bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from
Streptomyces davawensis
Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin
Authors: Grill Simon , Busenbender Simone , Pfeiffer Matthias , Köhler Uwe , Mack Matthias 
Published: December 2007

ABSTRACT

Streptomyces davawensi
s synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5′-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent
Sus scrofa
d
-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in
S. davawensi
s, which would not allow the formation of toxic RoFMN/RoFAD. The gene
ribC
from
S. davawensi
s was cloned. RibC from
S. davawensis
was overproduced in
Escherichia coli
and purified. Analysis of the flavokinase activity of RibC revealed that the
S. davawensis
enzyme is not riboflavin specific (roseoflavin,
k
cat
/

K
m

= 1.7 10
−2
μM
−1
s
−1
; riboflavin,
k
cat
/

K
m

= 7.5 10
−3
μM
−1
s
−1
). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium
Bacillus subtilis
(roseoflavin,
k
cat
/

K
m

= 1.3 10
−2
μM
−1
s
−1
; riboflavin,
k
cat
/

K
m

= 1.3 10
−2
μM
−1
s
−1
). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of
S. davawensis ribC
did not confer roseoflavin resistance to a
ribC
-defective
B. subtilis
strain.
The Target for the
Pseudomonas putida
Crc Global Regulator in the Benzoate Degradation Pathway Is the BenR Transcriptional Regulator
Authors: Moreno Renata , Rojo Fernando 
Published: December 2007

ABSTRACT

Crc protein is a global regulator involved in catabolite repression control of several pathways for the assimilation of carbon sources in pseudomonads when other preferred substrates are present. In
Pseudomonas putida
cells growing exponentially in a complete medium containing benzoate, Crc strongly inhibits the expression of the benzoate degradation genes. These genes are organized into several transcriptional units. We show that Crc directly inhibits the expression of the peripheral genes that transform benzoate into catechol (the
ben
genes) but that its effect on genes corresponding to further steps of the pathway (the
cat
and
pca
genes of the central catechol and β-ketoadipate pathways) is indirect, since these genes are not induced because the degradation intermediates, which act as inducers, are not produced. Crc inhibits the translation of target genes by binding to mRNA. The expression of the
ben
,
cat
, and
pca
genes requires the BenR, CatR, and PcaR transcriptional activators, respectively. Crc significantly reduced
benABCD
mRNA levels but did not affect those of
benR
. Crc bound to the 5′ end of
benR
mRNA but not to equivalent regions of
catR
and
pcaR
mRNAs. A translational fusion of the
benR
and
lacZ
genes was sensitive to Crc, but a transcriptional fusion was not. We propose that Crc acts by reducing the translation of
benR
mRNA, decreasing BenR levels below those required for the full expression of the
benABCD
genes. This strategy provides great metabolic flexibility, allowing the hierarchical assimilation of different structurally related compounds that share a common central pathway by selectively regulating the entry of each substrate into the central pathway.
Ammonia-Induced Formation of an AmtB-GlnK Complex Is Not Sufficient for Nitrogenase Regulation in the Photosynthetic BacteriumRhodobacter capsulatus
Authors: Tremblay Pier-Luc , Hallenbeck Patrick C. 
Published: December 2007

ABSTRACTA series ofRhodobacter capsulatusAmtB variants were created and assessed for effects on ammonia transport, formation of AmtB-GlnK complexes, and regulation of nitrogenase activity and NifH ADP-ribosylation. Confirming previous reports, H193 and H342 were essential for ammonia transport and the replacement of aspartate 185 with glutamate reduced ammonia transport. Several amino acid residues, F131, D334, and D335, predicted to be critical for AmtB activity, are shown here for the first time by mutational analysis to be essential for transport. Alterations of the C-terminal tail reduced methylamine transport, prevented AmtB-GlnK complex formation, and abolished nitrogenase switch-off and NifH ADP-ribosylation. On the other hand, D185E, with a reduced level of transport, was capable of forming an ammonium-induced complex with GlnK and regulating nitrogenase. This reinforces the notions that ammonia transport is not sufficient for nitrogenase regulation and that formation of an AmtB-GlnK complex is necessary for these processes. However, some transport-incompetent AmtB variants, i.e., F131A, H193A, and H342A, form ammonium-induced complexes with GlnK but fail to properly regulate nitrogenase. These results show that formation of an AmtB-GlnK complex is insufficient in itself for nitrogenase regulation and suggest that partial ammonia transport or occupation of the pore by ammonia is essential for this function.
The LysR-Type Transcriptional Regulator LeuO Controls Expression of Several Genes in
Salmonella enterica
Serovar Typhi
Authors: Hernández-Lucas I. , Gallego-Hernández A. L. , Encarnación S. , Fernández-Mora M. , Martínez-Batallar A. G. , Salgado H. , Oropeza R. , Calva E. 
Published: December 2007

