bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of various scinderin domains in chromaffin cells indicates that this protein acts as a molecular switch in the control of actin filament dynamics and exocytosis
Authors: Dumitrescu Pene Teodora , Rosé Sergio D. , Lejen Tatiana , Marcu Monica G. , Trifaró José‐Maria
Published: February 2005

Stimulation‐induced chromaffin cell cortical F‐actin disassembly allows the movement of vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+‐dependent protein, controls actin dynamics. Sc six domains have three actin, two PIP2 and two Ca2+‐binding sites. F‐actin severing activity of Sc is Ca2+‐dependent, whereas Sc‐evoked actin nucleation is Ca2+‐independent. Sc domain role in secretion was studied by co‐transfection of human growth hormone (hGH) reporter gene and green fluorescent protein (GFP)‐fusion Sc constructs. Cells over‐expressing actin severing Sc1‐6 or Sc1‐2 (first and second actin binding sites) constructs, increased F‐actin disassembly and hGH release upon depolarization. Over‐expression of nucleating Sc5‐6, Sc5 or ScABP3 (third actin site) constructs decreased F‐actin disassembly and hGH release upon stimulation. Over‐expression of ScL5‐6 or ScL5 (lack of third actin site) produced no changes. During secretion, actin sites 1 and 2 are involved in F‐actin severing, whereas site 3 is responsible for nucleation (polymerization). Sc functions as a molecular switch in the control of actin (disassembly ⇆ assembly) and release (facilitation ⇆ inhibition). The position of the switch (severing ⇆ nucleation) may be controlled by [Ca2+]i. Thus, increase in [Ca2+]i produced by stimulation‐induced Ca2+ entry would increase Sc‐evoked cortical F‐actin disassembly. Decrease in [Ca2+]i by either organelle sequestration or cell extrusion would favor Sc‐evoked actin nucleation.
Conformational changes at benzodiazepine binding sites of GABAA receptors detected with a novel technique
Authors: Berezhnoy Dmytro , Baur Roland , Gonthier Anne , Foucaud Bernard , Goeldner Maurice , Sigel Erwin
Published: February 2005

Benzodiazepines are widely used for their anxiolytic, sedative, myorelaxant and anticonvulsant properties. They allosterically modulate GABAA receptor function by increasing the apparent affinity of the agonist GABA. We studied conformational changes induced by channel agonists at the benzodiazepine binding site. We used the rate of covalent reaction between a benzodiazepine carrying a cysteine reactive moiety with mutated receptor having a cysteine residue in the benzodiazepine binding pocket, α1H101Cβ2γ2, as a sensor of its conformation. This reaction rate is sensitive to local conformational changes. Covalent reaction locks the receptor in the conformation stabilized by positive allosteric modulators. By using concatenated subunits we demonstrated that the covalent reaction occurs either exclusively at the α/γ subunit interface, or if it occurs in both α1 subunits, exclusively reaction at the α/γ subunit interface can modulate the receptor. We found evidence for an increased rate of reaction of activated receptors, whereas reaction rate with the desensitized state is slowed down. The benzodiazepine antagonist Ro15–1788 efficiently inhibited the covalent reaction in the presence of 100 µm GABA but only partially in its absence or in the presence of 10 µm GABA. It is concluded that Ro15–1788 efficiently protects activated and desensitized states, but not the resting state.
The expression of GLP‐1 receptor mRNA and protein allows the effect of GLP‐1 on glucose metabolism in the human hypothalamus and brainstem
Authors: Alvarez Elvira , Martínez M. Dolores , Roncero Isabel , Chowen Julie A. , García‐Cuartero Beatriz , Gispert Juan D. , Sanz Carmen , Vázquez Patricia , Maldonado Antonio , De Cáceres Javier , Desco Manuel , Pozo Miguel Angel , Blázquez Enrique
Published: February 2005

In the present work, several experimental approaches were used to determine the presence of the glucagon‐like peptide‐1 receptor (GLP‐1R) and the biological actions of its ligand in the human brain. In situ hybridization histochemistry revealed specific labelling for GLP‐1 receptor mRNA in several brain areas. In addition, GLP‐1R, glucose transporter isoform (GLUT‐2) and glucokinase (GK) mRNAs were identified in the same cells, especially in areas of the hypothalamus involved in feeding behaviour. GLP‐1R gene expression in the human brain gave rise to a protein of 56 kDa as determined by affinity cross‐linking assays. Specific binding of 125I‐GLP‐1(7–36) amide to the GLP‐1R was detected in several brain areas and was inhibited by unlabelled GLP‐1(7–36) amide, exendin‐4 and exendin (9–39). A further aim of this work was to evaluate cerebral‐glucose metabolism in control subjects by positron emission tomography (PET), using 2‐[F‐18] deoxy‐d‐glucose (FDG). Statistical analysis of the PET studies revealed that the administration of GLP‐1(7–36) amide significantly reduced (p < 0.001) cerebral glucose metabolism in hypothalamus and brainstem. Because FDG‐6‐phosphate is not a substrate for subsequent metabolic reactions, the lower activity observed in these areas after peptide administration may be due to reduction of the glucose transport and/or glucose phosphorylation, which should modulate the glucose sensing process in the GLUT‐2‐ and GK‐containing cells.
A truncated isoform of pyroglutamyl aminopeptidase II produced by exon extension has dominant‐negative activity
Authors: Chávez‐Gutiérrez Lucia , Bourdais Julie , Aranda Gonzalo , Vargas Miguel Angel , Matta‐Camacho Edna , Ducancel Frédéric , Segovia Lorenzo , Joseph‐Bravo Patricia , Charli Jean‐Louis
Published: February 2005

