bluuebunny/crossref_metadata_2025 huggingface dataset:
On the Nature of Critical Heat Flux in Microchannels
Authors: Bergles A. E. , Kandlikar S. G.
Published: February 2005

The critical heat flux (CHF) limit is an important consideration in the design of most flow boiling systems. Before the use of microchannels under saturated flow boiling conditions becomes widely accepted in cooling of high-heat-flux devices, such as electronics and laser diodes, it is essential to have a clear understanding of the CHF mechanism. This must be coupled with an extensive database covering a wide range of fluids, channel configurations, and operating conditions. The experiments required to obtain this information pose unique challenges. Among other issues, flow distribution among parallel channels, conjugate effects, and instrumentation need to be considered. An examination of the limited CHF data indicates that CHF in parallel microchannels seems to be the result of either an upstream compressible volume instability or an excursive instability rather than the conventional dryout mechanism. It is expected that the CHF in parallel microchannels would be higher if the flow is stabilized by an orifice at the entrance of each channel. The nature of CHF in microchannels is thus different than anticipated, but recent advances in microelectronic fabrication may make it possible to realize the higher power levels.
An Assessment of Module Cooling Enhancement With Thermoelectric Coolers
Authors: Simons R. E. , Ellsworth M. J. , Chu R. C.
Published: February 2005

The trend towards increasing heat flux at the chip and module level in computers is continuing. This trend coupled with the desire to increase performance by reducing chip operating temperatures presents a further challenge to thermal engineers. This paper will provide an assessment of the potential for module cooling enhancement with thermoelectric coolers. A brief background discussion of thermoelectric cooling is provided citing some of the early history of thermoelectrics as well as more recent developments from the literature. An example analyzing cooling enhancement of a multichip module package with a thermoelectric cooler is discussed. The analysis utilizes closed form equations incorporating both thermoelectric cooler parameters and package level thermal resistances to relate allowable module power to chip temperature. Comparisons are made of allowable module power with and without thermoelectric coolers based upon either air or water module level cooling. These results show that conventional thermoelectric coolers are inadequate to meet the requirements. Consideration is then given to improvements in allowable module power that might be obtained through increases in the thermoelectric figure of merit ZT or miniaturization of the thermoelectric elements.
Fis Regulates Transcriptional Induction of RpoS inSalmonella enterica
Authors: Hirsch Matthew , Elliott Thomas
Published: February 2005

The sigma factor RpoS is known to regulate at least 60 genes in response to environmental sources of stress or during growth to stationary phase (SP). Accumulation of RpoS relies on integration of multiple genetic controls, including regulation at the levels of transcription, translation, protein stability, and protein activity. Growth to SP in rich medium results in a 30-fold induction of RpoS, although the mechanism of this regulation is not understood. We characterized the activity of promoters servingrpoSinSalmonella entericaserovar Typhimurium and report that regulation of transcription during growth into SP depends on Fis, a DNA-binding protein whose abundance is high during exponential growth and very low in SP. Afismutant ofS. entericaserovar Typhimurium showed a ninefold increase in expression from the majorrpoSpromoter (PrpoS) during exponential growth, whereas expression during SP was unaffected. Increased transcription from PrpoSin the absence of Fis eliminated the transcriptional induction as cells enter SP. The mutant phenotype can be complemented by wild-typefiscarried on a single-copy plasmid. Fis regulation ofrpoSrequires the presence of a Fis site positioned at −50 with respect to PrpoS, and this site is bound by Fis in vitro. A model is presented in which Fis binding to this site allows repression ofrpoSspecifically during exponential growth, thus mediating transcriptional regulation ofrpoS.
The Metal Permease ZupT from
Escherichia coli
Is a Transporter with a Broad Substrate Spectrum
Authors: Grass Gregor , Franke Sylvia , Taudte Nadine , Nies Dietrich H. , Kucharski Lisa M. , Maguire Michael E. , Rensing Christopher
Published: February 2005

The
Escherichia coli zupT
(formerly
ygiE)
gene encodes a cytoplasmic membrane protein (ZupT) related to members of the eukaryotic ZIP family of divalent metal ion transporters. Previously, ZupT was shown to be responsible for uptake of zinc. In this study, we show that ZupT is a divalent metal cation transporter of broad substrate specificity. An
E. coli
strain with a disruption in all known iron uptake systems could grow in the presence of chelators only if
zupT
was expressed. Heterologous expression of
Arabidopsis thaliana
ZIP1 could also alleviate iron deficiency in this
E. coli
strain, as could expression of indigenous
mntH
or
feoABC
. Transport studies with intact cells showed that ZupT facilitates uptake of
55
Fe
2+
similarly to uptake of MntH or Feo. Other divalent cations were also taken up by ZupT, as shown using
57
Co
2+
. Expression of
zupT
rendered
E. coli
cells hypersensitive to Co
2+
and sensitive to Mn
2+
. ZupT did not appear to be metal regulated: expression of a Φ(
zupT-lacZ
) operon fusion indicated that
zupT
is expressed constitutively at a low level.
An Unusual Mutation Results in the Replacement of Diaminopimelate with Lanthionine in the Peptidoglycan of a Mutant Strain of
Mycobacterium smegmatis
Authors: Consaul Sandra A. , Wright Lori F. , Mahapatra Sebabrata , Crick Dean C. , Pavelka Martin S.
Published: February 2005

