bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Toll-Like Receptor Signaling in the Apoptotic Response of Macrophages toYersiniaInfection
Authors: Zhang Yue , Bliska James B.
Published: February 2003

Macrophages encode several Toll-like receptors (TLRs) that recognize bacterial components, such as lipoproteins (TLR2) or lipopolysaccharides (TLR4), and activate multiple signaling pathways. Activation of transcription factor NF-κB by TLR2 or TLR4 signaling promotes proinflammatory and cell survival responses. Alternatively, TLR2 or TLR4 signaling can promote apoptosis if the activation of NF-κB is blocked. The gram-negative bacterial pathogenYersinia pseudotuberculosissecretes into macrophages a protease (YopJ) that inhibits the activation of NF-κB and promotes apoptosis. We show that primary macrophages expressing constitutively active inhibitor κB kinase β (IKKβ) are completely resistant to YopJ-dependent apoptosis, indicating that YopJ inhibits signaling upstream of IKKβ. Apoptosis is reduced two- to threefold in TLR4−/−macrophages infected withY. pseudotuberculosis, while the apoptotic response of TLR2−/−macrophages toY. pseudotuberculosisinfection is equivalent to that of wild-type macrophages. Therefore, TLR4 is the primary source of apoptotic signaling inYersinia-infected macrophages. Our results also show that a small percentage of macrophages can die as a result of an apoptotic process that is YopJ dependent but does not require TLR2 or TLR4 signaling.
Effects of Ectopically Expressed Neuronal Wiskott-Aldrich Syndrome Protein Domains onRickettsia rickettsiiActin-Based Motility
Authors: Harlander Ronald S. , Way Michael , Ren Qun , Howe Dale , Grieshaber Scott S. , Heinzen Robert A.
Published: February 2003

Neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein 2/3 (Arp2/3) complex have emerged as critical host proteins that regulate pathogen actin-based motility. Actin tail formation and motility inListeria monocytogenesrequire the Arp2/3 complex but bypasses N-WASP signaling. Motility ofShigella flexneriand vaccinia virus requires both N-WASP and the Arp2/3 complex. Functional roles for these cytoskeletal regulatory proteins in actin-based motility ofRickettsia rickettsiihave not been established. In this study, functional domains of N-WASP tagged with green fluorescent protein that have characterized effects onShigellaand vaccinia virus actin-based motility were ectopically expressed in HeLa cells infected withR. rickettsiito assess their effects on rickettsial motility.S. flexneri-infected cells were used as a control. Expressed N-WASP domains did not localize toR. rickettsiior their actin tails. Expression of N-WASP missing the VCA domain (for “verprolin homology, cofilin homology, and acidic domains”), which acts as a dominant-negative form of N-WASP, completely inhibited actin-based motility ofS. flexneriwhile only moderately inhibiting motility ofR. rickettsii. Similarly, expression of the VCA domain, which acts as a dominant-negative with respect to Arp2/3 complex function, severely inhibited actin-based motility ofS. flexneri(no motility observed in the majority of expressing cells) but only moderately inhibitedR. rickettsiimotility. These results, taken together with the differential effects on motility observed upon expression of other N-WASP domains, suggest that actin-based motility ofR. rickettsiiis independent of N-WASP and the Arp2/3 complex.
Studies of the Mechanism of Action of the Aerolysin-Like Hemolysin of
Aeromonas sobria
in Stimulating T84 Cells To Produce Cyclic AMP
Authors: Fujii Yoshio , Nomura Tomohiko , Yokoyama Ritsuko , Shinoda Sumio , Okamoto Keinosuke
Published: February 2003

We previously reported that the aerolysin-like hemolysin of
Aeromonas sobria
stimulates T84 cells to produce cyclic AMP, which then emerges in the culture medium. In order to clarify the mechanism of action of the hemolysin, we examined the involvement of adenosine nucleotide. The results show that the hemolysin stimulates T84 cells to release ATP, which is then converted to adenosine by ectonucleotidase. The adenosine generated might stimulate the P1 adenosine receptors of T84 cells to produce cyclic AMP.
Expression of Variant Surface Antigens by
Plasmodium falciparum
Parasites in the Peripheral Blood of Clinically Immune Pregnant Women Indicates Ongoing Placental Infection
Authors: Ofori Michael F. , Staalsoe Trine , Bam Victoria , Lundquist Maja , David Kim P. , Browne Edmund N. L. , Akanmori Bartholomew D. , Hviid Lars
Published: February 2003

Placenta-sequestered
Plasmodium falciparum
parasites that cause pregnancy-associated malaria (PAM) in otherwise clinically immune women express distinct variant surface antigens (VSA
PAM
) not expressed by parasites in nonpregnant individuals. We report here that parasites from the peripheral blood of clinically immune pregnant women also express VSA
PAM
, making them a convenient source of VSA
PAM
expressors for PAM vaccine research.
Interleukin-12 Regulates Chemokine Gene Expression during the Early Immune Response toLeishmania major
Authors: Zaph Colby , Scott Phillip
Published: February 2003

Following infection withLeishmania major, the chemokines XCL1, CXCL10, and CCL2 were preferentially expressed in draining lymph nodes of resistant mice. Neutralization of interleukin 12 (IL-12) or gamma interferon in resistant mice resulted in decreased chemokine expression, while administration of IL-12 to susceptible mice resulted in an increase in the level of chemokine gene expression.
DsbA of
Pseudomonas aeruginosa
Is Essential for Multiple Virulence Factors
Authors: Ha Un-Hwan , Wang Yanping , Jin Shouguang
Published: February 2003