Thyrotropin‐releasing hormone is inactivated in the extracellular space by a membrane‐bound peptidase, pyroglutamyl aminopeptidase II (PPII), a member of the M1 family of zinc metallopeptidases. The functional significance of multiple PPII RNA species expression is unknown. We detected, in rat tissues, a RNA species derived from an alternative processing at the exon 14–intron 14 boundary. The alternatively processed RNA encoded a shorter version of PPII (PPII*), lacking part of the C‐terminal domain. PPII* was expressed in COS‐7 (or C6 glioma) cells but it did not exhibit any PPII activity. Co‐transfection of PPII and increasing amounts of PPII* expression vectors resulted in a dose‐dependent reduction in PPII activity and the formation of covalent PPII–PPII* heterodimers. PPII* is therefore a powerful dominant‐negative isoform of PPII, and heterodimerization may be its mechanism of action. Natural expression of shortened versions of M1 aminopeptidases may constitute a new mode of regulation of their activity.
Distinct roles of PKC isoforms in NCAM‐mediated neurite outgrowth
Authors: Kolkova Kateryna , Stensman Helena , Berezin Vladimir , Bock Elisabeth , Larsson Christer
Published: February 2005

The role of protein kinase C (PKC) isoforms in the neural cell adhesion molecule (NCAM)‐mediated neurite outgrowth was tested using a co‐culture system consisting of fibroblasts with or without NCAM expression upon which either primary cerebellar granular neurones (CGN) or pheochromocytoma (PC12‐E2) cells were grown. The latter transiently expressed various PKC isoforms and domains derived from selected PKCs. PKC inhibitors of various specificity inhibited NCAM‐stimulated neuritogenesis from CGN, indicating that PKC is involved in this process. Moreover, stimulation by the NCAM‐mimetic peptide, C3d, elicited phosphorylation of PKC in CGN. Expression of kinase‐deficient forms of PKCα, βI and βII blocked NCAM‐mediated neurite extension, but had no effect on nerve growth factor (NGF)‐mediated neurite outgrowth. Expression of two PKCɛ constructs: (i) a fragment from PKCɛ encompassing the pseudosubstrate, the C1a domain (including the actin‐binding site, ABS), and parts of the V3 region, or (ii) the PKCɛ‐specific ABS blocked NCAM‐mediated neurite extension in both cases. These two constructs also partially inhibited NGF‐stimulated neuritogenesis indicating that PKCɛ is a positive regulator of both NCAM‐ and NGF‐mediated differentiation. We suggest that PKCɛ is a common downstream mediator for several neuritogenic factors, whereas one or more conventional PKCs are specifically involved in NCAM‐stimulated neurite outgrowth.
Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase β and DNA‐ligase
Authors: Krishna T. Hari , Mahipal S. , Sudhakar A. , Sugimoto H. , Kalluri Raghu , Rao K. Subba
Published: February 2005

Synthetic deoxy‐oligo duplexes containing short gaps of 1 and 4 nucleotides were used as model substrates to assess the DNA gap repair ability of the neuronal extracts prepared from cerebral cortex of rats of different ages. Our results demonstrate that gap repair activity in neurons decreases markedly with age. The decreased activity could be restored by supplementing the neuronal extracts with pure recombinant rat liver DNA polymerase β. High levels of DNA polymerase β supplementation resulted in gap‐filling activity that proceeded essentially through addition of nucleotides through a slow distributive strand displacement mode to achieve full template length (32‐mer). However, at lower concentrations of DNA polymerase β, the gap repair takes place quickly through gap filling followed by ligation to downstream primer, in an energy efficient manner. For this to happen, the conditions required are the presence of 5′‐PO4 on the downstream primer and supplementation of aging neuronal extracts with DNA‐ligase in addition to recombinant DNA polymerase β. These results demonstrate that aging neurons are unable to affect base excision repair (BER) due to deficiency of DNA polymerase β and DNA‐ligase and fortifying aged neuronal extracts with these two factors can restore the lost BER activity.
Nuclear receptor peroxisome proliferator‐activated receptor‐γ is activated in rat microglial cells by the anti‐inflammatory drug HCT1026, a derivative of flurbiprofen
Authors: Bernardo Antonietta , Ajmone‐Cat Maria Antonietta , Gasparini Laura , Ongini Ennio , Minghetti Luisa
Published: February 2005