Mycobacterial peptidoglycan contains
l
-alanyl-
d
-
iso
-glutaminyl-
meso
-diaminopimelyl-
d-
alanyl-
d-
alanine peptides, with the exception of the peptidoglycan of
Mycobacterium leprae
, in which glycine replaces the
l-
alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the
meso
-diaminopimelate (DAP) moiety of one peptide and the penultimate
d-
alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of
Mycobacterium smegmatis
that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to β-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants,
M. smegmatis
strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the
cbs
gene, encoding cystathionine β-synthase, an enzyme that is a part of the cysteine biosynthetic pathway.
Transcriptional Polarity in rRNA Operons of
Escherichia coli nusA
and
nusB
Mutant Strains
Authors: Quan Selwyn , Zhang Ning , French Sarah , Squires Catherine L.
Published: February 2005

Synthesis of ribosomes in
Escherichia coli
requires an antitermination system that modifies RNA polymerase to achieve efficient transcription of the genes specifying 16S, 23S, and 5S rRNA. This modification requires nucleotide signals in the RNA and specific transcription factors, such as NusA and NusB. Transcription of
rrn
operons in strains lacking the ability to produce either NusA or NusB was examined by electron microscopy. The distribution and numbers of RNA polymerase molecules on
rrn
operons were determined for each mutant. Compared to the wild type, the 16S gene in the
nusB
mutant strain had an equivalent number of RNA polymerase molecules, but the number of RNA polymerase molecules was reduced 1.4-fold for the
nusA
mutant. For both mutant strains, there were twofold-fewer RNA polymerase molecules on the 23S RNA gene than for the wild type. Overall, the mutant strains each had 1.6-fold-fewer RNA polymerase molecules on their
rrn
operons than did the wild type. To determine if decreased transcription of the 23S gene observed by electron microscopy also affected the 30S/50S ribosomal subunit ratio, ribosome profiles were examined by sucrose gradient analysis. The 30S/50S ratio increased 2.5- to 3-fold for the
nus
mutant strains over that for wild-type cells. Thus, strains carrying either a
nusA
mutation or a
nusB
mutation have defects in transcription of 23S rRNA.
Activation of the
vrg6
Promoter of
Bordetella pertussis
by RisA
Authors: Cróinín Tadhg Ó. , Grippe Vanessa K. , Merkel Tod J.
Published: February 2005

The BvgAS two-component system positively regulates the expression of the virulence genes of
Bordetella pertussis
and negatively regulates a second set of genes whose function is unknown. The BvgAS-mediated regulation of the
bvg
-repressed genes is accomplished through the activation of expression of the negative regulator, BvgR. A second two-component regulatory system, RisAS, is required for expression of the
bvg
-repressed surface antigens VraA and VraB. We examined the roles of BvgR and RisA in the regulation of four
bvg
-repressed genes in
B. pertussis
. Our analyses demonstrated that all four genes are repressed by the product of the
bvgR
locus and are activated by the product of the
risA
locus. Deletion analysis of the
vrg6
promoter identified the upstream and downstream boundaries of the promoter and, in contrast to previously published results, demonstrated that sequences downstream of the start of transcription are not required for the regulation of expression of
vrg6
. Gel mobility-shift experiments demonstrated sequence-specific binding of RisA to the
vrg6
and
vrg18
promoters, and led to the identification of two putative RisA binding sites. Finally, transcriptional analysis and Western blot analysis demonstrated that BvgR regulates neither the expression nor the stability of RisA.
Compiling a Molecular Inventory for
Mycobacterium bovis
BCG at Two Growth Rates: Evidence for Growth Rate-Mediated Regulation of Ribosome Biosynthesis and Lipid Metabolism
Authors: Beste D. J. V. , Peters J. , Hooper T. , Avignone-Rossa C. , Bushell M. E. , McFadden J.
Published: February 2005

An experimental system of
Mycobacterium tuberculosis
growth in a carbon-limited chemostat has been established by the use of
Mycobacterium bovis
BCG as a model organism. For this model, carbon-limited chemostats with low concentrations of glycerol were used to simulate possible growth rates during different stages of tuberculosis. A doubling time of 23 h (
D
= 0.03 h
−1
) was adopted to represent cells during the acute phase of infection, whereas a lower dilution rate equivalent to a doubling time of 69 h (
D
= 0.01 h
−1
) was used to model mycobacterial persistence. This chemostat model allowed the specific response of the mycobacterial cell to carbon limitation at different growth rates to be elucidated. The macromolecular (RNA, DNA, carbohydrate, and lipid) and elemental (C, H, and N) compositions of the biomass were determined for steady-state cultures, revealing that carbohydrates and lipids comprised more than half of the dry mass of the BCG cell, with only a quarter of the dry weight consisting of protein and RNA. Consistent with studies of other bacteria, the specific growth rate impacts on the macromolecular content of BCG and the proportions of lipid, RNA, and protein increased significantly with the growth rate. The correlation of RNA content with the growth rate indicates that ribosome production in carbon-limited
M. bovis
BCG cells is subject to growth rate-dependent control. The results also clearly show that the proportion of lipids in the mycobacterial cell is very sensitive to changes in the growth rate, probably reflecting changes in the amounts of storage lipids. Finally, this study demonstrates the utility of the chemostat model of mycobacterial growth for functional genomic, physiology, and systems biology studies.
Two Novel Phycoerythrin-Associated Linker Proteins in the Marine CyanobacteriumSynechococcussp. Strain WH8102
Authors: Six Christophe , Thomas Jean-Claude , Thion Laurent , Lemoine Yves , Zal Frank , Partensky Frédéric
Published: February 2005

