bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaporation and instabilities of microscopic capillary bridges
Authors: Maeda Nobuo , Israelachvili Jacob N. , Kohonen Mika M.
Published: February 2003

The formation and disappearance of liquid bridges between two surfaces can occur either through equilibrium or nonequilibrium processes. In the first instance, the bridge molecules are in thermodynamic equilibrium with the surrounding vapor medium. In the second, chemical potential gradients result in material transfer; mechanical instabilities, because of van der Waals force jumps on approach or a Rayleigh instability on rapid separation, may trigger irreversible film coalescence or bridge snapping. We have studied the growth and disappearance mechanisms of laterally microscopic liquid bridges of three hydrocarbon liquids in slit-like pores. At rapid slit-opening rates, the bridges rupture by means of a mechanical instability described by the Young–Laplace equation. Noncontinuum but apparently reversible behavior is observed when a bridge is held at nanoscopic surface separations
H
close to the thermodynamic equilibrium Kelvin length, 2
r
K
cosθ, where
r
K
is the Kelvin radius and θ is the contact angle. During the course of slow evaporation (at
H
> 2
r
K
cosθ) and subsequent regrowth by capillary condensation (at
H
< 2
r
K
cosθ), the refractive index of the bridge may vary continuously and reversibly between that of the bulk liquid and vapor. The evaporation process becomes irreversible only at the very final stage of evaporation, when the refractive index of the fluid attains virtually that of the vapor. Measured refractive index profiles and the time-dependence of evaporating neck diameters also seem to differ from predictions based on a continuum picture of bridge evaporation far from the critical point. We discuss these findings in terms of the probable density profiles in evolving liquid bridges.
Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases
Authors: Hu Yanan , Chen Lan , Ha Sha , Gross Ben , Falcone Brian , Walker Deborah , Mokhtarzadeh Maryam , Walker Suzanne
Published: February 2003

MurG is an essential glycosyltransferase that forms the glycosidic linkage between
N-
acetyl muramyl pentapeptide and
N-
acetyl glucosamine in the biosynthesis of the bacterial cell wall. This enzyme is a member of a major superfamily of NDP-glycosyltransferases for which no x-ray structures containing intact substrates have been reported. Here we present the 2.5-Å crystal structure of
Escherichia coli
MurG in complex with its donor substrate, UDP-GlcNAc. Combined with genomic analysis of other superfamily members and site-specific mutagenesis of
E. coli
MurG, this structure sheds light on the molecular basis for both donor and acceptor selectivity for the superfamily. This structural analysis suggests that it will be possible to evolve new glycosyltransferases from prototypical superfamily members by varying two key loops while maintaining the overall architecture of the family and preserving key residues.
Global variation in terrestrial conservation costs, conservation benefits, and unmet conservation needs
Authors: Balmford Andrew , Gaston Kevin J. , Blyth Simon , James Alex , Kapos Val
Published: February 2003

Our ability to identify cost-efficient priorities for conserving biological diversity is limited by the scarcity of data on conservation costs, particularly at fine scales. Here we address this issue using data for 139 terrestrial programs worldwide. We find that the annual costs of effective field-based conservation vary enormously, across seven orders of magnitude, from <$0.1 to >$1,000,000 per km
2
. This variation can be closely predicted from positive associations between costs per unit area and an array of indices of local development. Corresponding measures of conservation benefit are limited but show opposing global trends, being higher in less developed parts of the world. The benefit-to-cost ratio of conservation is thus far greater in less developed regions, yet these are where the shortfall in current conservation spending is most marked. Substantially increased investment in tropical conservation is therefore urgently required if opportunities for cost-effective action are not to be missed.
Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice
Authors: Akamatsu Yoshiko , Monroe Robert , Dudley Darryll D. , Elkin Sheryl K. , Gärtner Frank , Talukder Sadiqur R. , Takahama Yousuke , Alt Frederick W. , Bassing Craig H. , Oettinger Marjorie A.
Published: February 2003

The recombination-activating gene (RAG)1 and RAG2 proteins comprise the lymphocyte-specific components of the V(D)J recombinase and are required for the assembly of antigen-receptor variable-region genes. A mutant truncated RAG2 protein (“core” RAG2) lacking the C-terminal 144 amino acids, together with core RAG1, is able to mediate the basic biochemical steps required for V(D)J recombinationin vitroand in transfected cell lines. Here we examine the effect of replacing the endogenousRAG2locus in mice with coreRAG2. These mice generate substantial numbers of B and T cells, demonstrating that the core RAG2 protein retains significantin vivofunction. However, core RAG2 mice display a reduction in the total number of B and T cells, reflecting impaired lymphocyte development at the progenitor stage associated with reduced chromosomal V(D)J recombination. We discuss potential roles of the RAG2 C terminus in mediating rearrangement of endogenous antigen-receptor loci.
Metabotropic mGlu5 receptors regulate adenosine A
2A
receptor signaling
Authors: Nishi Akinori , Liu Feng , Matsuyama Seiichiro , Hamada Miho , Higashi Hideho , Nairn Angus C. , Greengard Paul
Published: February 2003

