bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurospora crassa heat shock factor 1
Is an Essential Gene; a Second Heat Shock Factor-Like Gene,
hsf2
, Is Required for Asexual Spore Formation
Authors: Thompson Seona , Croft Nirvana J. , Sotiriou Antonis , Piggins Hugh D. , Crosthwaite Susan K.
Published: June 2008

Appropriate responses of organisms to heat stress are essential for their survival. In eukaryotes, adaptation to high temperatures is mediated by heat shock transcription factors (HSFs). HSFs regulate the expression of heat shock proteins, which function as molecular chaperones assisting in protein folding and stability. In many model organisms a great deal is known about the products of
hsf
genes. An important exception is the filamentous fungus and model eukaryote
Neurospora crassa
. Here we show that two
Neurospora crassa
genes whose protein products share similarity to known HSFs play different biological roles. We report that
heat shock factor 1
(
hsf1
) is an essential gene and that
hsf2
is required for asexual development. Conidiation may be blocked in the
hsf2
knockout (
hsf2

KO

) strain because HSF2 is an integral element of the conidiation pathway or because it affects the availability of protein chaperones. We report that genes expressed during conidiation, for example
fluffy
,
conidiation-10
, and
repressor of conidiation-1
show wild-type levels of expression in a
hsf2

KO

strain. However, consistent with the lack of macroconidium development, levels of
eas
are much reduced. Cultures of the
hsf2

KO

strain along with two other aconidial strains, the
fluffy
and
aconidial-2
strains, took longer than the wild type to recover from heat shock. Altered expression profiles of
hsp90
and a putative
hsp90
-associated protein in the
hsf2

KO

strain after exposure to heat shock may in part account for its reduced ability to cope with heat stress.
Critical Residues for Cofactor Binding and Catalytic Activity in the Aminoglycoside Resistance Methyltransferase Sgm
Authors: Savic Miloje , Ilic-Tomic Tatjana , Macmaster Rachel , Vasiljevic Branka , Conn Graeme L.
Published: June 2008

The 16S rRNA methyltransferase Sgm from “Micromonospora zionensis” confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of theS-adenosyl-l-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with aKD(binding constant) of 17.6 μM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.
Loss of Flagellum-Based Motility by
Listeria monocytogenes
Results in Formation of Hyperbiofilms
Authors: Todhanakasem Tatsaporn , Young Glenn M.
Published: June 2008

Biofilm formation by the gram-positive, motile, food-borne pathogen
Listeria monocytogenes
was demonstrated to occur by an ordered series of stages. Biofilm development involves flagellum-based motility, which when blocked decreases initial bacterial surface attachment but subsequently leads to the formation of hyperbiofilms, surface-attached communities reaching high density.
Genomic O Island 122, Locus for Enterocyte Effacement, and the Evolution of Virulent Verocytotoxin-ProducingEscherichia coli
Authors: Konczy Paulina , Ziebell Kim , Mascarenhas Mariola , Choi Aileen , Michaud Corinne , Kropinski Andrew M. , Whittam Thomas S. , Wickham Mark , Finlay Brett , Karmali Mohamed A.
Published: June 2008

The locus of enterocyte effacement (LEE) and genomic O island 122 (OI-122) are pathogenicity islands in verocytotoxin-producingEscherichia coli(VTEC) serotypes that are associated with outbreaks and serious disease. Composed of three modules, OI-122 may occur as “complete” (with all three modules) or “incomplete” (with one or two modules) in different strains. OI-122 encodes two non-LEE effector (Nle) molecules that are secreted by the LEE type III secretion system, and LEE and OI-122 are cointegrated in some VTEC strains. Thus, they are functionally linked, but little is known about the patterns of acquisition of these codependent islands. To examine this, we conducted a population genetics analysis, using multilocus sequence typing (MLST), with 72 VTEC strains (classified into seropathotypes A to E) and superimposed on the results the LEE and OI-122 contents of these organisms. The wide distribution of LEE and OI-122 modules among MLST clonal groups corroborates the hypothesis that there has been lateral transfer of both pathogenicity islands. Sequence analysis of apagC-like gene in OI-122 module 1 also revealed two nonsynonymous single-nucleotide polymorphisms that could help discriminate a subset of seropathotype C strains and determine the presence of the LEE. A nonsense mutation was found in this gene in five less virulent strains, consistent with a decaying or inactive gene. The modular nature of OI-122 could be explained by the acquisition of modules by lateral transfer, either singly or as a group, and by degeneration of genes within modules. Correlations between clonal group, seropathotype, and LEE and OI-122 content provide insight into the role of genomic islands in VTEC evolution.
The Striking Case of Tryptophan Provision in the Cedar Aphid
Cinara cedri
Authors: Gosalbes María José , Lamelas Araceli , Moya Andrés , Latorre Amparo
Published: June 2008

