bluuebunny/crossref_metadata_2025 huggingface dataset:
Trimetazidine Reduces Basal Cytosolic Ca2+ Concentration During Hypoxia in Single Xenopus Skeletal Myocytes
Authors: Stary C. M. , Kohin S. , Samaja M. , Howlett R. A. , Hogan M. C.
Published: July 2003

We tested the hypotheses that: (1) Ca2+ handling and force production would be irreversibly altered in skeletal muscle during steady‐state contractions when subjected to severe, prolonged hypoxia and subsequent reoxygenation; and (2) application of the cardio‐protective drug trimetazidine would attenuate these alterations. Single, living skeletal muscle fibres from Xenopus laevis were injected with the Ca2+ indicator fura 2, and incubated for 1 h prior to stimulation in 100 μM TMZ‐Ringer solution (TMZ; n = 6) or standard Ringer solution (CON; n = 6). Force and relative free cytosolic Ca2+ concentration ([Ca2+]c) were measured during continuous tetanic contractions produced every 5 s as fibres were sequentially perfused in the following manner: 3 min high extracellular PO2 (159 mmHg), 15 min hypoxic perfusion (3‐5 mmHg) then 3 min high PO2. Hypoxia caused a decrease in force and peak [Ca2+]c in both the TMZ and CON fibres, with no significant (P < 0.05) difference between groups. However, basal [Ca2+]c was significantly lower during hypoxia in the TMZ group vs. the CON group. While reoxygenation generated only modest recovery of relative force and peak [Ca2+]c in both groups, basal [Ca2+]c remained significantly less in the TMZ group. These results demonstrated that in contracting, single skeletal muscle fibres, TMZ prevented increases in basal [Ca2+]c generated during a severe hypoxic insult and subsequent reoxygenation, yet failed to protect the cell from the deleterious effects of prolonged hypoxia followed by reoxygenation.
Metabolic Heat Production, Heat Loss and the Circadian Rhythm of Body Temperature in the Rat
Authors: Refinetti Roberto
Published: July 2003

Metabolic heat production (calculated from oxygen consumption), dry heat loss (measured in a calorimeter) and body temperature (measured by telemetry) were recorded simultaneously at 6 min intervals over five consecutive days in rats maintained in constant darkness. Robust circadian rhythmicity (confirmed by chi square periodogram analysis) was observed in all three variables. The rhythm of heat production was phase‐advanced by about half an hour in relation to the body temperature rhythm, whereas the rhythm of heat loss was phase‐delayed by about half an hour. The balance of heat production and heat loss exhibited a daily oscillation 180 deg out of phase with the oscillation in body temperature. Computations indicated that the amount of heat associated with the generation of the body temperature rhythm (1.6 kJ) corresponds to less than 1% of the total daily energy budget (172 kJ) in this species. Because of the small magnitude of the fraction of heat balance associated with the body temperature rhythm, it is likely that the daily oscillation in heat balance has a very slow effect on body temperature, thus accounting for the 180 deg phase difference between the rhythms of heat balance and body temperature.
Nitric Oxide‐Dependent in vitro Secretion of Amylase from Innervated or Chronically Denervated Parotid Glands of the Rat in Response to Isoprenaline and Vasoactive Intestinal Peptide
Authors: Sayardoust Shariel , Ekström J.
Published: July 2003

The basal in vitro release of amylase was similar from rat parotid lobules of innervated and chronically denervated glands and was unaffected by the inhibitors used in this study. The secretion of amylase induced by isoprenaline or vasoactive intestinal peptide (VIP) was reduced by one‐third to one‐half from the lobules of the innervated glands and even more so from the lobules of the denervated glands by ODQ, an inhibitor of soluble guanyl cyclase which is activated by nitric oxide (NO) and catalyses the cGMP production. The use of N ω‐propyl‐L‐arginine (N‐PLA) revealed that the evoked secretion of amylase in the denervated glands depended on the activity of neuronal type NO synthase to synthesize NO. Since the denervated gland is virtually devoid of NO synthase‐containing nerve fibres, the neuronal type NO synthase was most probably of a non‐neuronal source. NO‐dependent amylase secretion was agonist related, since amylase secretion evoked by bethanechol and neuropeptide Y was not reduced by ODQ or N‐PLA. Hence, under physiological conditions, activation of β‐adrenoceptors (sympathetic activity) and VIP receptors (parasympathetic activity) is likely to cause secretion of parotid amylase partly through a NO/cGMP‐dependent intracellular pathway involving the activity of neuronal type NO synthase, possibly of acinar origin.
Axial Rotation of the Lower Human Spine by Rhythmic Torques Automatically Generated at the Resonant Frequency
Authors: Walsh E. Geoffrey
Published: July 2003