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is constitutively expressed in primary cultures of rat microglia, the main population of brain resident macrophages, and its ligand‐dependent activation leads to the repression of several microglial functions. A few non‐steroidal anti‐inflammatory drugs (NSAIDs), e.g. indomethacin and ibuprofen, show PPAR‐γ agonistic properties. It has been proposed that PPAR‐γ activation contributes to the potential benefits of the long‐term use of certain NSAIDs in delaying the progression of Alzheimer's disease (AD). Previous data have shown that the NSAID HCT1026 [2‐fluoro‐α‐methyl(1,1′‐biphenyl)4‐acetic acid‐4‐(nitrooxy)butyl ester], a derivative of flurbiprofen which releases nitric oxide (NO), reduces the number of reactive microglial cells in a variety of models. This evidence together with the chemical analogy with ibuprofen led us to investigate whether flurbiprofen and HCT1026 interact with PPAR‐γ and interfere with microglial activation. We found that a low concentration (1 µm) of HCT1026, but not flurbiprofen, activated PPAR‐γ in primary cultures of rat microglia, with kinetics similar to those of the synthetic agonist ciglitazone. The PPAR‐γ antagonist GW9662 (2‐chloro‐5‐nitrobenzanilide) prevented the activation of PPAR‐γ by HCT1026. Interestingly, unlike other NSAIDs that activate PPAR‐γ at concentrations higher than those required for cyclooxygenase inhibition, HCT1026 activated PPAR‐γ and inhibited prostaglandin E2 synthesis at the same low concentration (1 µm). The results suggest that HCT1026 may exert additional anti‐inflammatory actions through PPAR‐γ activation, allowing a more effective control of microglial activation and brain inflammation.
Activation of astrocytes in brain of conscious rats during acoustic stimulation: acetate utilization in working brain
Authors: Cruz Nancy F. , Lasater Amelia , Zielke H. Ronald , Dienel Gerald A.
Published: February 2005

To evaluate the response of astrocytes in the auditory pathway to increased neuronal signaling elicited by acoustic stimulation, conscious rats were presented with a unilateral broadband click stimulus and functional activation was assessed by quantitative autoradiography using three tracers to pulse label different metabolic pools in brain: [2‐14C]acetate labels the ‘small’ (astrocytic) glutamate pool, [1‐14C]hydroxybutyrate labels the ‘large’ glutamate pool, and [14C]deoxyglucose, reflects overall glucose utilization (CMRglc) in all brain cells. CMRglc rose during brain activation, and increased activity of the oxidative pathway in working astrocytes during acoustic stimulation was registered with [2‐14C]acetate. In contrast, the stimulation‐induced increase in metabolic activity was not reflected by greater trapping of products of [1‐14C]hydroxybutyrate. The [2‐14C]acetate uptake coefficient in the inferior colliculus and lateral lemniscus during acoustic stimulation was 15% and 18% (p < 0.01) higher in the activated compared to contralateral hemisphere, whereas CMRglc in these structures rose by 66% (p < 0.01) and 42% (p < 0.05), respectively. Calculated rates of brain utilization of blood‐borne acetate (CMRacetate) are about 15–25% of total CMRglc in non‐stimulated tissue and 10–20% of CMRglc in acoustically activated structures; they range from 28 to 115% of estimated rates of glucose oxidation in astrocytes. The rise in acetate utilization during acoustic stimulation is modest compared to total CMRglc, but astrocytic oxidative metabolism of ‘minor’ substrates present in blood can make a significant contribution to the overall energetics of astrocytes and astrocyte–neuron interactions in working brain.
Biochemical and functional evidence for heteromeric assembly of P2X1 and P2X4 subunits
Authors: Nicke Annette , Kerschensteiner Daniel , Soto Florentina
Published: February 2005

P2X receptors are ligand‐gated ion channels activated by extracellular ATP. In expression systems, P2X subunits form homo‐ and heterotrimeric receptors. Heteromerization is also likely to occur in vivo as (i) most P2X subunits show overlapping distribution in different tissues and (ii) the functional properties of many native P2X receptors differ from those of heterologously expressed homomeric receptors. Here, we used the Xenopus laevis oocyte expression system to test for heteromerization of P2X1 and P2X4 subunits. Upon co‐injection, P2X4 subunits were co‐purified with hexahistidyl‐tagged P2X1 subunits indicating heteromerization. Blue native polyacrylamide gel electrophoresis (BN‐PAGE) analysis of these P2X complexes excluded artificial aggregation and confirmed that both subunits were present in trimeric complexes of the same size. Two‐electrode voltage‐clamp experiments revealed functional P2X receptors with kinetic properties resembling homomeric P2X4 receptors and a pharmacological profile similar to homomeric P2X1 receptors. Thus, application of α,β‐methylene ATP evoked a slowly desensitizing current sensitive to the antagonists suramin and 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐ATP. This study provides for the first time biochemical and functional evidence for the formation of heteromeric P2X1+4 receptors. These receptors may account for native P2X mediated responses that until now could not be correlated with previously described recombinant P2X receptors.
Objective measurements of radiotherapy‐induced erythema
Authors: Nyström Josefina , Geladi Paul , Lindholm‐Sethson Britta , Rattfelt Jenny , Svensk Ann‐Christin , Franzen Lars
Published: February 2005

Background/aims: The development of acute radiation erythema is a common phenomenon among patients undergoing radiotherapy treatment. Because of the absence of reliable objective classification methods, the degree of skin reaction can at present mainly be judged subjectively in the clinic. This has motivated the present preliminary study, concerning the first steps in the development of an objective method for skin reaction classification.

