bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plasmid DNA transfer in Mycobacterium smegmatis involves novel DNA rearrangements in the recipient, which can be exploited for molecular genetic studies
Authors: Wang Jun , Derbyshire Keith M. 
Published: August 2004

SummaryThe establishment of molecular genetic techniques is essential for development of new treatments for mycobacterial infections. To this end, we recently described a novel DNA transfer process that occurs in the model mycobacterial organism Mycobacterium smegmatis. This transfer system is most like conjugal DNA transfer in that it requires two viable parents, is DNAse resistant and occurs between distinct donor and recipient strains. Cis‐acting sequences called bom, which confer transferability, are distinct from the prototypical oriT sites of conjugative plasmids, as they occur at multiple locations in the chromosome and require RecA in the recipient to mediate plasmid recircularization. Here, we show that a plasmid containing two of these bom regions can undergo several fates in the recipient cell, each of which require recipient recombination functions. The products of plasmid transfer that we observed provide further insights toward a model for DNA transfer. Furthermore, we have taken advantage of the recombination events that occur in the recipient to develop simple procedures for capturing, or replacing specific segments of the recipient chromosome. To demonstrate the potential of the system, we describe the capture and deletion of 25 kb of the M. smegmatis chromosome, and targeted‐allele exchange of the recipient recB and recD genes. Using these transfer‐mediated rearrangements, we demonstrate that homology with the recipient chromosome and RecB, but not RecD, are essential for DNA transfer.
Secretion of proteins with dimerization capacity by the haemolysin type I transport system ofEscherichia coli
Authors: Fraile Sofía , Muñoz Amalia , De Lorenzo Víctor , Fernández Luis Angel 
Published: August 2004

SummaryThe tolerance of the haemolysin transport system (Hly) for exporting dimeric protein substrates to the supernatants ofEscherichia colicultures was examined. A strong dimerization domain (i.e. an amphipathic α‐helix capable of forming a leucine zipper in the yeast transcription factor GCN4) was inserted into an epitope‐tagged version of the 23 kDa C‐terminal secretion signal of haemolysin (EHlyA). The zipper‐containing polypeptide (ZEHlyA) was effectively secreted byE. colicells carrying the HlyBD transporter and accumulated in the culture media as a stable dimer as determined by gel filtration chromatography.In vivoprotein cross‐linking experiments and coexpression with a secretion‐deficient derivative of ZEHlyA indicated that leucine zipper‐dependent dimerization occurs following secretion. To test whether dimerization allows the correct folding of the secreted polypeptide, immunoglobulin VHH‐domains obtained from camel antibodies were fused to EHlyA and ZEHlyA. Functional dimerization of the ZEHlyA hybrid was anticipated to increase the apparent binding affinity (i.e. avidity) of the VHHmoiety, thus becoming an excellent reporter of correct protein folding and dimerization. Both VHH‐EHlyA and VHH‐ZEHlyA hybrids were quantitatively secreted and found in the extracellular medium as active monomers and dimers respectively. When compared with their monomeric counterparts, the dimeric VHH‐ZEHlyA molecules showed superior binding properties to their cognate antigen, with a 10‐fold increase in their avidity. These data reveal a non‐anticipated permissiveness of the Hly type I transport machinery for the secretion of substrates with dimerization capacity.
MicroReview: Small molecules that regulate lifespan: evidence for xenohormesis
Authors: Lamming Dudley W. , Wood Jason G. , Sinclair David A. 
Published: August 2004

SummaryBarring genetic manipulation, the diet known as calorie restriction (CR) is currently the only way to slow down ageing in mammals. The fact that CR works on most species, even microorganisms, implies a conserved underlying mechanism. Recent findings in the yeast Saccharomyces cerevisiae indicate that CR extends lifespan because it is a mild biological stressor that activates Sir2, a key component of yeast longevity and the founding member of the sirtuin family of deacetylases. The sirtuin family appears to have first arisen in primordial eukaryotes, possibly to help them cope with adverse conditions. Today they are found in plants, yeast, and animals and may underlie the remarkable health benefits of CR. Interestingly, a class of polyphenolic molecules produced by plants in response to stress can activate the sirtuins from yeast and metazoans. At least in the case of yeast, these molecules greatly extend lifespan by mimicking CR. One explanation for this surprising observation is the ‘xenohormesis hypothesis’, the idea that organisms have evolved to respond to stress signalling molecules produced by other species in their environment. In this way, organisms can prepare in advance for a deteriorating environment and/or loss of food supply.
Structure of the Pseudomonas aeruginosa acyl‐homoserinelactone synthase LasI
Authors: Gould Ty A. , Schweizer Herbert P. , Churchill Mair E. A. 
Published: August 2004