Subjects sat on a ‘Balans’ chair supported by the shaft of a large torque generator. The lower but not the upper part of the body was free to make to/fro movements maintained by feeding back a modified velocity signal to the generator. The stiffness could be increased by the use of a position signal. Using the equations for a torsion pendulum the good linearity observed in relationship to the applied forces allowed stiffness, inertia, damping, critical damping and the damping factor to be determined in absolute terms. The method enables parameters relevant to back function and problems to be evaluated. Eleven adults acted as subjects.
Role of GABA and NO in the Paraventricular Nucleus‐Mediated Reflex Inhibition of Renal Sympathetic Nerve Activity Following Stimulation of Right Atrial Receptors in the Rat
Authors: Yang Zhuo , Coote John H.
Published: July 2003

The aim of this study was to determine the site within the brain at which inhibition of renal sympathetic nerve activity (RSNA) occurs following right atrial receptor stimulation. The atrial receptors were stimulated by inflating a balloon at the right vena cava‐atrium junction and the reflex effect was observed before and during application of neurotransmitter agonists and antagonists into the paraventricular nucleus (PVN), or intrathecally to the spinal cord. Balloon inflation reduced RSNA by 29.1 ± 3% without changing blood pressure in anaesthetised Wistar rats. Microinjection of the GABAA receptor antagonist bicuculline (0.025 mM, 100 nl) into the PVN increased RSNA by 42.3 ± 5% and this was changed little by balloon inflation when PVN increased RSNA by 50.6 ± 6.3%. Microinjection of the nitric oxide synthase (NOS) inhibitors L‐NAME (0.1 mM, 100 nl) or L‐NMMA (0.2 mM, 100 nl) into PVN elicited increases in RSNA of 36 ± 8% or 54 ± 10%, respectively. Balloon inflation during PVN stimulation plus NOS inhibition resulted in RSNA activity of 8 ± 4% or ‐1 ± 1%, respectively, compared to baseline control. Baseline RSNA was similar throughout this series of tests ranging from 9.1 ± 1.3 to 11.5 ± 1.1 spike counts s−1. To rule out the possibility that the atrial reflex inhibition was in part dependent on a dopamine‐mediated PVN‐spinal projection pathway inhibiting RSNA at a spinal locus, a dopamine D1 receptor antagonist SCH 23390 was intrathecally applied to the spinal cord. The effect of subsequent balloon inflation on RSNA was not significantly reduced. It was concluded that atrial receptor activation causes an inhibition of RSNA at the PVN and that this effect is mediated by GABA.
Chill Induction of the SigB-Dependent General Stress Response inBacillus subtilisand Its Contribution to Low-Temperature Adaptation
Authors: Brigulla Matthias , Hoffmann Tamara , Krisp Andrea , Völker Andrea , Bremer Erhard , Völker Uwe
Published: July 2003

A variety of environmental and metabolic cues trigger the transient activation of the alternative transcription factor SigB ofBacillus subtilis, which subsequently leads to the induction of more than 150 general stress genes. This general stress regulon provides nongrowing and nonsporulated cells with a multiple, nonspecific, and preemptive stress resistance. By a proteome approach we have detected the expression of the SigB regulon during continuous growth at low temperature (15°C). Using a combination of Western blot analysis and SigB-dependent reporter gene fusions, we provide evidence for high-level and persistent induction of thesigBoperon and the SigB regulon, respectively, in cells continuously exposed to low temperatures. In contrast to all SigB-activating stimuli described thus far, induction by low temperatures does not depend on the positive regulatory protein RsbV or its regulatory phosphatases RsbU and RsbP, indicating the presence of an entirely new pathway for the activation of SigB by chill stress inB. subtilis. The physiological importance of the induction of the general stress response for the adaptation ofB. subtilisto low temperatures is emphasized by the observation that growth of asigBmutant is drastically impaired at 15°C. Inclusion of the compatible solute glycine betaine in the growth medium not only improved the growth of the wild-type strain but rescued the growth defect of thesigBmutant, indicating that the induction of the general stress regulon and the accumulation of glycine betaine are independent means by whichB. subtiliscells cope with chill stress.
Membrane Binding by MinD Involves Insertion of Hydrophobic Residues within the C-Terminal Amphipathic Helix into the Bilayer
Authors: Zhou Huaijin , Lutkenhaus Joe
Published: July 2003