Methods: Three non‐invasive techniques were used: near‐infrared (NIR) spectroscopy, laser Doppler perfusion imaging and digital photography. The NIR spectra were analysed with principal component analysis (PCA), and the results from the other two with traditional univariate methods. Measurements were made on breast cancer patients who had been exposed to different irradiation doses. A total of 28 breast cancer patients participated one to three times each; 12 were treated with photons at 4 or 6 MeV and 16 were treated with high‐energy electrons between 10 and 20 MeV to a maximum dose of 50 Gy.

Results: PCA of NIR spectra shows that information on radiation dose lies mainly in the first principal component. It is observed that the higher the dose the higher the score value. The results from the laser Doppler measurements show that in 79% of the cases the perfusion increases significantly with radiation dose. Analysis of the digital photography shows that a proposed skin redness index (SRI), increases with a higher radiation dose. However, the increase in most cases is not significant. By combining all data, correlation to radiation doses was seen for 74% of the patients who participated more than once.

Conclusion: All three non‐invasive methods correlate with the radiation dose but to various degrees. NIR spectroscopy, laser Doppler and a combination of the three techniques are the most promising methods for characterising erythema.
Terahertz Vibration of Short Carbon Nanotubes Modeled as Timoshenko Beams
Authors: Yoon J. , Ru C. Q. , Mioduchowski A.
Published: February 2005

Short carbon nanotubes of smaller aspect ratio (say, between 10 and 50) are finding significant application in nanotechnology. This paper studies vibration of such short carbon nanotubes whose higher-order resonant frequencies fall within terahertz range. Because rotary inertia and shear deformation are significant for higher-order modes of shorter elastic beams, the carbon nanotubes studied here are modeled as Timoshenko beams instead of classical Euler beams. Detailed results are demonstrated for double-wall carbon nanotubes of aspect ratio 10, 20, or 50 based on the Timoshenko-beam model and the Euler-beam model, respectively. Comparisons between different single-beam or double-beam models indicate that rotary inertia and shear deformation, accounted for by the Timoshenko-beam model, have a substantial effect on higher-order resonant frequencies and modes of double-wall carbon nanotubes of small aspect ratio (between 10 and 20). In particular, Timoshenoko-beam effects are significant for both large-diameter and small-diameter double-wall carbon nanotubes, while double-beam effects characterized by noncoaxial deflections of the inner and outer tubes are more significant for small-diameter than large-diameter double-wall carbon nanotubes. This suggests that the Timoshenko-beam model, rather than the Euler-beam model, is relevant for terahertz vibration of short carbon nanotubes.
Statistical Damage Mechanics— Part I: Theory
Authors: Krajcinovic D. , Rinaldi A.
Published: February 2005

Statistical damage mechanics in this work establishes the connection between damaged random heterogeneous micromaterial and the system macroparameter. Renormalization group theory provides the bridge from the microscale to the macroscale. Delaunay lattices, which simulate and capture the role of the disordered microstructure in damage process, substitute a polycrystal specimen assuming that microcracks are grain-boundaries cracks. The macroparameters of the system, in the form of algebraic functions, are obtained applying the Family–Vicsek scaling relation on simulation data.
A Fast Boundary Element Method for the Analysis of Fiber-Reinforced Composites Based on a Rigid-Inclusion Model
Authors: Liu Y. J. , Nishimura N. , Otani Y. , Takahashi T. , Chen X. L. , Munakata H.
Published: February 2005

A new boundary element method (BEM) is developed for three-dimensional analysis of fiber-reinforced composites based on a rigid-inclusion model. Elasticity equations are solved in an elastic domain containing inclusions which can be assumed much stiffer than the host elastic medium. Therefore the inclusions can be treated as rigid ones with only six rigid-body displacements. It is shown that the boundary integral equation (BIE) in this case can be simplified and only the integral with the weakly-singular displacement kernel is present. The BEM accelerated with the fast multipole method is used to solve the established BIE. The developed BEM code is validated with the analytical solution for a rigid sphere in an infinite elastic domain and excellent agreement is achieved. Numerical examples of fiber-reinforced composites, with the number of fibers considered reaching above 5800 and total degrees of freedom above 10 millions, are solved successfully by the developed BEM. Effective Young’s moduli of fiber-reinforced composites are evaluated for uniformly and “randomly” distributed fibers with two different aspect ratios and volume fractions. The developed fast multipole BEM is demonstrated to be very promising for large-scale analysis of fiber-reinforced composites, when the fibers can be assumed rigid relative to the matrix materials.
Analysis of the Bridgman Procedure to Characterize the Mechanical Behavior of Materials in the Tensile Test: Experiments and Simulation
Authors: Celentano Diego J. , Cabezas and Eduardo E. , Garcı´a Claudio M.
Published: February 2005