Buchnera aphidicola
BCc has lost its symbiotic role as the tryptophan supplier to the aphid
Cinara cedri
. We report the presence of a plasmid in this endosymbiont that contains the
trpEG
genes. The remaining genes for the pathway (
trpDCBA
) are located on the chromosome of the secondary endosymbiont “
Candidatus
Serratia symbiotica.” Thus, we propose that a symbiotic consortium is necessary to provide tryptophan.
Cloning and Characterization of the Tetrocarcin A Gene Cluster from
Micromonospora chalcea
NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics
Authors: Fang Jie , Zhang Yiping , Huang Lijuan , Jia Xinying , Zhang Qi , Zhang Xu , Tang Gongli , Liu Wen
Published: June 2008

Tetrocarcin A (TCA), produced by
Micromonospora chalcea
NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of
d
-tetronitrose,
l
-amicetose, and
l
-digitoxose may begin with
d
-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and
trans
-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene
tcaD3
(with the functions of
chlD3
and
chlD4
) to Δ
chlD3
and Δ
chlD4
in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.
CcpN Controls Central Carbon Fluxes in
Bacillus subtilis
Authors: Tännler Simon , Fischer Eliane , Le Coq Dominique , Doan Thierry , Jamet Emmanuel , Sauer Uwe , Aymerich Stéphane
Published: June 2008

The transcriptional regulator CcpN of
Bacillus subtilis
has been recently characterized as a repressor of two gluconeogenic genes,
gapB
and
pckA
, and of a small noncoding regulatory RNA,
sr1
, involved in arginine catabolism. Deletion of
ccpN
impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose,
gapB
and
pckA
are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of
pckA
under glycolytic condition causes the growth defect observed in the
ccpN
mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a
ccpN
mutant. The high flux through the PP pathway in the
ccpN
mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of
sr1
, the third known target of CcpN, plays only a marginal role in
ccpN
mutant phenotypes.
Vibrio choleraeVciB Promotes Iron Uptake via Ferrous Iron Transporters
Authors: Mey Alexandra R. , Wyckoff Elizabeth E. , Hoover Lindsey A. , Fisher Carolyn R. , Payne Shelley M.
Published: June 2008

Vibrio choleraeuses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein inV. cholerae, VciB. ThevciBgene and its linked gene,vciA, were isolated in a screen forV. choleraegenes that permitted growth of anEscherichia colisiderophore mutant in low-iron medium. ThevciABoperon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation ofE. coliandShigella flexneristrains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen whenvciBwas expressed in anE. coliorS. flexneristrain defective for the ferrous iron transporter Feo. Supplying theE. coli feomutant with a plasmid encoding eitherE. coliorV. choleraeFeo, or theS. flexneriferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systemsV. choleraeFbp andHaemophilus influenzaeHit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. AV. cholerae vciBmutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.
Conversion of
Bacillus subtilis
OhrR from a 1-Cys to a 2-Cys Peroxide Sensor
Authors: Soonsanga Sumarin , Lee Jin-Won , Helmann John D.
Published: June 2008

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by
Bacillus subtilis
OhrR) and 2-Cys (represented by
Xanthomonas campestris
OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The
B. subtilis
1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the
X. campestris
2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys
B. subtilis
OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the
X. campestris
OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.
Role of Spore Coat Proteins in the Resistance of
Bacillus subtilis
Spores to
Caenorhabditis elegans
Predation
Authors: Laaberki Maria-Halima , Dworkin Jonathan
Published: June 2008

Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode
Caenorhabditis elegans
, a likely predator of
Bacillus
spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type
Bacillus subtilis
spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using
B. subtilis
strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to
C. elegans
digestion depending on the extent of the spore coat structure modifications.
The Plant-Associated BacteriumStenotrophomonas rhizophilaExpresses a New Enzyme for the Synthesis of the Compatible Solute Glucosylglycerol
Authors: Hagemann Martin , Ribbeck-Busch Kathrin , Klähn Stephan , Hasse Dirk , Steinbruch Robert , Berg Gabriele
Published: June 2008