MinD binds to phospholipid vesicles in the presence of ATP and is released by MinE, which stimulates the MinD ATPase. Membrane binding requires a short conserved C-terminal region, which has the potential to form an amphipathic helix. This finding has led to a model in which the binding of ATP regulates the formation or accessibility of this helix, which then embeds in the membrane bilayer. To test this model, we replaced each of the four hydrophobic residues within this potential helix with tryptophan or a charged residue. Introduction of a negatively charged amino acid decreased membrane binding of MinD and its ability to activate MinC. In contrast, mutants with tryptophan substitutions retained the ability to bind to the membrane and activate MinC. Fluorescence emission spectroscopy analysis of the tryptophan mutants F263W, L264W, and L267W confirmed that these tryptophan residues did insert into the hydrophobic interior of the bilayer. We conclude that membrane binding by MinD involves penetration of the hydrophobic residues within the C-terminal amphipathic helix into the hydrophobic interior of the bilayer.
Molecular Characterization and Quantitative Analysis of Superoxide Dismutases in Virulent and Avirulent Strains of
Aeromonas salmonicida
subsp.
salmonicida
Authors: Dacanay A. , Johnson S. C. , Bjornsdottir R. , Ebanks R. O. , Ross N. W. , Reith M. , Singh R. K. , Hiu J. , Brown L. L.
Published: July 2003

Aeromonas salmonicida
subsp.
salmonicida
is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which
A
.
salmonicida
evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs),
sodA
and
sodB
. The
sodA
gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The
sodB
gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O
2
−
-catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative
sodA
were consistent with these differences. We could not identify other SOD isozymes such as
sodC
either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of
A
.
salmonicida
subsp.
salmonicida
strain A449 when cultured in vitro and in vivo.
Gene Expression Analysis of Energy Metabolism Mutants of
Desulfovibrio vulgaris
Hildenborough Indicates an Important Role for Alcohol Dehydrogenase
Authors: Haveman Shelley A. , Brunelle Véronique , Voordouw Johanna K. , Voordouw Gerrit , Heidelberg John F. , Rabus Ralf
Published: July 2003

Comparison of the proteomes of the wild-type and Fe-only hydrogenase mutant strains of
Desulfovibrio vulgaris
Hildenborough, grown in lactate-sulfate (LS) medium, indicated the near absence of open reading frame 2977 (ORF2977)-coded alcohol dehydrogenase in the
hyd
mutant. Hybridization of labeled cDNA to a macroarray of 145 PCR-amplified
D. vulgaris
genes encoding proteins active in energy metabolism indicated that the
adh
gene was among the most highly expressed in wild-type cells grown in LS medium. Relative to the wild type, expression of the
adh
gene was strongly downregulated in the
hyd
mutant, in agreement with the proteomic data. Expression was upregulated in ethanol-grown wild-type cells. An
adh
mutant was constructed and found to be incapable of growth in media in which ethanol was both the carbon source and electron donor for sulfate reduction or was only the carbon source, with hydrogen serving as electron donor. The
hyd
mutant also grew poorly on ethanol, in agreement with its low level of
adh
gene expression. The
adh
mutant grew to a lower final cell density on LS medium than the wild type. These results, as well as the high level of expression of
adh
in wild-type cells on media in which lactate, pyruvate, formate, or hydrogen served as the sole electron donor for sulfate reduction, indicate that ORF2977 Adh contributes to the energy metabolism of
D. vulgaris
under a wide variety of metabolic conditions. A hydrogen cycling mechanism is proposed in which protons and electrons originating from cytoplasmic ethanol oxidation by ORF2977 Adh are converted to hydrogen or hydrogen equivalents, possibly by a putative H
2
-heterodisulfide oxidoreductase complex, which is then oxidized by periplasmic Fe-only hydrogenase to generate a proton gradient.
Molecular Characterization and Expression in
Escherichia coli
of Three β-1,3-Glucanase Genes from
Lysobacter enzymogenes
Strain N4-7
Authors: Palumbo Jeffrey D. , Sullivan Raymond F. , Kobayashi Donald Y.
Published: July 2003

Lysobacter enzymogenes
strain N4-7 produces multiple biochemically distinct extracellular β-1,3-glucanase activities. The
gluA
,
gluB
, and
gluC
genes, encoding enzymes with β-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of β-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of
gluA
and
gluC
gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the
gluC
product. Analysis of the
gluB
gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in
Escherichia coli
resulted in the production of proteins with β-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45°C and that GluB is most active between pH 4.5 and 5.0 at 41°C. Activity of recombinant proteins against various β-1,3 glucan substrates indicates that GluA and GluC are most active against linear β-1,3 glucans, while GluB is most active against the insoluble β-1,3 glucan substrate zymosan A. These data suggest that the contribution of β-1,3-glucanases to the biocontrol activity of
L. enzymogenes
may be due to complementary activities of these enzymes in the hydrolysis of β-1,3 glucans from fungal cell walls.
TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)
Authors: Schröder Gunnar , Lanka Erich
Published: July 2003