This note presents an experimental analysis and a numerical simulation of the mechanical behavior experienced by cylindrical specimens of pure copper during the tensile test. A set of experiments has been carried out in order to derive the hardening parameters that characterize the material response. The simulation of the deformation process during the whole test is performed with a finite element large strain elastoplasticity-based formulation. The results of the simulation show that the mechanical characterization involving the classical Bridgman correction factor, defined in terms of logarithmic strains and aimed at predicting the stress distribution at the necking zone, cannot properly describe the hardening response for this material. Therefore, the use of a different correction factor, which consequently leads to another set of hardening parameters, is proposed. Finally, an adequate experimental validation of the numerical results is obtained for this last case.
Exact Critical Loads for a Pinned Half-Sine Arch Under End Couples
Authors: Chen Jen-San , Lin Jian-San
Published: February 2005

In this note we show that for a pinned half-sine arch under end couples snap-through buckling will occur unsymmetrically if the initial height of the shallow arch is greater than 6.5466r, where r is the radius of gyration of the cross section. The closed-form expression for the critical couple can be obtained analytically.
A Plane Stress Perfectly Plastic Mode I Crack Solution With Continuous Stress Field
Authors: Unger David J.
Published: February 2005

A statically admissible solution for a perfectly plastic material in plane stress is presented for the mode I crack problem. The yield condition employed is an alternative type first proposed by von Mises in order to approximate his original yield condition for plane stress while eliminating most of the elliptic region as pertaining to partial differential equations. This yield condition is composed of two intersecting parabolas rather than a single ellipse in the principal stress space. The attributes of this particular solution of the mode I problem over that previously obtained are that it contains neither stress discontinuities nor compressive stresses anywhere in the field.
The High-Affinity Maltose/Trehalose ABC Transporter in the Extremely Thermophilic Bacterium
Thermus thermophilus
HB27 Also Recognizes Sucrose and Palatinose
Authors: Silva Zélia , Sampaio Maria-Manuel , Henne Anke , Böhm Alex , Gutzat Ruben , Boos Winfried , da Costa Milton S. , Santos Helena
Published: February 2005

We have studied the transport of trehalose and maltose in the thernophilic bacterium
Thermus thermophilus
HB27, which grows optimally in the range of 70 to 75°C. The

K
m

values at 70°C were 109 nM for trehalose and 114 nM for maltose; also, a high

K
m

(424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while
d
-galactose,
d
-fucose,
l
-rhamnose,
l
-arabinose, and
d
-mannose were not competitive inhibitors. In the recently published genome of
T. thermophilus
HB27, two gene clusters designated
malEFG
1 (TTC1627 to -1629) and
malEFG
2 (TTC1288 to -1286) and two monocistronic genes designated
malK1
(TTC0211) and
malK2
(TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the
malE1
and
malE2
genes, lacking the sequence encoding the signal peptides, were expressed in
Escherichia coli
. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (

K
d

values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either
malF1
or
malK1
were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in
malK2
or
malF2
showed no growth defect on any of these sugars. Therefore,
malEFG1
encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and
malK1
encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90°C for MalE1 and 105°C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that
malEFG1
and
malK1
constitute the high-affinity ABC transport system of
T. thermophilus
HB27 for trehalose, maltose, sucrose, and palatinose.
A Protein Secretion Pathway Critical for
Mycobacterium tuberculosis
Virulence Is Conserved and Functional in
Mycobacterium smegmatis
Authors: Converse Scott E. , Cox Jeffery S.
Published: February 2005

The Snm protein secretion system is a critical determinant of
Mycobacterium tuberculosis
virulence. However, genes encoding components of this pathway are conserved among all mycobacteria, including the nonpathogenic saprophyte
Mycobacterium smegmatis
. We show that the Snm system is operational in
M. smegmatis
and that secretion of its homologous ESAT-6 and CFP-10 substrates is regulated by growth conditions. Importantly, we show that Snm secretion in
M. smegmatis
requires genes that are homologous to those required for secretion in
M. tuberculosis
. Using a gene knockout strategy in
M. smegmatis
, we have also discovered four new gene products that are essential for Snm secretion, including the serine protease mycosin 1. Despite the evolutionary distance between
M. smegmatis
and
M. tuberculosis
, the
M. smegmatis
Snm system can secrete the
M. tuberculosis
ESAT-6 and CFP-10 proteins, suggesting that substrate recognition is also conserved between the two species.
M. smegmatis
, therefore, represents a powerful system to study the multicomponent Snm secretory machine and to understand the role of this conserved system in mycobacterial biology.
AU-Rich Sequences within 5′ Untranslated Leaders Enhance Translation and Stabilize mRNA in
Escherichia coli
Authors: Komarova Anastassia V. , Tchufistova Ludmila S. , Dreyfus Marc , Boni Irina V.
Published: February 2005

