bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pyruvate:Quinone Oxidoreductase from
Corynebacterium glutamicum
: Purification and Biochemical Characterization
Authors: Schreiner Mark E. , Eikmanns Bernhard J. 
Published: January 2005

ABSTRACT

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO
2
with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in
Escherichia coli
. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer
Corynebacterium glutamicum
. The activity was highest (0.055 ± 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type
C. glutamicum
, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (
k
cat
= 37.8 ± 3 s
−1
;

K
m

= 30 ± 3 mM) and 2-oxobutyrate (
k
cat
= 33.2 ± 3 s
−1
;

K
m

= 90 ± 8 mM). Thiamine pyrophosphate (

K
m

= 1 μM) and certain divalent metal ions such as Mg
2+
(

K
m

= 29 μM), Mn
2+
(

K
m

= 2 μM), and Co
2+
(

K
m

= 11 μM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol,
p
-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (

K
m

= 106 μM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in
C. glutamicum
.
Heat Shock Proteome Analysis of Wild-Type
Corynebacterium glutamicum
ATCC 13032 and a Spontaneous Mutant Lacking GroEL1, a Dispensable Chaperone
Authors: Barreiro Carlos , González-Lavado Eva , Brand Sven , Tauch Andreas , Martín Juan F. 
Published: January 2005

ABSTRACT

Proteome analysis of
Corynebacterium glutamicum
ATCC 13032 showed that levels of several proteins increased drastically in response to heat shock. These proteins were identified as DnaK, GroEL1, GroEL2, ClpB, GrpE, and PoxB, and their heat response was in agreement with previous transcriptomic results. A major heat-induced protein was absent in the proteome of strain 13032B of
C. glutamicum
, used for genome sequencing in Germany, compared with the wild-type ATCC 13032 strain. The missing protein was identified as GroEL1 by matrix-assisted laser desorption ionization-time of flight peptide mass fingerprinting, and the mutation was found to be due to an insertion sequence, Is
Cg1
, that was integrated at position 327 downstream of the translation start codon of the
groEL1
gene, resulting in a truncated transcript of this gene, as shown by Northern analysis. The GroEL1 chaperone is, therefore, dispensable in
C. glutamicum
. On the other hand, GroEL2 appears to be essential for growth. Based on these results, the role of the duplicate
groEL1
and
groEL2
genes is analyzed.
Trimeric Structure of Major Outer Membrane Proteins Homologous to OmpA inPorphyromonas gingivalis
Authors: Nagano Keiji , Read Erik K. , Murakami Yukitaka , Masuda Takashi , Noguchi Toshihide , Yoshimura Fuminobu 
Published: January 2005

ABSTRACTThe major outer membrane proteins Pgm6 (41 kDa) and Pgm7 (40 kDa) ofPorphyromonas gingivalisATCC 33277 are encoded by open reading framespg0695andpg0694, respectively, which form a single operon. Pgm6 and Pgm7 (Pgm6/7) have a high degree of similarity toEscherichia coliOmpA in the C-terminal region and are predicted to form eight-stranded β-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular masses of 40 and 120 kDa, with and without a reducing agent, suggesting a monomer and trimer, respectively. To verify the predicted trimeric structure and function of Pgm6/7, we constructed three mutants withpg0695,pg0694, or both deleted. The double mutant produced no Pgm6/7. The single-deletion mutants appeared to contain less Pgm7 and Pgm6 and to form homotrimers that migrated slightly faster (115 kDa) and slower (130 kDa), respectively, than wild-type Pgm6/7 under nonreducing conditions. N-terminal amino acid sequencing and mass spectrometry analysis of partially digested Pgm6/7 detected only fragments from Pgm6 and Pgm7. Two-dimensional, diagonal electrophoresis and chemical cross-linking experiments with or without a reducing agent clearly showed that Pgm6/7 mainly form stable heterotrimers via intermolecular disulfide bonds. Furthermore, growth retardation and arrest of the three mutants and increased permeability of their outer membranes indicated that Pgm6/7 play an important role in outer membrane integrity. Based on results of liposome swelling experiments, these proteins are likely to function as a stabilizer of the cell wall rather than as a major porin in this organism.
Transcriptional Regulation ofsitABCDofSalmonella entericaSerovar Typhimurium by MntR and Fur
Authors: Ikeda Jack S. , Janakiraman Anuradha , Kehres David G. , Maguire Michael E. , Slauch James M. 
Published: January 2005