SummaryThe LasI/LasR quorum‐sensing system plays a pivotal role in virulence gene regulation of the opportunistic human pathogen, Pseudomonas aeruginosa. Here we report the crystal structure of the acyl‐homoserine lactone (AHL) synthase LasI that produces 3‐oxo‐C12‐AHL from the substrates 3‐oxo‐C12‐acyl‐carrier protein (acyl‐ACP) and S‐adenosyl‐L‐methionine. The LasI six‐stranded beta sheet platform, buttressed by three alpha helices, forms a V‐shaped substrate‐binding cleft that leads to a tunnel passing through the enzyme that can accommodate the acyl‐chain of acyl‐ACP. This tunnel places no apparent restriction on acyl‐chain length, in contrast to a restrictive hydrophobic pocket seen in the AHL‐synthase EsaI. Interactions of essential conserved N‐terminal residues, Arg23, Phe27 and Trp33, suggest that the N‐terminus forms an enclosed substrate‐binding pocket for S‐adenosyl‐L‐methionine. Analysis of AHL‐synthase surface residues identified a binding site for acyl‐ACP, a role that was supported by in vivo reporter assay analysis of the mutated residues, including Arg154 and Lys150. This structure and the novel explanation of AHL‐synthase acyl‐chain‐length selectivity promise to guide the design of Pseudomonas aeruginosa‐specific quorum‐sensing inhibitors as antibacterial agents.
Enforcement of Property Rights and Underdevelopment
Authors: Dabla‐Norris Era , Freeman Scott 
Published: August 2004

AbstractThe authors formalize the role of legal infrastructure in economic development in a general‐equilibrium model with endogenously determined property rights enforcement. The mutual importance of property rights protection and market production is illustrated by the model's multiplicity of equilibria. In one equilibrium, property rights are enforced, and market activity unhampered. In the other, property rights are not enforced, discouraging economic activity, which leaves the economy without the resources and the incentives to enforce property rights. Even identically endowed economies may therefore find themselves in very different equilibria.
Specialization, Transaction Efficiency, and Firm Size: Empirical Evidence
Authors: Lam Kit‐Chun , Liu Pak‐Wai 
Published: August 2004

AbstractThe paper provides some empirical evidence for the relationship between average firm size and the relative transaction efficiency and per capita income as hypothesized by Liu and Yang in Journal of Economic Behavior and Organization 42(2) (2000):145–65. The empirical data of 11 industries in Hong Kong from 1982 to 1999 are found to be broadly consistent with the predictions of Liu and Yang.
Calcium currents and transients in co‐cultured contracting normal and Duchenne muscular dystrophy human myotubes
Authors: Imbert Nathalie , Vandebrouck Clarisse , Duport Gérard , Raymond Guy , Hassoni Abdul A. , Constantin Bruno , Cullen Michael J. , Cognard Christian 
Published: August 2004

