bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual control of cardiac Na+–Ca2+ exchange by PIP2: electrophysiological analysis of direct and indirect mechanisms
Authors: Yaradanakul Alp , Feng Siyi , Shen Chengcheng , Lariccia Vincenzo , Lin Mei‐Jung , Yang Jinsong , Kang T. M.  , Dong Ping , Yin Helen L. , Albanesi Joseph P. , Hilgemann Donald W.
Published: June 2007

Cardiac Na+–Ca2+ exchange (NCX1) inactivates in excised membrane patches when cytoplasmic Ca2+ is removed or cytoplasmic Na+ is increased. Exogenous phosphatidylinositol‐4,5‐bis‐phosphate (PIP2) can ablate both inactivation mechanisms, while it has no effect on inward exchange current in the absence of cytoplasmic Na+. To probe PIP2 effects in intact cells, we manipulated PIP2 metabolism by several means. First, we used cell lines with M1 (muscarinic) receptors that couple to phospholipase C's (PLCs). As expected, outward NCX1 current (i.e. Ca2+ influx) can be strongly inhibited when M1 agonists induce PIP2 depletion. However, inward currents (i.e. Ca2+ extrusion) without cytoplasmic Na+ can be increased markedly in parallel with an increase of cell capacitance (i.e. membrane area). Similar effects are incurred by cytoplasmic perfusion of GTPγS or the actin cytoskeleton disruptor latrunculin, even in the presence of non‐hydrolysable ATP (AMP‐PNP). Thus, G‐protein signalling may increase NCX1 currents by destabilizing membrane cytoskeleton–PIP2 interactions. Second, to increase PIP2 we directly perfused PIP2 into cells. Outward NCX1 currents increase as expected. But over minutes currents decline substantially, and cell capacitance usually decreases in parallel. Third, using BHK cells with stable NCX1 expression, we increased PIP2 by transient expression of a phosphatidylinositol‐4‐phosphate‐5‐kinase (hPIP5KIβ) and a PI4‐kinase (PI4KIIα). NCX1 current densities were decreased by > 80 and 40%, respectively. Fourth, we generated transgenic mice with 10‐fold cardiac‐specific overexpression of PI4KIIα. This wortmannin‐insensitive PI4KIIα was chosen because basal cardiac phosphoinositides are nearly insensitive to wortmannin, and surface membrane PI4‐kinase activity, defined functionally in excised patches, is not blocked by wortmannin. Both phosphatidylinositol‐4‐phosphate (PIP) and PIP2 were increased significantly, while NCX1 current densities were decreased by 78% with no loss of NCX1 expression. Most mice developed cardiac hypertrophy, and immunohistochemical analysis suggests that NCX1 is redistributed away from the outer sarcolemma. Cholera toxin uptake was increased 3‐fold, suggesting that clathrin‐independent endocytosis is enhanced. We conclude that direct effects of PIP2 to activate NCX1 can be strongly modulated by opposing mechanisms in intact cells that probably involve membrane cytoskeleton remodelling and membrane trafficking.
Toward Automated Analysis of Biofilm Architecture: Bias Caused by Extraneous Confocal Laser Scanning Microscopy Images
Authors: Merod Robin T. , Warren Jennifer E. , McCaslin Hope , Wuertz Stefan
Published: June 2007

An increasing number of studies utilize confocal laser scanning microscopy (CLSM) for in situ visualization of biofilms and rely on the use of image analysis programs to extract quantitative descriptors of architecture. Recently, designed programs have begun incorporating procedures to automatically determine threshold values for three-dimensional CLSM image stacks. We have found that the automated threshold calculation is biased when a stack contains images lacking pixels of biological significance. Consequently, we have created the novel program Auto PHLIP-ML to resolve this bias by iteratively excluding extraneous images based on their area coverage of biomass. A procedure was developed to identify the optimal percent area coverage value used for extraneous image removal (PACVEIR). The optimal PACVEIR was defined to occur when the standard deviation of mean thickness, determined from replicate image stacks, was at a maximum, because it more accurately reflected inherent structural variation. Ten monoculture biofilms of either
Ralstonia eutropha
JMP228n::
gfp
or
Acinetobacter
sp. strain BD413 were tested to verify the routine. All biofilms exhibited an optimal PACVEIR between 0 and 1%. Prior to the exclusion of extraneous images, JMP228n::
gfp
appeared to develop more homogeneous biofilms than BD413. However, after the removal of extraneous images, JMP228n::
gfp
biofilms were found to form more heterogeneous biofilms. Similarly, JMP228n::
gfp
biofilms grown on glass surfaces vis-à-vis polyethylene membranes produced significantly different architectures after extraneous images had been removed but not when such images were included in threshold calculations. This study shows that the failure to remove extraneous images skewed a seemingly objective analysis of biofilm architecture and significantly altered statistically derived conclusions.
Engineering of
Saccharomyces cerevisiae
for Efficient Anaerobic Alcoholic Fermentation of
l
-Arabinose
Authors: Wisselink H. Wouter , Toirkens Maurice J. , del Rosario Franco Berriel M. , Winkler Aaron A. , van Dijken Johannes P. , Pronk Jack T. , van Maris Antonius J. A.
Published: June 2007