The rhizobacteriumStenotrophomonas rhizophilaaccumulates the compatible solutes glucosylglycerol (GG) and trehalose under salt stress conditions. The complete gene for the GG synthesis enzyme was cloned and sequenced. This enzyme fromS. rhizophilarepresented a novel fusion protein composed of a putative C-terminal GG-phosphate synthase domain and an N-terminal putative GG-phosphate phosphatase domain, which was named GgpPS. A similar gene was cloned fromPseudomonassp. strain OA146. TheggpPSgene was induced after a salt shock inS. rhizophilacells. After the salt-loaded cells reached stationary phase, theggpPSmRNA content returned to the low level characteristic of the control cells, and GG was released into the medium. The completeggpPSgene and a truncated version devoid of the phosphatase part were obtained as recombinant proteins. Enzyme activity tests revealed the expected abilities of the full-length protein to synthesize GG and the truncated GgpPS to synthesize GG-phosphate. However, dephosphorylation of GG-phosphate was detected only with the complete GgpPS protein. These enzyme activities were confirmed by complementation experiments using defined GG-defective mutants of the cyanobacteriumSynechocystissp. strain PCC 6803. Genes coding for proteins very similar to the newly identified fusion protein GgpPS for GG synthesis inS. rhizophilawere found in genome sequences of related bacteria, where these genes are often linked to a gene coding for a transporter of the Mfs superfamily.
Function and Redundancy of the Chaplin Cell Surface Proteins in Aerial Hypha Formation, Rodlet Assembly, and Viability in
Streptomyces coelicolor
Authors: Di Berardo Christina , Capstick David S. , Bibb Maureen J. , Findlay Kim C. , Buttner Mark J. , Elliot Marie A.
Published: June 2008

The chaplins are a family of eight secreted proteins that are critical for raising aerial hyphae in
Streptomyces coelicolor
. These eight chaplins can be separated into two main groups: the long chaplins (ChpA to -C) and the short chaplins (ChpD to -H). The short chaplins can be further subdivided on the basis of their abilities to form intramolecular disulfide bonds: ChpD, -F, -G, and -H contain two Cys residues, while ChpE has none. A “minimal chaplin strain” containing only
chpC
,
chpE
, and
chpH
was constructed and was found to raise a substantial aerial mycelium. This strain was used to examine the roles of specific chaplins. Within this strain, the Cys-containing ChpH was identified as the major polymerization unit contributing to aerial hypha formation and assembly of an intricate rodlet ultrastructure on the aerial surfaces, and the two Cys residues were determined to be critical for its function. ChpC augmented aerial hypha formation and rodlet assembly, likely by anchoring the short chaplins to the cell surface, while ChpE was essential for the viability of wild-type
S. coelicolor
. Interestingly, the lethal effects of a
chpE
null mutation could be suppressed by the loss of the other chaplins, the inactivation of the twin arginine translocation (Tat) secretion pathway, or the loss of the rodlins.
Synthetic Lethality with the
dut
Defect in
Escherichia coli
Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation
Authors: Ting Helen , Kouzminova Elena A. , Kuzminov Andrei
Published: June 2008

Synthetic lethality is inviability of a double-mutant combination of two fully viable single mutants, commonly interpreted as redundancy at an essential metabolic step. The
dut-1
defect in
Escherichia coli
inactivates dUTPase, causing increased uracil incorporation in DNA and known synthetic lethalities [SL(
dut
) mutations]. According to the redundancy logic, most of these SL(
dut
) mutations should affect nucleotide metabolism. After a systematic search for SL(
dut
) mutants, we did identify a single defect in the DNA precursor metabolism, inactivating thymidine kinase (
tdk
), that confirmed the redundancy explanation of synthetic lethality. However, we found that the bulk of mutations interacting genetically with
dut
are in DNA repair, revealing layers of damage of increasing complexity that uracil-DNA incorporation sends through the chromosomal metabolism. Thus, we isolated mutants in functions involved in (i) uracil-DNA excision (
ung
,
polA
, and
xthA
); (ii) double-strand DNA break repair (
recA
,
recBC
, and
ruvABC
); and (iii) chromosomal-dimer resolution (
xerC
,
xerD
, and
ftsK
). These mutants in various DNA repair transactions cannot be redundant with dUTPase and instead reveal “defect-damage-repair” cycles linking unrelated metabolic pathways. In addition, two SL(
dut
) inserts (
phoU
and
degP
) identify functions that could act to support the weakened activity of the Dut-1 mutant enzyme, suggesting the “compensation” explanation for this synthetic lethality. We conclude that genetic interactions with
dut
can be explained by redundancy, by defect-damage-repair cycles, or as compensation.
Engineering of Functional Replication Protein A Homologs Based on Insights into the Evolution of Oligonucleotide/ Oligosaccharide-Binding Folds
Authors: Lin Yuyen , Lin Li-Jung , Sriratana Palita , Coleman Kelli , Ha Taekjip , Spies Maria , Cann Isaac K. O.
Published: June 2008