The recent availability of the whole genome ofSynechococcussp. strain WH8102 allows us to have a global view of the complex structure of the phycobilisomes of this marine picocyanobacterium. Genomic analyses revealed several new characteristics of these phycobilisomes, consisting of an allophycocyanin core and rods made of one type of phycocyanin and two types of phycoerythrins (I and II). Although the allophycocyanin appears to be similar to that found commonly in freshwater cyanobacteria, the phycocyanin is simpler since it possesses only one complete set of α and β subunits and two rod-core linkers (CpcG1 and CpcG2). It is therefore probably made of a single hexameric disk per rod. In contrast, we have found two novel putative phycoerythrin-associated linker polypeptides that appear to be specific for marineSynechococcusspp. The first one (SYNW2000) is unusually long (548 residues) and apparently results from the fusion of a paralog of MpeC, a phycoerythrin II linker, and of CpeD, a phycoerythrin-I linker. The second one (SYNW1989) has a more classical size (300 residues) and is also an MpeC paralog. A biochemical analysis revealed that, like MpeC, these two novel linkers were both chromophorylated with phycourobilin. Our data suggest that they are both associated (partly or totally) with phycoerythrin II, and we propose to name SYNW2000 and SYNW1989 MpeD and MpeE, respectively. We further show that acclimation of phycobilisomes to high light leads to a dramatic reduction of MpeC, whereas the two novel linkers are not significantly affected. Models for the organization of the rods are proposed.
Structural and Spectroscopic Properties of a Reaction Center Complex from the Chlorosome-Lacking Filamentous Anoxygenic Phototrophic BacteriumRoseiflexus castenholzii
Authors: Yamada Mitsunori , Zhang Hui , Hanada Satoshi , Nagashima Kenji V. P. , Shimada Keizo , Matsuura Katsumi
Published: February 2005

The photochemical reaction center (RC) complex ofRoseiflexus castenholzii, which belongs to the filamentous anoxygenic phototrophic bacteria (green filamentous bacteria) but lacks chlorosomes, was isolated and characterized. The genes coding for the subunits of the RC and the light-harvesting proteins were also cloned and sequenced. The RC complex was composed of L, M, and cytochrome subunits. The cytochrome subunit showed a molecular mass of approximately 35 kDa, contained hemesc, and functioned as the electron donor to the photo-oxidized special pair of bacteriochlorophylls in the RC. The RC complex appeared to contain three molecules of bacteriochlorophyll and three molecules of bacteriopheophytin, as in the RC preparation fromChloroflexus aurantiacus.Phylogenetic trees based on the deduced amino acid sequences of the RC subunits suggested thatR. castenholziihad diverged fromC. aurantiacusvery early after the divergence of filamentous anoxygenic phototrophic bacteria from purple bacteria. AlthoughR. castenholziiis phylogenetically related toC. aurantiacus, the arrangement of itspufgenes, which code for the light-harvesting proteins and the RC subunits, was different from that inC. aurantiacusand similar to that in purple bacteria. The genes are found in the orderpufB,-A,-L, -M, and-C, with thepufLandpufMgenes forming one continuous open reading frame. Since the photosynthetic apparatus and genes ofR. castenholziihave intermediate characteristics between those of purple bacteria andC. aurantiacus, it is likely that they retain many features of the common ancestor of purple bacteria and filamentous anoxygenic phototrophic bacteria.
Divergent Regulatory Pathways Control A and S Motility in
Myxococcus xanthus
through FrzE, a CheA-CheY Fusion Protein
Authors: Li Yinuo , Bustamante Víctor H. , Lux Renate , Zusman David , Shi Wenyuan
Published: February 2005

Myxococcus xanthus
moves on solid surfaces by using two gliding motility systems, A motility for individual-cell movement and S motility for coordinated group movements. The
frz
genes encode chemotaxis homologues that control the cellular reversal frequency of both motility systems. One of the components of the core Frz signal transduction pathway, FrzE, is homologous to both CheA and CheY from the enteric bacteria and is therefore a novel CheA-CheY fusion protein. In this study, we investigated the role of this fusion protein, in particular, the CheY domain (FrzE
CheY
). FrzE
CheY
retains all of the highly conserved residues of the CheY superfamily of response regulators, including Asp709, analogous to phosphoaccepting Asp57 of
Escherichia coli
CheY. While in-frame deletion of the entire
frzE
gene caused both motility systems to show a hyporeversal phenotype, in-frame deletion of the FrzE
CheY
domain resulted in divergent phenotypes for the two motility systems: hyperreversals of the A-motility system and hyporeversals of the S-motility system. To further investigate the role of FrzE
CheY
in A and S motility, point mutations were constructed such that the putative phosphoaccepting residue, Asp709, was changed from D to A (and was therefore never subject to phosphorylation) or E (possibly mimicking constitutive phosphorylation). The D709A mutant showed hyperreversals for both motilities, while the D709E mutant showed hyperreversals for A motility and hyporeversal for S motility. These results show that the FrzE
CheY
domain plays a critical signaling role in coordinating A and S motility. On the basis of the phenotypic analyses of the
frzE
mutants generated in this study, a model is proposed for the divergent signal transduction through FrzE in controlling and coordinating A and S motility in
M. xanthus
.
Multiple Extracellular Loops Contribute to Substrate Binding and Transport by the
Escherichia coli
Cobalamin Transporter BtuB
Authors: Fuller-Schaefer Cynthia A. , Kadner Robert J.
Published: February 2005