DsbA is a periplasmic thiol:disulfide oxidoreductase which contributes to the process of protein folding by catalyzing the formation of disulfide bonds. In this study, we demonstrate that the
dsbA
gene is required for the expression of the type III secretion system under low-calcium inducing conditions, intracellular survival of
P. aeruginosa
upon infection of HeLa cells, and twitching motility. The diverse phenotypes of the
dsbA
mutant are likely due to its defect in the folding of proteins that are involved in various biological processes, such as signal sensing, protein secretion, and defense against host clearing. In light of its effect on various virulence factors, DsbA could be an important target for the control of
P. aeruginosa
infections.
Effect of Vaccination with Refined Components of the Organism on Infection of Mice with
Mycobacterium leprae
Authors: Ngamying Maeya , Sawanpanyalert Pathom , Butraporn Raywadee , Nikasri Junjira , Cho Sang-Nae , Levy Louis , Brennan Patrick J.
Published: February 2003

Only native products of
Mycobacterium leprae
, whether cell wall, cytosol, or membrane derived, can confer protective immunity against challenge in the mouse footpad. Previously, recombinant proteins were shown to be ineffective. The cell wall skeleton—the mycolyl-arabinogalactan-peptidoglycan complex—devoid of proteins is not protective.
CheZ-Mediated Dephosphorylation of the
Escherichia coli
Chemotaxis Response Regulator CheY: Role for CheY Glutamate 89
Authors: Silversmith Ruth E. , Guanga Gerald P. , Betts Laurie , Chu Carolyn , Zhao Rui , Bourret Robert B.
Published: February 2003

The swimming behavior of
Escherichia coli
at any moment is dictated by the intracellular concentration of the phosphorylated form of the chemotaxis response regulator CheY, which binds to the base of the flagellar motor. CheY is phosphorylated on Asp57 by the sensor kinase CheA and dephosphorylated by CheZ. Previous work (Silversmith et al., J. Biol. Chem. 276:18478, 2001) demonstrated that replacement of CheY Asn59 with arginine resulted in extreme resistance to dephosphorylation by CheZ despite proficient binding to CheZ. Here we present the X-ray crystal structure of CheYN59R in a complex with Mn
2+
and the stable phosphoryl analogue BeF
3
−
. The overall folding and active site architecture are nearly identical to those of the analogous complex containing wild-type CheY. The notable exception is the introduction of a salt bridge between Arg59 (on the β3α3 loop) and Glu89 (on the β4α4 loop). Modeling this structure into the (CheY-BeF
3
−
-Mg
2+
)
2
CheZ
2
structure demonstrated that the conformation of Arg59 should not obstruct entry of the CheZ catalytic residue Gln147 into the active site of CheY, eliminating steric interference as a mechanism for CheZ resistance. However, both CheYE89A and CheYE89Q, like CheYN59R, conferred clockwise flagellar rotation phenotypes in strains which lacked wild-type CheY and displayed considerable (≈40-fold) resistance to dephosphorylation by CheZ. CheYE89A and CheYE89Q had autophosphorylation and autodephosphorylation properties similar to those of wild-type CheY and could bind to CheZ with wild-type affinity. Therefore, removal of Glu89 resulted specifically in CheZ resistance, suggesting that CheY Glu89 plays a role in CheZ-mediated dephosphorylation. The CheZ resistance of CheYN59R can thus be largely explained by the formation of the salt bridge between Arg59 and Glu89, which prevents Glu89 from executing its role in catalysis.
A Two-Component Regulator of Universal Stress Protein Expression and Adaptation to Oxygen Starvation inMycobacterium smegmatis
Authors: O'Toole Ronan , Smeulders Marjan J. , Blokpoel Marian C. , Kay Emily J. , Lougheed Kathryn , Williams Huw D.
Published: February 2003

We identified a response regulator inMycobacterium smegmatiswhich plays an important role in adaptation to oxygen-starved stationary phase. The regulator exhibits strong sequence similarity to DevR/Rv3133c ofM. tuberculosis. The structural gene is present on a multigene locus, which also encodes a sensor kinase. AdevRmutant ofM. smegmatiswas adept at surviving growth arrest initiated by either carbon or nitrogen starvation. However, its culturability decreased several orders of magnitude below that of the wild type under oxygen-starved stationary-phase conditions. Two-dimensional gel analysis revealed that a number of oxygen starvation-inducible proteins were not expressed in thedevRmutant. Three of these proteins are universal stress proteins, one of which is encoded directly upstream ofdevR. Another protein closely resembles a proposed nitroreductase, while a fifth protein corresponds to the α-crystallin (HspX) orthologue ofM. smegmatis. None of the three universal stress proteins or nitroreductase, and a considerably lower amount of HspX was detected in carbon-starved wild-type cultures. A fusion of thehspXpromoter togfpdemonstrated that DevR directs gene expression whenM. smegmatisenters stationary phase brought about, in particular, by oxygen starvation. To our knowledge, this is the first time a role for a two-component response regulator in the control of universal stress protein expression has been shown. Notably, thedevRmutant was 104-fold more sensitive than wild type to heat stress. We conclude that DevR is a stationary-phase regulator required for adaptation to oxygen starvation and resistance to heat stress inM. smegmatis.
Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in
Lactococcus lactis
MG1363
Authors: Solem Christian , Koebmann Brian J. , Jensen Peter R.
Published: February 2003