The bacterial single-stranded DNA-binding protein (SSB) and the archaeal/eukaryotic functional homolog, replication protein A (RPA), are essential for most aspects of DNA metabolism. Structural analyses of the architecture of SSB and RPA suggest that they are composed of different combinations of a module called the oligonucleotide/oligosaccharide-binding (OB) fold. Members of the domains
Bacteria
and
Eukarya
, in general, contain one type of SSB or RPA. In contrast, organisms in the archaeal domain have different RPAs made up of different organizations of OB folds. Interestingly, the euryarchaeon
Methanosarcina acetivorans
harbors multiple functional RPAs named MacRPA1 (for
M. acetivorans
RPA 1), MacRPA2, and MacRPA3. Comparison of MacRPA1 with related proteins in the publicly available databases suggested that intramolecular homologous recombination might play an important role in generating some of the diversity of OB folds in archaeal cells. On the basis of this information, from a four-OB-fold-containing RPA, we engineered chimeric modules to create three-OB-fold-containing RPAs to mimic a novel form of RPA found in
Methanococcoides burtonii
and
Methanosaeta thermophila
. We further created two RPAs that mimicked the RPAs in
Methanocaldococcus jannaschii
and
Methanothermobacter thermautotrophicus
through fusions of modules from MacRPA1 and
M. thermautotrophicus
RPA. Functional studies of these engineered proteins suggested that fusion and shuffling of OB folds can lead to well-folded polypeptides with most of the known properties of SSB and RPAs. On the basis of these results, different models that attempt to explain how intramolecular and intermolecular homologous recombination can generate novel forms of SSB or RPAs are proposed.
Primary aldosteronism and aldosterone-associated hypertension
Authors: Rayner B
Published: June 2008

The field of primary aldosteronism (PA) and aldosterone-related hypertension has undergone rapid evolution. From a relatively rare curiosity PA has become a common problem particularly in selected hypertensive populations. Patients with PA and aldosterone-related hypertension appear to be at higher cardiovascular and renal risk than comparable patients with essential hypertension probably due to the pleiotropic effects of aldosterone. Aldosterone is also linked to metabolic syndrome and diabetes.

The aldosterone-to-renin ratio (ARR) has allowed the widespread screening for PA, but the exact cut-off values may vary in different population groups. All patients with hypertension and hypokalaemia, and young patients with hypertension, hypertension with an incidental adrenal mass, and severe or resistant hypertension should be screened. The use of the ARR to screen all hypertensives for PA is controversial as the test lacks specificity and many patients with false-positive tests will undergo complex and expensive testing to confirm the diagnosis. The fludrocortisone suppression test, the saline infusion test or 24-hour aldosterone excretion may be used to confirm PA in patients with a positive ARR. Adrenal venous sampling is the most reliable test to detect the presence of an aldosterone-producing adenoma, but spiral CT scan or adrenocortical scintigraphy may be useful in centres without facilities for adrenal venous sampling.

Spironolactone is emerging as an important antihypertensive agent in patients with resistant hypertension and aldosterone-related hypertension. The ARR may be a useful guide to drug selection in hypertensives patients, but further research is needed to make more definitive recommendations.
Displaced granulosa cells in the fallopian tube mimicking small cell carcinoma
Authors: Vydianath B , Ganesan R , McCluggage W G
Published: June 2008

A case is reported where displaced non-neoplastic ovarian granulosa cells within the fallopian tube mimicked a small cell carcinoma. This peculiar phenomenon of displaced granulosa cells has been described previously in the ovary as a rare diagnostic pitfall which may be misinterpreted as metastatic carcinoma. This is believed to be the first documentation of its occurrence in the fallopian tube. Awareness of this rare phenomenon and immunostaining for markers of sex cord differentiation assist in diagnosis and in preventing a false positive diagnosis of malignancy.
RuttOpt — a decision support system for routing of logging trucks
Authors: Andersson Gert , Flisberg Patrik , Lidén Bertil , Rönnqvist Mikael
Published: June 2008