Dopamine, by activating dopamine D1-type receptors, and adenosine, by activating adenosine A
2A
receptors, stimulate phosphorylation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of
M
r
32,000) at Thr-34. In this study, we investigated the effect of metabotropic glutamate (mGlu) receptors on DARPP-32 phosphorylation at Thr-34 in neostriatal slices. A broad-spectrum mGlu receptor agonist,
trans
-ACPD, and a group I mGlu receptor agonist, DHPG, stimulated DARPP-32 phosphorylation at Thr-34. Studies with mGlu receptor antagonists revealed that the effects of
trans
-ACPD and DHPG were mediated through activation of mGlu5 receptors. The action of mGlu5 receptors required activation of adenosine A
2A
receptors by endogenous adenosine. Conversely, the action of adenosine A
2A
receptors required activation of mGlu5 receptors by endogenous glutamate. Coactivation of mGlu5 and adenosine A
2A
receptors by exogenous agonists synergistically increased DARPP-32 phosphorylation. mGlu5 receptors did not require activation of dopamine D1-type receptors by endogenous dopamine, nor did dopamine D1-type receptors require activation of mGlu5 receptors by endogenous glutamate. DHPG potentiated the effect of forskolin, but not that of 8-bromo-cAMP, and stimulated DARPP-32 phosphorylation in the presence of the phosphodiesterase inhibitor IBMX, suggesting that mGlu5 receptors stimulate the rate of cAMP formation coupled to adenosine A
2A
receptors. The action of mGlu5 receptors was attenuated by inhibitors of extracellular signal-regulated kinase, but not by inhibitors of phospholipase C, p38, casein kinase 1, or Cdk5. The results demonstrate that mGlu5 receptors potentiate adenosine A
2A
/DARPP-32 signaling by stimulating the adenosine A
2A
receptor-mediated formation of cAMP in an extracellular signal-regulated kinase-dependent manner.
The maltase-glucoamylase gene: Common ancestry to sucrase-isomaltase with complementary starch digestion activities
Authors: Nichols Buford L. , Avery Stephen , Sen Partha , Swallow Dallas M. , Hahn Dagmar , Sterchi Erwin
Published: February 2003

Brush-border maltase-glucoamylase (MGA) activity serves as the final step of small intestinal digestion of linear regions of dietary starch to glucose. Brush-border sucrase-isomaltase (SI) activity is complementary, through digestion of branched starch linkages. Here we report the cloning and sequencing of human MGA gene and demonstrate its close evolutionary relationship to SI. The gene is ≈82,000 bp long and located at chromosome 7q34. Forty-eight exons were identified. The 5′ gene product, when expressed as the N-terminal protein sequence, hydrolyzes maltose and starch, but not sucrose, and is thus distinct from SI. The catalytic residue was identified by mutation of an aspartic acid and was found to be identical with that described for SI. The exon structures of MGA and SI were identical. This homology of genomic structure is even more impressive than the previously reported 59% amino acid sequence identity. The shared exon structures and peptide domains, including proton donors, suggest that MGA and SI evolved by duplication of an ancestral gene, which itself had already undergone tandem gene duplication. The complementary human enzyme activities allow digestion of the starches of plant origin that make up two-thirds of most diets.
Origin-specific unwinding of herpes simplex virus 1 DNA by the viral UL9 and ICP8 proteins: Visualization of a specific preunwinding complex
Authors: Makhov Alexander M. , Lee Sam S.-K. , Lehman I. Robert , Griffith Jack D.
Published: February 2003

Herpes simplex virus 1 contains three origins of replication; two copies of oriS and one of a similar sequence, oriL. Here, the combined action of multiple factors known or thought to influence the opening of oriS are examined. These include the viral origin-binding protein, UL9, and single-strand binding protein ICP8, host cell topoisomerase I, and superhelicity of the DNA template. By using electron microscopy, it was observed that when ICP8 and UL9 proteins were added together to oriS-containing supertwisted DNA, a discrete preunwinding complex was formed at oriS on 40% of the molecules, which was shown by double immunolabeling electron microscopy to contain both proteins. This complex was relatively stable to extreme dilution. Addition of ATP led to the efficient unwinding of ≈50% of the DNA templates. Unwinding proceeded until the acquisition of a high level of positive supertwists in the remaining duplex DNA inhibited further unwinding. Addition of topoisomerase I allowed further unwinding, opening >1 kb of DNA around oriS.
Understanding missense mutations in the
BRCA1
gene: An evolutionary approach
Authors: Fleming Melissa A. , Potter John D. , Ramirez Christina J. , Ostrander Gary K. , Ostrander Elaine A.
Published: February 2003

The role of missense changes in
BRCA1
in breast cancer susceptibility has been difficult to establish. We used comparative evolutionary methods to identify potential functionally important amino acid sites in exon 11 and missense changes likely to disrupt gene function, aligning sequences from 57 eutherian mammals and categorizing amino acid sites by degree of conservation. We used Bayesian phylogenetic analyses to determine relationships among orthologs and identify codons evolving under positive selection. Most conserved residues occur in a region with the highest concentration of protein-interacting domains. Rapidly evolving residues are concentrated in the RAD51-interacting domain, suggesting that selection is acting most strongly on the role of
BRCA1
in DNA repair. Investigation of the functional role of missense changes in breast-cancer susceptibility should focus on 38 missense changes in conserved and 3 in rapidly evolving regions of exon 11.
The mouse organellar biogenesis mutant buff results from a mutation inVps33a, a homologue of yeastvps33andDrosophilacarnation
Authors: Suzuki Tamio , Oiso Naoki , Gautam Rashi , Novak Edward K. , Panthier Jean-Jacques , Suprabha P. G. , Vida Thomas , Swank Richard T. , Spritz Richard A.
Published: February 2003