ABSTRACT

LeuO is a LysR-type transcriptional regulator that has been implicated in the bacterial stringent response and in the virulence of
Salmonella
. A genomic analysis with
Salmonella enterica
serovar Typhi revealed that LeuO is a positive regulator of OmpS1, OmpS2, AssT, and STY3070. In contrast, LeuO down-regulated the expression of OmpX, Tpx, and STY1978. Transcriptional fusions supported the positive and negative LeuO regulation. Expression of
ompS1
,
assT
, and STY3070 was induced in an
hns
mutant, consistent with the notion that H-NS represses these genes; transcriptional activity was lower for
tpx
and STY1978 in an
hns
background, suggesting that this global regulatory protein has a positive effect. In contrast,
ompS2
and
ompX
expression appeared to be H-NS independent. LeuO specifically bound to the 5′ intergenic regions of
ompS2
,
assT
, STY3070,
ompX
, and
tpx
, while it was not observed to bind to the promoter region of STY1978, suggesting that LeuO regulates in direct and indirect ways. In this work, a novel set of genes belonging to the LeuO regulon are described; interestingly, these genes are involved in a variety of biological processes, suggesting that LeuO is a global regulator in
Salmonella
.
LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence
Authors: Nijvipakul Sarayut , Wongratana Janewit , Suadee Chutintorn , Entsch Barrie , Ballou David P. , Chaiyen Pimchai 
Published: December 2007

ABSTRACT

The
luxG
gene is part of the
lux
operon of marine luminous bacteria.
luxG
has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study,
luxG
from
Photobacterium leiognathi
TH1 was cloned and expressed in
Escherichia coli
in both native and C-terminal His
6
-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0,

K
m

for NADH,

K
m

for FMN, and
k
cat
were calculated to be 15.1 μM, 2.7 μM, and 1.7 s
−1
, respectively. Coupled assays between LuxG and luciferases from
P. leiognathi
TH1 and
Vibrio campbellii
also showed that LuxG could supply FMNH
−
for light emission in vitro. A
luxG
gene knockout mutant of
P. leiognathi
TH1 exhibited a much dimmer luminescent phenotype compared to the native
P. leiognathi
TH1, implying that LuxG is the most significant source of FMNH
−
for the luminescence reaction in vivo.
Genetic Analysis of Lipooligosaccharide Core Biosynthesis in
Campylobacter jejuni
81-176
Authors: Kanipes Margaret I. , Tan Xuan , Akelaitis Aurel , Li Jiajun , Rockabrand David , Guerry Patricia , Monteiro Mario A. 
Published: December 2007

ABSTRACT

We report isolation and characterization of
Campylobacter jejuni
81-176
lgtF
and
galT
lipooligosaccharide (LOS) core mutants. It has been suggested that the
lgtF
gene of
C. jejuni
encodes a two-domain glucosyltransferase that is responsible for the transfer of a β-1,4-glucose residue on heptosyltransferase I (Hep I) and for the transfer of a β-1,2-glucose residue on Hep II. A site-specific mutation in the
lgtF
gene of
C. jejuni
81-176 resulted in expression of a truncated LOS, and complementation of the mutant in
trans
restored the core mobility to that of the wild type. Mass spectrometry and nuclear magnetic resonance of the truncated LOS confirmed the loss of two glucose residues, a β-1,4-glucose on Hep I and a β-1,2-glucose on Hep II. Mutation of another gene,
galT
, encoding a glycosyltransferase, which maps outside the region defined as the LOS biosynthetic locus in
C. jejuni
81-176, resulted in loss of the β-(1,4)-galactose residue and all distal residues in the core. Both mutants invaded intestinal epithelial cells in vitro at levels comparable to the wild-type levels, in marked contrast to a deeper inner core
waaC
mutant. These studies have important implications for the role of LOS in the pathogenesis of
Campylobacter
-mediated infection.
lfnA
from
Pseudomonas aeruginosa
O12 and
wbuX
from
Escherichia coli
O145 Encode Membrane-Associated Proteins and Are Required for Expression of 2,6-Dideoxy-2-Acetamidino-
l
-Galactose in Lipopolysaccharide O Antigen
Authors: King Jerry D. , Mulrooney Erin F. , Vinogradov Evgeny , Kneidinger Bernd , Mead Kristen , Lam Joseph S. 
Published: December 2007