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has previously been suggested to have almost absolute control over the glycolytic flux in
Lactococcus lactis
(B. Poolman, B. Bosman, J. Kiers, and W. N. Konings, J. Bacteriol. 169:5887-5890, 1987). Those studies were based on inhibitor titrations with iodoacetate, which specifically inhibits GAPDH, and the data suggested that it should be possible to increase the glycolytic flux by overproducing GAPDH activity. To test this hypothesis, we constructed a series of mutants with GAPDH activities from 14 to 210% of that of the reference strain MG1363. We found that the glycolytic flux was unchanged in the mutants overproducing GAPDH. Also, a decrease in the GAPDH activity had very little effect on the growth rate and the glycolytic flux until 25% activity was reached. Below this activity level, the glycolytic flux decreased proportionally with decreasing GAPDH activity. These data show that GAPDH activity has no control over the glycolytic flux (flux control coefficient = 0.0) at the wild-type enzyme level and that the enzyme is present in excess capacity by a factor of 3 to 4. The early experiments by Poolman and coworkers were performed with cells resuspended in buffer, i.e., nongrowing cells, and we therefore analyzed the control by GAPDH under similar conditions. We found that the glycolytic flux in resting cells was even more insensitive to changes in the GAPDH activity; in this case GAPDH was also present in a large excess and had no control over the glycolytic flux.
Multiple Light Inputs Control Phototaxis in
Synechocystis
sp. Strain PCC6803
Authors: Ng Wing-On , Grossman Arthur R. , Bhaya Devaki
Published: February 2003

The phototactic behavior of individual cells of the cyanobacterium
Synechocystis
sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a
taxD1
mutant (Cyanobase accession no.
sll0041
; also called
pisJ1
) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A Δ
taxD1
mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in
Synechocystis
sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.
Chromosomal Expression of the
Haemophilus influenzae
Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis
Authors: Fink Doran L. , St. Geme Joseph W.
Published: February 2003

The
Haemophilus influenzae
Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation. Hap consists of a 45-kDa outer membrane translocator domain called Hap
β
and a 110-kDa extracellular passenger domain called Hap
S
. All adhesive activity resides within Hap
S
, which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis. In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation. We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in
H. influenzae
clinical isolates. Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis. In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis. These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap.
Structural Model for 12-Helix Transporters Belonging to the Major Facilitator Superfamily
Authors: Hirai Teruhisa , Heymann Jürgen A. W. , Maloney Peter C. , Subramaniam Sriram
Published: February 2003

The major facilitator superfamily includes a large collection of evolutionarily related proteins that have been implicated in the transport of a variety of solutes and metabolites across the membranes of organisms ranging from bacteria to humans. We have recently reported the three-dimensional structure, at 6.5 Å resolution, of the oxalate transporter, OxlT, a representative member of this superfamily. In the oxalate-bound state, 12 helices surround a central cavity to form a remarkably symmetrical structure that displays a well-defined pseudo twofold axis perpendicular to the plane of the membrane as well as two less pronounced, mutually perpendicular pseudo twofold axes in the plane of the membrane. Here, we combined this structural information with sequence information from other members of this protein family to arrive at models for the arrangement of helices in this superfamily of transport proteins. Our analysis narrows down the number of helix arrangements from about a billion starting possibilities to a single probable model for the relative spatial arrangement for the 12 helices, consistent both with our structural findings and with the majority of previous biochemical studies on members of this superfamily.
AUTOMATIC TEST DATA GENERATION FOR PROGRAM PATHS USING GENETIC ALGORITHMS
Authors: BUENO PAULO MARCOS SIQUEIRA , JINO MARIO
Published: February 2003

A new technique and tool are presented for test data generation for path testing. They are based on the dynamic technique and on a Genetic Algorithm, which evolves a population of input data towards reaching and solving the predicates along the program paths. We improve the performance of test data generation by using past input data to compose the initial population for the search. An experiment was done to assess the performance of the techniques compared to that of random data generation.
WHAT INFORMATION IS RELEVANT WHEN SELECTING SOFTWARE TESTING TECHNIQUES?
Authors: VEGAS SIRA , JURISTO NATALIA , BASILI VICTOR
Published: February 2003

One of the main problems in software testing is the development of a suitable set of test cases so that the effectiveness of the test is maximised with a minimum number of test cases. A lot of testing techniques are now available for developing test cases. However, some of them are misused, others are never used and only a few are applied again and again. When developers have to decide what testing techniques(s) they should use in a project, they have little (if any) experiential information about the available testing techniques, their usefulness and, in general, how suited they are to the project. This paper presents the results of developing a characterization scheme for test technique selection. When instantiated for different techniques, the scheme should provide developers with enough information for choosing the best suited to their project. Thus, their decisions would be based on sound knowledge of the techniques, instead of perceptions, suppositions and assumptions.
The Power and Prospects of Fluorescence Microscopies and Spectroscopies
Authors: Michalet Xavier , Kapanidis Achillefs N. , Laurence Ted , Pinaud Fabien , Doose Soeren , Pflughoefft Malte , Weiss Shimon
Published: February 2003

▪ Abstract  Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.
NEW INSIGHTS INTO THE DIVERSITY AND FUNCTION OF NEURONAL IMMUNOGLOBULIN SUPERFAMILY MOLECULES
Authors: Rougon Geneviève , Hobert Oliver
Published: February 2003

