bluuebunny/crossref_metadata_2025 huggingface dataset:
An Internally Located RNA Hairpin Enhances Replication of
Tomato Bushy Stunt Virus
RNAs
Authors: Ray Debashish , White K. Andrew
Published: December 2002

Defective interfering (DI) RNAs of
Tomato bushy stunt virus
(TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotide-long sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3′ terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientation-independent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5′-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5′-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.
The RNA Replication Enhancer Element of Tombusviruses Contains Two Interchangeable Hairpins That Are Functional during Plus-Strand Synthesis
Authors: Panavas T. , Nagy P. D.
Published: December 2002

Replication of the RNA genomes of tombusviruses, which are small plus-sense RNA viruses of plants, may be regulated by
cis
-acting elements, including promoters and replication enhancers that are present in the RNA templates. Using a partially purified RNA-dependent RNA polymerase (RdRp) preparation (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), we demonstrate that the minus-strand templates of tombusviruses contain a replication enhancer, which can upregulate RNA synthesis initiating from the minimal plus-strand initiation promoter by 10- to 20-fold in an in vitro assay. Dissection of the sequence of the replication enhancer element revealed that the two stem-loop structures present within the ∼80-nucleotide-long enhancer region have interchangeable roles in upregulating RNA synthesis. The single-stranded sequence located between the two stem-loops also plays an important role in stimulation of RNA synthesis. We also demonstrate that one of the two hairpins, both of which are similar to the hairpin of the minus-strand initiation promoter, can function as a promoter in vitro in the presence of short cytidylate-containing initiation sites. Overall, the in vitro data presented are consistent with previous in vivo results (D. Ray and K. A. White, Virology 256:162-171, 1999) and they firmly establish the presence of a replication enhancer on the minus-stranded RNA of tombusviruses.
Mutations in Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Causing Increased Receptor Binding Activity and Resistance to the Transition State Sialic Acid Analog 4-GU-DANA (Zanamivir)
Authors: Murrell Matthew , Porotto Matteo , Weber Thomas , Greengard Olga , Moscona Anne
Published: December 2002

Entry and fusion of human parainfluenza virus type 3 (HPF3) require the interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-GU-DANA, a potent inhibitor of influenza virus neuraminidase, inhibits not only HPF3 neuraminidase but also the receptor binding activity of HPF3 HN and thus its ability to promote attachment and fusion. We previously generated a 4-GU-DANA-resistant HPF3 virus variant (ZM1) with a markedly fusogenic plaque morphology that harbored two HN gene mutations resulting in amino acid alterations. The present study using cells that express the individual mutations of ZM1 HN shows that one of these mutations is responsible for the increases in receptor binding and neuraminidase activities as well as the diminished sensitivity of both activities to the inhibitory effect of 4-GU-DANA. To examine the hypothesis that increased receptor binding avidity underlies 4-GU-DANA resistance, parallel studies were carried out on the high-affinity HN variant virus C22 and cells expressing the C22 variant HN. This variant also exhibited reduced sensitivity to 4-GU-DANA in terms of receptor binding and infectivity but without concomitant changes in the neuraminidase activity of HN. Another high-affinity HN variant, C0, was not resistant in terms of infectivity; however, a small increase in the receptor binding activity of C0 HN and a partial resistance of this activity to 4-GU-DANA were revealed by sensitive methods that we developed. In each virus variant, one mutation in HN accounted for both increased receptor binding avidity and 4-GU-DANA resistance; the higher affinity for the receptor overcomes the inhibitory effect of 4-GU-DANA. Thus, in contrast to influenza viruses for which 4-GU-DANA escape variants include hemagglutinin mutants with decreased receptor binding avidity that promotes virion release, for HPF3, HN mutants with increased receptor binding avidity are those that can escape the growth inhibitory effect of 4-GU-DANA.
Role of Protein Kinase C βII in Influenza Virus Entry via Late Endosomes
Authors: Sieczkarski Sara B. , Brown H. Alex , Whittaker Gary R.
Published: December 2002