The
Escherichia coli
outer membrane TonB-dependent transporters for iron complexes and cobalamins recognize their multiple and diverse substrates with high specificity and affinity. The X-ray crystallographic structures of several transporters show that the substrate-binding surfaces are comprised of residues from the internal globular domain and multiple extracellular loops. The extracellular loops on the N-terminal half of the transmembrane beta-barrel of the cobalamin transporter BtuB participate in binding of the cofactor calcium atoms and undergo substantial conformation changes upon substrate binding. The functional relevance of the five C-terminal loops was examined by examining the effects of short in-frame deletions. Each loop contributed in different ways to the binding of BtuB substrates. Deletions in loops 7, 8, 9, and 11 strongly decreased cobalamin binding and transport, whereas deletions in loops 8, 9, and 10 affected binding and entry of phage BF23. None of the loops were essential for the action of colicin E1 or E3, which is consistent with the crystallographic observation that the colicin E3 receptor-binding domain can contact almost all of the loops. A deletion in loop 9 or 11 eliminated the ability of cobalamin to inhibit the action of colicin E1. These phenotypes show that there are multiple independent binding elements and point out similarities and differences in binding properties among the TonB-dependent transporters.
Genome-Wide Analyses of
Escherichia coli
Gene Expression Responsive to the BaeSR Two-Component Regulatory System
Authors: Nishino Kunihiko , Honda Takeshi , Yamaguchi Akihito
Published: February 2005

The BaeSR two-component regulatory system controls expression of exporter genes conferring drug resistance in
Escherichia coli
(S. Nagakubo, K. Nishino, T. Hirata, and A. Yamaguchi, J. Bacteriol.
184:
4161-4167, 2002; N. Baranova and H. Nikaido, J. Bacteriol.
184:
4168-4176, 2002). To understand the whole picture of BaeSR regulation, a DNA microarray analysis of the effect of BaeR overproduction was performed. BaeR overproduction activated 59 genes related to two-component signal transduction, chemotactic responses, flagellar biosynthesis, maltose transport, and multidrug transport, and BaeR overproduction also repressed the expression of the
ibpA
and
ibpB
genes. All of the changes in the expression levels were also observed by quantitative real-time reverse transcription-PCR analysis. The expression levels of 15 of the 59 BaeR-activated genes were decreased by deletion of
baeSR
. Of 11 genes induced by indole (a putative inducer of the BaeSR system), 10 required the BaeSR system for induction. Combination of the expression data sets revealed a BaeR-binding site sequence motif, 5′-TTTTTCTCCATDATTGGC-3′ (where D is G, A, or T). Several genes up-regulated by BaeR overproduction, including genes for maltose transport, chemotactic responses, and flagellar biosynthesis, required an intact PhoBR or CreBC two-component regulatory system for up-regulation. These data indicate that there is cross-regulation among the BaeSR, PhoBR, and CreBC two-component regulatory systems. Such a global analysis should reveal the regulatory network of the BaeSR system.
Structure of the Nucleotide Complex of PyrR, the
pyr
Attenuation Protein from
Bacillus caldolyticus
, Suggests Dual Regulation by Pyrimidine and Purine Nucleotides
Authors: Chander Preethi , Halbig Kari M. , Miller Jamie K. , Fields Christopher J. , Bonner Heather K. S. , Grabner Gail K. , Switzer Robert L. , Smith Janet L.
Published: February 2005

PyrR is a protein that regulates the expression of genes and operons of pyrimidine nucleotide biosynthesis (
pyr
genes) in many bacteria. PyrR acts by binding to specific sequences on
pyr
mRNA and causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated. PyrR from
Bacillus subtilis
has been purified and extensively studied. In this work, we describe the purification to homogeneity and characterization of recombinant PyrR from the thermophile
Bacillus caldolyticus
and the crystal structures of unliganded PyrR and a PyrR-nucleotide complex. The
B. caldolyticus pyrR
gene was previously shown to restore normal regulation of the
B. subtilis pyr
operon in a
pyrR
deletion mutant. Like
B. subtilis
PyrR,
B. caldolyticus
PyrR catalyzes the uracil phosphoribosyltransferase reaction but with maximal activity at 60°C. Crystal structures of
B. caldolyticus
PyrR reveal a dimer similar to the
B. subtilis
PyrR dimer and, for the first time, binding sites for nucleotides. UMP and GMP, accompanied by Mg
2+
, bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg
2+
binding differs. GMP binding was unexpected. The protein bound specific sequences of
pyr
RNA 100 to 1,000 times more tightly than
B. subtilis
PyrR, depending on the RNA tested and the assay method; uridine nucleotides enhanced RNA binding, but guanosine nucleotides antagonized it. The new findings of specific GMP binding and its antagonism of RNA binding suggest cross-regulation of the
pyr
operon by purines.
Screening for Quorum-Sensing Inhibitors (QSI) by Use of a Novel Genetic System, the QSI Selector
Authors: Rasmussen Thomas Bovbjerg , Bjarnsholt Thomas , Skindersoe Mette Elena , Hentzer Morten , Kristoffersen Peter , Köte Manuela , Nielsen John , Eberl Leo , Givskov Michael
Published: February 2005