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as
l
-arabinose, is required. Wild-type strains of
Saccharomyces cerevisiae
, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of
l
-arabinose into ethanol by engineered
S. cerevisiae
strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of
l
-arabinose by an engineered
S. cerevisiae
strain. This fermentation was achieved by combining the expression of the structural genes for the
l
-arabinose utilization pathway of
Lactobacillus plantarum
, the overexpression of the
S. cerevisiae
genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting
S. cerevisiae
strain exhibited high rates of arabinose consumption (0.70 g h
−1
g [dry weight]
−1
) and ethanol production (0.29 g h
−1
g [dry weight]
−1
) and a high ethanol yield (0.43 g g
−1
) during anaerobic growth on
l
-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.
Quantitative Analysis of Population Heterogeneity of the Adaptive Salt Stress Response and Growth Capacity of
Bacillus cereus
ATCC 14579
Authors: den Besten Heidy M. W. , Ingham Colin J. , van Hylckama Vlieg Johan E. T. , Beerthuyzen Marke M. , Zwietering Marcel H. , Abee Tjakko
Published: June 2007

Bacterial populations can display heterogeneity with respect to both the adaptive stress response and growth capacity of individual cells. The growth dynamics of
Bacillus cereus
ATCC 14579 during mild and severe salt stress exposure were investigated for the population as a whole in liquid culture. To quantitatively assess the population heterogeneity of the stress response and growth capacity at a single-cell level, a direct imaging method was applied to monitor cells from the initial inoculum to the microcolony stage. Highly porous Anopore strips were used as a support for the culturing and imaging of microcolonies at different time points. The growth kinetics of cells grown in liquid culture were comparable to those of microcolonies grown upon Anopore strips, even in the presence of mild and severe salt stress. Exposure to mild salt stress resulted in growth that was characterized by a remarkably low variability of microcolony sizes, and the distributions of the log
10
-transformed microcolony areas could be fitted by the normal distribution. Under severe salt stress conditions, the microcolony sizes were highly heterogeneous, and this was apparently caused by the presence of both a nongrowing and growing population. After discriminating these two subpopulations, it was shown that the variability of microcolony sizes of the growing population was comparable to that of non-salt-stressed and mildly salt-stressed populations. Quantification of population heterogeneity during stress exposure may contribute to an optimized application of preservation factors for controlling growth of spoilage and pathogenic bacteria to ensure the quality and safety of minimally processed foods.
Agrobacterium tumefaciens fur
Has Important Physiological Roles in Iron and Manganese Homeostasis, the Oxidative Stress Response, and Full Virulence
Authors: Kitphati Worawan , Ngok-ngam Patchara , Suwanmaneerat Sukanya , Sukchawalit Rojana , Mongkolsuk Skorn
Published: June 2007

In
Agrobacterium tumefaciens
, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A
fur
mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the
fur
mutant was more sensitive than the wild type to an iron chelator, 2,2′-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations.
A. tumefaciens sitA
, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (
sitABCD
), was strongly repressed by Mn
2+
and, to a lesser extent, by Fe
2+
, and this regulation was Fur dependent. Moreover, the
fur
mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the
fur
mutant showed constitutive up-expression of the manganese uptake
sit
operon. Fur
At
showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The
fur
mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the
fur
mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.
Isolation and Characterization of a Novel Haloacid Permease from
Burkholderia cepacia
MBA4
Authors: Yu Manda , Faan Yun-Wing , Chung Wilson Y. K. , Tsang Jimmy S. H.
Published: June 2007