ABSTRACTSalmonella entericaserovar Typhimurium has two manganese transport systems, MntH and SitABCD. MntH is a bacterial homolog of the eukaryotic natural resistance-associated macrophage protein 1 (Nramp1), and SitABCD is an ABC-type transporter. Previously we showed thatmntHis negatively controlled at the transcriptional level by thetrans-acting regulatory factors, MntR and Fur. In this study, we examined the transcriptional regulation ofsitABCDand compared it to the transcriptional regulation ofmntHby constructinglacZfusions to the promoter regions with and without mutations in putative MntR and/or Fur binding sites. The presence of Mn caused transcriptional repression of thesitABCDandmntHpromoters primarily via MntR, but Fur was also capable of some repression in response to Mn. Likewise, Fe in the medium repressed transcription of bothsitandmntHprimarily via Fur, although MntR was also involved in this response. Transcriptional control by MntR and Fur was disrupted by site-specific mutations in the putative MntR and Fur binding sites, respectively. Transcription of thesitoperon was also affected by the oxygen level and growth phase, but the increased expression observed under high oxygen conditions and higher cell densities is consistent with decreased availability of metals required for repression by the metalloregulatory proteins.
Markerless Mutagenesis inMethanococcus maripaludisDemonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease
Authors: Moore Brian C. , Leigh John A. 
Published: January 2005

ABSTRACTAmong the archaea,Methanococcus maripaludishas the unusual ability to usel- ord-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools forM.maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of theM.maripaludis hptgene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of theuptgene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow ond-alanine. The wild-type gene for alanine dehydrogenase, incorporated into theuptsite, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for thelisomer, and the racemase converts thedisomer to thelisomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.
Functional Interactions between the Carbon and Iron Utilization Regulators, Crp and Fur, in
Escherichia coli
Authors: Zhang Zhongge , Gosset Guillermo , Barabote Ravi , Gonzalez Claudio S. , Cuevas William A. , Saier Milton H. 
Published: January 2005

ABSTRACT

In
Escherichia coli
, the ferric uptake regulator (Fur) controls expression of the iron regulon in response to iron availability while the cyclic AMP receptor protein (Crp) regulates expression of the carbon regulon in response to carbon availability. We here identify genes subject to significant changes in expression level in response to the loss of both Fur and Crp. Many iron transport genes and several carbon metabolic genes are subject to dual control, being repressed by the loss of Crp and activated by the loss of Fur. However, the
sodB
gene, encoding superoxide dismutase, and the
aceBAK
operon, encoding the glyoxalate shunt enzymes, show the opposite responses, being activated by the loss of Crp and repressed by the loss of Fur. Several other genes including the
sdhA-D
,
sucA-D
, and
fumA
genes, encoding key constituents of the Krebs cycle, proved to be repressed by the loss of both transcription factors. Finally, the loss of both Crp and Fur activated a heterogeneous group of genes under σ
S
control encoding, for example, the cyclopropane fatty acid synthase, Cfa, the glycogen synthesis protein, GlgS, the 30S ribosomal protein, S22, and the mechanosensitive channel protein, YggB. Many genes appeared to be regulated by the two transcription factors in an apparently additive fashion, but apparent positive or negative cooperativity characterized several putative Crp/Fur interactions. Relevant published data were evaluated, putative Crp and Fur binding sites were identified, and representative results were confirmed by real-time PCR. Molecular explanations for some, but not all, of these effects are provided.
Multiple Regulators Control Expression of the Entner-Doudoroff Aldolase (Eda) of
Escherichia coli
Authors: Murray Elizabeth L. , Conway Tyrrell 
Published: January 2005