TraG-like proteins are essential components of type IV secretion systems. During secretion, TraG is thought to translocate defined substrates through the inner cell membrane. The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity. TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA. TrwB (R388) is a hexameric TraG-like protein that binds ATP. Both proteins, however, lack NTPase activity under in vitro conditions. We characterized derivatives of TraG and TrwB truncated by the N-terminal membrane anchor (TraGΔ2 and TrwBΔ1) and/or containing a point mutation at the putative nucleotide-binding site (TraGΔ2K187T and TraGK187T). Unlike TraG and TrwB, truncated derivatives behaved as monomers without the tendency to form oligomers or aggregates. Surface plasmon resonance analysis with immobilized relaxase showed that mutant TraGK187T was as good a binding partner as the wild-type protein, whereas truncated TraG monomers were unable to bind relaxase. TraGΔ2 and TrwBΔ1 bound ATP and, with similar affinity, ADP. Binding of ATP and ADP was strongly inhibited by the presence of Mg2+or single-stranded DNA and was competed for by other nucleotides. Compared to the activity of TraGΔ2, the ATP- and ADP-binding activity of the point mutation derivative TraGΔ2K187T was significantly reduced. Each TraG derivative bound DNA with an affinity similar to that of the native protein. DNA binding was inhibited or competed for by ATP, ADP, and, most prominently, Mg2+. Thus, both nucleotide binding and DNA binding were sensitive to Mg2+and were competitive with respect to each other.
YjdE (AdiC) Is the Arginine:Agmatine Antiporter Essential for Arginine-Dependent Acid Resistance in
Escherichia coli
Authors: Gong Shimei , Richard Hope , Foster John W.
Published: July 2003

To survive in extremely acidic conditions,
Escherichia coli
has evolved three adaptive acid resistance strategies thought to maintain internal pH. While the mechanism behind acid resistance system 1 remains enigmatic, systems 2 and 3 are known to require external glutamate (system 2) and arginine (system 3) to function. These latter systems employ specific amino acid decarboxylases and putative antiporters that exchange the extracellular amino acid substrate for the intracellular by-product of decarboxylation. Although GadC is the predicted antiporter for system 2, the antiporter specific for arginine/agmatine exchange has not been identified. A computer-based homology search revealed that the
yjdE
(now called
adiC
) gene product shared an overall amino acid identity of 22% with GadC. A series of
adiC
mutants isolated by random mutagenesis and by targeted deletion were shown to be defective in arginine-dependent acid resistance. This defect was restored upon introduction of an

adiC
+

-containing plasmid. An
adiC
mutant proved incapable of exchanging extracellular arginine for intracellular agmatine but maintained wild-type levels of arginine decarboxylase protein and activity. Western blot analysis indicated AdiC is an integral membrane protein. These data indicate that the arginine-to-agmatine conversion defect of
adiC
mutants was at the level of transport. The
adi
gene region was shown to be organized into two transcriptional units,
adiAY
and
adiC
, which are coordinately regulated but independently transcribed. The data also illustrate that the AdiA decarboxylase:AdiC antiporter system is designed to function only at acid levels sufficient to harm the cell.
Characterization of
Staphylococcus aureus
SarA Binding Sites
Authors: Sterba Kristen M. , Mackintosh Samuel G. , Blevins Jon S. , Hurlburt Barry K. , Smeltzer Mark S.
Published: July 2003

The staphylococcal accessory regulator locus (
sarA
) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and
cis
elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene
cna
. The results suggest the existence of multiple high-affinity binding sites within the
cna
promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including
cna
,
fnbA
, and
sspA
. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the
Staphylococcus aureus
genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the
S. aureus
genes
spa
,
fnbA
,
sspA
,
agr
,
hla
, and
cna
. With the exception of
cna
, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.
The Core Dimerization Domains of Histidine Kinases Contain Recognition Specificity for the Cognate Response Regulator
Authors: Ohta Noriko , Newton Austin
Published: July 2003

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the
Caulobacter crescentus
cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.
Genetic Variation at thevlsELocus ofBorrelia burgdorferiwithin Ticks and Mice over the Course of a Single Transmission Cycle
Authors: Ohnishi Jun , Schneider Brad , Messer William B. , Piesman Joseph , de Silva Aravinda M.
Published: July 2003

The Lyme disease spirochete,Borrelia burgdorferi, causes a persistent infection in the vertebrate host even though infected animals mount an active immune response against the spirochete. One strategy used by the spirochete to evade vertebrate host immunity is to vary the structure and expression of outer membrane antigens. ThevlsElocus represents the best-studied example of antigenic variation inB. burgdorferi. During vertebrate host infection, recombination between the activevlsElocus and silent, partialvlsEcopies leads to gene conversion events and the generation of novel alleles at the expression site. In the present study, we followed a population ofB. burgdorferiorganisms moving through vertebrate host and tick stages to complete one transmission cycle. The major goal of the study was to determine if thevlsElocus was subject to different selective pressure and/or recombination frequency at different stages of the spirochete's life cycle. We report here that thevlsEgenetic diversity generated within the rodent host was maintained through the larval and nymphal tick stages. Therefore, naturally infected ticks are likely to transmit spirochete populations with multiplevlsEalleles into naive vertebrate hosts. AlthoughvlsEgenetic diversity in mice was maintained through tick stages, the dominantvlsEalleles were different between tick stages as well as between individual ticks. We propose that population-level bottlenecks experienced by spirochetes, especially during the larval-to-nymphal molt, are responsible for individual infected ticks harboring different dominantvlsEalleles. AlthoughvlsEgenetic diversity is maintained through tick stages, the VlsE protein is unlikely to be of functional importance in the vector, because the protein was expressed by very few (<1%) bacteria in the vector.
Purification and Biochemical Characterization of the F
1
F
o
-ATP Synthase from Thermoalkaliphilic
Bacillus
sp. Strain TA2.A1
Authors: Cook Gregory M. , Keis Stefanie , Morgan Hugh W. , von Ballmoos Christoph , Matthey Ulrich , Kaim Georg , Dimroth Peter
Published: July 2003

