferating autologous responder cells was also higher (P &lt; 0.001) in AML than that mediated by control Treg. Using Transwell inserts, we showed that interleukin-10 and transforming growth factor-β1 production as well as cell-to-cell contact were necessary for Treg-mediated suppression. Also, the pretreatment Treg frequency predicted response to chemotherapy. Unexpectedly, patients who achieved complete remission still had elevated frequency of Treg, which mediated high levels of suppressor activity.Conclusions: Treg accumulating in the peripheral circulation of AML patients mediate vigorous suppression via contact-dependent and contact-independent mechanisms. Patients with lower Treg frequency at diagnosis have a better response to induction chemotherapy. During the post-induction period, the Treg frequency and suppressive activity remain elevated in complete remission, suggesting that Treg are resistant to conventional chemotherapy.