▪ Abstract  Immunoglobulin superfamily (IgSF) proteins are implicated in diverse steps of brain development, including neuronal migration, axon pathfinding, target recognition and synapse formation, as well as in the maintenance and function of neuronal networks in the adult. We provide here a review of recent findings on the diversity and the role of transmembrane and secreted members of IgSF proteins in the nervous system. We illustrate that the complexity of IgSF protein function results from various different levels of regulation including regulation of gene expression, protein localization, and protein interactions.
Spironolactone and Its Main Metabolite, Canrenoic Acid, Block Human Ether-a-Go-Go–Related Gene Channels
Authors: Caballero Ricardo , Moreno Ignacio , González Teresa , Arias Cristina , Valenzuela Carmen , Delpón Eva , Tamargo Juan
Published: February 2003

Background—

It has been demonstrated that spironolactone (SP) decreases the QT dispersion in chronic heart failure. In this study, the effects of SP and its metabolite, canrenoic acid (CA), on human ether-a-go-go–related gene (HERG) currents were analyzed.

Methods and Results—

HERG currents elicited in stably transfected Chinese hamster ovary cells were measured with the whole-cell patch-clamp technique. SP decreased HERG currents in a concentration-dependent manner (IC
50
=23.0±1.5 μmol/L) and shifted the midpoint of the activation curve to more negative potentials (Vh=−13.1±3.4 versus −18.9±3.6 mV,
P
<0.05) without modifying the activation and deactivation kinetics. SP-induced block (1 μmol/L) appeared at the range of membrane potentials coinciding with that of channel activation, and thereafter, it remained constant, reaching 24.7±3.8% at +60 mV (n=6,
P
<0.05). CA (0.01 nmol/L to 500 μmol/L) blocked HERG channels in a voltage- and frequency-independent manner. CA at 1 nmol/L shifted the midpoint of the activation curve to −19.9±1.8 mV and accelerated the time course of channel activation (τ=1064±125 versus 820±93 ms, n=11,
P
<0.01). The envelope of the tail test demonstrated that at the very beginning of the pulses to +40 mV (25 ms), a certain amount of block was apparent (31.3±9.9%). CA did not modify the voltage-dependence of HERG channel inactivation (Vh=−60.8±5.6 versus −62.9±3.1 mV, n=6,
P
>0.05) or the kinetics of the reactivation process at any potential tested. CA and aldosterone also blocked the native
I
Kr
in guinea-pig ventricular myocytes.

Conclusions—

At concentrations reached after administration of therapeutic doses of SP, CA blocked the HERG channels by binding to both the closed and open states of the channel.
Induction of Coxsackievirus-Adenovirus–Receptor Expression During Myocardial Tissue Formation and Remodeling
Authors: Fechner Henry , Noutsias Michel , Tschoepe Carsten , Hinze Kerstin , Wang Xiaomin , Escher Felicitas , Pauschinger Matthias , Dekkers Dick , Vetter Roland , Paul Martin , Lamers Jos , Schultheiss Heinz-Peter , Poller Wolfgang
Published: February 2003

Background—

The coxsackievirus-adenovirus receptor (CAR) was cloned as a receptor for both viruses, but its primary biological functions and regulatory mechanisms are unknown. CAR was low in healthy adult myocardium, whereas strong CAR reexpression was observed in human dilated cardiomyopathy. The molecular mechanisms of CAR induction in cardiomyocytes are unknown.

Methods and Results—

We report on CAR regulation during development, CAR induction after myocardial infarction, and cell-to-cell contact–dependent CAR regulation in the rat. The high CAR expression during development in various organs decreased up to 190-fold after birth. After infarction resulting in severe cardiac dysfunction (dP/dt
max
, −53%; dP/dt
min
, −58%; left ventricular pressure, −45%), CAR was induced locally in cardiomyocytes of the infarct zone, where it was also expressed by capillary-like CD31
+
structures and CD18
+
interstitial cells, whereas it remained confined to subendothelial layers of arterioles and venules. In cultured cardiomyocytes, endothelin-1, cardiotrophin-1, leukemia-inhibiting factor, and cyclic stretch had no effect on CAR, whereas at high versus low cell density, CAR was suppressed up to 10-fold (
P
=0.006). Conditioned media from low- or high-density cardiomyocytes or cardiofibroblasts had no effect.

Conclusions—

The locally confined CAR upregulation after infarction makes induction by various humoral factors unlikely, because cardiac dysfunction results in high activities of sympathetic and renin-angiotensin systems and cytokines. The cell culture experiments identify a cell-to-cell contact–dependent mechanism of CAR regulation. Further characterization of the signals linking cell-to-cell interactions to CAR gene expression may provide insight into mechanisms and functional consequences of the generalized CAR induction in dilated cardiomyopathy, and of its local induction after myocardial infarction.
Rapid Stimulation of
l
-Arginine Transport by
d
-Glucose Involves p42/44
mapk
and Nitric Oxide in Human Umbilical Vein Endothelium
Authors: Flores Carlos , Rojas Susana , Aguayo Claudio , Parodi Jorge , Mann Giovanni , Pearson Jeremy D. , Casanello Paola , Sobrevia Luis
Published: February 2003