ABSTRACT

The rare sugar 2,6-dideoxy-2-acetamidino-
l
-galactose (
l
-FucNAm) is found only in bacteria and is a component of cell surface glycans in a number of pathogenic species, including the O antigens of
Pseudomonas aeruginosa
serotype O12 and
Escherichia coli
O145.
P
.
aeruginosa
is an important opportunistic pathogen, and the O12 serotype is associated with multidrug-resistant epidemic outbreaks. O145 is one of the classic non-O157 serotypes associated with Shiga toxin-producing, enterohemorrhagic
E
.
coli
. The acetamidino (NAm) moiety of
l
-FucNAm is of interest, because at neutral pH it contributes a positive charge to the cell surface, and we aimed to characterize the biosynthesis of this functional group. The pathway is not known, but expression of NAm-modified sugars coincides with the presence of a
pseA
homologue in the relevant biosynthetic locus. PseA is a putative amidotransferase required for synthesis of a NAm-modified sugar in
Campylobacter jejuni
. In
P
.
aeruginosa
O12 and
E
.
coli
O145, the
pseA
homologues are
lfnA
and
wbuX
, respectively, and we hypothesized that these genes function in
l
-FucNAm biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and nuclear magnetic resonance analysis of the
lfnA
mutant O-antigen structure indicated that the mutant expresses 2,6-dideoxy-2-acetamido-
l
-galactose (
l
-FucNAc) in place of
l
-FucNAm. The mutation could be complemented by expression of either His
6
-tagged
lfnA
or
wbuX
in
trans
, confirming that these genes are functional homologues and that they are required for NAm moiety synthesis. Both proteins retained their activity when fused to a His
6
tag and localized to the membrane fraction. These data will assist future biochemical investigation of this pathway.
Altered Utilization ofN-Acetyl-d-Galactosamine byEscherichia coliO157:H7 from the 2006 Spinach Outbreak
Authors: Mukherjee Amit , Mammel Mark K. , LeClerc J. Eugene , Cebula Thomas A. 
Published: December 2007

ABSTRACTIn silico analyses of previously sequenced strains ofEscherichia coliO157:H7, EDL933 and Sakai, localized the gene cluster for the utilization ofN-acetyl-d-galactosamine (Aga) andd-galactosamine (Gam). This gene cluster encodes the Aga phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) and other catabolic enzymes responsible for transport and catabolism of Aga. As the complete coding sequences for enzyme IIA (EIIA)Aga/Gam, EIIBAga, EIICAga, and EIIDAgaof the Aga PTS are present,E. coliO157:H7 strains normally are able to utilize Aga as a sole carbon source. The Gam PTS complex, in contrast, lacks EIICGam, and consequently,E. coliO157:H7 strains cannot utilize Gam. Phenotypic analyses of 120 independent isolates ofE. coliO157:H7 from our culture collection revealed that the overwhelming majority (118/120) displayed the expected Aga+Gam−phenotype. Yet, when 194 individual isolates, derived from a 2006 spinach-associatedE. coliO157:H7 outbreak, were analyzed, all (194/194) displayed an Aga−Gam−phenotype. Comparison ofaga/gamsequences from two spinach isolates with those of EDL933 and Sakai revealed a single nucleotide change (G:C→A:T) in theagaFgene in the spinach-associated isolates. The base substitution inagaF, which encodes EIIAAga/Gamof the PTS, changes a conserved glycine residue to serine (Gly91Ser). Pyrosequencing of this region showed that all spinach-associatedE. coliO157:H7 isolates harbored this same G:C→A:T substitution. Notably, whenagaF+was cloned into an expression vector and transformed into six spinach isolates, all (6/6) were able to grow on Aga, thus demonstrating that the Gly91Ser substitution underlies the Aga−phenotype in these isolates.
Diagnostic and prognostic significance of cyclin A expression in low-grade astrocytomas: comparison with astrogliosis and high-grade tumours
Authors: Hara A , Saegusa M , Ichinoe M , Okayasu I 
Published: December 2007