Burkholderia cepacia
MBA4 is a bacterium that can utilize 2-haloacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease mediates the uptake of haloacid. In this paper, we report the first cloning and characterization of such a haloacid permease. The structural gene, designated
deh4p
, was found 353 bases downstream of the dehalogenase gene
deh4a
. Quantitative analysis of the expression of
deh4p
showed that it was induced by monochloroacetate (MCA), to a level similar to the MCA-induced level of
deh4a
. The nucleotide sequence of
deh4p
was determined, and an open reading frame of 1,656 bp encoding a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. Deh4p has the signatures of sugar transport proteins and integral membrane proteins of the major facilitator superfamily. Uptake of [
14
C]MCA into the cell was Deh4p dependent. Deh4p has apparent

K
m

s of 5.5 and 8.9 μM and
V
max
s of 9.1 and 23.1 nmol mg
−1
min
−1
for acetate and MCA, respectively. A mutant with a transposon-inactivated haloacid operon failed to grow on MCA even when
deh4a
was provided in
trans
.
NCgl2620 Encodes a Class II Polyphosphate Kinase in
Corynebacterium glutamicum
Authors: Lindner Steffen N. , Vidaurre Dolores , Willbold Sabine , Schoberth Siegfried M. , Wendisch Volker F.
Published: June 2007

Corynebacterium glutamicum
is able to accumulate up to 600 mM cytosolic phosphorus in the form of polyphosphate (poly P). Granular poly P (volutin) can make up to 37% of the internal cell volume. This bacterium lacks the classic enzyme of poly P synthesis, class I polyphosphate kinase (PPK1), but it possesses two genes,
ppk2A
(corresponds to NCgl0880) and
ppk2B
(corresponds to NCgl2620), for putative class II (PPK2) PPKs. Deletion of
ppk2B
decreased PPK activity and cellular poly P content, while overexpression of
ppk2B
increased both PPK activity and cellular poly P content. Neither deletion nor overexpression of
ppk2A
changed specific activity of PPK or cellular poly P content significantly. Purified PPK2B of
C. glutamicum
is active as a homotetramer and formed poly P with an average chain length of about 125, as determined with
31
P nuclear magnetic resonance. The catalytic efficiency of
C. glutamicum
PPK2B was higher in the poly P-forming direction than for nucleoside triphosphate formation from poly P. The
ppk2B
deletion mutant, which accumulated very little poly P and grew as
C. glutamicum
wild type under phosphate-sufficient conditions, showed a growth defect under phosphate-limiting conditions.
Monitoring and Source Tracking of Tetracycline Resistance Genes in Lagoons and Groundwater Adjacent to Swine Production Facilities over a 3-Year Period
Authors: Koike S. , Krapac I. G. , Oliver H. D. , Yannarell A. C. , Chee-Sanford J. C. , Aminov R. I. , Mackie R. I.
Published: June 2007

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc
r
) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (
n
= 124) and lagoon (
n
= 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc
r
genes [
tet
(M),
tet
(O),
tet
(Q),
tet
(W),
tet
(C),
tet
(H), and
tet
(Z)]. The concentration of Tc
r
genes was quantified by real-time quantitative PCR. To confirm the Tc
r
gene source in groundwater, comparative analysis of
tet
(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc
r
genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc
r
genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of
tet
(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.
The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Cacciapuoti Giovanna , Porcelli Marina , Moretti Maria Angela , Sorrentino Francesca , Concilio Luigi , Zappia Vincenzo , Liu Zhi-Jie , Tempel Wolfram , Schubot Florian , Rose John P. , Wang Bi-Cheng , Brereton Phillip S. , Jenney Francis E. , Adams Michael W. W.
Published: June 2007

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile
Pyrococcus furiosus
was cloned and expressed in
Escherichia coli
, and the recombinant protein was purified to homogeneity.
P. furiosus
ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the
k
cat
/

K
m

value.
P. furiosus
ACAPT is thermoactive and thermostable with an apparent melting temperature of 108°C that increases to 112°C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-Å resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of
P. furiosus
showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and
N
-(3-aminopropyl)cadaverine (APC). This is the first report in
Archaea
of an unusual polyamine APC that is proposed to play a role in stress adaptation.
Structure and Function of Cold Shock Proteins in Archaea
Authors: Giaquinto Laura , Curmi Paul M. G. , Siddiqui Khawar S. , Poljak Anne , DeLong Ed , DasSarma Shiladitya , Cavicchioli Ricardo
Published: June 2007

Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g.,
Methanogenium frigidum
). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from
M. frigidum. csp
genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of
csp
genes from psychrophilic and mesophilic
Euryarchaeota
and
Crenarchaeota
to complement a cold-sensitive growth defect in
Escherichia coli
. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the
E. coli
growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in
E. coli
CspA. The
M. frigidum
Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover,
M. frigidum
Csp was characterized as binding
E. coli
single-stranded RNA, consistent with its ability to complement function in
E. coli
. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the
Archaea
to be able to function effectively in the
Bacteria
and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of
M. frigidum
Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.
Regulation of Carbon and Nitrogen Utilization by CbrAB and NtrBC Two-Component Systems in
Pseudomonas aeruginosa
Authors: Li Wei , Lu Chung-Dar
Published: June 2007

The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in
Pseudomonas aeruginosa
was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the
cbrAB
deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the
cbr
mutant were selected from minimal medium containing
l
-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an
ntrB
allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these
ntrB
alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these
ntrB
mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the Δ
cbrAB ntrB
(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in
P. aeruginosa
. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.
Protein Modulator of Multidrug Efflux Gene Expression inPseudomonas aeruginosa
Authors: Daigle Denis M. , Cao Lily , Fraud Sebastien , Wilke Mark S. , Pacey Angela , Klinoski Rachael , Strynadka Natalie C. , Dean Charles R. , Poole Keith
Published: June 2007

nalCmultidrug-resistant mutants ofPseudomonas aeruginosashow enhanced expression of themexAB-oprMmultidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor ofmexAB-oprMexpression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [KD], 158.0 ± 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting thatmexAB-oprMhyperexpression innalCmutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression ofmexAtranscription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in analCstrain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamedarmR(antirepressor forMexR). A representative “noninteracting” mutant MexR protein, MexRI104F, was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced;KD, 853.2 ± 151.1 nM). Consistent with this, MexRI104Frepressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal α-helix of the protein that alone is sufficient for interaction with MexR.
The Intracellular Concentration of Acetyl Phosphate in
Escherichia coli
Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators
Authors: Klein Adam H. , Shulla Ana , Reimann Sylvia A. , Keating David H. , Wolfe Alan J.
Published: June 2007

Acetyl phosphate, the intermediate of the AckA-Pta pathway, acts as a global signal in
Escherichia coli
. Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer.
Role of Staphylococcal Phage and SaPI Integrase in Intra- and Interspecies SaPI Transfer
Authors: Maiques Elisa , Úbeda Carles , Tormo María Ángeles , Ferrer María Desamparados , Lasa Íñigo , Novick Richard P. , Penadés José R.
Published: June 2007

SaPIbov2 is a member of the SaPI family of staphylococcal pathogenicity islands and is very closely related to SaPIbov1. Typically, certain temperate phages can induce excision and replication of one or more of these islands and can package them into special small phage-like particles commensurate with their genome sizes (referred to as the excision-replication-packaging [ERP] cycle). We have studied the phage-SaPI interaction in some depth using SaPIbov2, with special reference to the role of its integrase. We demonstrate here that SaPIbov2 can be induced to replicate by different staphylococcal phages. After replication, SaPIbov2 is efficiently encapsidated and transferred to recipient organisms, including different non-
Staphylococcus aureus
staphylococci, where it integrates at a SaPI-specific attachment site,

att
C

, by means of a self-coded integrase (Int). Phages that cannot induce the SaPIbov2 ERP cycle can transfer the island by
recA
-dependent classical generalized transduction and can also transfer it by a novel mechanism that requires the expression of SaPIbov2
int
in the recipient but not in the donor. It is suggested that this mechanism involves the encapsidation of standard transducing fragments containing the intact island followed by
int
-mediated excision, circularization, and integration in the recipient.
New Complexes of Molybdenum(II) and Tungsten(II) with a C,N-Chelated Stannylene
Authors: Padělková Zdeňka , Císařová Ivana , Fejfarová Karla , Holubová Jana , Růžička Aleš , Holeček Jaroslav
Published: June 2007

Two new complexes of [Sn{2-[(CH3)2NCH2]C6H4}2] with [Mo(η5-C5H5)(CO)3Cl] and [W(η5-C5H5)(CO)3Cl], respectively, were prepared via insertion of stannylene into the Mo-Cl or W-Cl bond forming thus the transition metal-tin bond. These complexes were studied by IR, NMR and X-ray diffraction techniques. The tin atom in these complexes is five-coordinated with the carbon atoms of ligands and transition metal in equatorial plane and the chlorine and the nitrogen donor atoms in axial positions. The attempts to photolyse, reduce or fluorinate the prepared complexes and characterize the products of these reactions failed.
E/Z Isomerization of 3-Hydrazonocamphor Promoted by Coordination to Palladium or Platinum
Authors: Carvalho M. Fernanda N. N. , Ferreira Ana S. D. , Ferreira da Silva João L. , Veiros Luís F.
Published: June 2007