In the mouse, more than 16 loci are associated with mutant phenotypes that include defective pigmentation, aberrant targeting of lysosomal enzymes, prolonged bleeding, and immunodeficiency, the result of defective biogenesis of cytoplasmic organelles: melanosomes, lysosomes, and various storage granules. Many of these mouse mutants are homologous to the human Hermansky–Pudlak syndrome (HPS), Chediak–Higashi syndrome, and Griscelli syndrome. We have mapped and positionally cloned one of these mouse loci, buff (bf), which has a mutant phenotype similar to that of human HPS. Mousebfresults from a mutation inVps33aand thus is homologous to the yeast vacuolar protein-sorting mutantvps33andDrosophilacarnation (car). This is the first found defect of the class C vacuole/prevacuole-associated target solubleN-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) complex in mammals and the first mammalian mutant found that is directly homologous to avpsmutation of yeast.VPS33Athus is a good candidate gene for a previously uncharacterized form of human HPS.
Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation
Authors: Zhan Yifan , Purton Jared F. , Godfrey Dale I. , Cole Timothy J. , Heath William R. , Lew Andrew M.
Published: February 2003

Peripheral activation can cause bystander thymocyte death by eliciting a “cytokine storm.” This event complicatesin vivostudies using exogenous ligand-induced models of negative selection. A stable transgenic model that selectively eliminates peripheral CD4 cells has allowed us to analyze negative selection as direct cognate events in two T cell receptor transgenic mice, OT-II and DO11. Whereas cognate peptide induced a massive deletion in double-positive (DP) cells in mice with peripheral CD4 cells, this DP deletion was modest in mice lacking peripheral CD4 cells. Using BrdUrd and annexin V staining, we found that negative selection primarily occurs in a cohort of DP cells and the absence of single-positive (SP) cells is largely caused by reduction in the cohort of DP precursors. Moreover, the fates of DP cells and SP cells after antigen exposure were vastly different. Whereas SP cells up-regulated uniformly their CD69 and CD44 levels, increased their cell size, and survived after antigen exposure, DP cells had less CD69 and CD44 up-regulation, no size change, and promptly died. Thus, negative selection represents an “abortive” activation different from activation-induced cell death of mature T cells.
The critical regulator of embryonic hematopoiesis, SCL, is vital in the adult for megakaryopoiesis, erythropoiesis, and lineage choice in CFU-S
12
Authors: Hall Mark A. , Curtis David J. , Metcalf Donald , Elefanty Andrew G. , Sourris K. , Robb Lorraine , Göthert Joachim R. , Jane Stephen M. , Begley C. Glenn
Published: February 2003

Gene targeting studies have shown that the transcription factor SCL is critically important for embryonic hematopoiesis, but the early lethality of SCL null mice has precluded the genetic analysis of its function in the adult. We have now generated a conditional knockout of SCL by using Cre/Lox technology and an IFN-inducible Cre transgenic mouse. Deletion of SCL in adult mice perturbed megakaryopoiesis and erythropoiesis with the loss of early progenitor cells in both lineages. This led to a blunted response to the hematopoietic stress induced by polyinosinic-polycytidylic acid, with a persistently low platelet count and hematocrit compared with controls. In contrast, progenitors of granulocyte and macrophage lineages were not affected, even in the setting of stress. Immature progenitor cells (day 12 colony-forming unit spleen) with multilineage capacity were still present in the SCL null bone marrow, but these progenitors had lost the capacity to generate erythroid and megakaryocyte cells, and colonies were composed of only myeloid cells. These results suggest that SCL is critical for megakaryopoiesis and erythropoiesis, but is dispensable for production of myeloid cells during adult hematopoiesis.
Sodium ion cycling mediates energy coupling between complex I and ATP synthase
Authors: Gemperli Anja C. , Dimroth Peter , Steuber Julia
Published: February 2003

We show here sodium ion cycling between complex I from
Klebsiella pneumoniae
and the F
1
F
0
ATP synthase from
Ilyobacter tartaricus
in a reconstituted proteoliposome system. In the course of NADH oxidation by complex I, an electrochemical sodium ion gradient was established and served as a driving force for the synthesis of ATP from ADP and phosphate. In the opposite direction, the Δμ̃

Na
+

generated by ATP hydrolysis could be coupled to NADH formation by reversed electron transfer from ubiquinol to NAD. For reverse electron transfer, a transmembrane voltage larger than 30 mV was obligatory. No NADH-driven proton transport into the lumen of proteoliposomes was detected. We conclude that Na
+
is used as the exclusive coupling ion by the enterobacterial complex I.
Identification of nucleocytoplasmic cycling as a remote sensor in cellular signaling by databased modeling
Authors: Swameye I. , Müller T. G. , Timmer J. , Sandra O. , Klingmüller U.
Published: February 2003