We describe the decision support system RuttOpt, which is developed for scheduling logging trucks in the forest industry. The system is made up of a number of modules. One module is the Swedish road database NVDB, which consists of detailed information of all of the roads in Sweden. This also includes a tool to compute distances between locations. A second module is an optimization routine that finds a schedule, i.e., set of routes for all trucks. This is based on a two-phase algorithm where linear programming and a standard tabu search method are used. A third module is a database storing all relevant information. At the center of the system is a user interface where information and results can be viewed on maps, Gantt schedules, and result reports. The RuttOpt system has been used in a number of case studies and we describe four of these. The case studies have been made in both forest companies and hauling companies. The cases range from 10 to 110 trucks and with a planning horizon ranging between 1 and 5 days. The results show that the system can be used to solve large case studies and that the potential savings are in the range 5%–30%.
Lower capacitance response and capillary fluid absorption in women to defend central blood volume in response to acute hypovolemic circulatory stress
Authors: Lindenberger M. , Olsen H. , Länne T.
Published: June 2008

Acute hemorrhage is a leading cause of death in trauma, and women are more susceptible to hypovolemic circulatory stress than men. The mechanisms underlying the susceptibility are not clear, however. The aim of the present study was to examine the compensatory mechanisms to defend central blood volume during experimental hypovolemia in women and men. Twenty-two women (23.1 ± 0.4 yr) and 16 men (23.2 ± 0.5 yr) were included. A lower body negative pressure (LBNP) of 11–44 mmHg induced experimental hypovolemic circulatory stress. The volumetric technique was used to assess the capacitance response (redistribution of peripheral venous blood to the central circulation) as well as to assess net capillary fluid transfer from tissue to blood in the arm. Plasma norepinephrine (NE) and forearm blood flow were measured before and during hypovolemia, and forearm vascular resistance (FVR) was calculated. LBNP created comparable hypovolemia in women and men. FVR increased less in women during hypovolemic stress, and no association between plasma NE and FVR was seen in women ( R2 = 0.01, not significant), in contrast to men ( R2 = 0.59, P < 0.05). Women demonstrated a good initial capacitance response, but this was not maintained with time, in contrast to men [e.g., decreased by 24 ± 4% (women) vs. 4 ± 5% (men), LBNP of 44 mmHg, P < 0.01], and net capillary fluid absorption from tissue to blood was lower in women (0.086 ± 0.007 vs. 0.115 ± 0.011 ml·100 ml−1·min−1, P < 0.05). In conclusion, women showed impaired vasoconstriction, reduced capacitance response with time, and reduced capillary fluid absorption during acute hypovolemic circulatory stress, indicating less efficiency to defend central blood volume than men.
Differential effects of cyclic and static stretch on coronary microvascular endothelial cell receptors and vasculogenic/angiogenic responses
Authors: Zheng Wei , Christensen Lance P. , Tomanek Robert J.
Published: June 2008

Mechanical stretch, an important growth stimulus, results not only from pulsatile blood flow and diastolic stretch of the ventricles [cyclic stretch (CS)] but also from tissue expansion during growth [constant static stretch (SS)]. We compared growth factor receptor expression and vasculogenic/angiogenic responses of rat coronary microvascular endothelial cells (ECs) by exposing cells to CS (10% elongation at 30 cycles/min) and SS (constant 10% elongation). Both CS and SS increased VEGF receptor (VEGF-R)2 protein levels and the extent of tube formation and branching. Moreover, both CS and SS enhanced VEGF-induced cell proliferation and tube formation, indicating that both types of stretch increase the sensitivity of ECs to VEGF. Blockade of VEGF-R2 prevented the increases in EC proliferation and aggregate tube length. However, CS but not SS enhanced EC Tie-2 protein and migration. CS affected a greater increase in tube length and branch formation than did SS. A unique finding was that SS but not CS increased VEGFR-1 in ECs. Our study is the first to distinguish between the effects of CS and SS on growth factor receptor expression and rat coronary microvascular EC proliferation, migration, and tube formation. In conclusion, EC angiogenic responses to these two types of stretch display both differences and similarities, but both CS and SS are dependent on VEGF-R2 signaling for their vasculogenic/angiogenic effects.
Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP
Authors: Hanson Madelyn S. , Stephenson Alan H. , Bowles Elizabeth A. , Sridharan Meera , Adderley Shaquria , Sprague Randy S.
Published: June 2008