With the widespread appearance of antibiotic-resistant bacteria, there is an increasing demand for novel strategies to control infectious diseases. Furthermore, it has become apparent that the bacterial life style also contributes significantly to this problem. Bacteria living in the biofilm mode of growth tolerate conventional antimicrobial treatments. The discovery that many bacteria use quorum-sensing (QS) systems to coordinate virulence and biofilm development has pointed out a new, promising target for antimicrobial drugs. We constructed a collection of screening systems, QS inhibitor (QSI) selectors, which enabled us to identify a number of novel QSIs among natural and synthetic compound libraries. The two most active were garlic extract and 4-nitro-pyridine-
N
-oxide (4-NPO). GeneChip-based transcriptome analysis revealed that garlic extract and 4-NPO had specificity for QS-controlled virulence genes in
Pseudomonas aeruginosa
. These two QSIs also significantly reduced
P. aeruginosa
biofilm tolerance to tobramycin treatment as well as virulence in a
Caenorhabditis elegans
pathogenesis model.
Role of Penicillin-Binding Protein 2 (PBP2) in the Antibiotic Susceptibility and Cell Wall Cross-Linking of
Staphylococcus aureus
: Evidence for the Cooperative Functioning of PBP2, PBP4, and PBP2A
Authors: Łęski Tomasz A. , Tomasz Alexander
Published: February 2005

Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of
Staphylococcus aureus
, was used to select a spontaneous resistant mutant of
S. aureus
strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The
pbpB
gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of
pbpD
, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of
pbpD
also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne
mecA
gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native
S. aureus
transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.
Light-Induced Carotenogenesis inStreptomyces coelicolorA3(2): Identification of an Extracytoplasmic Function Sigma Factor That Directs Photodependent Transcription of the Carotenoid Biosynthesis Gene Cluster
Authors: Takano Hideaki , Obitsu Saemi , Beppu Teruhiko , Ueda Kenji
Published: February 2005

Carotenoids are produced by a variety of organisms, but the mechanisms that regulate gene expression leading to carotenoid biosynthesis have been characterized for only a few organisms. In this study, we found thatStreptomyces coelicolorA3(2), a gram-positive filamentous bacterium, produces carotenoids under blue light induction. The carotenoid fraction isolated from the cell extract contained multiple compounds, including isorenieratene and β-carotene. The carotenoid biosynthesis gene cluster ofS. coelicolorconsists of two convergent operons,crtEIBVandcrtYTU, as previously shown forStreptomyces griseus. ThecrtEIBVnull mutant completely lost its ability to produce carotenoids. Thecrtgene cluster is flanked by a regulatory region that consists of two divergent operons,litRQandlitSAB. Thelit(light-induced transcription) genes encode a MerR-type transcriptional regulator (LitR), a possible oxidoreductase (LitQ), an extracytoplasmic function sigma factor (σLitS), a putative lipoprotein (LitA), and a putative anti-sigma factor (LitB). S1 protection assay revealed that the promoters precedingcrtE(PcrtE),crtY(PcrtY),litR(PlitR), andlitS(PlitS) are activated upon illumination. AlitSmutant lost both the ability to produce carotenoids and the activities of PcrtE, PcrtY, and PlitS, which suggested that σLitSdirects light-induced transcription from these promoters. An RNA polymerase holocomplex containing purified σLitSrecombinant protein generated specific PcrtEand PcrtYtranscripts in an in vitro runoff transcriptional assay. AlitRmutant that had an insertion of the kanamycin resistance gene was defective both in the ability to produce carotenoids and in all of the light-dependent promoter activities. Overexpression oflitSresulted in constitutive carotenoid production in both the wild type and thelitRmutant. These results indicate that σLitSacts as a light-induced sigma factor that directs transcription of thecrtbiosynthesis gene cluster, whose activity is controlled by an unknown LitR function. This is the first report to describe light-inducible gene expression inStreptomyces.
DNA Bridging: a Property Shared among H-NS-Like Proteins
Authors: Dame Remus T. , Luijsterburg Martijn S. , Krin Evelyne , Bertin Philippe N. , Wagner Rolf , Wuite Gijs J. L.
Published: February 2005

The nucleoid-associated protein H-NS is thought to play an essential role in the organization of bacterial chromatin in
Escherichia coli
. Homologues, often with very low sequence identity, are found in most gram-negative bacteria. Microscopic analysis reveals that, despite limited sequence identity, their structural organization results in similar DNA binding properties.
The Promoter Region Rather than Its Downstream Inverted Repeat Sequence Is Responsible for Low-Level Transcription of the Thermostable Direct Hemolysin-Related Hemolysin (
trh
) Gene of
Vibrio parahaemolyticus
Authors: Nakaguchi Yoshitsugu , Nishibuchi Mitsuaki
Published: February 2005