Considerable progress has been made in identifying the molecular composition of complex signaling networks controlling cell proliferation, differentiation, and survival. However, to discover general building principles and predict the dynamic behavior of signaling networks, it is necessary to develop quantitative models based on experimental observations. Here we report a mathematical model of the core module of the Janus family of kinases (JAK)–signal transducer and activator of transcription (STAT) signaling pathway based on time-resolved measurements of receptor and STAT5 phosphorylation. Applying the fitted model, we can determine the quantitative behavior of STAT5 populations not accessible to experimental measurement. By
in silico
investigations, we identify the parameters of nuclear shuttling as the most sensitive to perturbations and verify experimentally the model prediction that inhibition of nuclear export results in a reduced transcriptional yield. The model reveals that STAT5 undergoes rapid nucleocytoplasmic cycles, continuously coupling receptor activation and target gene transcription, thereby forming a remote sensor between nucleus and receptor. Thus, dynamic modeling of signaling pathways can promote functional understanding at the systems level.
Beneficial effects of antioxidants and
l
-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress
Authors: de Nigris Filomena , Lerman Lilach O. , Ignarro Sharon Williams , Sica Giacomo , Lerman Amir , Palinski Wulf , Ignarro Louis J. , Napoli Claudio
Published: February 2003

Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and
l
-arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by disturbed flow
in vitro
and
in vivo
. Both human endothelial cells exposed to shear stress and high atherosclerosis-prone areas of hypercholesterolemic low-density lipoprotein receptor knockout (LDLR
−/−
) mice showed increased activities of redox-transcription factors (ELK-1,
p
-Jun, and
p
-CREB) and decreased expression of eNOS. Intervention with antioxidants and
l
-arginine reduced the activation of redox-transcription factors and increased eNOS expression in cells and
in vivo
. These results demonstrate that atherogenic effects induced by turbulent shear stress can be prevented by cotreatment with antioxidants and
l
-arginine. The therapeutic possibility to modulate shear stress-response genes may have important implications for the prevention of atherosclerosis and its clinical manifestations.
Diffusion-limited kinetics of the solution–solid phase transition of molecular substances
Authors: Petsev Dimiter N. , Chen Kai , Gliko Olga , Vekilov Peter G.
Published: February 2003

For critical tests of whether diffusion-limited kinetics is an option for the solution–solid phase transition of molecular substances or whether they are determined exclusively by a transition state, we performed crystallization experiments with ferritin and apoferritin, a unique pair of proteins with identical shells but different molecular masses. We find that the kinetic coefficient for crystallization is identical (accuracy ≤7%) for the pair, indicating diffusion-limited kinetics of crystallization. Data on the kinetics of this phase transition in systems ranging from small-molecule ionic to protein and viri suggest that the kinetics of solution-phase transitions for broad classes of small-molecule and protein materials are diffusion-limited.
Blue light activates calcium-permeable channels inArabidopsismesophyll cells via the phototropin signaling pathway
Authors: Stoelzle Sonja , Kagawa Takatoshi , Wada Masamitsu , Hedrich Rainer , Dietrich Petra
Published: February 2003

Light is a central regulator of plant growth and development. Among the processes triggered by blue and UV-A light, phototropism, stomatal movement, and chloroplast orientation rely on the activation of blue-light receptors known as phototropins. So far, these photoreceptors constitute a class of light receptor kinases unique to the plant kingdom. InArabidopsis thaliana, the two members phot1 and phot2 have been shown to display partially overlapping functions. Up to now little is known about the signaling cascade, which links these phototropins to the physiological responses downstream of blue-light perception. Here, we show that on illumination with blue light, but not red light, voltage-dependent and calcium-permeable channels activate in the plasma membrane of mesophyll cells. Blue-light stimulation in the presence of the photosynthetic electron transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicates that blue-light receptors rather than photosynthesis control channel activity. Sensitivity toward the protein kinase inhibitor K252a further pointed to the possible involvement of light receptor kinases. In support of this hypothesis, in the photoreceptor mutantphot1-5, blue-light induction of calcium currents was dramatically reduced and was eliminated in the double mutantphot1-5 phot2-1. By contrast, incry1-304 cry2-1, anArabidopsismutant lacking another class of plant blue-light receptors, the channel remained sensitive to blue light. We thus conclude that blue light triggers calcium fluxes via the phototropin-activated calcium-permeable channel.
Multiple mutations and cancer
Authors: Loeb Lawrence A. , Loeb Keith R. , Anderson Jon P.
Published: February 2003

Most human tumors are highly heterogenous. We have hypothesized that this heterogeneity results from a mutator phenotype. Our premise is that normal mutation rates are insufficient to account for the multiple mutations found in human cancers, and, instead, that cancers must exhibit a mutator phenotype early during their evolution. Here, we examine the current status and implications of the mutator phenotype hypothesis for the prognosis, treatment, and prevention of human cancers.
The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation
Authors: Seidah Nabil G. , Benjannet Suzanne , Wickham Louise , Marcinkiewicz Jadwiga , Jasmin Stéphanie Bélanger , Stifani Stefano , Basak Ajoy , Prat Annik , Chrétien Michel
Published: February 2003