Increases in the second messenger cAMP are associated with receptor-mediated ATP release from erythrocytes. In other signaling pathways, cAMP-specific phosphodiesterases (PDEs) hydrolyze this second messenger and thereby limit its biological actions. Although rabbit and human erythrocytes possess adenylyl cyclase and synthesize cAMP, their PDE activity is poorly characterized. It was reported previously that the prostacyclin analog iloprost stimulated receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the PDEs that hydrolyze erythrocyte cAMP synthesized in response to iloprost were not identified. PDE3 inhibitors were reported to augment increases in cAMP stimulated by prostacyclin analogs in platelets and pulmonary artery smooth muscle cells. Additionally, PDE3 activity was identified in embryonic avian erythrocytes, but the presence of this PDE in mammalian erythrocytes has not been investigated. Here, using Western blot analysis, we determined that PDE3B is a component of rabbit and human erythrocyte membranes. In addition, we report that the preincubation of rabbit and human erythrocytes with the PDE3 inhibitors milrinone and cilostazol potentiates iloprost-induced increases in cAMP. In addition, cilostamide, the parent compound of cilostazol, potentiated iloprost-induced increases in cAMP in human erythrocytes. These findings demonstrate that PDE3B is present in rabbit and human erythrocytes and are consistent with the hypothesis that PDE3 activity regulates cAMP levels associated with a signaling pathway activated by iloprost in these cells.
Tissue-specific pyruvate dehydrogenase complex deficiency causes cardiac hypertrophy and sudden death of weaned male mice
Authors: Sidhu Sukhdeep , Gangasani Ashish , Korotchkina Lioubov G. , Suzuki Gen , Fallavollita James A. , Canty John M. , Patel Mulchand S.
Published: June 2008

Pyruvate dehydrogenase complex (PDC) plays an important role in energy homeostasis in the heart by catalyzing the oxidative decarboxylation of pyruvate derived primarily from glucose and lactate. Because various pathophysiological states can markedly alter cardiac glucose metabolism and PDC has been shown to be altered in response to chronic ischemia, cardiac physiology of a mouse model with knockout of the α-subunit of the pyruvate dehydrogenase component of PDC in heart/skeletal muscle (H/SM-PDCKO) was investigated. H/SM-PDCKO mice did not show embryonic lethality and grew normally during the preweaning period. Heart and skeletal muscle of homozygous male mice had very low PDC activity (∼5% of wild-type), and PDC activity in these tissues from heterozygous females was ∼50%. Male mice did not survive for >7 days after weaning on a rodent chow diet. However, they survived on a high-fat diet and developed left ventricular hypertrophy and reduced left ventricular systolic function compared with wild-type male mice. The changes in the heterozygote female mice were of lesser severity. The deficiency of PDC in H/SM-PDCKO male mice greatly compromises the ability of the heart to oxidize glucose for the generation of energy (and hence cardiac function) and results in cardiac pathological changes. This mouse model demonstrates the importance of glucose oxidation in cardiac energetics and function under basal conditions.
Chemical ablation of the Purkinje system causes early termination and activation rate slowing of long-duration ventricular fibrillation in dogs
Authors: Dosdall Derek J. , Tabereaux Paul B. , Kim Jong J. , Walcott Gregory P. , Rogers Jack M. , Killingsworth Cheryl R. , Huang Jian , Robertson Peter G. , Smith William M. , Ideker Raymond E.
Published: June 2008

Endocardial mapping has suggested that Purkinje fibers may play a role in the maintenance of long-duration ventricular fibrillation (LDVF). To determine the influence of Purkinje fibers on LDVF, we chemically ablated the Purkinje system with Lugol solution and recorded endocardial and transmural activation during LDVF. Dog hearts were isolated and perfused, and the ventricular endocardium was exposed and treated with Lugol solution ( n = 6) or normal Tyrode solution as a control ( n = 6). The left anterior papillary muscle endocardium was mapped with a 504-electrode (21 × 24) plaque with electrodes spaced 1 mm apart. Transmural activation was recorded with a six-electrode plunge needle on each side of the plaque. Ventricular fibrillation (VF) was induced, and perfusion was halted. LDVF spontaneously terminated sooner in Lugol-ablated hearts than in control hearts (4.9 ± 1.5 vs. 9.2 ± 3.2 min, P = 0.01). After termination of VF, both the control and Lugol hearts were typically excitable, but only short episodes of VF could be reinduced. Endocardial activation rates were similar during the first 2 min of LDVF for Lugol-ablated and control hearts but were significantly slower in Lugol hearts by 3 min. In control hearts, the endocardium activated more rapidly than the epicardium after 4 min of LDVF with wave fronts propagating most often from the endocardium to epicardium. No difference in transmural activation rate or wave front direction was observed in Lugol hearts. Ablation of the subendocardium hastens VF spontaneous termination and alters VF activation sequences, suggesting that Purkinje fibers are important in the maintenance of LDVF.
Intramitochondrial signaling: interactions among mitoKATP, PKCε, ROS, and MPT
Authors: Costa Alexandre D. T. , Garlid Keith D.
Published: June 2008