The goal of the present study was to investigate differences in calcium movements between normal and Duchenne muscular dystrophy (DMD) human contracting myotubes co‐cultured with explants of rat spinal cord with attached dorsal root ganglia. Membrane potential, variations of intracellular calcium concentration and T‐ and L‐type calcium currents were recorded. Further, a descriptive and quantitative study by electron microscopy of the ultrastructure of the co‐cultures was carried out.
The resting membrane potential was slightly less negative in DMD (‐61.4 ± 1.1 mV) than in normal myotubes (‐65.5 ± 0.9 mV). Both types of myotube displayed spontaneous action potentials (mean firing frequency, 0.42 and 0.16 Hz, respectively), which triggered spontaneous calcium transients measured with Indo‐1.
The time integral under the spontaneous Ca2+ transients was significantly greater in DMD myotubes (97 ± 8 nm s) than in normal myotubes (67 ± 13 nm s).
The L‐ and T‐type current densities estimated from patch‐clamp recordings were smaller in DMD cells (2.0 ± 0.5 and 0.90 ± 0.19 pA pF−1, respectively) than in normal cells (3.9 ± 0.7 and 1.39 ± 0.30 pA pF−1, respectively).
The voltage‐dependent inactivation relationships revealed a shift in the conditioning potential at which inactivation is half‐maximal (Vh,0.5) of the T‐ and L‐type currents towards less negative potentials, from ‐72.1 ± 0.7 and ‐53.7 ± 1.5 mV in normal cells to ‐61.9 ± 1.4 and ‐29.2 ± 1.4 mV in DMD cells, respectively.
Both descriptive and quantitative studies by electron microscopy suggested a more advanced development of DMD myotubes as compared to normal ones. This conclusion was supported by the significantly larger capacitance of the DMD myotubes (408 ± 45 pF) than of the normal myotubes (299 ± 34 pF) of the same apparent size.
Taken together, these results show that differences in T‐ and L‐type calcium currents between normal and DMD myotubes cannot simply explain all observed alterations in calcium homeostasis in DMD myotubes, thus suggesting that other transmembrane calcium transport mechanisms must also be altered in DMD myotubes compared with normal myotubes.
Is myosin phosphatase regulated in vivo by inhibitor‐1? Evidence from inhibitor‐1 knockout mice
Authors: Carr Andrew N. , Sutliff Roy L. , Weber Craig S. , Allen Patrick B. , Greengard Paul , de Lanerolle Primal , Kranias Evangelia G. , Paul Richard J. 
Published: August 2004

The Ca2+ sensitivity of smooth muscle contractility is modulated via regulation of phosphatase activity. Protein phosphatase inhibitor‐1 (I‐1) is the classic type‐1 phosphatase inhibitor, but its presence and role in cAMP‐dependent protein kinase (PKA) modulation of smooth muscle is unclear. To address the relevance of I‐1 in vivo, we investigated smooth muscle function in a mouse model lacking the I‐1 protein (I‐1(‐/‐) mice).
Significant amounts of I‐1 protein were detected in the wild‐type (WT) mouse aorta and could be phosphorylated by PKA, as indicated by 32P‐labelled aortic extracts from WT mice.
Despite the significant presence of I‐1 in WT aorta, phenylephrine and KCl concentration‐ isometric force relations in the presence or absence of the PKA pathway activator isoproterenol (isoprenaline) were unchanged compared to I‐1(‐/‐) aorta. cGMP‐dependent protein kinase (PKG) relaxation pathways were also not different. Consistent with these findings, dephosphorylation rates of the 20 kDa myosin light chains (MLC20), measured in aortic extracts, were nearly identical between WT and I‐1(‐/‐) mice.
In the portal vein, I‐1 protein ablation was associated with a significant (P < 0.05) rightward shift in the EC50 of isoproterenol relaxation (EC50= 10.4 ± 1.4 nm) compared to the WT value (EC50= 3.5 ± 0.2 nm). Contraction in response to acetylcholine as well as Ca2+ sensitivity were similar between WT and I‐1(‐/‐) aorta.
Despite the prevalence of I‐1 and its activation by PKA in the aorta, I‐1 does not appear to play a significant role in contractile or relaxant responses to any pharmacomechanical or electromechanical agonists used. I‐1 may play a role as a fine‐tuning mechanism involved in regulating portal vein responsiveness to β‐adrenergic agonists.
Regulation of a G protein‐gated inwardly rectifying K+ channel by a Ca2+‐independent protein kinase C
Authors: Leaney Joanne L. , Dekker Lodewijk V. , Tinker Andrew 
Published: August 2004