Seven secretory mammalian kexin-like subtilases have been identified that cleave a variety of precursor proteins at monobasic and dibasic residues. The recently characterized pyrolysin-like subtilase SKI-1 cleaves proproteins at nonbasic residues. In this work we describe the properties of a proteinase K-like subtilase, neural apoptosis-regulated convertase 1 (NARC-1), representing the ninth member of the secretory subtilase family. Biosynthetic and microsequencing analyses of WT and mutant enzyme revealed that human and mouse pro-NARC-1 are autocatalytically and intramolecularly processed into NARC-1 at the (
Y
,
I
)V
V
(V,L)(
L
,
M
)↓ motif, a site that is representative of its enzymic specificity.
In vitro
peptide processing studies and/or Ala substitutions of the P1–P5 sites suggested that hydrophobic/aliphatic residues are more critical at P1, P3, and P5 than at P2 or P4. NARC-1 expression is highest in neuroepithelioma SK-N-MCIXC, hepatic BRL-3A, and in colon carcinoma LoVo-C5 cell lines.
In situ
hybridization and Northern blot analyses of NARC-1 expression during development in the adult and after partial hepatectomy revealed that it is expressed in cells that have the capacity to proliferate and differentiate. These include hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons. Accordingly, transfection of NARC-1 in primary cultures of embryonic day 13.5 telencephalon cells led to enhanced recruitment of undifferentiated neural progenitor cells into the neuronal lineage, suggesting that NARC-1 is implicated in the differentiation of cortical neurons.
Developmental loss of miniature
N
-methyl-
d
-aspartate receptor currents in NR2A knockout mice
Authors: Townsend Matthew , Yoshii Akira , Mishina M. , Constantine-Paton Martha
Published: February 2003

The
N
-methyl-
d
-aspartate (NMDA) glutamate receptor (NMDAR), long implicated in developmental plasticity, shows decay time kinetics that shorten postnatally as NR2A subunits are added to the receptor. Neither the mechanism nor immediate effect of this change is known. We studied developing NMDAR currents by using visual neurons in slices from NR2A knockout (NR2AKO) and WT mice. Both strains show increased dendritic levels of synaptic density scaffolding protein PSD-95 with age. Dendritic levels of NR2A increased at the same time in WT and immunoprecipitated with PSD-95. PSD-95/NMDAR binding was significantly decreased in the NR2AKO. Moreover, NMDAR miniature currents (minis) were lost and rise times of NMDAR evoked currents increased in mutant mice. Age-matched WT cells showed NR2A-rich receptors predominating in minis, yet slow NR2B mediated currents persisted in evoked currents. Disrupting photoreceptor activation of retinal ganglion cells eliminated increases in PSD-95 and NR2A in superior collicular dendrites of WT mice and slowed the loss of miniature NMDAR currents in NR2AKOs. These data demonstrate that NMDARs that respond to single quantal events mature faster during development by expressing the NR2A subunit earlier than NMDARs that respond to evoked release. We hypothesize that NR2A-rich NMDARs may be localized to the center of developing synapses by an activity-dependent process that involves the targeting of PSD-95 to the postsynaptic density. Neonatal receptors become restricted to perisynpatic or extrasynaptic sites, where they participate primarily in evoked currents.
The genome sequence of
Clostridium tetani
, the causative agent of tetanus disease
Authors: Brüggemann Holger , Bäumer Sebastian , Fricke Wolfgang Florian , Wiezer Arnim , Liesegang Heiko , Decker Iwona , Herzberg Christina , Martínez-Arias Rosa , Merkl Rainer , Henne Anke , Gottschalk Gerhard
Published: February 2003

Tetanus disease is one of the most dramatic and globally prevalent diseases of humans and vertebrate animals, and has been reported for over 24 centuries. The manifestation of the disease, spastic paralysis, is caused by the second most poisonous substance known, the tetanus toxin, with a human lethal dose of ≈1 ng/kg. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid; nevertheless, according to the World Health Organization, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The causative agent of tetanus disease is
Clostridium tetani
, an anaerobic spore-forming bacterium, whose natural habitat is soil, dust, and intestinal tracts of various animals. Here we report the complete genome sequence of toxigenic

C
.
tetani

E88, a variant of strain Massachusetts. The genome consists of a 2,799,250-bp chromosome encoding 2,372 ORFs. The tetanus toxin and a collagenase are encoded on a 74,082-bp plasmid, containing 61 ORFs. Additional virulence-related factors could be identified, such as an array of surface-layer and adhesion proteins (35 ORFs), some of them unique to

C
.
tetani

. Comparative genomics with the genomes of
Clostridium perfringens
, the causative agent of gas gangrene, and
Clostridium acetobutylicum
, a nonpathogenic solvent producer, revealed a remarkable capacity of

C
.
tetani

: The organism can rely on an extensive sodium ion bioenergetics. Additional candidate genes involved in the establishment and maintenance of a pathogenic lifestyle of

C
.
tetani

are presented.
Essential role for Atox1 in the copper-mediated intracellular trafficking of the Menkes ATPase
Authors: Hamza Iqbal , Prohaska Joseph , Gitlin Jonathan D.
Published: February 2003