d
-Glucose infusion and gestational diabetes induce vasodilatation in humans and increase
l
-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells. High
d
-glucose (25 mmol/L, 2 minutes) induced membrane hyperpolarization and an increase of
l
-arginine transport (V
max
6.1±0.7 versus 4.4±0.1 pmol/μg protein per minute) with no change in transport affinity (
K
m
105±9 versus 111±16 μmol/L).
l
-[
3
H]Citrulline formation and intracellular cGMP, but not intracellular Ca
2+
, were increased by high
d
-glucose. The effects of
d
-glucose were mimicked by levcromakalim (ATP-sensitive K
+
channel blocker), paralleled by p42/p44
mapk
and Ser
1177
–endothelial NO synthase phosphorylation, inhibited by
N
G
-nitro-
l
-arginine methyl ester (L-NAME; NO synthesis inhibitor), glibenclamide (ATP-sensitive K
+
channel blocker), KT-5823 (protein kinase G inhibitor), PD-98059 (mitogen-activated protein kinase kinase 1/2 inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor), but they were unaffected by calphostin C (protein kinase C inhibitor). Elevated
d
-glucose did not alter superoxide dismutase activity. Our findings demonstrate that the human fetal endothelial
l
-arginine/NO signaling pathway is rapidly activated by elevated
d
-glucose via NO and p42/44
mapk
. This could be determinant in pathologies in which rapid fluctuations of plasma
d
-glucose may occur and may underlie the reported vasodilatation in early stages of diabetes mellitus.
Hibernating Myocardium Retains Metabolic and Contractile Reserve Despite Regional Reductions in Flow, Function, and Oxygen Consumption at Rest
Authors: Fallavollita James A. , Malm Brian J. , Canty John M.
Published: February 2003

Hibernating myocardium, characterized by reductions in flow and function at rest, has limited contractile reserve in response to increases in external workload. We hypothesized that this attenuation of function reflects an adaptive downregulation that prevents the development of metabolic evidence of ischemia during stress. To test this hypothesis, pigs were chronically instrumented with a proximal left anterior descending artery stenosis for 3 months, resulting in severe anteroapical hypokinesis with reduced resting perfusion (0.78±0.05 versus 0.94±0.07 mL · min
−1
· g
−1
in remote,
P
<0.01; and 0.99±0.08 in controls,
P
<0.05). Open-chest studies confirmed resting dysfunction compared with normal controls (segment shortening 9.2±2.2% versus 23.5±1.1%,
P
<0.05). Resting myocardial oxygen consumption was reduced (63±3 versus 77±6 μL · g
−1
· min
−1
in controls,
P
<0.05), yet lactate consumption was normal. Although subendocardial perfusion failed to increase during graded, intravenous epinephrine infusion (n=8), peak segment shortening (to 17.3±3.1%,
P
<0.05) and oxygen consumption (to 90±6 μL · g
−1
· min
−1
,
P
<0.01) increased from the depressed resting levels. There was no lactate production in hibernating myocardium, and lactate uptake increased during stress (0.7±0.1 to 1.2±0.1 μmol · g
−1
· min
−1
,
P
<0.05). The absence of metabolic evidence of ischemia was also confirmed during atrial pacing to a rate of 120 bpm (n=8). Thus, despite reductions in function and oxygen consumption at rest, hibernating myocardium retains the ability to increase metabolism without the development of acute ischemia. This supports the hypothesis that the downregulation of oxygen consumption and function in hibernating myocardium is an adaptive response that prevents a supply-demand imbalance during submaximal increases in cardiac workload when coronary flow reserve is limited.
A Cardiac Sodium Channel Mutation Cosegregates With a Rare Connexin40 Genotype in Familial Atrial Standstill
Authors: Groenewegen W. Antoinette , Firouzi Mehran , Bezzina Connie R. , Vliex Saskia , van Langen Irene M. , Sandkuijl Lodewijk , Smits Jeroen P.P. , Hulsbeek Miriam , Rook Martin B. , Jongsma Habo J. , Wilde Arthur A.M.
Published: February 2003

Atrial standstill (AS) is a rare arrhythmia that occasionally appears to be genetically determined. This study investigates the genetic background of this arrhythmogenic disorder in a large family. Forty-four family members were clinically evaluated. One deceased and three living relatives were unambiguously affected by AS. All other relatives appeared unaffected. Candidate gene screening revealed a novel mutation in the cardiac sodium channel gene SCN5A (D1275N) in all three affected living relatives and in five unaffected relatives, and the deceased relative was an obligate carrier. In addition, two closely linked polymorphisms were detected within regulatory regions of the gene for the atrial-specific gap junction protein connexin40 (Cx40) at nucleotides −44 (G→A) and +71 (A→G). Eight relatives were homozygous for both polymorphisms, which occurred in only ≈7% of control subjects, and three of these relatives were affected by AS. The three living AS patients exclusively coinherited both the rare Cx40 genotype and the SCN5A-D1275N mutation. SCN5A-D1275N channels showed a small depolarizing shift in activation compared with wild-type channels. Rare Cx40 genotype reporter gene analysis showed a reduction in reporter gene expression compared with the more common Cx40 genotype. In this study, familial AS was associated with the concurrence of a cardiac sodium channel mutation and rare polymorphisms in the atrial-specific Cx40 gene. We propose that, although the functional effect of each genetic change is relatively benign, the combined effect of genetic changes eventually progresses to total AS.
Spatiotemporal Characteristics of Junctional and Nonjunctional Focal Ca
2+
Release in Rat Atrial Myocytes
Authors: Woo Sun-Hee , Cleemann Lars , Morad Martin
Published: February 2003