We have shown previously that when the
Escherichia coli
chromosomal
lacZ
gene is put under the control of an extended Shine-Dalgarno (SD) sequence (10 or 6 nucleotides in length), the translation efficiency can be highly variable, depending on the presence of AU-rich targets for ribosomal protein S1 in the mRNA leader. Here, the same strains have been used to examine the question of how strong ribosome binding to extended SD sequences affects the stability of
lacZ
mRNAs translated with different efficiencies. The steady-state concentration of the
lacZ
transcripts has been found to vary over a broad range, directly correlating with translation efficiency but not with the SD duplex stability. The observed strain-to-strain variations in
lacZ
mRNA level became far less marked in the presence of the
rne-1
mutation, which partially inactivates RNase E. Together, the results show that (i) an SD sequence, even one that is very long, cannot stabilize the
lacZ
mRNA in
E. coli
if translation is inefficient; (ii) inefficiently translated
lacZ
transcripts are sensitive to RNase E; and (iii) AU-rich elements inserted upstream of a long SD sequence enhance translation and stabilize mRNA, despite the fact that they constitute potential RNase E sites. These data strongly support the idea that the
lacZ
mRNA in
E. coli
can be stabilized only by translating, and not by stalling, ribosomes.
The Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase from
Acinetobacter
sp. Strain ADP1: Characterization of a Novel Type of Acyltransferase
Authors: Stöveken Tim , Kalscheuer Rainer , Malkus Ursula , Reichelt Rudolf , Steinbüchel Alexander
Published: February 2005

The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium
Acinetobacter
sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant
Escherichia coli
Rosetta (DE3)pLysS (pET23a::
atfA
). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol acyltransferase (MGAT) activity. The
sn
-1 and
sn
-3 positions of acylglycerols are accepted with higher specificity than the
sn
-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from
Acinetobacter
sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria.
Protein Isoaspartate Methyltransferase Is a Multicopy Suppressor of Protein Aggregation in
Escherichia coli
Authors: Kern Renée , Malki Abderrahim , Abdallah Jad , Liebart Jean-Claude , Dubucs Catherine , Yu Myeong Hee , Richarme Gilbert
Published: February 2005

We used preS2-S′-β-galactosidase, a three-domain fusion protein that aggregates extensively at 43°C in the cytoplasm of
Escherichia coli
, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S′-β-galactosidase at 37 and 43°C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43°C. First, we demonstrate that the differential solubility of preS2-S′-β-galactosidase results in a lactose-positive phenotype at 37°C as opposed to a lactose-negative phenotype at 43°C. We searched for multicopy suppressors of preS2-S′-β-galactosidase aggregation by selecting pink lactose-positive colonies on a background of white lactose-negative colonies at 43°C after transformation of bacteria with an
E. coli
gene bank. We discovered that protein isoaspartate methyltransferase (PIMT) is a multicopy suppressor of preS2-S′-β-galactosidase aggregation at 43°C. Overexpression of PIMT reduces the amount of preS2-S′-β-galactosidase found in inclusion bodies at 43°C and increases its amount in soluble fractions. It reduces the level of isoaspartate formation in preS2-S′-β-galactosidase and increases its thermal stability in
E. coli
crude extracts without increasing the thermostability of a control protein, citrate synthase, in the same extracts. We could not detect any induction of the heat shock response resulting from PIMT overexpression, as judged from amounts of DnaK and GroEL, which were similar in the PIMT-overproducing and control strains. These results suggest that PIMT might be overburdened in some physiological conditions and that its overproduction may be beneficial in conditions in which protein aggregation occurs, for example, during biotechnological protein overproduction or in protein aggregation diseases.
The Flag-2 Locus, an Ancestral Gene Cluster, Is Potentially Associated with a Novel Flagellar System from
Escherichia coli
Authors: Ren Chuan-Peng , Beatson Scott A. , Parkhill Julian , Pallen Mark J.
Published: February 2005

Escherichia coli
K-12 possesses two adjacent, divergent, promoterless flagellar genes,
fhiA
-
mbhA
, that are absent from
Salmonella enterica
. Through bioinformatics analysis, we found that these genes are remnants of an ancestral 44-gene cluster and are capable of encoding a novel flagellar system, Flag-2. In enteroaggregative
E. coli
strain 042, there is a frameshift in
lfgC
that is likely to have inactivated the system in this strain. Tiling path PCR studies showed that the Flag-2 cluster is present in 15 of 72 of the well-characterized ECOR strains. The Flag-2 system resembles the lateral flagellar systems of
Aeromonas
and
Vibrio
, particularly in its apparent dependence on RpoN. Unlike the conventional Flag-1 flagellin, the Flag-2 flagellin shows a remarkable lack of sequence polymorphism. The Flag-2 gene cluster encodes a flagellar type III secretion system (including a dedicated flagellar sigma-antisigma combination), thus raising the number of distinct type III secretion systems in
Escherichia
/
Shigella
to five. The presence of the Flag-2 cluster at identical sites in
E. coli
and its close relative
Citrobacter rodentium
, combined with its absence from
S. enterica
, suggests that it was acquired by horizontal gene transfer after the former two species diverged from
Salmonella
. The presence of Flag-2-like gene clusters in
Yersinia pestis
,
Yersinia pseudotuberculosis
, and
Chromobacterium violaceum
suggests that coexistence of two flagellar systems within the same species is more common than previously suspected. The fact that the Flag-2 gene cluster was not discovered in the first 10
Escherichia
/
Shigella
genome sequences studied emphasizes the importance of maintaining an energetic program of genome sequencing for this important taxonomic group.
DNA Binding: a Novel Function of
Pseudomonas aeruginosa
Type IV Pili
Authors: van Schaik Erin J. , Giltner Carmen L. , Audette Gerald F. , Keizer David W. , Bautista Daisy L. , Slupsky Carolyn M. , Sykes Brian D. , Irvin Randall T.
Published: February 2005