The metallochaperone Atox1 directly interacts with the copper-transporting ATPases and plays a critical role in perinatal copper homeostasis. To determine the cell biological mechanisms of Atox1 function, intracellular copper metabolism, and Menkes ATPase abundance, localization and trafficking were examined in immortalized fibroblast cell lines derived from
Atox1
+/+
and
Atox1
−/−
embryos. Consistent with the proposed role for Atox1 in copper delivery to the secretory pathway, a marked increase in intracellular copper content secondary to impaired copper efflux was observed in Atox1-deficient cells. Although the localization of the Menkes ATPase was identical in
Atox1
+/+
and
Atox1
−/−
cells under conditions of equivalent intracellular copper content, a significant impairment in copper-mediated Menkes ATPase trafficking was observed in the absence of Atox1. When quantitative confocal immunofluorescence was used, significant differences in the time and dose-dependent trafficking of the Menkes ATPase from the Golgi compartment in response to copper were observed between
Atox1
+/+
and
Atox1
−/−
cells. These data reveal an essential role for Atox1 in establishing the threshold for copper-dependent movement of the copper-transporting ATPases within the secretory compartment and that, in the absence of Atox1, this movement alone is not sufficient to restore normal copper efflux. Taken together, these findings provide a cell biological model for the role of this metallochaperone under the physiological conditions of copper limitation in mammalian cells.
Conformational dynamics of the Na
+
/K
+
-ATPase probed by voltage clamp fluorometry
Authors: Geibel Sven , Kaplan Jack H. , Bamberg Ernst , Friedrich Thomas
Published: February 2003

The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active Na
+
/K
+
-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5–M6 loop of the sheep α
1
-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in
Xenopus
oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5–M6 loop by showing large, ouabain-sensitive fluorescence changes (≈5%) on addition of extracellular K
+
. When the enzyme was subjected to voltage jumps under Na
+
/Na
+
-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the E
1
P–E
2
P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of Na
+
by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating K
+
concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing K
+
concentrations. As a result, the stationary distribution between E
1
and E
2
states and voltage-dependent relaxation times can be determined at any time and membrane potential under Na
+
/Na
+
exchange as well as Na
+
/K
+
turnover conditions.
Promoters of the murine embryonic β-like globin genes
Ey
and β
h1
do not compete for interaction with the β-globin locus control region
Authors: Hu Xiao , Bulger Michael , Roach Julia N. , Eszterhas Susan K. , Olivier Emmanuel , Bouhassira Eric E. , Groudine Mark T. , Fiering Steven
Published: February 2003

Mammalian β-globin loci contain multiple β-like genes that are expressed at different times during development. The murine β-globin locus contains two genes expressed during the embryo stage, Ey and βh1, and two genes expressed at both the fetal and postnatal stages, β-major and β-minor. Studies of transgenic human β-like globin loci in mice have suggested that expression of one gene at the locus will suppress expression of other genes at the locus. To test this hypothesis we produced mouse lines with deletions of either the Ey or βh1 promoter in the endogenous murine β-globin locus. Promoter deletion eliminated expression of the mutant gene but did not affect expression of the remaining embryonic gene or the fetal/adult β-globin genes on the mutant allele. These results demonstrate a lack of competitive effects between individual mouse embryonic β-globin gene promoters and other genes in the locus. The implication of these findings for models of β-globin gene expression are discussed.
The missing link between thermodynamics and structure in F
1
-ATPase
Authors: Yang W. , Gao Y. Q. , Cui Q. , Ma J. , Karplus M.
Published: February 2003

F
1
F
o
-ATP synthase is the enzyme responsible for most of the ATP synthesis in living systems. The catalytic domain F
1
of the F
1
F
o
complex, F
1
-ATPase, has the ability to hydrolyze ATP. A fundamental problem in the development of a detailed mechanism for this enzyme is that it has not been possible to determine experimentally the relation between the ligand binding affinities measured in solution and the different conformations of the catalytic β subunits (β
TP
, β
DP
, β
E
) observed in the crystal structures of the mitochondrial enzyme, MF
1
. Using free energy difference simulations for the hydrolysis reaction ATP+H
2
O → ADP+P
i
in the β
TP
and β
DP
sites and unisite hydrolysis data, we are able to identify β
TP
as the “tight” (
K
D
= 10
−12
M, MF
1
) binding site for ATP and β
DP
as the “loose” site. An energy decomposition analysis demonstrates how certain residues, some of which have been shown to be important in catalysis, modulate the free energy of the hydrolysis reaction in the β
TP
and β
DP
sites, even though their structures are very similar. Combined with the recently published simulations of the rotation cycle of F
1
-ATPase, the present results make possible a consistent description of the binding change mechanism of F
1
-ATPase at an atomic level of detail.
Hair-bundle movements elicited by transepithelial electrical stimulation of hair cells in the sacculus of the bullfrog
Authors: Bozovic D. , Hudspeth A. J.
Published: February 2003