Atrial myocytes have two functionally separate Ca
2+
release sites: those in peripheral sarcoplasmic reticulum (SR) adjacent to the Ca
2+
channels of surface membrane and those in central SR not associated with Ca
2+
channels. Recently, we have reported on the gating of these two different Ca
2+
release sites by Ca
2+
current. In the present study, we report on the spatiotemporal properties of focal Ca
2+
releases (sparks) occurring spontaneously in central and peripheral sites of voltage-clamped rat atrial myocytes, using rapid 2-dimensional (2-D) confocal Ca
2+
imaging. Peripheral and central sparks were similar in size and release time (≈300 000 Ca
2+
ions for ≅12 ms), but significantly larger and longer than ventricular sparks. Both sites were resistant to Cd
2+
and inhibited by ryanodine. Peripheral sparks were brighter and flattened against surface membrane, had ≈5-fold higher frequency, ≈2 times faster diffusion coefficient, and dissipated abruptly. Central sparks, in contrast, occurred less frequently, were elongated along the cellular longitudinal axis, and dissipated slowly. Compound sparks (composed of 2 to 5 unitary focal releases) aligned longitudinally and occurred more frequently at the center. The diversity of peripheral and central sparks with respect to shape, frequency, and speed of spatial development and decay is consistent with regional ultrastructural heterogeneity of SR. The retarded dissipation of central atrial sparks, and high prevalence of compound sparks in cell center may be critical in facilitating the propagation of Ca
2+
waves in atrial myocytes lacking t-tubular system and provide the atrial myocytes with functional Ca
2+
signaling diversity. The full text of this article is available at http://www.circresaha.org.
High-Density Lipoproteins Protect Isolated Rat Hearts From Ischemia-Reperfusion Injury by Reducing Cardiac Tumor Necrosis Factor-α Content and Enhancing Prostaglandin Release
Authors: Calabresi Laura , Rossoni Giuseppe , Gomaraschi Monica , Sisto Francesca , Berti Ferruccio , Franceschini Guido
Published: February 2003

The incidence and severity of primary cardiac events are inversely related to the plasma concentration of high-density lipoproteins (HDLs). We investigated whether HDLs may exert a direct cardioprotection in buffer-perfused isolated rat hearts, which underwent a 20-minute low-flow ischemia followed by a 30-minute reperfusion. The administration of HDLs at physiological concentrations (0.5 and 1.0 mg/mL) during the 10 minutes immediately before ischemia rapidly and remarkably improved postischemic functional recovery and decreased creatine kinase release in the coronary effluent. Reconstituted HDLs containing apolipoprotein A-I (apoA-I) and phosphatidylcholine, but not lipid-free apoA-I or phosphatidylcholine liposomes, were also effective in protecting the heart from ischemia-reperfusion injury. HDLs at reperfusion were less effective than when given before ischemia. HDLs caused a dose-dependent reduction of ischemia-induced cardiac tumor necrosis factor-α (TNF-α) expression and content, which correlated with the improved functional recovery. A parallel increase of TNF-α release in the coronary effluent was observed, due to a direct binding of cardiac TNF-α to HDLs. Taken together, these findings argue for a cause-effect relationship between the HDL-mediated removal of TNF-α from the ischemic myocardium and the HDL-induced cardioprotection. Indeed, etanercept, a recombinant TNF-α–blocking protein, caused a dose-dependent improvement of postischemic functional recovery. HDLs also enhanced ischemia-induced prostaglandin release, which may contribute to the cardioprotective effect. A low plasma HDL level may expose the heart to excessive ischemia-reperfusion damage, and HDL-targeted therapies may be helpful to induce immediate or delayed myocardial protection from ischemia-reperfusion injury.
Depletion of Intracellular Ca
2+
Stores Sensitizes the Flow-Induced Ca
2+
Influx in Rat Endothelial Cells
Authors: Kwan Hiu-Yee , Leung Pan-Cheung , Huang Yu , Yao Xiaoqiang
Published: February 2003

Hemodynamic shear stress elicits a rise in endothelial [Ca
2+
]
i
, which may serve as a key second messenger to regulate many flow-associated physiological and biochemical processes. In the present study, we used Mn
2+
quenching of fluorescent dye Fluo3 as an assay to investigate the Ca
2+
influx of rat aortic endothelial cells in response to flow. We found that the Ca
2+
signaling in response to flow could be greatly influenced by the status of intracellular Ca
2+
stores. Depletion of intracellular Ca
2+
stores by thapsigargin (4 μmol/L) or cyclopiazonic acid (10 μmol/L) drastically sensitized the Ca
2+
influx in response to flow. Ca
2+
-mobilizing agonist bradykinin (100 nmol/L) or ATP (100 μmol/L) had similar sensitizing effect. The effect of bradykinin or ATP was blocked by
Xestospongin C
and U73122, suggesting that the sensitization was related to the IP
3
-mediated store depletion. On the other hand, the Mn
2+
quenching in response to flow was greatly reduced by ochratoxin A (100 nmol/L), an agent that could increase the filling state of intracellular Ca
2+
stores. In addition, we found that depletion-sensitized Ca
2+
influx in response to flow was mediated by a PKG-inhibitable cation channel and that the influx was affected by membrane potential and K
+
channel activity. In conclusion, the present study argues for a critical role of intracellular Ca
2+
status in determining the Ca
2+
signaling in response to flow and it provides a general mechanistic explanation for the stimulatory role of blood-borne agonists on flow-induced Ca
2+
influx.
Sarcoplasmic Reticulum Ca
2+
Release Is Not a Dominating Factor in Sinoatrial Node Pacemaker Activity
Authors: Honjo H. , Inada S. , Lancaster M.K. , Yamamoto M. , Niwa R. , Jones S.A. , Shibata N. , Mitsui K. , Horiuchi T. , Kamiya K. , Kodama I. , Boyett M.R.
Published: February 2003