Many viruses take advantage of receptor-mediated endocytosis in order to enter target cells. We have utilized influenza virus and Semliki Forest virus (SFV) to define a role for protein kinase C βII (PKCβII) in endocytic trafficking. We show that specific PKC inhibitors prevent influenza virus infection, suggesting a role for classical isoforms of PKC. We also examined virus entry in cells overexpressing dominant-negative forms of PKCα and -β. Cells expressing a phosphorylation-deficient form of PKCβII (T500V), but not an equivalent mutant form of PKCα, inhibited successful influenza virus entry—with the virus accumulating in late endosomes. SFV, however, believed to enter cells from the early endosome, was unaffected by PKCβII T500V expression. We also examined the trafficking of two cellular ligands, transferrin and epidermal growth factor (EGF). PKCβII T500V expression specifically blocked EGF receptor trafficking and degradation, without affecting transferrin receptor recycling. As with influenza virus, in PKCβII kinase-dead cells, EGF receptor was trapped in a late endosome compartment. Our findings suggest that PKCβII is an important regulator of a late endosomal sorting event needed for influenza virus entry and infection.
Ribavirin Causes Error Catastrophe during Hantaan Virus Replication
Authors: Severson William E. , Schmaljohn Connie S. , Javadian Ali , Jonsson Colleen B.
Published: December 2002

Except for ribavirin, no other antiviral drugs for treating hantaviral diseases have been identified. It is well established that ribavirin will inhibit the production of infectious Hantaan virus (HTNV); however, its mechanism of action is unknown. To characterize the inhibitory effect of ribavirin on HTNV, the levels of viral RNAs, proteins, and infectious particles were measured for 3 days posttreatment of HTNV-infected Vero E6 cells. HTNV-infected cells treated with ribavirin showed a slight reduction in the levels of cRNA, viral RNA, and mRNA populations on the first day postinfection. The amount of cRNA and viral RNA increased to that observed for untreated HTNV-infected cells on day 2, whereas mRNA levels were more greatly reduced on days 2 and 3. Despite the finding of S-segment mRNA, albeit low, three of the viral proteins—nucleocapsid (N) protein and glycoproteins G1 and G2—could not be detected by immunohistochemistry in ribavirin-treated cells. To test the hypothesis that these effects were caused by incorporation of ribavirin into nascent RNA and a resultant “error catastrophe” was occurring, we cloned and sequenced the S-segment cRNA/mRNA from ribavirin-treated or untreated cells from day 3. We found a high mutation frequency (9.5/1,000 nucleotides) in viral RNA synthesized in the presence of ribavirin. Hence, the transcripts produced in the presence of the drug were not functional. These results suggest that ribavirin's mechanism of action lies in challenging the fidelity of the hantavirus polymerase, which causes error catastrophe.
Partial Molecular Characterization of Two Simian Immunodeficiency Viruses (SIV) from African Colobids: SIVwrc from Western Red Colobus (
Piliocolobus badius
) and SIVolc from Olive Colobus (
Procolobus verus
)
Authors: Courgnaud Valerie , Formenty Pierre , Akoua-Koffi Chantal , Noe Ronald , Boesch Christophe , Delaporte Eric , Peeters Martine
Published: December 2002

In order to study primate lentivirus evolution in the
Colobinae
subfamily, in which only one simian immunodeficiency virus (SIV) has been described to date, we screened additional species from the three different genera of African colobus monkeys for SIV infection. Blood was obtained from 13 West African colobids, and HIV cross-reactive antibodies were observed in 5 of 10
Piliocolobus badius
, 1 of 2
Procolobus verus,
and 0 of 1
Colobus polykomos
specimens. Phylogenetic analyses of partial
pol
sequences revealed that the new SIVs were more closely related to each other than to the other SIVs and especially did not cluster with the previously described SIVcol from
Colobus guereza
. This study presents evidence that the three genera of African colobus monkeys are naturally infected with an SIV and indicates also that there was no coevolution between virus and hosts at the level of the
Colobinae
subfamily.
Differential Cytokine Responses following Marek's Disease Virus Infection of Chickens Differing in Resistance to Marek's Disease
Authors: Kaiser Pete , Underwood Greg , Davison Fred
Published: December 2002

The production of cytokine mRNAs, in addition to viral DNA, was quantified by real-time quantitative reverse transcription-PCR (RT-PCR) (cytokines) or PCR (virus) in splenocytes during the course of Marek's disease virus (MDV) infection in four inbred chicken lines: two resistant (lines 61and N) and two susceptible (lines 72and P). Virus loads were only different after 10 days postinfection (dpi), increasing in susceptible lines and decreasing in resistant lines. Gamma interferon (IFN-γ) mRNA was expressed by splenocytes from all infected birds between 3 and 10 dpi, associated with increasing MDV loads. For other cytokines, differences between lines were only seen for interleukin-6 (IL-6) and IL-18, with splenocytes from susceptible birds expressing high levels of both transcripts during the cytolytic phase of infection, whereas splenocytes from resistant birds expressed neither transcript. These results indicate that these two cytokines could play a crucial role in driving immune responses, which in resistant lines maintain MDV latency but in susceptible lines result in lymphomas.
OPTIMAL SNAP-STABILIZING NEIGHBORHOOD SYNCHRONIZER IN TREE NETWORKS
Authors: JOHNEN COLETTE , ALIMA LUC O. , DATTA AJOY K. , TIXEUIL SÉBASTIEN
Published: December 2002