We determined the transcriptional start site of the thermostable direct hemolysin-related hemolysin gene (
trh
) of
Vibrio parahaemolyticus
by using a PCR-based method and identified the promoter. Mutagenic analysis indicated that the promoter-bearing region rather than its downstream inverted repeat sequence was responsible for the low-revel of
trh
transcription.
DNA Content and Nucleoid Distribution in
Methanothermobacter thermautotrophicus
Authors: Majerník Alan I. , Lundgren Magnus , McDermott Paul , Bernander Rolf , Chong James P. J.
Published: February 2005

Flow cytometry and epifluorescence microscopy results for the euryarchaeon
Methanothermobacter thermautotrophicus
were consistent with filaments containing multiple cells. Filaments of one to four cells contained two to eight nucleoids. Single chromosome-containing cells were not observed. Filaments containing multiple genome copies displayed synchronous DNA replication initiation. Chromosome segregation occurred during replication or rapidly after replication termination.
Involvement of P1 Adhesin in Gliding Motility of
Mycoplasma pneumoniae
as Revealed by the Inhibitory Effects of Antibody under Optimized Gliding Conditions
Authors: Seto Shintaro , Kenri Tsuyoshi , Tomiyama Tetsuo , Miyata Makoto
Published: February 2005

To examine the participation of P1 adhesin in gliding of
Mycoplasma pneumoniae
, we examined the effects of an anti-P1 monoclonal antibody on individual gliding mycoplasmas. The antibody reduced the gliding speed and removed the gliding cells from the glass over time in a concentration-dependent manner but had only a slight effect on nongliding cells, suggesting that the conformational changes of P1 adhesin and its displacement are involved in the gliding mechanism.
In vitro and in vivo effect of fluoxetine on the permeability of3H-serotonin across rat intestine
Authors: Martel Fátima , Monteiro Rosário , Lemos Clara , Vieira-Coelho M A
Published: February 2005

The aim of this work was to characterize the mucosal-to-serosal (apical to basolateral; AP-BL) and serosal-to-mucosal (basolateral to apical; BL-AP) transport of serotonin (5-HT) across rat jejunum, ileum, and colon, and to determine the influence of serotonin neuronal transporter inhibitors on this transport. The AP-BL apparent permeability (Papp) of3H-5-HT increased in the order colon = jejunum < ileum, and the BL-AP Pappof3H-5-HT increased in the order colon < jejunum = ileum. In vitro, neither fluoxetine (0.02 or 0.2 µmol/L) nor desipramine (0.4 or 4 µmol/L) had a significant effect upon the AP-BL or BL-AP Pappof3H-5-HT in any of the intestinal regions. However, fluoxetine (0.2 µmol/L) decreased the accumulation of3H-5-HT in the ileum (to 65% of control) in the BL-AP experiments. In vivo, chronic fluoxetine (10 mg/kg daily administered orally for 15 days), as assessed in the ileum, significantly increased (to ±180% of control levels) the BL-AP Pappof3H-5-HT and tended to increase the AP-BL Pappof3H-5-HT. In conclusion, the increase in the Pappof3H-5-HT after chronic administration of fluoxetine suggests that this treatment is able to increase the extracellular concentration of3H-5-HT at the intestinal level.Key words: fluoxetine, serotonin, rat intestine, permeability.
Differential participation of protein kinase C and Rho kinase in α1-adrenoceptor mediated contraction in rat arteries
Authors: Mueed Irem , Bains Perminder , Zhang Lili , MacLeod Kathleen M
Published: February 2005

The major functional α1-adrenoceptor in the rat aorta is of the α1Dsubtype and that in the caudal artery is of the α1Asubtype. In the present study, the participation of protein kinase C (PKC) and Rho kinase (RhoK) in contractile responses to stimulation of the α1-adrenoceptors in these two arteries was investigated. Both the PKC inhibitor Ro-318220 and the RhoK inhibitor Y-27632 significantly blocked contractile responses of the aorta to phenylephrine (PE) and the selective α1A-adrenoceptor agonist A61603. When used in combination, the inhibitors had an additive blocking effect. In the caudal artery, Y-27632 but not Ro-318220 inhibited contractile responses to PE and A61603, and, in combination, the antagonism produced was no greater than that by Y-27632 alone. Contractile responses to direct activation of PKC with phorbol 12,13-dibutyrate were much smaller and levels of CPI-17 (PKC-activated protein phosphatase inhibitor of 17 kDa) were much lower in the caudal artery than the aorta. The results suggest that both PKC and RhoK contribute independently to contractile responses to stimulation of α1D-adrenoceptors in the aorta. However, RhoK, but not PKC, participates in contractile responses to stimulation of α1A-adrenoceptors in the caudal artery. This difference may largely be due to differences between the two arteries in the extent to which PKC participates in contraction.Key words: vascular smooth muscle, α1-adrenoceptors, protein kinase C, rho kinase, phenylephrine.
Differential effects of sodium tungstate and vanadyl sulfate on vascular responsiveness to vasoactive agents and insulin sensitivity in fructose-fed rats
Authors: Al-Awwadi Najim , Bichon-Laurent Florence , Dimo Théophile , Michel Alain , Portet Karine , Cros Gérard , Poucheret Patrick
Published: February 2005