Activation of protein kinase Cε (PKCε), opening of mitochondrial ATP-sensitive K+channels (mitoKATP), and increased mitochondrial reactive oxygen species (ROS) are key events in the signaling that underlies cardioprotection. We showed previously that mitoKATPis opened by activation of a mitochondrial PKCε, designated PKCε1, that is closely associated with mitoKATP. mitoKATPopening then causes an increase in ROS production by complex I of the respiratory chain. This ROS activates a second pool of PKCε, designated PKCε2, which inhibits the mitochondrial permeability transition (MPT). In the present study, we measured mitoKATP-dependent changes in mitochondrial matrix volume to further investigate the relationships among PKCε, mitoKATP, ROS, and MPT. We present evidence that 1) mitoKATPcan be opened by H2O2and nitric oxide (NO) and that these effects are mediated by PKCε1 and not by direct actions on mitoKATP, 2) superoxide has no effect on mitoKATPopening, 3) exogenous H2O2or NO also inhibits MPT opening, and both compounds do so independently of mitoKATPactivity via activation of PKCε2, 4) mitoKATPopening induced by PKG, phorbol ester, or diazoxide is not mediated by ROS, and 5) mitoKATP-generated ROS activates PKCε1 and induces phosphorylation-dependent mitoKATPopening in vitro and in vivo. Thus mitoKATP-dependent mitoKATPopening constitutes a positive feedback loop capable of maintaining the channel open after the stimulus is no longer present. This feedback pathway may be responsible for the lasting protective effect of preconditioning, colloquially known as the memory effect.
The acute effect of atrioventricular pacing on sympathetic nerve activity in patients with normal and depressed left ventricular function
Authors: Segerson Nathan M. , Wasmund Stephen L. , Daccarett Marcos , Fabela Manuel L. , Hammond Christopher H. , Stoddard Gregory , Smith Michael L. , Hamdan Mohamed H.
Published: June 2008

Although modest elevations in pacing rate improve cardiac output and induce reflex sympathoinhibition, the threshold rate above which hemodynamic perturbations induce reflex sympathoexcitation remains unknown. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressures (MAP) and sympathetic nerve activity (SNA) were measured during normal sinus rhythm (NSR) and atrioventricular (AV) sequential pacing in 25 patients. Pacing was performed at 100, 120, and 140 beats/min with an AV interval of 100 ms. Patients were divided into two groups based on normal or abnormal left ventricular ejection fraction (LVEF): group 1 ( n = 11; mean LVEF, 55%) and group 2 ( n = 14; mean LVEF, 31%). In group 1, relative to NSR, SBP decreased an average of 2%, 3%, and 8% at 100, 120, and 140 beats/min ( P < 0.001), respectively. DBP and MAP increased 9%, 15%, and 15% ( P = 0.001) and 3%, 6%, and 5% [ P = not significant (NS)], respectively. In group 2, SBP reductions were even greater, with an average decrease of 4%, 8%, and 16% ( P < 0.001). Whereas DBP increased 9%, 9%, and 8% at 100, 120, and 140 beats/min ( P = NS), MAP increased 3% and 2% at 100 and 120 beats/min but decreased 3% at 140 beats/min ( P = 0.001). SNA recordings were obtained in 11 patients (6 in group 1 and 5 in group 2). In group 1, SNA decreased during all rates, with a mean 21% reduction. In group 2, however, SNA decreased at 100 and 120 beats/min (49% and 38%) but increased 24% at 140 beats/min. Patients with depressed LVEF exhibited altered hemodynamic and sympathetic responses to rapid sequential pacing. The implications of these findings in device programming and arrhythmia rate control await future studies.
Molecular and physiological characterization of RV remodeling in a murine model of pulmonary stenosis
Authors: Urashima Takashi , Zhao Mingming , Wagner Roger , Fajardo Giovanni , Farahani Sara , Quertermous Tom , Bernstein Daniel
Published: June 2008