3-Hydrazonocamphor, 3-(RR1NN)C10H14O (R = Me, R1 = H), undergoes intramolecular hydrogen bridging by coordination to platinum or palladium. This effect is evidenced by considerable decrease in the ν(C=O) frequency (compared to the free ligand) in the IR spectra of the complexes [MCl2L2] (M = Pd, Pt; L = 3-(RR1NN)C10H14O) as well as by the magnetic non- equivalence of the two ligands, as revealed by 13C NMR. DFT calculations indicate that coordination of 3-(Me(H)NN)C10H14O promotes E/Z isomerization of the hydrazono group of the ligand, inducing formation of intramolecular hydrogen bonding and corresponding stabilization of the complex. Characterization of the complexes [MCl2L2] (M = Pt; L: R, R1 = Me (1), R = Me, R1 = H (2) and M = Pd; L: R = Me, R1 = H (3)) was performed by analytical and spectroscopic techniques. Redox properties of the 3-hydazonocamphors and their complexes were studied by cyclic voltammetry. The structure of trans-[PtCl2{3-(Me2NN)C10H14O}2] was determined by single-crystal X-ray diffraction analysis. The complex has square-planar geometry and crystallizes in the tetragonal P43 space group.
Synthesis of Ferrierite from Gels Containing a Mixture of Two Templates
Authors: Pinar Ana Belén , García Raquel , Pérez-Pariente Joaquín
Published: June 2007

Ferrierite synthesis from gels containing a mixture of two template molecules, in the absence of inorganic cations and in fluoride media, is described. One of them is a bulky structure-directing agent (SDA), 1-benzyl-1-methylpyrrolidinium cation (bmp), and the other one is a small organic cation acting as a co-template. We present here the results obtained with tetramethylammonium (TMA) and quinuclidinium (Qui) compounds as co-templates in a gel containing bmp cation as SDA. It has been observed that in both cases co-templates exert a marked effect on the nature of the synthesis products.
A comparative study of the quality of DNA obtained from fresh frozen and formalin-fixed decalcified paraffin-embedded bone marrow trephine biopsy specimens using two different methods
Authors: Talaulikar D , Gray J X , Shadbolt B , McNiven M , Dahlstrom J E
Published: June 2007

Background:

Given its prognostic value, there is renewed interest in molecular staging in non-Hodgkin’s lymphoma (NHL) using immunoglobulin heavy and light chain (IgH, IgL) gene rearrangements.

Aims:

To compare the efficiency of DNA amplification from fresh frozen and formalin-fixed decalcified paraffin-embedded (FFDPE) bone marrow trephines for use in molecular staging using two methods.

Methods:

After manually extracting DNA from 13 FFDPE and 14 fresh frozen trephine biopsy specimens, two methods were used to test for amplifiability: use of the amplification control master mix supplied in the In Vivo Scribe immunoglobulin heavy chain (IgH) clonality kit, which creates 5 amplicons between 96–600 base pairs (bp); and real-time amplification of the β-globin gene.

Results:

Using the first method, the mean maximum length of amplicons generated from FFDPE trephines was statistically lower at 300 bp compared to fresh frozen samples, all of which generated amplicons up to 600 bp in size (p<0.001). Real-time amplification of the β-globin gene showed that the mean crossing threshold of fresh frozen samples was statistically lower than that of FFDPE samples (23.48 (95% CI 22.47 to 24.48) vs 33.64 (95% CI 32.15 to 35.12); p<0.001).

Conclusions:

Although amplifiable DNA can be extracted from both fresh-frozen and FFDPE trephine samples for IgH/IgL analysis, freshly frozen specimens are superior as a source of template DNA, especially for higher base pair PCR products.
My mother, the smoker
Authors: Levy Kirsten
Published: June 2007

Watching a frail parent, with a pack-a-day smoking habit, decline into dementia, I wrote a blog and morphed it into a narrative of my mother’s smoking, paralleling her habit with the rise and fall of smoking through the ages. I imagine that some readers might be grappling with similar anguish and I offer this part memoir, part history, part health ethics discussion to understand what is so compelling about her experience with tobacco.
“To observe well … and thence to make himself rules”: John Locke’s principles and practice of child healthcare
Authors: Williams A N
Published: June 2007