We describe here purification and biochemical characterization of the F
1
F
o
-ATP synthase from the thermoalkaliphilic organism
Bacillus
sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F
1
F
o
-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F
1
subunits α, β, γ, δ, and ε and F
o
subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F
1
F
o
holoenzyme or the isolated F
1
moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F
1
. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide
m
-chlorophenylhydrazone, while the electroneutral Na
+
/H
+
antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na
+
ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic
Bacillus
species. The ATP synthase was specifically modified at its c subunits by
N
,
N
′-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.
Novel Psychrophilic and Thermolabile
l
-Threonine Dehydrogenase from Psychrophilic
Cytophaga
sp. Strain KUC-1
Authors: Kazuoka Takayuki , Takigawa Shouhei , Arakawa Noriaki , Hizukuri Yoshiyuki , Muraoka Ikuo , Oikawa Tadao , Soda Kenji
Published: July 2003

A psychrophilic bacterium,
Cytophaga
sp. strain KUC-1, that abundantly produces a NAD
+
-dependent
l
-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the
Cytophaga
enzyme is distinct from other
l
-threonine dehydrogenases that have thus far been studied.
l
-Threonine and
dl
-
threo
-3-hydroxynorvaline were the substrates, and NAD
+
and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45°C. The kinetic parameters of the enzyme are highly influenced by temperatures. The

K
m

for
l
-threonine was lowest at 20°C. Dead-end inhibition studies with pyruvate and adenosine-5′-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD
+
binds to an enzyme prior to
l
-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into
Escherichia coli
, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from
Staphylococcus aureus
and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast,
l
-threonine dehydrogenase from
E. coli
belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the
Cytophaga
enzyme.
l
-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the
E. coli
and human UDP-glucose 4-epimerases are highly conserved in the
Cytophaga
enzyme, except for the residues participating in the substrate binding.
Essential Nature of the
mreC
Determinant of
Bacillus subtilis
Authors: Lee Joong-Chul , Stewart George C.
Published: July 2003

The
mre
genes of
Escherichia coli
and
Bacillus subtilis
are cell shape determination genes. Mutants affected in
mre
function are spheres instead of the normal rods. Although the
mre
determinants are not required for viability in
E
.
coli
, the
mreB
determinant is an essential gene in
B
.
subtilis
. Conflicting results have been reported as to whether the two membrane-associated proteins MreC and MreD are essential proteins. Furthermore, although the MreB protein has been studied in some detail, the roles of the MreC and MreD proteins in cell shape determination are unknown. We constructed a strain of
B
.
subtilis
in which expression of the
mreC
determinant is dependent upon the addition of isopropyl-β-
d
-thiogalactopyranoside to the culture medium. Utilizing this conditional strain, it was shown that
mreC
is an essential gene in
B
.
subtilis
. Furthermore, it was shown that cells lacking sufficient quantities of MreC undergo morphological changes, namely, swelling and twisting of the cells, which is followed by cell lysis. Electron microscopy was utilized to demonstrate that a polymeric material accumulated at one side of the division septum of the cells and that the presence of this material correlated with the bending of the cell. The best explanation for the results is that the MreC protein is involved in the control of septal versus long-axis peptidoglycan synthesis, that cells lacking MreC perform aberrant septal peptidoglycan synthesis, and that lysis results from a deficiency in long-axis peptidoglycan synthesis.
Lateral Flagellar Gene System of
Vibrio parahaemolyticus
Authors: Stewart Bonnie J. , McCarter Linda L.
Published: July 2003