The opportunistic pathogen
Pseudomonas aeruginosa
produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from
P. aeruginosa
strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of
P. aeruginosa
type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.
The Hfq Homolog inLegionella pneumophilaDemonstrates Regulation by LetA and RpoS and Interacts with the Global Regulator CsrA
Authors: McNealy Tamara L , Forsbach-Birk Vera , Shi Chunwei , Marre Reinhard
Published: February 2005

A gene inLegionella pneumophilathat has significant homology to publishedhfqgenes demonstrated regulation by RpoS and the transcriptional regulator LetA. Additionally, Hfq has a positive effect on the presence of transcripts of the genes for CsrA and the ferric uptake regulator Fur. Mutants lackinghfqdemonstrate defects in growth and pigmentation and slight defects in virulence in both amoeba and macrophage infection models. Hfq appears to play a major role in exponential-phase regulatory cascades ofL. pneumophila.
COMPARATIVE STRUCTURAL INVESTIGATION OF xFe2O3·(100-x)[3B2O3·MO] (MO⇒KClorCaCl2) GLASS SYSTEMS BY RAMAN SPECTROSCOPY
Authors: PǍŞCUŢǍ P. , ARDELEAN I.
Published: February 2005

Two vitreous systems, x Fe 2 O 3·(100-x)[3 B 2 O 3· MO ] ( MO ⇒ KCl or CaCl 2) with 0≤x≤5 mol %, were characterized by Raman spectroscopy in attempt to determine the local structure of glasses. The mode of which glass structure is influenced by Fe 2 O 3 and CaCl 2-replacement of KCl , is established. The results show that BO 3 and BO 4 are the main structural units of the glass systems.
ANALYTICAL SOLUTIONS OF ARBITRARY ORDERS TO THE CLASSICAL AND QUANTUM OSCILLATORS WITH VELOCITY-DEPENDENT QUARTIC ANHARMONICITIES
Authors: MANDAL SWAPAN
Published: February 2005

The classical oscillator with velocity-dependent anharmonicity (COVDA) arises when the velocity of the oscillator is reasonably high. By using an intuitive approach, we obtain an approximate analytical solution of arbitrary order to the problem of a COVDA. In addition to the third harmonic generation manifested by the nonlinear interaction, it is found that the solution contains the secular terms since the intuitive approach basically depends on the perturbation method. By assuming the small anharmonic constant, the secular terms are summed up for all orders and we obtain the renormalization of the frequency. The frequency of the oscillator decreases with the increase of the anharmonic constant. Interestingly, the magnitude of the shifts of the frequency of the oscillator with velocity-dependent quartic anharmonicity is identical with those of the oscillator with q-dependent quartic anharmonicity. However, the sign of the shifts for those two types of anharmonic oscillator is opposite in nature. These results indicate that the frequency shifts of the oscillator are actually the resultant effects (shifts) due to the q-dependent and the velocity-dependent anharmonicities. Finally, with the help of the correspondence principle, the solution of a quantum oscillator with velocity-dependent quartic anharmonicity (QOVDA) is obtained from the knowledge of the solution of its classical counterpart.
CMOS INTEGRATED CIRCUIT CONTROLLERS FOR SWITCHING POWER CONVERTERS
Authors: ALARCÓN EDUARD , VILLAR GERARD , POVEDA ALBERTO
Published: February 2005

Two case examples of high-speed CMOS microelectronic implementations of high-performance controllers for switching power converters are presented. The design and implementation of a current-programmed controller and a general-purpose feedforward one-cycle controller are described. The integrated circuit controllers attain high-performance by means of using current-mode analog signal processing, hence allowing high switching frequencies that extend the operation margin compared to previous designs. Global layout-extracted transistor-level simulation results for 0.8 μm and 0.35 μm standard CMOS technologies confirm both the correct operation of the circuits in terms of bandwidth as well as their functionality for the control of switching power converters. The circuits may be used either as standalone IC controllers or as controller circuits that are technology-compatible with on-chip switching power converters and on-chip loads for future powered systems-on-chip.
CRITICAL CHOICES IN A SYSTEM FOR OPTIMIZED DESIGN OF ARBITRARY WAVEFORM TRANSFORMERS
Authors: BRESLIN J. G. , HURLEY W. G.
Published: February 2005