It is often forgotten that the philosopher John Locke (1632–1704) was a highly regarded physician with a lifelong interest in medicine and was frequently consulted on medical matters, including the health of children. This child health aspect in Locke’s history has been largely ignored, with even modern commentaries on Locke and medicine giving it only a cursory mention. However, it is clear that, in child health, Locke’s influence is far more substantial than GF Still’s and George Jackson’s opinions, which limited Locke solely toThoughts concerning education(1692/3). That a fundamental reappraisal of Locke’s role in child healthcare is necessary and that his place as a pioneer of modern child healthcare needs to be proclaimed are emphasised here. As modern day child healthcare has evolved to embrace advocacy and learning disability, Locke’s importance through his influence on paediatrics, child healthcare and human rights becomes more evident. Locke’s influence in child healthcare comes not only through his other celebrated philosophical writings, but also through extensive personal correspondence and case records. As well as throwing light onto the 17th century aspects of child healthcare, Locke, through his enquiry and self-evident humility in his correspondence on medical matters, inspires and educates us with his pragmatic approach to the practice of medicine.
Early European attitudes towards “good death”: Eugenios Voulgaris,Treatise on euthanasia, St Petersburg, 1804
Authors: Galanakis E , Dimoliatis I D K
Published: June 2007

Eugenios Voulgaris (Corfu, Greece, 1716; St Petersburg, Russia, 1806) was an eminent theologian and scholar, and bishop of Kherson, Ukraine. He copiously wrote treatises in theology, philosophy and sciences, greatly influenced the development of modern Greek thought, and contributed to the perception of Western thought throughout the Eastern Christian world. In hisTreatise on euthanasia(1804), Voulgaris tried to moderate the fear of death by exalting the power of faith and trust in the divine providence, and by presenting death as a universal necessity, a curative physician and a safe harbour. Voulgaris presented his views in the form of a consoling sermon, abundantly enriched with references to classical texts, the Bible and the Church Fathers, as well as to secular sources, including vital statistics from his contemporary England and France. Besides euthanasia, he introduced terms such as dysthanasia, etoimothanasia and prothanasia.

TheTreatise on euthanasiais one of the first books, if not the very first, devoted to euthanasia in modern European thought and a remarkable text for the study of the very early European attitudes towards “good death”. In theTreatise, euthanasia is clearly meant as a spiritual preparation and reconciliation with dying rather than a physician-related mercy killing, as the term progressed to mean during the 19th and the 20th centuries. This early text is worthy of study not only for the historian of medical ethics or of religious ethics, but for everybody who is trying to courageously confront death, either in private or in professional settings.
The effects of flooding regime on the rare Atlantic coastal plain species Hydrocoytle umbellata
Authors: Dawe Christine E. , Reekie Edward G.
Published: June 2007

The hydrological regime on many lakes is changing through climate change, construction of dams, and land-use changes. We examined the effects of flooding regime on growth and development of the endangered semiaquatic species, Hydrocotyle umbellata L. (water pennywort), in a greenhouse experiment. Growth was twice as fast under dry, above-water (terrestrial) conditions as under flood conditions. Depth of flooding (15 or 30 cm) had little impact. Short-term floods at either the middle or end of the growing season were just as damaging as continuous flooding. The decrease in growth under flood conditions was largely due to a temporary reduction in unit leaf rate (growth per unit leaf area) rather than to a reduction in leaf area ratio (leaf area per unit mass). Flooded plants allocated more resources to the production of petioles at the expense of reproductive structures and to a lesser extent, belowground structures. They also produced fewer, larger leaves with more stomata on the upper than on the lower surface. These changes in structure were largely restricted to new leaves produced under flood conditions; most of the mature leaves produced in a terrestrial environment senesced upon flooding. Chemical analysis of tubers produced by plants grown under terrestrial conditions indicates that these structures function in carbohydrate storage. The likely impact of changes in hydrological regime on this endangered species is discussed in light of these findings.
A hypoplastic constitutive model for clays with meta-stable structure
Authors: Maín David
Published: June 2007

A new hypoplastic model for clays with meta-stable structure is presented in this paper. A new method for incorporation of structure effects into hypoplastic models based on the modification of barotropy and pyknotropy factors is proposed and applied to an existing hypoplastic model for reconstituted clays. The new model is characterized by a simple calibration procedure and a small number of parameters. This makes the model particularly suitable for practical applications. The model is evaluated using experimental data for two natural soft clays. Thanks to the incrementally nonlinear character of the hypoplastic equation, the proposed model predicts behaviour of overconsolidated clays comparably to advanced kinematic hardening elastoplastic models.Key words: constitutive relations, hypoplasticity, clays, structure of soils.
Effects of net stress and suction history on the small strain stiffness of a compacted clayey silt
Authors: Vassallo Roberto , Mancuso Claudio , Vinale Filippo
Published: June 2007