Electrically evoked otoacoustic emission is a manifestation of reverse transduction by the inner ear. We present evidence for a single-cell correlate of this phenomenon, hair-bundle movement driven by transepithelial electrical stimulation of the frog's sacculus. Responses could be observed at stimulus frequencies up to 1 kHz, an order of magnitude higher than the organ's natural range of sensitivity to acceleration or sound. Measurements at high-stimulus frequencies and pharmacological treatments allow us to distinguish two mechanisms that mediate the electrical responses: myosin-based adaptation and Ca
2+
-dependent reclosure of transduction channels. These mechanisms also participate in the active process that amplifies and tunes the mechanical responses of this receptor organ. Transient application of the channel blocker gentamicin demonstrated the crucial role of mechanoelectrical transduction channels in the rapid responses to electrical stimulation. A model for electrically driven bundle motion that incorporates the negative stiffness of the hair bundle as well as its two mechanisms of motility captures the essential features of the measured responses.
Emergence and evolution of
Vibrio cholerae
O139
Authors: Faruque Shah M. , Sack David A. , Sack R. Bradley , Colwell Rita R. , Takeda Yoshifumi , Nair G. Balakrish
Published: February 2003

The emergence of
Vibrio cholerae
O139 Bengal during 1992–1993 was associated with large epidemics of cholera in India and Bangladesh and, initially, with a total displacement of the existing
V. cholerae
O1 strains. However, the O1 strains reemerged in 1994 and initiated a series of disappearance and reemergence of either of the two serogroups that was associated with temporal genetic and phenotypic changes sustained by the strains. Since the initial emergence of the O139 vibrios, new variants of the pathogen derived from multiple progenitors have been isolated and characterized. The clinical and epidemiological characteristics of these strains have been studied. Rapid genetic reassortment in O139 strains appears to be a response to the changing epidemiology of
V. cholerae
O1 and also a strategy for persistence in competition with strains of the O1 serogroup. The emergence of
V. cholerae
O139 has provided a unique opportunity to witness genetic changes in
V. cholerae
that may be associated with displacement of an existing serogroup by a newly emerging one and, thus, provide new insights into the epidemiology of cholera. The genetic changes and natural selection involving both environmental and host factors are likely to influence profoundly the genetics, epidemiology, and evolution of toxigenic
V. cholerae
, not only in the Ganges Delta region of India and Bangladesh, but also in other areas of endemic and epidemic cholera.
The hippocampus mediates glucocorticoid-induced impairment of spatial memory retrieval: Dependence on the basolateral amygdala
Authors: Roozendaal Benno , Griffith Qyana K. , Buranday Jason , de Quervain Dominique J.-F. , McGaugh James L.
Published: February 2003

Previous studies have indicated that stress-activated glucocorticoid hormones induce temporary memory retrieval impairment. The present study examined whether adrenal steroid receptors in the hippocampus mediate such glucocorticoid effects on spatial memory retrieval. The specific glucocorticoid receptor (GR) agonist 11β, 17β-dihydroxy-6,21-dimethyl-17α-pregna-4,6-trien-20yn-3-one (RU 28362; 5 or 15 ng) infused into the hippocampus of male Sprague–Dawley rats 60 min before water-maze retention testing, 24 h after training, dose-dependently impaired probe-trial retention performance, as assessed both by time spent in the training quadrant and initial latency to cross the platform location. The GR agonist did not affect circulating corticosterone levels immediately after the probe trial, indicating that RU 28362 infusions did not influence retention by altering glucocorticoid feedback mechanisms. As infusions of the GR agonist into the hippocampus 60 min before training did not influence water-maze acquisition or immediate recall, the findings indicated that the GR agonist-induced retention impairment was induced selectively by an influence on information retrieval. In contrast, pretest infusions of the GR agonist administered into the basolateral complex of the amygdala (BLA; 2 or 6 ng) did not alter retention performance in the water maze. However,
N
-methyl-
d
-aspartate-induced lesions of the BLA, made 1 week before training, blocked the memory retrieval impairment induced by intrahippocampal infusions of RU 28362 given 60 min before the retention test. These findings indicate that the effects of glucocorticoids on retrieval of long-term spatial memory depend on the hippocampus and, additionally, that neuronal input from the BLA is critical in enabling hippocampal glucocorticoid effects on memory retrieval.
Contrasting roles of axonal (pyramidal cell) and dendritic (interneuron) electrical coupling in the generation of neuronal network oscillations
Authors: Traub Roger D. , Pais Isabel , Bibbig Andrea , LeBeau Fiona E. N. , Buhl Eberhard H. , Hormuzdi Sheriar G. , Monyer Hannah , Whittington Miles A.
Published: February 2003

Electrical coupling between pyramidal cell axons, and between interneuron dendrites, have both been described in the hippocampus. What are the functional roles of the two types of coupling? Interneuron gap junctions enhance synchrony of γ oscillations (25–70 Hz) in isolated interneuron networks and also in networks containing both interneurons and principal cells, as shown in mice with a knockout of the neuronal (primarily interneuronal) connexin36. We have recently shown that pharmacological gap junction blockade abolishes kainate-induced γ oscillations in connexin36 knockout mice; without such gap junction blockade, γ oscillations do occur in the knockout mice, albeit at reduced power compared with wild-type mice. As interneuronal dendritic electrical coupling is almost absent in the knockout mice, these pharmacological data indicate a role of axonal electrical coupling in generating the γ oscillations. We construct a network model of an experimental γ oscillation, known to be regulated by both types of electrical coupling. In our model, axonal electrical coupling is required for the γ oscillation to occur at all; interneuron dendritic gap junctions exert a modulatory effect.
HLA-G gene repression is reversed by demethylation
Authors: Moreau Philippe , Mouillot Gaël , Rousseau Philippe , Marcou Céline , Dausset Jean , Carosella Edgardo D.
Published: February 2003