Aim:Definitive distinction between low-grade astrocytoma and astrogliosis is a long-standing difficulty due to their similar histopathological characteristics. To clarify differences in biological significance, this study focused on various components of the cell cycle machinery and proliferation as key parameters, comparing expression in astrogliosis, as well as low- and high-grade astrocytomas.Methods:The expression of p16, p21 and p27, and cyclin A, cyclin D1, cyclin E, Rb and Ki-67 was immunohistochemically examined in 40 cases of astrogliosis and 48 cases of low-grade astrocytomas (grade II), as well as 50 high-grade tumours (grades III and IV). The results were also compared with survival data for the astrocytomas.Results:Cell proliferation determined by Ki-67 immunoreactivity did not differ between astrogliosis and low-grade tumours. Average labelling indices (LIs) for p16, p21, Rb, cyclin A and cyclin E showed a stepwise increase from astrogliosis, through low- to high-grade astrocytomas, indicating the possibility that over 9%, 6% and 4% of LIs for p16, p21 and cyclin A, respectively, may be useful predictors in the case of the latter, in contrast to significant decrease in p27 LIs. Significantly higher mean LI values for cyclin D1 were also evident in astrogliosis (12.42) as compared with astrocytomas (low grade, 2.26; high grade, 4.60). Positive correlations between LIs for Rb and Ki-67 were observed with astrogliosis and low- but not high-grade tumours. In addition, high cyclin A LI values were independently associated with poor outcome in low-grade tumours.Conclusion:These findings provide evidence that expression of cell-cycle-related molecules may be a reliable parameter for differential diagnosis of low-grade astrocytomas and astrogliosis. Moreover, detection of cyclin A appears to be useful for predicting behaviour of low-grade astrocytomas.
Reduction of E-cadherin expression is associated with non-lobular breast carcinomas of basal-like and triple negative phenotype
Authors: Mahler-Araujo B , Savage K , Parry S , Reis-Filho J S 
Published: December 2007

Aim:E-cadherin inactivation in breast cancer has been shown to be strongly associated with lobular breast cancer. However, little is known about the levels of E-cadherin expression according to the breast cancer “molecular” subtypes. The aim of this study was to address the distribution of E-cadherin expression according to the different molecular subtypes of breast cancer.Methods:E-cadherin expression was immunohistochemically analysed in a tissue microarray containing duplicate cores of 245 invasive breast carcinomas, of which 182 cases were of non-lobular histology, using a semi-quantitative scoring system based on the percentage of cells showing membrane immunopositivity.Results:In non-lobular breast carcinomas, reduced and/or negative E-cadherin expression was significantly associated with lack of oestrogen receptor expression, low levels ofCCND1expression, positivity for cytokeratins 5/6 and 17, epidermal growth factor receptor and caveolins 1 and 2, p53 expression, high MIB-1 proliferation indices, basal-like phenotype and triple negative phenotype.Conclusion:This study demonstrates that in the group of non-lobular breast cancers, reduction/lack of E-cadherin expression is preferentially found in basal-like breast carcinomas.
Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets, and leukocytes
Authors: Tjwa Marc , Bellido-Martin Lola , Lin Yuan , Lutgens Esther , Plaisance Stéphane , Bono Françoise , Delesque-Touchard Nathalie , Hervé Caroline , Moura Rute , Billiau An D. , Aparicio Cristina , Levi Marcel , Daemen Mat , Dewerchin Mieke , Lupu Florea , Arnout Jef , Herbert Jean-Marc , Waer Mark , García de Frutos Pablo , Dahlbäck Björn , Carmeliet Peter , Hoylaerts Marc F. , Moons Lieve 
Published: December 2007