An experimental study was carried out to investigate the effects of the mean net stress and suction history on the initial shear stiffness, G0, of a compacted clayey silt. Isotropic tests were performed using two suction-controlled devices, a triaxial cell and a resonant column torsional shear (RCTS) cell, so as to investigate the volumetric behaviour of this material. As for saturated soils, one can expect to find a strong correlation among stress history, volumetric state, and G0. Initial shear stiffness was measured almost continuously along various isotropic stress paths, including compressions and drying–wetting single stages or cycles, by using the RCTS cell. The collected data demonstrate a strong dependency of G0 on mean net stress (p – ua) and suction (ua – uw). Cycles of suction, in particular increasing suction beyond the past maximum value, induce significant accumulation of irreversible strains and increase of stiffness, confirming that G0 is not univocally related to the stress state (p – ua, ua – uw).Key words: unsaturated, compacted, small strain, stiffness, volumetric behaviour, stress history.
The ligament of Marshall as a parasympathetic conduit
Authors: Ulphani Joseph S. , Arora Rishi , Cain Jack H. , Villuendas Roger , Shen Sharon , Gordon David , Inderyas Firdous , Harvey Laura A. , Morris Alexander , Goldberger Jeffrey J. , Kadish Alan H.
Published: June 2007

The objective of the study was to investigate the morphology, distribution, and electrophysiological profile of the autonomic fibers that innervate the ligament of Marshall (LOM). Gross anatomical dissections were performed in 10 dogs. Sections of the left vagus nerve, left stellate ganglion, and the LOM were immunostained to identify adrenergic and cholinergic nerves. Hearts were also stained for acetylcholinesterase to identify epicardial cholinergic nerves. In vivo electropyhsiological studies were performed in another 10 dogs before and after LOM ablation. The anatomical examination revealed that the LOM is innervated by a branch of the left vagus. Immunohistochemistry confirmed that these nerve bundles are predominantly cholinergic (cholinergic-to-adrenergic ratio of 12.6 ± 3.9:1). Cholinergic nerves originating in the LOM were found to innervate surrounding left atrial structures, including the pulmonary veins, left atrial appendage, coronary sinus, and posterior left atrial fat pad. Ablation of the LOM significantly attenuated effective refractory period shortening at distant sites, such as pulmonary veins and left atrial appendage, in response to vagal stimulation (vagal-induced ERP decrease in the left atrium: baseline vs. postablation = 17 vs. 4%; P = 0.0056). In conclusion, the LOM contains a predominance of cholinergic nerve fibers. Cholinergic fibers arising from the LOM innervate surrounding structures and contribute to the electrophysiological profile of the left atrium. These findings may provide a basis for the role of the LOM in the genesis and maintenance of atrial fibrillation.
Activation of hypoxia-inducible factor-1 via prolyl-4 hydoxylase-2 gene silencing attenuates acute inflammatory responses in postischemic myocardium
Authors: Natarajan Ramesh , Salloum Fadi N. , Fisher Bernard J. , Ownby Evan D. , Kukreja Rakesh C. , Fowler Alpha A.
Published: June 2007