ABSTRACT

The
Escherichia coli eda
gene, which encodes the Entner-Doudoroff aldolase, is central to the catabolism of several sugar acids. Here, we show that Eda synthesis is induced by growth on gluconate, glucuronate, or methyl-β-
d
-glucuronide; phosphate limitation; and carbon starvation. Transcription of
eda
initiates from three promoters, designated P1, P2, and P4, each of which is responsible for induction under different growth conditions. P1 controls
eda
induction on gluconate and is regulated by GntR. P2 controls
eda
induction on glucuronate and galacturonate and is regulated by KdgR. P4 is active under conditions of phosphate starvation and is directly controlled by PhoB. In addition, CsrA activates Eda synthesis, apparently by an indirect mechanism that may be involved in the modest changes in expression level that are associated with carbon starvation. The complex regulation of
eda
is discussed with respect to its several physiological roles, which apparently accommodate not only sugar acid catabolism but also detoxification of metabolites that could accumulate during starvation-induced stress.
Von Willebrand factor accelerates platelet adhesion and thrombus formation on a collagen surface in platelet-reduced blood under flow conditions
Authors: Tomokiyo Kazuhiko , Kamikubo Yuichi , Hanada Takako , Araki Tatsuya , Nakatomi Yasushi , Ogata Yoichi , Jung Stephanie M. , Nakagaki Tomohiro , Moroi Masaaki 
Published: January 2005

AbstractPlasma von Willebrand factor (VWF) has been identified as an indispensable factor for platelet adhesion and thrombus formation on a collagen surface under flow conditions. VWF binds to collagen and then tethers platelets to the collagen surface through interaction with platelet glycoprotein Ib and also contributes to the thrombus formation on the collagen surface. In the present study, we demonstrated that the addition of VWF/factor VIII complex or purified VWF (&gt; 2 ristocetin cofactor activity units/mL) increased platelet adhesion to the collagen surface in platelet-reduced blood (∼ 5 × 104 platelets/μL) to the normal level. VWF had no stimulatory effect when it was allowed to bind to the collagen surface before blood flow was initiated. Addition of an excess of FITC (fluorescein-5-isothiocyanate)–abeled VWF to platelet-reduced blood under these flow conditions demonstrated that the VWF was mainly incorporated into the platelet aggregates. These results indicated that the supplemented VWF stimulates the platelet adhesion onto the collagen surface by enhancing platelet aggregation in the platelet-reduced condition. This also suggests a possibility that supplementation of VWF to individuals with thrombocytopenia might be effective for increasing their hemostatic potential.
A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease
Authors: Fiedler Walter , Serve Hubert , Döhner Hartmut , Schwittay Michael , Ottmann Oliver G. , O'Farrell Anne-Marie , Bello Carlo L. , Allred Randy , Manning William C. , Cherrington Julie M. , Louie Sharianne G. , Hong Weiru , Brega Nicoletta M. , Massimini Giorgio , Scigalla Paul , Berdel Wolfgang E. , Hossfeld Dieter K. 
Published: January 2005

AbstractFifteen patients with refractory AML were treated in a phase 1 study with SU11248, an oral kinase inhibitor of fms-like tyrosine kinase 3 (Flt3), Kit, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) receptors. Separate cohorts of patients received SU11248 for 4-week cycles followed by either a 2- or a 1-week rest period. At the starting dose level of 50 mg (n = 13), no dose-limiting toxicities were observed. The most frequent grade 2 toxicities were edema, fatigue, and oral ulcerations. Two fatal bleedings possibly related to the disease, one from a concomitant lung cancer and one cerebral bleeding, were observed. At the 75 mg dose level (n = 2), one case each of grade 4 fatigue, hypertension, and cardiac failure was observed, and this dose level was abandoned. All patients with FLT3 mutations (n = 4) had morphologic or partial responses compared with 2 of 10 evaluable patients with wild-type FLT3. Responses, although longer in patients with mutated FLT3, were of short duration. Reductions of cellularity and numbers of Ki-67+, phospho-Kit+, phospho–kinase domain–containing receptor–positive (phospho-KDR+), phospho–signal transducer and activator of transcription 5–positive (phospho-STAT5+), and phospho-Akt+ cells were detected in bone marrow histology analysis. In summary, monotherapy with SU11248 induced partial remissions of short duration in acute myeloid leukemia (AML) patients. Further evaluation of this compound, for example in combination with chemotherapy, is warranted.
Ligands for natural killer cell–activating receptors are expressed upon the maturation of normal myelomonocytic cells but at low levels in acute myeloid leukemias
Authors: Nowbakht Pegah , Ionescu Mihai-Constantin S. , Rohner Andreas , Kalberer Christian P. , Rossy Emmanuel , Mori Lucia , Cosman David , De Libero Gennaro , Wodnar-Filipowicz Aleksandra 
Published: January 2005