Vibrio parahaemolyticus
possesses dual flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (i.e., swimming) with a motor that is powered by the sodium motive force. Multiple proton-driven lateral flagella enable translocation over surfaces (i.e., swarming). The polar flagellum is produced continuously, while production of lateral flagella is induced when the organism is grown on surfaces. This work describes the isolation of mutants with insertions in the structural and regulatory
laf
genes. A Tn
5
-based
lux
transcriptional reporter transposon was constructed and used for mutagenesis and subsequent transcriptional analysis of the
laf
regulon. Twenty-nine independent insertions were distributed within 16
laf
genes. DNA sequence analysis identified 38
laf
genes in two loci. Among the mutants isolated, 11 contained surface-induced
lux
fusions. A hierarchy of
laf
gene expression was established following characterization of the
laf
::
lux
transcriptional fusion strains and by mutational and primer extension analyses of the
laf
regulon. The
laf
system is like many enteric systems in that it is a proton-driven, peritrichous flagellar system; however,
laf
regulation was different from the
Salmonella
-
Escherichia coli
paradigm. There is no apparent
flhDC
counterpart that encodes master regulators known to control flagellar biosynthesis and swarming in many enteric bacteria. A potential σ
54
-dependent regulator, LafK, was demonstrated to control expression of early genes, and a lateral-specific σ
28
factor controls late flagellar gene expression. Another notable feature was the discovery of a gene encoding a MotY-like product, which previously had been associated only with the architecture of sodium-type polar flagellar motors.
Phase Variation in the Phage Growth Limitation System ofStreptomyces coelicolorA3(2)
Authors: Sumby Paul , Smith Margaret C. M.
Published: July 2003

The phase-variable phage growth limitation (Pgl) system ofStreptomyces coelicolorA3(2) is an unusual bacteriophage resistance mechanism that confers protection against the temperate phage φC31 and homoimmune relatives. Pgl is subject to phase variation, and data presented here show that this is at least partially due to expansion and contraction of a polyguanine tract present within the putative adenine-specific DNA methyltransferase gene,pglX. Furthermore, thepglXparalogue SC6G9.02, here renamedpglS, was shown to be able to interfere with the Pgl phenotype, suggesting that PglS could provide an alternative activity to that conferred by PglX.
Isolation and Analysis of Mutants of Double-Stranded-RNA Bacteriophage φ6 with Altered Packaging Specificity
Authors: Qiao Jian , Qiao Xueying , Sun Yang , Mindich Leonard
Published: July 2003

The genomes of bacteriophage φ6 and its relatives are packaged through a mechanism that involves the recognition and translocation of the three different plus strand transcripts of the segmented double-stranded RNA genomes into preformed polyhedral structures called procapsids or inner cores. This packaging requires hydrolysis of nucleoside triphosphates and takes place in the order S-M-L. Packaging is dependent on unique sequences of about 200 nucleotides near the 5′ ends of plus strand transcripts of the three genomic segments. Changes in the
pac
sequences lead to loss of packaging ability but can be suppressed by second-site changes in RNA or amino acid changes in protein P1, the major structural protein of the procapsid. It appears that P1 is the determinant of the RNA binding sites, and it is suggested that the binding sites overlap or are conformational changes of the same domains.
Molecular and Functional Analysis of thelepBGene, Encoding a Type I Signal Peptidase fromRickettsia rickettsiiandRickettsia typhi
Authors: Rahman M. Sayeedur , Simser Jason A. , Macaluso Kevin R. , Azad Abdu F.
Published: July 2003

The type I signal peptidaselepBgenes fromRickettsia rickettsiiandRickettsia typhi, the etiologic agents of Rocky Mountain spotted fever and murine typhus, respectively, were cloned and characterized. Sequence analysis of the clonedlepBgenes fromR. rickettsiiandR. typhishows open reading frames of 801 and 795 nucleotides, respectively. Alignment analysis of the deduced amino acid sequences reveals the presence of highly conserved motifs that are important for the catalytic activity of bacterial type I signal peptidase. Reverse transcription-PCR and Northern blot analysis demonstrated that thelepBgene ofR. rickettsiiis cotranscribed in a polycistronic message with the putativenuoF(encoding NADH dehydrogenase I chain F),secF(encoding protein export membrane protein), andrnc(encoding RNase III) genes in asecF-nuoF-lepB-rnccluster. The clonedlepBgenes fromR. rickettsiiandR. typhihave been demonstrated to possess signal peptidase I activity inEscherichia colipreprotein processing in vivo by complementation assay.
Biochemical Function of
msl5
(
pks8
plus
pks17
) in
Mycobacterium tuberculosis
H37Rv: Biosynthesis of Monomethyl Branched Unsaturated Fatty Acids
Authors: Dubey Vinod S. , Sirakova Tatiana D. , Cynamon Michael H. , Kolattukudy Pappachan E.
Published: July 2003