High fructose feeding induces insulin resistance, impaired glucose tolerance, and hypertension in rats and mimics most of the features of the metabolic syndrome X. The effects of a 6-week treatment with the transition metals administered in drinking water, vanadium (VOSO4·5H2O, 0.75 mg/mL) or tungsten (Na2O4W, 2 g/mL), were investigated on the reactivity to norepinephrine (NEPI) or acetylcholine (ACh) of thoracic aorta rings isolated from fructose (60%) or standard chow fed rats. Maximal effect (Emax) and pD2(–log EC50) values were determined in each case in the presence or absence of endothelium, while the degree of insulin resistance was determined using the euglycemic hyper insulinemic glucose clamp technique. Aortic segments isolated from 6-week fructose-fed animals were characterized by NEPI hyperresponsiveness (increase in Emax) and endothelium-dependent NEPI supersensitivity (increase in pD2) without any change in the reactivity to ACh. Vanadium or tungsten administered in fructose-fed animals prevented both hypertension and NEPI hyperresponsiveness, while vanadium, but not tungsten, reduced NEPI supersensitivity. Vanadium, but not tungsten, increased the relaxing activity of ACh, both in control and fructose-fed animals. Insulin resistance associated with high fructose feeding was reversed by vanadium but not by tungsten treatment. The differential effects of the two transition metals on vascular responsiveness to NEPI or ACh may be explained by their differential effects on insulin sensitivity.Key words: vanadium, tungsten, aorta, hypertension, fructose, glucose clamp.
Effects of valproic acid on cardiac metabolism
Authors: Daniels Thomas , Gallagher Maureen , Tremblay George , Rodgers Robert L
Published: February 2005

We investigated whether the antiepileptic valproic acid (VPA) might interfere with oxidative metabolism in heart, as it does in liver. We administered VPA to working rat hearts perfused with radiolabeled carbohydrate and fatty acid fuels. Measurements included oxidation rates of (i) glucose, pyruvate, or lactate in the presence of palmitate and (ii) palmitate, octanoate, or butyrate in the presence of glucose. Oxidation rates were quantified as the rate of appearance of14CO2or3H2O from14C- or3H-labeled substrates. In hearts perfused with palmitate, VPA (1 mmol/L) strongly inhibited the oxidation of pyruvate and lactate but slightly stimulated the oxidation of glucose. VPA also inhibited lactate or pyruvate uptake into erythrocytes in vitro. In hearts perfused with glucose, VPA strongly inhibited the oxidation of palmitate and octanoate but had no effect on butyrate oxidation. The absence of valproate CoA ligase activity in cell-free homogenates indicated that the inhibition of fatty acid oxidation by VPA did not require prior activation to valproyl-CoA. The results are consistent with the hypothesis that VPA selectively interferes with myocardial fuel oxidation by mechanisms that are independent of conversion to the CoA thioester.Key words: myocardial, glucose, lactate, pyruvate, palmitate, octanoate, butyrate, metabolism, medium-chain acyl-CoA ligase.
Effects of growth hormone on leucine absorption, intestinal morphology, and ultrastructure of the goldish intestine
Authors: Walker R L , Buret A G , Jackson C L , Scott K G.-E , Bajwa R , Habibi H R
Published: February 2005

The mechanisms whereby exogenous growth hormone modulates intestinal structure and function in fish were investigated. Goldfish (Carassius auratus) were fed commercial flake diet sprayed with recombinant carp growth hormone (cGH) daily for 1 month. Control animals received food sprayed with the vehicle. After 1 month of daily feedings, body mass and length were determined, and animals were sacrificed to study intestinal characteristics. Sections of foregut were removed after determination of total gut length for measurement of leucine uptake, histology, and epithelial ultrastructure. Oral administration of cGH for 1 month resulted in a 40% increase in body mass and an 8% increase in body length above controls. Gut length was 43% greater and the gut length to body length ratio was 32% greater as a result of the cGH treatment. Feeding with cGH also resulted in a significant increase in leucine uptake and increased gut mucosal thickness. Analysis of transmission electron micrographs revealed significant increases in the microvillous height and density and epithelial surface area. The findings indicate that growth hormone added to feed may increase growth in fish, in part by significantly increasing gut length, mucosal thickness, and epithelial brush border surface area, leading to enhanced epithelial absorption.Key words: growth hormone, goldfish, intestine, morphology, leucine.
Digastric muscle activities in anoxic infant rats
Authors: Saiki Chikako , Matsumoto Shigeji
Published: February 2005