AbstractNatural killer (NK) cell–mediated cytolytic activity against tumors requires the engagement of activating NK receptors by the tumor-associated ligands. Here, we have studied the role of NKG2D and natural cytotoxicity receptors (NCRs) in the recognition of human leukemia. To detect as-yet-unknown cell-surface molecules recognized by NCRs, we developed soluble forms of NKp30, NKp44, and NKp46 as staining reagents binding the putative cognate ligands. Analysis of UL16-binding protein-1 (ULBP1), ULBP2, and ULBP3 ligands for NKG2D and of potential ligands for NKp30, NKp44, and NKp46 in healthy hematopoietic cells demonstrated the ligand-negative phenotype of bone marrow–derived CD34+ progenitor cells and the acquisition of cell-surface ligands during the course of myeloid differentiation. In acute myeloid leukemia (AML), leukemic blasts from approximately 80% of patients expressed very low levels of ULBPs and NCR-specific ligands. Treatment with differentiation-promoting myeloid growth factors, together with interferon-γ, upregulated cell-surface levels of ULBP1 and putative NCR ligands on AML blasts, conferring an increased sensitivity to NK cell–mediated lysis. We conclude that the ligand-negative/low phenotype in AML is a consequence of cell maturation arrest on malignant transformation and that defective expression of ligands for the activating NKG2D and NCR receptors may compromise leukemia recognition by NK cells.
Involvement of connective tissue–type mast cells in Th1 immune responses via Stat4 expression
Authors: Kataoka Tatsuki R. , Komazawa Nobuyasu , Morii Eiichi , Oboki Keisuke , Nakano Toru 
Published: January 2005

AbstractMast cells are the sentinels of immune systems and, like other immuno-competent cells, they are produced by hematopoietic stem cells. We analyzed the expression of signal transducer and activator of transcription 4 (Stat4), and investigated its role in mast cells. Murine mast cells are usually divided into 2 distinct populations by their distribution and contents of their granules: mucosal mast cells (MMCs) and connective tissue–type mast cells (CTMCs). Stat4 protein was detected in CTMCs but not in MMCs. The absence of Stat4 expression in cultured mast cells was due to the presence of Stat6. In T-helper (Th) cells, Stat4 plays an important role in Th1 shift by inducing a set of genes, such as interferon gamma (IFN-γ) and interleukin-18 receptor α subunit (IL-18Rα). As in Th1 shift, we found that Stat4 trans-activated these genes in the Stat4-expressing cultured mast cells, namely, microphthalmia transcription factor (MITF)–deficient cultured MMCs, Stat6-deficient cultured MMCs, and cultured CTMCs. Stat4 also enhanced expression of nitric oxide synthase 2 (NOS2) in CTMCs, which brought about increased levels of NO-dependent cytotoxic activity. These data indicate that expression of Stat4 in CTMCs plays an important role on Th1 immune responses.
A neurotrophin axis in myeloma: TrkB and BDNF promote tumor-cell survival
Authors: Pearse Roger N. , Swendeman Steven L. , Li Ying , Rafii Dahlia , Hempstead Barbara L. 
Published: January 2005

AbstractMultiple myeloma (MM) is a B-cell neoplasm that is characterized by the clonal expansion of malignant plasma cells and is frequently associated with chromosomal translocations placing an oncogene under the control of the immunoglobulin heavy chain enhancer. Despite these pathogenic translocations, MM cells remain dependent on external cues for survival. We present evidence that brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of growth factors, and its high-affinity receptor, tropomyosin receptor kinase B (TrkB), contribute to these survival cues. MM cells express TrkB, and respond to BDNF by activating mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase–a PI3K target (PI3K/Akt) signaling cascades. Addition of BDNF protects human MM cell lines (HMCLs) from apoptosis induced by dexamethasone or bortezomib and prolongs the survival of primary MM cells cultured alone or with human bone marrow (BM) stroma. As BDNF and TrkB are expressed by osteoblasts, stromal cells, and endothelial cells within the BM microenvironment, a BDNF-TrkB axis may be critical to the interactions of MM with bone and stroma that contribute to MM tumor progression. Finally, BDNF is expressed by malignant plasma cells isolated from a subset of patients with MM, as well as by most HMCLs, suggesting a potential role for this neurotrophin axis in autocrine as well as paracrine support of MM.
Activity and specificity of toxin-related mouse T cell ecto–ADP-ribosyltransferase ART2.2 depends on its association with lipid rafts
Authors: Bannas Peter , Adriouch Sahil , Kahl Sarah , Braasch Fenja , Haag Friedrich , Koch-Nolte Friedrich 
Published: January 2005