Recent work on isolated sinoatrial node cells from rabbit has suggested that sarcoplasmic reticulum Ca
2+
release plays a dominant role in the pacemaker potential, and ryanodine at a high concentration (30 μmol/L blocks sarcoplasmic reticulum Ca
2+
release) abolishes pacemaking and at a lower concentration abolishes the chronotropic effect of β-adrenergic stimulation. The aim of the present study was to test this hypothesis in the intact sinoatrial node of the rabbit. Spontaneous activity and the pattern of activation were recorded using a grid of 120 pairs of extracellular electrodes. Ryanodine 30 μmol/L did not abolish spontaneous activity or shift the position of the leading pacemaker site, although it slowed the spontaneous rate by 18.9±2.5% (n=6). After ryanodine treatment, β-adrenergic stimulation still resulted in a substantial chronotropic effect (0.3 μmol/L isoproterenol increased spontaneous rate by 52.6±10.5%, n=5). In isolated sinoatrial node cells from rabbit, 30 μmol/L ryanodine slowed spontaneous rate by 21.5±2.6% (n=13). It is concluded that sarcoplasmic reticulum Ca
2+
release does not play a dominating role in pacemaking in the sinoatrial node. The full text of this article is available at http://www.circresaha.org.
Conditional Expression of a Dominant-Negative c-Myb in Vascular Smooth Muscle Cells Inhibits Arterial Remodeling After Injury
Authors: You Xiao-Mang , Mungrue Imran N. , Kalair Waseem , Afroze Talat , Ravi Bheeshma , Sadi Al Muktafi , Gros Robert , Husain Mansoor
Published: February 2003

Inhibiting activity of the c-Myb transcription factor attenuates G
1
to S phase cell cycle transitions in vascular smooth muscle cells (SMCs) in vitro. To determine the effects of arterial SMC-specific expression of a dominant-negative c-Myb molecule (
Myb-Engrailed
) on vascular remodeling in vivo, we performed carotid artery wire-denudation in 2 independent lines of binary transgenic mice with SM22α promoter-defined Doxycycline-suppressible expression of
Myb-Engrailed
. Adult mice with arterial SMC-specific expression of
Myb-Engrailed
were overtly normal in appearance and did not display any changes in cardiovascular structure or physiology. However, bromodeoxyuridine-defined arterial SMC proliferation, neointima formation, medial hyperplasia, and arterial remodeling were markedly decreased in mice expressing arterial SMC-restricted
Myb-Engrailed
after arterial injury. These data suggest that c-Myb activity in arterial SMCs is not essential for arterial structure or function during development, but is involved in the proliferation of arterial SMCs as occurs in vascular pathology, and that the expression of a dominant-negative c-Myb can dramatically reduce adverse arterial remodeling in an in vivo model of restenosis. As such, this model represents a novel tissue-specific strategy for the potential gene therapy of diseases characterized by arterial SMC proliferation.
Divergent Roles of Angiotensin II AT
1
and AT
2
Receptors in Modulating Coronary Microvascular Function
Authors: Zhang Cuihua , Hein Travis W. , Wang Wei , Kuo Lih
Published: February 2003

Angiotensin II (Ang II) is a potent vasoconstrictor in the peripheral circulation and has been implicated in many cardiovascular diseases associated with elevated oxidative stress. However, its direct vasomotor action and its linkage to oxidative stress–induced vascular dysfunction in the coronary microcirculation remain elusive. In this study, we directly assessed the vasomotor action of Ang II in isolated porcine coronary arterioles and also examined whether Ang II can modulate endothelium-dependent nitric oxide (NO)-mediated dilation via superoxide production. Ang II evoked vasoconstriction at a low concentration (1 nmol/L) and dilations at higher concentrations (>10 nmol/L). Ang II type 1 (AT
1
) receptor antagonist losartan abolished vasoconstriction, whereas Ang II type 2 (AT
2
) receptor antagonist PD 123319 eliminated vasodilation. Adenosine stimulated a significant arteriolar NO production and dilation. NO synthase inhibitor
N
G
-monomethyl-
l
-arginine (L-NMMA) abolished stimulated NO production and attenuated vasodilation. Pretreating vessels with a subvasomotor concentration of Ang II (0.1 nmol/L, 60 minutes) mimicked inhibitory effects of L-NMMA. Ang II–mediated inhibition was not observed in the presence of L-NMMA or after endothelial removal but was prevented by losartan, superoxide scavenger TEMPOL, or NADPH oxidase inhibitor apocynin. Dihydroethidium staining showed that Ang II elicited losartan- and TEMPOL-sensitive superoxide production in arterioles. These results demonstrate that Ang II evokes AT
1
receptor–mediated vasoconstriction and AT
2
receptor–mediated vasodilation of coronary arterioles. Ang II at a subvasomotor level impairs endothelium-dependent NO-mediated dilation attributable to elevated superoxide production via AT
1
receptor activation of NADPH oxidase. These data may partly explain the impaired coronary flow regulation in heart diseases associated with an upregulated renin-angiotensin system.
Differential Actions of PAR
2
and PAR
1
in Stimulating Human Endothelial Cell Exocytosis and Permeability
Authors: Klarenbach Scott W. , Chipiuk Adam , Nelson Randy C. , Hollenberg Morley D. , Murray Allan G.
Published: February 2003