Members of the Kir3.0 family of inwardly rectifying K+ channels are expressed in neuronal, atrial and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate and modulating hormone release. They are activated directly by Gβγ subunits released in response to Gi/o‐coupled receptor stimulation. However, it is not clear to what extent this process can be dynamically regulated by other cellular signalling systems. In this study we have explored pathways activated by the Gq/11‐coupled M1 and M3 muscarinic receptors and their role in the regulation of Kir3.1+3.2A neuronal‐type channels stably expressed in the human embryonic kidney cell line HEK293.
We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M1 and M3 receptors. This contrasts with the simple stimulation seen through activation of M2 and M4 receptors.
Channel stimulation via M1 but not M3 receptors was sensitive to pertussis toxin whereas channel inhibition through both M1 and M3 receptors was insensitive. In contrast over‐expression of the C‐terminus of phospholipase Cβ1 or a Gq/11‐specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase.
The inhibitory effects of M1 and M3 receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4αphorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M3 receptor.
The receptor‐mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0‐669 nm).
The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by immunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation of the novel PKC isoforms PKCδ and PKCε.
We also demonstrate that activation of such a pathway via both receptor‐mediated and receptor‐independent means profoundly attenuated subsequent channel stimulation by Gi/o‐coupled receptors.
Our data support a role for a Ca2+‐independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M1 and M3 muscarinic receptor stimulation.
Differential regulation of Ca2+‐activated Cl− currents in rabbit arterial and portal vein smooth muscle cells by Ca2+‐calmodulin‐dependent kinase
Authors: Greenwood Iain A. , Ledoux Jonathan , Leblanc Normand 
Published: August 2004

Ca2+‐activated chloride currents (ICl(Ca)) were recorded from smooth muscle cells isolated from rabbit pulmonary (PA) and coronary artery (CA) as well as rabbit portal vein (PV). The characteristics and regulation by Ca2+‐calmodulin‐dependent kinase II (CaMKII) were compared between the three cell types.
In PA and CA myocytes dialysed and superfused with K+‐free media, pipette solutions containing fixed levels of free Ca2+ in the range of 250 nm to 1 μm evoked well sustained, outwardly rectifying ICl(Ca) currents in about 90 % of cells. The CaMKII inhibitor KN‐93 (5 μm) increased the amplitude of ICl(Ca) in PA and CA myocytes. However, the threshold intracellular Ca2+ concentration for detecting this effect was different in the two arterial cell types. KN‐93 also enhanced the rate of activation of the time‐dependent current during depolarising steps, slowed the kinetics of the tail current following repolarisation, and induced a negative shift of the steady‐state activation curve.
In PA myocytes, the effects of KN‐93 were not mirrored by its inactive analogue KN‐92 but were reproduced by the inclusion of autocamtide‐2‐related CaMKII inhibitory peptide (ARIP) in the pipette solution. Cell dialysis with constitutively active CaMKII (30 nm) significantly reduced ICl(Ca) evoked by 500 nm Ca2+.
In PV myocytes, ICl(Ca) was evoked by pipette solutions containing up to 1 μm free Ca2+ in less than 40 % of cells. Application of KN‐93 to cells where ICl(Ca) was sustained produced a small inhibition (≈25 %) of the current in 70 % of the cells.
The present study shows that regulation of Ca2+‐dependent Cl− channels by CaMKII differs between arterial and portal vein myocytes.
Ionic selectivity of native ATP‐activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia
Authors: Liu Dong‐Mei , Adams David J. 
Published: August 2004

The relative permeability of the native P2X receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements of ATP‐evoked currents in parasympathetic neurones dissociated from rat submandibular ganglia using the dialysed whole‐cell patch clamp technique.
The P2X receptor‐channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Na+ > Li+ > Cs+ > Rb+ > K+, and permeability ratios relative to Cs+ (PX/PCs) ranging from 1.11 to 0.86.
The selectivity for the divalent alkaline earth cations was also weak with the sequence Ca2+ > Sr2+ > Ba2+ > Mn2+ > Mg2+. ATP‐evoked currents were strongly inhibited when the extracellular divalent cation concentration was increased.
The calculated permeability ratios of different ammonium cations are higher than those of the alkali metal cations. The permeability sequence obtained for the saturated organic cations is inversely correlated with the size of the cation. The unsaturated organic cations have a higher permeability than that predicted by molecular size.
Acidification to pH 6.2 increased the ATP‐induced current amplitude twofold, whereas alkalization to 8.2 and 9.2 markedly reduced current amplitude. Cell dialysis with either anti‐P2X2 and/or anti‐P2X4 but not anti‐P2X1 antibodies attenuated the ATP‐evoked current amplitude. Taken together, these data are consistent with homomeric and/or heteromeric P2X2 and P2X4 receptor subtypes expressed in rat submandibular neurones.
The permeability ratios for the series of monovalent organic cations, with the exception of unsaturated cations, were approximately related to the ionic size. The relative permeabilities of the monovalent inoganic and organic cations tested are similar to those reported previously for cloned rat P2X2 receptors expressed in mammalian cells.
Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin
Authors: Hamilton Sara G. , McMahon Stephen B. , Lewin Gary R. 
Published: August 2004