The digastric muscle acts for both feeding (including mastication and swallowing) and respiration. In this study, we examined whether or not the muscle activity is detectable during anoxia in developing rats. Rats at 4 different ages, days 5, 10, 16, and 24, were exposed to 100% N2 under pentobarbital or ketamine–xylazine anesthesia, and the electromyograms of digastric muscles (dEMG) and the diaphragm (diaEMG) were examined simultaneously. Prior to the anoxic exposure, at all ages, the dEMG was similar to or less apparent than the diaEMG, which was detected at each inspiratory movement. In anoxia, we first observed dEMG activity, mostly sporadic (days 5 and 10) or mostly tonic (days 16 and 24), when diaEMG activity was temporarily suppressed (we termed it Phase 1). Second, synchronous phasic or tonic dEMG and phasic diaEMG were recorded temporarily before terminal apnea (we termed it Phase 2). These phenomena were also obtained in vagotomized rats (all ages) or in rats injected with the N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate) (days 16 and 24). In conclusion, our results suggest that in anoxia, dEMG activity is detectable during diaEMG suppression in early anoxia, irrespective of the developmental age, the anesthetic (pentobarbital or ketamine–xylazine), vagotomy, or MK-801 injections.Key words: newborn, hypoxia, apnea, swallowing, trigeminal nerve.
Physiological (osmotic fragility) and morphological effects on red blood cells: action of phytic acid and stannous fluoride
Authors: Lima-Filho Guilherme L , Lima Glaydes M.T , Moreno Silvana R.F , Aleixo Luiz C.M , Santos-Filho Sebastião D , Freitas Rosimeire S , Melo Vilma G.B , Bernardo-Filho Mario
Published: February 2005

Phytic acid occurs in foods derived from plants. We have investigated the possibility that phytic acid and stannous fluoride are capable of altering the physiological properties (osmotic fragility) and morphological properties of red blood cells (RBC). Osmotic fragility was unchanged by the presence of phytic acid and stannous fluoride in the studied concentrations, but RBC morphology was modified in the presence of the studied substances. In conclusion, the alterations to RBC morphology were not sufficient to promote modifications in osmotic fragility. Our results suggest that the chelating properties of phytic acid could be responsible for the observed effects.Key words: phytic acid, stannous fluoride, morphology, osmotic fragility.
Pattern of insulin delivery affects hepatic insulin sensitizing substance (HISS) action and insulin resistance
Authors: Reid Maria A. G , Lautt W Wayne
Published: February 2005

Hepatic insulin sensitizing substance (HISS) action accounts for 55% of the glucose disposal effect of a bolus of insulin in the fed state. To determine the effect of continuous versus pulsatile insulin delivery on HISS action in male Sprague–Dawley rats, insulin sensitivity was assessed using the rapid insulin sensitivity test (RIST) before and after a continuous, pulsatile, or bolus insulin (60 mU/kg i.v.) delivery. There was a significant difference in the RIST index after a continuous insulin infusion (247.9 mg/kg before, 73.2 mg/kg after) but not after 3 pulses where insulin action returned to baseline between pulses (211.6 mg/kg before, 191.0 mg/kg after) or single bolus (205.8 mg/kg before, 189.9 mg/kg after) insulin infusion. If a 3-pulse infusion was timed so that insulin action did not return to baseline between pulses, HISS action was suppressed. Continuous insulin infusion (10–30 min) showed progressive postinfusion blockade of HISS action. To maintain HISS-dependent insulin action, continuous insulin infusions should be avoided.Key words: pulsatile, glucose uptake, RIST, euglycemic clamp, insulin sensitivity.
Spontaneous mitochondrial membrane potential change during apoptotic induction by quercetin in K562 and K562/adr cells
Authors: Kothan S , Dechsupa S , Leger G , Moretti J L , Vergote J , Mankhetkorn S
Published: February 2005

Natural products from plants such as flavonoids are potential drugs to overcome multidrug resistance (MDR) in cancer treatments. However, their modes of action are still unclear. In this study, the effects of quercetin on mitochondrial membrane potential (ΔΨm) change as well as quercetin's ability to induce apoptosis and inhibit Pgp-mediated efflux of 99mTc-MIBI in K562/adr cells were investigated. Quercetin exhibits cytotoxicity against erythroleukemic cells: IC50 are 11.0 ± 2.0 µmol/L and 5.0 ± 0.4 µmol/L for K562 and K562/adr, respectively. Quercetin induces cell death via apoptosis in both K562 and K562/adr cells and does not inhibit Pgp-mediated efflux of 99mTc-MIBI. Quercetin (10 µmol/L, 3 h) and etoposide (100 µmol/L, 24 h) induce similar levels of apoptosis in K562 and K562/adr cells. Quercetin induces an increase followed by a decrease in |ΔΨm| value depending on its concentration. A decrease in the |ΔΨm| value is associated with an increase in the percentage of early apoptotic cells. It is clearly shown that quercetin results in a spontaneous ΔΨm change during apoptotic induction. Therefore, quercetin is potentially an apoptotic-inducing agent, which reacts at the mitochondrial level.Key words: multidrug resistance (MDR), quercetin, apoptosis, 99mTc-Annexin V, mitochondrial membrane potential (ΔΨm), 99mTc-MIBI.
Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction
Authors: Ren Bin , Shao Qiming , Ganguly Pallab K , Tappia Paramjit S , Takeda Nobuakira , Dhalla Naranjan S
Published: February 2005

Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ATPase and Na+–Ca2+exchanger as well as SR Ca2+pump ATPase, Ca2+release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg·kg–1·day–1) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemody nam ically and the heart and lung were examined morphologically. Some hearts were immediately frozen at –70 °C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ATPase and increased SL Na+–Ca2+exchange activities as well as reduced SR Ca2+pump and SR Ca2+release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ATPase isoforms as well as Na+–Ca2+exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+pump proteins and Ca2+release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression.Key words: sarcolemmal Na+/K+ATPase, Na+–Ca2+exchange, sarcoplasmic reticulum, heart failure, ACE inhibition.