Traditionally, magnetic component design has been based on power frequency transformers with sinusoidal excitation. However, the movement towards higher density integrated circuits means that reductions in the size of magnetic components must be achieved by operating at higher frequencies, mainly through nonsinusoidal switching circuits. As this trend continues, computing tools are required to carry out designs of magnetic components that also allow evaluation of the high frequency losses in these components. A computer design package is described here that implements a robust transformer design methodology allowing customizable transformer geometries. The concept of a critical frequency is a vital part of this methodology. In addition, the winding choice at high frequencies is optimized to give the most accurate results for the best possible speed. This paper includes a description of the software design processes used and describes the main aspects that were incorporated into the system.
Single-Channel Properties Support a Potential Contribution of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels and
I
f
to Cardiac Arrhythmias
Authors: Michels Guido , Er Fikret , Khan Ismail , Südkamp Michael , Herzig Stefan , Hoppe Uta C.
Published: February 2005

Background—

The pacemaker current
I
f
is present in atrial and ventricular myocytes. However, it remains controversial whether
I
f
overexpression in diseased states might play a role for arrhythmogenesis, because first
I
f
activation in whole-cell recordings hardly overlapped the diastolic voltage of working myocardium.

Methods and Results—

To obtain further insight into
I
HCN
and
I
f
properties, we provide for the first time detailed single-channel analysis of heterologously expressed hyperpolarization-activated cyclic nucleotide-gated (HCN) isoforms and native human
I
f
. HCN subtypes differed significantly in single-channel amplitude, conductance, and activation kinetics. Interestingly, threshold potentials of HCN isoforms were more positive than would have been expected from whole-cell measurements. Single-channel properties of cells cotransfected with HCN2 and HCN4 were distinct from cells expressing HCN2 or HCN4 alone, demonstrating that different HCN isoforms can influence current properties of a single HCN channel complex, thus providing direct functional evidence for HCN heteromerization. Pooled data of homomeric and heteromeric HCN channels and of native
I
f
extrapolated from maximum likelihood fits indicated a multistate gating scheme comprising 5 closed- and 4 open-channel states. Single-channel characteristics of
I
f
in human atrial myocytes closely resembled those of HCN4 or HCN2+HCN4, supporting the hypothesis that native
I
f
channels in atrial myocardium are heteromeric complexes composed of HCN4 and/or HCN2. Most interestingly, half-maximal activation of single-channel atrial
I
f
(−68.3±4.9 mV; k=−9.9±1.5; n=8) was well within the diastolic voltage range of human atrial myocardium.

Conclusions—

These observations support a potential contribution of HCN/
I
f
to the arrhythmogenesis of working myocardium under pathological conditions.
Mineralocorticoid Receptor Inhibition Ameliorates the Transition to Myocardial Failure and Decreases Oxidative Stress and Inflammation in Mice With Chronic Pressure Overload
Authors: Kuster Gabriela M. , Kotlyar Eugene , Rude Mary K. , Siwik Deborah A. , Liao Ronglih , Colucci Wilson S. , Sam Flora
Published: February 2005

Background—

Although aldosterone, acting via mineralocorticoid receptors, causes left ventricular (LV) hypertrophy in experimental models of high-aldosterone hypertension, little is known about the role of aldosterone or mineralocorticoid receptors in mediating adverse remodeling in response to chronic pressure overload.

Methods and Results—

We used the mineralocorticoid receptor–selective antagonist eplerenone (EPL) to test the role of mineralocorticoid receptors in mediating the transition from hypertrophy to failure in mice with chronic pressure overload caused by ascending aortic constriction (AAC). One week after AAC, mice were randomized to regular chow or chow containing EPL (200 mg/kg per day) for an additional 7 weeks. EPL had no significant effect on systolic blood pressure after AAC. Eight weeks after AAC, EPL treatment improved survival (94% versus 65%), attenuated the increases in LV end-diastolic (3.4±0.1 versus 3.7±0.1 mm) and end-systolic (2.0±0.1 versus 2.5±0.2 mm) dimensions, and ameliorated the decrease in fractional shortening (42±2% versus 34±4%). EPL also decreased myocardial fibrosis, myocyte apoptosis, and the ratio of matrix metalloproteinase-2/tissue inhibitor of matrix metalloproteinase-2. These beneficial effects of EPL were associated with less myocardial oxidative stress, as assessed by 3-nitrotyrosine staining, reduced expression of the adhesion molecule intercellular adhesion molecule-1, and reduced infiltration by macrophages.

Conclusions—

Mineralocorticoid receptors play an important role in mediating the transition from LV hypertrophy to failure with chronic pressure overload. The effects of mineralocorticoid receptor stimulation are associated with alterations in the interstitial matrix and myocyte apoptosis and may be mediated, at least in part, by oxidative stress and inflammation.