Right ventricular (RV) dysfunction is a common long-term complication in patients after the repair of congenital heart disease. Previous investigators have examined the cellular and molecular mechanisms of left ventricular (LV) remodeling, but little is known about the stressed RV. Our purpose was to provide a detailed physiological characterization of a model of RV hypertrophy and failure, including RV-LV interaction, and to compare gene alterations between afterloaded RV versus LV. Pulmonary artery constriction was performed in 86 mice. Mice with mild and moderate pulmonary stenosis (PS) developed stable hypertrophy without decompensation. Mice with severe PS developed edema, decreased RV function, and high mortality. Tissue Doppler imaging demonstrated septal dyssynchrony and deleterious RV-LV interaction in the severe PS group. Microarray analysis showed 196 genes with increased expression and 1,114 with decreased expression. Several transcripts were differentially increased in the afterloaded RV but not in the afterloaded LV, including clusterin, neuroblastoma suppression of tumorigenicity 1, Dkk3, Sfrp2, formin binding protein, annexin A7, and lysyl oxidase. We have characterized a murine model of RV hypertrophy and failure, providing a platform for studying the physiological and molecular events of RV remodeling. Although the molecular responses of the RV and LV to afterload stress are mostly concordant, there are several key differences, which may represent targets for RV failure-specific therapy.
Effect of β2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A
Authors: Kaur Manminder , Holden Neil S. , Wilson Sylvia M. , Sukkar Maria B. , Chung Kian Fan , Barnes Peter J. , Newton Robert , Giembycz Mark A.
Published: June 2008

In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, PGE2, forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) β2-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1β in ASM cells. IL-1β-induced IL-8 release was also repressed by PGE2and forskolin, whereas the β2-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKIα, a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-κB-dependent genes, we used an adenoviral-delivered NF-κB-dependent luciferase reporter to examine the effects of forskolin and the β2-adrenoceptor agonists on NF-κB activation. There was no effect on luciferase activity measured in the presence of forskolin or β2-adrenoceptor agonists. This finding is consistent with the observation that IL-1β-induced expression of IL-6, a known NF-κB-dependent gene in ASM, was also unaffected by β2-adrenoceptor agonists, forskolin, PGE2, 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1β-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including β2-adrenoceptor agonists, may not occur through a generic effect on NF-κB.
TIMAP is a positive regulator of pulmonary endothelial barrier function
Authors: Csortos Csilla , Czikora Istvan , Bogatcheva Natalia V. , Adyshev Djanybek M. , Poirier Christophe , Olah Gabor , Verin Alexander D.
Published: June 2008

TGF-β-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the β-isoform of the catalytic subunit of PP1 (PP1cβ) from pulmonary artery EC. As PP1cβ, but not PP1cα, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.
Activation of AMPK attenuates neutrophil proinflammatory activity and decreases the severity of acute lung injury
Authors: Zhao Xia , Zmijewski Jaroslaw W. , Lorne Emmanuel , Liu Gang , Park Young-Jun , Tsuruta Yuko , Abraham Edward
Published: June 2008

AMP-activated protein kinase (AMPK) is activated by increases in the intracellular AMP-to-ATP ratio and plays a central role in cellular responses to metabolic stress. Although activation of AMPK has been shown to have anti-inflammatory effects, there is little information concerning the role that AMPK may play in modulating neutrophil function and neutrophil-dependent inflammatory events, such as acute lung injury. To examine these issues, we determined the effects of pharmacological activators of AMPK, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) and barberine, on Toll-like receptor 4 (TLR4)-induced neutrophil activation. AICAR and barberine dose-dependently activated AMPK in murine bone marrow neutrophils. Exposure of LPS-stimulated neutrophils to AICAR or barberine inhibited release of TNF-α and IL-6, as well as degradation of IκBα and nuclear translocation of NF-κB, compared with findings in neutrophil cultures that contained LPS without AICAR or barberine. Administration of AICAR to mice resulted in activation of AMPK in the lungs and was associated with decreased severity of LPS-induced lung injury, as determined by diminished neutrophil accumulation in the lungs, reduced interstitial pulmonary edema, and diminished levels of TNF-α and IL-6 in bronchoalveolar lavage fluid. These results suggest that AMPK activation reduces TLR4-induced neutrophil activation and diminishes the severity of neutrophil-driven proinflammatory processes, including acute lung injury.
Prices in Mecca During the Mamluk Period
Authors: Mortel Richard T.
Published: June 2008

The political, social and economic history of western Arabia during the medieval period still remains terra incognita for the great majority of Islamicists, in spite of the intrinsic importance of the subject and the existence of a corpus of first-rate source materials. The goal of this article is to contribute to a deeper understanding of the economic history of Mecca through a detailed study of the available information regarding the prices of cereal grains and other foodstuffs there during the Mamluk period. So that the maximum advantage may be derived from the discussion, it will be preceeded by a short outline of the political history of Mecca during Mamluk times, as well as the salient features of its economy.