AbstractThe role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation.
The MMSET protein is a histone methyltransferase with characteristics of a transcriptional corepressor
Authors: Marango Jotin , Shimoyama Manabu , Nishio Hitomi , Meyer Julia A. , Min Dong-Joon , Sirulnik Andres , Martinez-Martinez Yolanda , Chesi Marta , Bergsagel P. Leif , Zhou Ming-Ming , Waxman Samuel , Leibovitch Boris A. , Walsh Martin J. , Licht Jonathan D. 
Published: December 2007

Abstract
MMSET, identified by its fusion to the IgH locus in t(4;14)-associated multiple myeloma, possesses domains found within chromatin regulators, including the SET domain. MMSET protein is overexpressed and highly associated with chromatin in myeloma cell lines carrying t(4;14). MMSET possesses methyltransferase activity for core histone H3 lysine 4 and histone 4 lysine 20, whereas MMSET made in cells only modified H4. Segments of MMSET fused to the Gal4 DNA binding domain repressed transcription of a chromatin-embedded Gal4 reporter gene. MMSET-mediated repression was associated with increased H4K20 methylation gene and loss of histone acetylation. Consistent with this repressive activity, MMSET could form a complex with HDAC1 and HDAC2, mSin3a, and the histone demethylase LSD1, suggesting that it is a component of corepressor complexes. Furthermore, MMSET coexpression enhances HDAC1- and HDAC2-mediated repression in transcriptional reporter assays. Finally, shRNA-mediated knockdown of MMSET compromised viability of a myeloma cell line, suggesting a biologic role for the protein in malignant cell growth. Collectively, these data suggest that, by acting directly as a modifier of chromatin as well as through binding of other chromatin-modifying enzymes, MMSET influences gene expression and potentially acts as a pathogenic agent in multiple myeloma.
Genetic endothelial systems biology of sickle stroke risk
Authors: Chang Milbauer Liming , Wei Peng , Enenstein Judy , Jiang Aixiang , Hillery Cheryl A. , Scott J. Paul , Nelson Stephen C. , Bodempudi Vidya , Topper James N. , Yang Ruey-Bing , Hirsch Betsy , Pan Wei , Hebbel Robert P. 
Published: December 2007

AbstractGenetic differences in endothelial biology could underlie development of phenotypic heterogeneity among persons afflicted with vascular diseases. We obtained blood outgrowth endothelial cells from 20 subjects with sickle cell anemia (age, 4-19 years) shown to be either at-risk (n = 11) or not-at-risk (n = 9) for ischemic stroke because of, respectively, having or not having occlusive disease at the circle of Willis. Gene expression profiling identified no significant single gene differences between the 2 groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were predetermined to survey each of 9 biologic systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches. Correspondingly, subsequent biologic testing showed significantly exaggerated RelA activation on the part of blood outgrowth endothelial cells from the at-risk subjects in response to stimulation with interleukin-1β/tumor necrosis factorα. We conclude that the pathobiology of circle of Willis disease in the child with sickle cell anemia predominantly involves inflammation biology, which could reflect differences in genetically determined endothelial biology that account for differing host responses to inflammation.
IL6/sIL6R complex contributes to emergency granulopoietic responses in G-CSF– and GM-CSF–deficient mice
Authors: Walker Francesca , Zhang Hui-Hua , Matthews Vance , Weinstock Janet , Nice Edouard C. , Ernst Matthias , Rose-John Stefan , Burgess Antony W. 
Published: December 2007

AbstractMice defective in both granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have severely impaired neutrophil production and function, yet these mice respond to acute pathogen challenge with a significant neutrophil response. We have recently reported the development of an in vitro system to detect granulopoietic cytokines secreted from cells isolated from G-CSF, GM-CSF double knockout mice. The conditioned media produced by these cells after stimulation with lipopolysaccharide or Candida albicans supports the production and differentiation of granulocytes (ie, the conditioned media contains neutrophil promoting activity [NPA]). We now show that the NPA in the G-CSF−/−/GM-CSF−/− conditioned media requires interleukin-6 (IL6), is abolished by soluble gp130, and can be specifically immunodepleted by an anti-IL6R antibody. NPA effects on bone marrow cells are also mimicked by Hyper-IL6, and the soluble IL6R is present in NPA. These results show that the IL6/sIL6R complex is the major effector of NPA. NPA production by mice defective for both G-CSF and GM-CSF uncovers an alternative pathway to granulocyte production, which is activated after exposure to pathogens.