We show that the disruption of one of the mycocerosic acid synthase (
mas
)-like genes,
msl5
(
pks8
plus
pks17
) in
Mycobacterium tuberculosis
H37Rv generates a mutant incapable of producing monomethyl branched unsaturated C
16
to C
20
fatty acids that are minor constituents of acyltrehaloses and sulfolipids. The
msl5
mutation did not cause any significant change in the acyl lipid composition and also did not affect growth in culture, in mouse alveolar macrophage cell line MH-S, or in the murine lung.
A 65-Kilobase Pathogenicity Island Is Unique to Philadelphia-1 Strains of
Legionella pneumophila
Authors: Brassinga Ann Karen C. , Hiltz Margot F. , Sisson Gary R. , Morash Michael G. , Hill Nathan , Garduno Elizabeth , Edelstein Paul H. , Garduno Rafael A. , Hoffman Paul S.
Published: July 2003

Nucleotide sequence analysis of an ∼80-kb genomic region revealed an ∼65-kb locus that bears hallmarks of a pathogenicity island. This locus includes homologues of a type IV secretion system, mobile genetic elements, and known virulence factors. Comparative studies with other
Legionella pneumophila
strains and serogroups indicated that this ∼65-kb locus is unique to
L
.
pneumophila
serogroup 1 Philadelphia-1 strains.
Microarray Transcription Analysis of Clinical
Staphylococcus aureus
Isolates Resistant to Vancomycin
Authors: Mongodin Emmanuel , Finan Jon , Climo Michael W. , Rosato Adriana , Gill Steven , Archer Gordon L.
Published: July 2003

The transcriptomes of vancomycin intermediate-resistance
Staphylococcus aureus
(VISA) clinical isolates HIP5827 and Mu50 (MIC = 8 μg/ml) were compared to those of highly vancomycin-resistant
S. aureus
(VRSA; MIC = 32 μg/ml) passage derivatives by microarray. There were 35 genes with increased transcription and 16 genes with decreased transcription in common between the two VRSAs compared to those of their VISA parents. Of the 35 genes with increased transcription, 15 involved purine biosynthesis or transport, and the regulator (
purR
) of the major purine biosynthetic operon (
purE-purD
) was mutant. We hypothesize that increased energy (ATP) is required to generate the thicker cell walls that characterize resistant mutants.
Transcriptional Expression ofEscherichia coliGlutamate-Dependent Acid Resistance GenesgadAandgadBCin anhns rpoSMutant
Authors: Waterman Scott R. , Small P. L. C.
Published: July 2003

Resistance to being killed by acidic environments with pH values lower than 3 is an important feature of both pathogenic and nonpathogenicEscherichia coli. The most potentE. coliacid resistance system utilizes two isoforms of glutamate decarboxylase encoded bygadAandgadBand a putative glutamate:γ-aminobutyric acid antiporter encoded bygadC. Thegadsystem is controlled by two repressors (H-NS and CRP), one activator (GadX), one repressor-activator (GadW), and two sigma factors (σSand σ70). In contrast to results of previous reports, we demonstrate thatgadtranscription can be detected in anhns rpoSmutant strain ofE. coliK-12, indicating thatgadpromoters can be initiated by σ70in the absence of H-NS.
Spatial Variation Effects on Quantum Theory of the Micromaser with Ultra-Cold Λ-Type Three-Level Atom
Authors: Obada Abdel-Shafy F. , Abdel-Aty Mahmoud
Published: July 2003

We generalize a study of the behavior of a three-level atom in a single-mode mazer. Our formulation takes into account the interaction region and the spatial variation along the cavity axis. A general analytic time-dependent solution is obtained, and some special cases reduce to very simple expressions. When propagation effects are taken into consideration, the emission probability is influenced significantly. We provide the necessary arguments to justify the validity of our conclusions for emission probability and micromaser whose dynamics is governed by the wave function. Our main conclusion is that in the present system, the inclusion of the spatial dependence of the cavity region is necessary and important. We provide numerous examples to illustrate this correspondence, and provide evidence of its usefulness.
Effects of Field-Induced Coherence on Laser Without Population Inversion and on Absorptionless Dispersion for a V-Type Three Level System
Authors: Mandal Swapan
Published: July 2003

A V-type three level system with two closely spaced upper levels interacting with a single mode of the electromagnetic field is studied for the absorptive and dispersive lineshapes. A semiclassical description for the atom-field interaction is being adopted to obtain the usual optical Bloch equations. The approximate analytical solutions for the density matrix elements are obtained by solving coupled optical Bloch equations. Through the off-diagonal density matrix elements, the introduction of phase angles between the levels participating in dipole allowed transitions are automatic. It is shown that these phases are appreciable if the applied electromagnetic field is strong. These field dependent phases (coherence) are responsible for the reduction of the inversionless gain and the absorptionless dispersion. For large decay constants, it is found that the energy gap between the upper two levels has no role to play on the laser without inversion and on the absorptionless dispersion. However, for large population per unit decay, a significant increase of the absorptionless dispersion and the inversionless gain is obtained. The condition for large population decay and a possible experimental realization of the field dependent phases are discussed.
Spiked Harmonic Oscillator: N-1 Expansion
Authors: Datta S. , Bhattacharjee J. K.
Published: July 2003