ATP can elicit pain in humans and, together with other P2X channel agonists, can produce nocifensive responses in rodents. We used the rat in vitro skin‐nerve preparation to quantify primary afferent responses to ATP and its stable analogue α,β‐methylene ATP in normal and carrageenan‐inflamed skin.
Both ATP and α,β‐methylene ATP were found to specifically activate the peripheral terminals of Aδ and C‐fibre nociceptors in the skin. Thirty‐nine per cent of the nociceptors tested responded to the maximal dose of α,β‐methylene ATP (5 mm). In contrast, non‐nociceptive, low‐threshold mechano‐sensitive fibres were never activated by the same agonist concentrations.
Amongst the nociceptor population, C‐mechanoheat fibres (C‐MH or polymodal nociceptors) were markedly more responsive to P2X agonists than mechanonociceptors (C‐M nociceptors) with Aδ‐ or C‐fibre axons. Both C‐mechanoheat and C‐mechanonociceptors were activated by α,β‐methylene ATP doses as low as 50 μm.
In skin inflamed with carrageenan 3‐4 h before recording both the number of responsive C‐fibre nociceptors and their response magnitude increased. The increased neural response under inflammatory conditions was largely observed in C‐mechanoheat or polymodal nociceptors. After low doses of P2X agonists C‐MH fibres but not C‐M fibres developed elevated ongoing activity and this effect was only seen after carrageenan inflammation. The time course of α,β‐methylene ATP‐evoked discharges in nociceptors was found to correlate well with the time course of behavioural nocifensive responses in rats to the same agonist described in a previous study (Hamilton et al. 1999).
We conclude that the rapid increase in the number of α,β‐methylene ATP responsive nociceptors and the increased magnitude of the neural response following carrageenan inflammation explains why very low concentrations of such agonists can cause pain in inflammatory states.
Minimal force‐frequency modulation of inotropy and relaxation of in situ murine heart
Authors: Georgakopoulos Dimitrios , Kass David A. 
Published: August 2004

The normal influence of heart rate (HR) on cardiac contraction and relaxation in the mouse remains uncertain despite its importance in interpreting many genetically engineered models. Prior in vivo data have repeatedly shown positive effects only at subphysiological heart rates, yet depressed basal conditions and use of load‐dependent parameters probably have an impact on these results.
Open‐chest mice of various strains (n = 16, etomidate/urethane anaesthesia) were instrumented with a miniaturized pressure‐volume catheter employing absolute left ventricular (LV) volume calibration. HR was slowed (< 400 beats min−1) using ULFS‐49, and atrial or ventricular pacing was achieved via an intra‐oesophageal catheter. Pressure‐volume data yielded cardiac‐specific contractile indexes minimally altered by vascular load.
At a resting HR of 600 beats min−1, peak pressure‐rise rate (dP/dtmax) was 16 871 ± 2941 mmHg s−1 (mean ±s.d.) and the relaxation time constant was 3.9 ± 0.8 ms, similar to values in conscious animals. Within the broad physiological range (500‐850 beats min−1), load‐insensitive contractile indexes and relaxation rate varied minimally, whereas dP/dtmax peaked at 600 ± 25 beats min−1 and decreased at higher rates due to preload sensitivity. Contraction and relaxation were enhanced modestly (13‐15 %) at HRs of between 400 and 500 beats min−1.
The minimal force‐frequency dependence was explained by rapid calcium cycling kinetics, with a mechanical restitution time constant of 9 ± 2.7 ms, and by dominant sarcoplasmic reticular buffering (recirculation fraction of 93 ± 1 %).
The mouse normally has a very limited force‐frequency reserve at physiological HRs, unlike larger mammals and man. This is important to consider when studying disease evolution and survival of genetic models that alter calcium homeostasis and SR function.