Emerging research suggests that oxidant-driven transcription of key cytokine/chemokine networks within the myocardium plays a crucial role in producing ischemia-reperfusion (I/R) injury. We recently showed that activation of hypoxia-inducible factor-1 (HIF-1) attenuated cardiac I/R injury. Diminished injury in these prior studies was associated with significant reductions in circulating interleukin-8 levels, suggesting that HIF-1 may play an important role in modulating postischemic cardiac inflammation. In the current study, we examined the role of HIF-1 activation in modulating proinflammatory chemokine [macrophage inflammatory protein (MIP)-2, cytokine-induced neutrophil chemoattractant factor (KC), and lipopolysaccharide-induced CXC chemokine (LIX)] and adhesion molecule [intercellular adhesion molecule (ICAM)-1] expression in murine cardiomyocytes in vitro (HL-1 cell line) and in intact murine hearts following in vivo I/R injury. Our results show that HIF-1 activation induced both pharmacologically by the prolyl hydroxylase inhibitor dimethyloxallyl glycine and via small-interfering RNA (siRNA)-mediated prolyl-4 hydroxylase-2 (P4HA2) gene silencing significantly attenuated tumor necrosis factor-α-induced chemokine (KC and LIX) and ICAM-1 expression in cardiomyocytes. In vivo, postischemic hearts obtained from animals receiving the P4HA2 siRNA (HIF-1 activation) exhibited significantly reduced CXC chemokine (MIP-2, KC, and LIX), CC chemokine (monocyte chemoattractant protein-1), and ICAM-1 expression when compared with postischemic hearts from either saline I/R controls or postischemic hearts from animals receiving a nontargeting control siRNA (no HIF-1 activation). Diminished chemokine and adhesion molecule expression in HIF-1-activated postischemic hearts was associated with significantly reduced polymorphonuclear leukocyte infiltration and myocardial infarct size (>60% reduction P4HA2 siRNA I/R vs. saline I/R, P < 0.001, n = 6). In conclusion, these results demonstrate for the first time that HIF-1 activation following infusion of siRNA to P4HA2 plays a key role in modulating I/R-associated cardiac inflammatory responses.
Influence of channel subunit composition on L-type Ca2+ current kinetics and cardiac wave stability
Authors: Gudzenko Vadim , Shiferaw Yohannes , Savalli Nicoletta , Vyas Roshni , Weiss James N. , Olcese Riccardo
Published: June 2007

Previous studies have demonstrated that the slope of the function relating the action potential duration (APD) and the diastolic interval, known as the APD restitution curve, plays an important role in the initiation and maintenance of ventricular fibrillation. Since the APD restitution slope critically depends on the kinetics of the L-type Ca2+ current, we hypothesized that manipulation of the subunit composition of these channels may represent a powerful strategy to control cardiac arrhythmias. We studied the kinetic properties of the human L-type Ca2+ channel (Cav1.2) coexpressed with the α2δ-subunit alone (α1C + α2δ) or in combination with β2a, β2b, or β3 subunits (α1C + α2δ + β), using Ca2+ as the charge carrier. We then incorporated the kinetic properties observed experimentally into the L-type Ca2+ current mathematical model of the cardiac action potential to demonstrate that the APD restitution slope can be selectively controlled by altering the subunit composition of the Ca2+ channel. Assuming that β2b most closely resembles the native cardiac L-type Ca2+ current, the absence of β, as well as the coexpression of β2a, was found to flatten restitution slope and stabilize spiral waves. These results imply that subunit modification of L-type Ca2+ channels can potentially be used as an antifibrillatory strategy.
Heart failure progression is accelerated following myocardial infarction in type 2 diabetic rats
Authors: Chandler Margaret P. , Morgan Eric E. , McElfresh Tracy A. , Kung Theodore A. , Rennison Julie H. , Hoit Brian D. , Young Martin E.
Published: June 2007

Clinical studies have shown a greater incidence of myocardial infarction in diabetic patients, and following an infarction, diabetes is associated with an increased risk for the development of left ventricular (LV) dysfunction and heart failure. The goal of this study was to determine if the progression of heart failure following myocardial infarction in type 2 diabetic (T2D) rats is accelerated compared with nondiabetic rats. Male nondiabetic Wistar-Kyoto (WKY) and T2D Goto-Kakizaki (GK) rats underwent coronary artery ligation or sham surgery to induce heart failure. Postligation (8 and 20 wk), two-dimensional echocardiography and LV pressure measurements were made. Heart failure progression, as assessed by enhanced LV remodeling and contractile dysfunction, was accelerated 8 wk postligation in the T2D animals. LV remodeling was evident from increased end-diastolic and end-systolic diameters and areas in the GK compared with the WKY infarcted group. Furthermore, enhanced LV contractile dysfunction was evident from a greater deterioration in fractional shortening and enhanced myocardial performance index (an index of global LV dysfunction) in the GK infarcted group. This accelerated progression was accompanied by greater increases in atrial natriuretic factor and skeletal α-actin (gene markers of heart failure and hypertrophy) mRNA levels in GK infarcted hearts. Despite similar decreases in metabolic gene expression (i.e., peroxisome proliferator-activated receptor-α-regulated genes associated with fatty acid oxidation) between infarcted WKY and GK rat hearts, myocardial triglyceride levels were elevated in the GK hearts only. These results, demonstrating enhanced remodeling and LV dysfunction 8 wk postligation provide evidence of an accelerated progression of heart failure in T2D rats.