Endothelial cell proteinase activated receptors (PARs) belong to a family of heterotrimeric G protein-coupled receptors that are implicated in leukocyte accumulation and potentiation of reperfusion injury. We characterized the effect and the signal transduction pathways recruited after stimulation of endothelial PAR
2
. We used von Willebrand Factor (vWF) release and monolayer permeability to peroxidase to report Weibel-Palade body (WPB) exocytosis and pore formation, respectively. Human umbilical vein endothelial cells (HUVECs) were stimulated with the selective PAR
2
agonist peptide SLIGRL-NH
2
or PAR
1
agonist peptide TFLLR-NH
2
. PAR
2
stimulation resulted in WPB exocytosis like PAR
1
stimulation but, unlike PAR
1
, failed to increase monolayer permeability. BAPTA-AM inhibited PAR
2
-induced exocytosis, indicating a PAR
2
calcium-dependent signal in ECs. Moreover, PAR
2
-like PAR
1
-stimulated exocytosis requires actin cytoskeleton remodeling, because vWF release is inhibited if the cells were pretreated with Jasplakinolide. Rho-GTPase activity is required for PAR-stimulated exocytosis, because inactivation of this family of actin-regulatory proteins with
Clostridium difficile
toxin B blocked exocytosis. Expression of dominant-negative mutant Cdc42
17N
inhibited exocytosis whereas neither dominant-negative Rac
17N
expression nor C3 exotoxin treatment affected vWF release. PAR
2
stimulated RhoA-GTP weakly compared with the PAR
1
agonist. We conclude that both PAR
2
and PAR
1
elicit WP body exocytosis in a calcium and Cdc42 GTPase-dependent manner. In contrast, the differential effect of PAR
1
versus PAR
2
activation to increase monolayer permeability correlates with weak RhoA activation by the PAR
2
agonist.
Mechanism of Pacemaking in
I
K1
-Downregulated Myocytes
Authors: Silva Jonathan , Rudy Yoram
Published: February 2003

Biological pacemakers were recently created by genetic suppression of inward rectifier potassium current,
I
K1
, in guinea pig ventricular cells. We simulated these cells by adjusting
I
K1
conductance in the Luo-Rudy model of the guinea pig ventricular myocyte. After 81%
I
K1
suppression, the simulated cell reached steady state with pacemaker period of 594 ms. Pacemaking current is carried by the Na
+
-Ca
2+
exchanger,
I
NaCa
, which depends on the intracellular calcium concentration [Ca
2+
]
i
. This [Ca
2+
]
i
dependence suggests responsiveness (increase in rate) to β-adrenergic stimulation (βAS), as observed experimentally. Simulations of βAS demonstrate such responsiveness, which depends on
I
NaCa
expression. However, a simultaneous βAS-mediated increase in the slow delayed rectifier,
I
Ks
, limits βAS sensitivity.
VE-Based Training Strategies for Acquiring Survey Knowledge
Authors: Witmer Bob G. , Sadowski Wallace J. , Finkelstein Neal M.
Published: February 2003

Virtual environments (VEs) have been used successfully to train wayfinders to navigate through buildings and learn their layout. However, at the same time, for many, the VE deficiencies have reduced the effectiveness of VEs for training spatial tasks. In an effort to improve VE effectiveness, we conducted research to determine if certain unique capabilities of VEs could compensate for its deficiencies. Research participants were required to learn the layout or configuration of one floor of an office building as portrayed in a VE. To improve spatial learning, we developed three VE navigation training aids: local and global orientation cues, aerial views, and a themed environment enhanced with sights and sounds and divided into four distinct sectors. The navigation aids were provided during the training but were not available during testing of survey knowledge. Of the three training aids investigated, only the aerial views were effective in improving performance on the survey knowledge tests. The effectiveness of the navigation aids seemed to depend on how they were used during training. A retention test given one week after training indicated that spatial knowledge acquired in a VE diminished little over the one-week retention interval.
Perceived Orientation in Physical and Virtual Environments: Changes in Perceived Orientation as a Function of Idiothetic Information Available
Authors: Lathrop William B. , Kaiser Mary K.
Published: February 2003

Two experiments examined perceived spatial orientation in a small environment as a function of experiencing that environment under three conditions: real-world, desktop-display (DD), and head-mounted display (HMD). Across the three conditions, participants acquired two targets located on a perimeter surrounding them, and attempted to remember the relative locations of the targets. Subsequently, participants were tested on how accurately and consistently they could point in the remembered direction of a previously seen target. Results showed that participants were significantly more consistent in the real-world and HMD conditions than in the DD condition. Further, it is shown that the advantages observed in the HMD and real-world conditions were not simply due to nonspatial response strategies. These results suggest that the additional idiothetic information afforded in the realworld and HMD conditions is useful for orientation purposes in our presented task domain. Our results are relevant to interface design issues concerning tasks that require spatial search, navigation, and visualization.