The ground state of the spiked harmonic oscillator with the potential [Formula: see text] has been obtained variationally for α < 3 and recently by a numerical procedure for α > 3. Due to the Klauder phenomemon at α = 3, analytic techniques do not smoothly interpolate between α < 3 and α > 3. Here we use the N-1 expansion, where N is the dimensionality of space, to set up an analytic scheme that can be continued across α = 3.
Positive and Negative Circuits in Dynamical Systems
Authors: Gouzé Jean-Luc
Published: July 2003

We state precisely and demonstrate two conjectures of R. Thomas following which (a) the existence of a positive circuit in the oriented interaction graph of a differential system is a necessary condition for the existence of several steady states, and (b) the existence of a negative non-oriented circuit of length at least two is a necessary condition for the existence of a stable periodic orbit.
Oscillations in a Prey-Predator-Superpredator System
Authors: Boudjellaba H. , Sari T.
Published: July 2003

The behaviour of a food chain with three populations of the type prey-predator-superpredator is studied in the case where the growth rates of the three populations are highly diversified. The analysis shows that the system can have high-frequency oscillations, due to the interaction between prey and predator, which arise during a low-frequency cycle, due to the interaction between predator and superpredator. Using an averaging method, the dynamics of the superpredator is determined, during the high-frequency oscillations of the prey and predator.
Kinetic Models of Lipophilic Compound Uptake and Bioelimination in Amphibians During the Aquatic Stages
Authors: Venturino Andrés , Bergoc Rosa M. , Pechen Ana M.
Published: July 2003

During embryonic development, amphibians are particularly sensitive to lipophilic toxic compounds, given their tendency to bioaccumulate from the aquatic environment. On the basis of a series of toxicokinetic experiments with the organophosphorus malathion, we have developed several biomathematical models to explain the behavior of the toxin in an aquatic environment, as well as its biotransformation in amphibian larval stages.

Malathion in medium displayed uniexponential bioelimination, while it rapidly accumulated in larvae, decaying according to complex kinetics. Both kinetics were satisfactorily predicted by a model of two compartments in series. In the first compartment, which accounts for bioaccumulation in lipidic tissues, an exponential decline in capacity correlated with progressive depletion of larval lipid reserves was considered. In the second, metabolic biotransformation by several degradative enzymes was considered as a function of the mass of compound present in it. The rapid mass equilibrium between medium and lipidic compartment justifies the apparent uniexponential decay in the first, driven by the overall biotransformation rate constant from the metabolic compartment.
Analysis of Ant Foraging Algorithms
Authors: Britton N. F. , Stickland T. R. , Franks N. R.
Published: July 2003

We consider some models of ant foraging and recruitment behaviour that depend on each individual ant following a simple algorithm. Self-organisation enables the colony as a whole to establish a foraging strategy in the absence of any hierarchical control. In this paper we investigate how the effectiveness of such a foraging strategy depends on the persistence of the signals used by the individual ants and on the errors they make in following such signals. The use of inhibitory as well as excitatory signals is considered, and shown to be extremely effective in certain circumstances. This is interesting, as such signals have never been observed in real ant colonies. Such models are often investigated by simulation, but we approach them from the point of view of statistical mechanics. Looked at another way, which yields some insight, we approximate the stochastic process that models the system by a diffusion process with small diffusion parameter. This approach does not replace simulation, but supplements it. Its advantage is that it can elucidate the role of parameters more clearly and using much less computer time than simulation, but its disadvantage is that many simplifying assumptions must be made before the problem is amenable to analytic treatment.
Modelling the Dynamics of Leishmaniasis Considering Human, Animal Host and Vector Populations
Authors: Burattini Marcelo N. , Coutinho Francisco A. B. , Lopez Luis F. , Massad Eduardo
Published: July 2003

Some of the vector-borne infections with public health importance involve an animal reservoir. This work describes, through a compartimental model, the dynamics of leishmaniasis considering the interactions between the three populations involved, namely the sandfly, the domestic dog (as the principal intermediate host), and the human population. An expression for the Basic Reproduction Ratio (R0), which takes into account the time delay representing the extrinsic incubation period of the parasite in the vector, is presented. Numerical simulation, equilibrium and stability analysis provided insights into the transmission dynamics of the infection that can help in the design of control strategies.
A Method to Treat Some Dynamical Statistical Models
Authors: Ciuperca Gabriela
Published: July 2003

In this paper we present a method for the estimation of the parameters of models described by a nonlinear system of differential equations: we study the maximum likelihood estimator and the jackknife estimator for parameters of the system and for the covariance matrix of the state variables and we seek possible linear relations between parameters. We take into account the difficulty due to the small number of observations. The optimal experimental design for this kind of problem is determined. We give an application of this method for the glucose metabolism of goats.