AbstractAdenosine diphosphate (ADP)–ribosyl-transferases (ARTs) transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD) onto target proteins. T cells express ART2.2, a toxin-related, glycosylphosphatidylinositol (GPI)–anchored ecto-enzyme. After the release of NAD from cells, ART2.2 ADP-ribosylates the P2X7 purinoceptor, lymphocyte function–associated antigen (LFA-1), and other membrane. Using lymphoma transfectants expressing either ART2.2 with its native GPI anchor (ART2.2-GPI) or ART2.2 with a grafted transmembrane anchor (ART2.2-Tm), we demonstrated that ART2.2-GPI but not ART2.2-Tm associated with glycosphingolipid-enriched microdomains (lipid rafts). At limiting substrate concentrations, ART2.2-GPI exhibited more than 10-fold higher activity than ART2.2-Tm. On intact cells, ART2.2-GPI ADP-ribosylated a small number of distinct target proteins. Strikingly, the disruption of lipid rafts by cyclodextrin or membrane solubilization by Triton X-100 increased the spectrum of modified target proteins. However, ART2.2 itself was a prominent target for ADP-ribosylation only when GPI anchored. Furthermore, cholesterol depletion or detergent solubilization abolished the auto-ADP-ribosylation of ART2.2. These findings imply that ART2.2-GPI, but not ART2.2-Tm, molecules are closely associated on the plasma membrane and lend support to the hypothesis that lipid rafts exist on living cells as platforms to which certain proteins are admitted and others are excluded. Our results further suggest that raft association focuses ART2.2 on specific targets that constitutively or inducibly associate with lipid rafts.
PD166326, a novel tyrosine kinase inhibitor, has greater antileukemic activity than imatinib mesylate in a murine model of chronic myeloid leukemia
Authors: Wolff Nicholas C. , Veach Darren R. , Tong William P. , Bornmann William G. , Clarkson Bayard , Ilaria Robert L. 
Published: January 2005

AbstractImatinib mesylate is highly effective in newly diagnosed chronic myeloid leukemia (CML), but BCR/ABL (breakpoint cluster region/abelson murine leukemia)–positive progenitors persist in most patients with CML treated with imatinib mesylate, indicating the need for novel therapeutic approaches. In this study, we have used the murine CML-like myeloproliferative disorder as a platform to characterize the pharmacokinetic, signal transduction, and antileukemic properties of PD166326, one of the most potent members of the pyridopyrimidine class of protein tyrosine kinase inhibitors. In mice with the CML-like disease, PD166326 rapidly inhibited Bcr/Abl kinase activity after a single oral dose and demonstrated marked antileukemic activity in vivo. Seventy percent of PD166326-treated mice achieved a white blood cell (WBC) count less than 20.0 × 109/L (20 000/μL) at necropsy, compared with only 8% of imatinib mesylate–treated animals. Further, two thirds of PD166326-treated animals had complete resolution of splenomegaly, compared with none of the imatinib mesylate–treated animals. Consistent with its more potent antileukemic effect in vivo, PD166326 was also superior to imatinib mesylate in inhibiting the constitutive tyrosine phosphorylation of numerous leukemia-cell proteins, including the src family member Lyn. PD166326 also prolonged the survival of mice with imatinib mesylate–resistant CML induced by the Bcr/Abl mutants P210/H396P and P210/M351T. Altogether, these findings demonstrate the potential of more potent Bcr/Abl inhibitors to provide more effective antileukemic activity. Clinical development of PD166326 or a related analog may lead to more effective drugs for the treatment of de novo and imatinib mesylate–resistant CML.