The HLA-G molecule plays an important role in immune tolerance, protecting the fetus from maternal immune attack, and probably contributes to graft tolerance and tumor escape from the host immune system. HLA-G expression is tightly regulated and involves mechanisms acting in part at the transcriptional level. Nevertheless, almost all regulatory sequences that govern constitutive and inducible HLA class I gene transcription are disrupted in the HLA-G gene promoter, suggesting an unusual regulatory process. In further investigating the molecular mechanisms of HLA-G gene activation, we evaluated the influence of epigenetic mechanisms on seven HLA-G-negative cell lines that exhibit various phenotypes. Exposure of cells to histone deacetylase inhibitors, or to the demethylating agent 5-aza-2′-deoxycytidine, revealed that HLA-G gene transcription is inhibited by DNA methylation. Reversal of methylation-mediated repression may directly induce HLA-G cell-surface expression, supporting the idea that HLA-G might be activated by such a mechanism during malignancy, inflammation, and allogenic reactions.
Abrogation of postentry restriction of HIV-1-based lentiviral vector transduction in simian cells
Authors: Kootstra Neeltje A. , Münk Carsten , Tonnu Nina , Landau Nathaniel R. , Verma Inder M.
Published: February 2003

HIV-1 replication in simian cells is restricted at an early postentry step because of the presence of an inhibitory cellular factor. This block reduces the usefulness of HIV-1-based lentiviral vectors in primate animal models. Here, we demonstrate that substitution of the cyclophilin A (CyPA) binding region in the capsid of an HIV-1-based lentiviral vector (LV) with that of the macrophage tropic HIV-1 Ba-L resulted in a vector that was resistant to the inhibitory effect and efficiently transduced simian cells. Notably, the chimeric
gag
LV efficiently transduced primary simian hematopoietic progenitor cells, a critical cellular target in gene therapy. The alterations in the CyPA binding region did not affect CyPA incorporation; however, transduction by the
gag
chimeric LV seemed to be relatively insensitive to cyclosporin A, indicating that it does not require CyPA for early postentry steps. In dual infection experiments, the
gag
chimeric LV failed to remove the block to transduction of the WT LV, suggesting that the
gag
chimeric LV did not saturate the inhibitory simian cellular factor. These data suggest that the CyPA binding region of capsid contains a viral determinant involved in the postentry restriction of HIV-1-based lentiviral vectors. Overall, the findings demonstrate that the host range of HIV-1-based LV can be altered by modifications in the packaging construct.
Evolution of feedback-inhibited β/α barrel isoenzymes by gene duplication and a single mutation
Authors: Hartmann Markus , Schneider Thomas R. , Pfeil Andrea , Heinrich Gabriele , Lipscomb William N. , Braus Gerhard H.
Published: February 2003

The β/α barrel is the common protein fold of numerous enzymes and was proposed recently to be the result of gene duplication and fusion of an ancient half-barrel. The initial enzyme of shikimate biosynthesis possesses the additional feature of feedback regulation. The crystal structure and kinetic studies on chimera and mutant proteins of yeast 3-deoxy-
d
-arabino-heptulosonate-7-phosphate (DAHP) synthase from
Saccharomyces cerevisiae
inhibited by phenylalanine (Aro3p) and DAHP synthase
S. cerevisiae
inhibited by tyrosine (Aro4p) give insight into important regions for regulation in the enzyme: The loop, which is connecting the two half-barrels, and structural elements added to the barrel are prerequisites for regulation and form a cavity on the N-terminal side of the β/α barrel. In the cavity of Aro4p at position 226 is a glycine residue, which is highly conserved in all other tyrosine-regulated DAHP synthases as well. Sequence alignments with phenylalanine-regulated DAHP synthases including Aro3p show a highly conserved serine residue at this position. An exchange of glycine to serine and vice versa leads to a complete change in the regulation pattern. Therefore the evolution of these differently feedback-inhibited isoenzymes required gene duplication and a single mutation within the internal extra element. Numerous additional amino acid substitutions present in the contemporary isoenzymes are irrelevant for regulation and occurred independently.
Mechanistic studies of β-arylsulfotransferase IV
Authors: Chapman Eli , Bryan Marian C. , Wong Chi-Huey
Published: February 2003

Sulfotransferases are an important class of enzymes that catalyze the transfer of a sulfuryl group to a hydroxyl or amine moiety on various molecules including small-molecule drugs, steroids, hormones, carbohydrates, and proteins. They have been implicated in a number of disease states but remain poorly understood, complicating the design of specific, small-molecule inhibitors. A linear free-energy analysis in both the forward and reverse directions indicates that the transfer of a sulfuryl group to an aryl hydroxyl group catalyzed by β-arylsulfotransferase IV likely proceeds by a dissociative (sulfotrioxide-like) mechanism. Values for the Brønsted coefficients (β
nuc
and β
lg
) are +0.33 and −0.45, giving Leffler α values of 0.19 and 0.61 for the forward and reverse reactions, respectively.