We propose a snap-stabilizing synchronization technique, called the Neighborhood Synchronizer[Formula: see text] that synchronizes nodes with their neighbors in a tree network. The [Formula: see text] scheme has optimal memory requirement — only one bit per processor. [Formula: see text] is snap-stabilizing [11], meaning that it always behaves according to its specification. The proposed synchronizer being snap-stabilizing is optimal in terms of stabilization time. We show an application of the synchronizer by designing an efficient broadcast algorithm [Formula: see text] in tree networks. [Formula: see text] is also snap-stabilizing and needs only 2h + 2m - 1 rounds to broadcast m messages, where h is the height of the tree.
EFFICIENT PARALLEL ALGORITHMS FOR TEMPLATE MATCHING
Authors: RAJASEKARAN SANGUTHEVAR
Published: December 2002

The parallel complexity of template matching has been well studied. In this paper we present more work-efficient algorithms than the existing ones. Our algorithms are based on FFT primitives. We consider the following models of computing: PRAM and the hypercube.
TheIn VitroInhibitory Effects of Crude Extracts of Traditional Chinese Herbs on 3-Hydroxy-3-Methylglutaryl-Coenzyme a Reductase on Vero Cells
Authors: Liu Ju-Chi , Chan Paul , Hsu Feng-Lin , Chen Yi-Jen , Hsieh Ming-Hsiung , Lo Ming-Yu , Lin Jung-Yaw
Published: December 2002

Cardiovascular disease is still the leading cause of death in Western countries. Epidemiological studies have shown that hypercholesterolemia is a major risk factor for coronary artery disease. Clinical trials of lipid lowering therapy with 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co-A) reductase inhibitor have been shown to decrease coronary events and mortality. Flavonoids are polyphenolic natural antioxidants occurring in natural products such as traditional Chinese herbs, fruits and beverages such as tea and wine. The aim of this study was to evaluate the effects of crude extracts from traditional Chinese herbs on HMG Co-A reductase. The methods for analysis of specific inhibitors of mevalonate biosynthesis have been well-established by using Vero cells, a cell line obtained from kidneys of African green monkeys. Crude extracts from different traditional Chinese herbs were dissolved in 1% Dulbecco's modified Eagle's medium and incubated with Vero cells with or without the addition of 1 mM mevalonate or 5 mM sodium acetate for 24 hours in order to observe cell growth. Pravastatin, a specific HMG Co-A reductase inhibitor, was used as a positive control which inhibits Vero cells growth effectively and cell growth inhibition was reversible after 1 mM mevalonate. Among 100 traditional Chinese herbs used for the study, only two herbs: Curcuma zedoaria Roscoe and Poncirus trifoliata Raf. showed significant growth inhibition of Vero cells. This study shows that some crude extracts isolated from traditional medicinal herbs were effective HMG Co-A reductase inhibitors which might be developed into new hypocholesterolemic agents.
The Evaluation of Reno- and Hepato-Protective Effects of Huai-Shan-Yao (Rhizome Dioscoreae)
Authors: Lee Shih-Chang , Tsai Chin-Chun , Chen Jung-Chou , Lin Chun-Ching , Hu Miao-Lin , Lu Shin
Published: December 2002

Huai-Shan-Yao (Chinese yam; Rhizome Dioscoreae) is a common food in china. In the present study, we evaluated the protective effects of the crude extract of Huai-Shan-Yao on acute kidney and liver injuries in rats induced by ethanol. Results of pharmacological, biochemical and pathologic observations all showed that rats treated with the extract of Huai-Shan-Yao had decreased damage in renal tubules as well as decreased inflammation in the central vein and necrosis in the liver tissue.
The Cardiorespiratory Response and Energy Expenditure of Tai-Chi-Qui-Gong
Authors: Chao Yann-Fen C. , Chen Ssu-Yuan , Lan Ching , Lai Jin-Shin
Published: December 2002

The purpose of this study was to evaluate the cardiorespiratory response and energy expenditure during the practice of Tai-Chi-Qui-Gong (TCQG). Forty-seven TCQG practitioners with a mean age of 60.7 ± 9.9 years participated in this study. The cardiorespiratory responses and energy expenditure were measured by a K4 telemetry system. TCQG has 54 motions and can be divided into 3 sets. The result indicated that the exercise intensity of each motion is about 3 metabolic equivalents (MET) and the energy expenditure of each set is about 60 Kcal. The estimated intensity of TCQG in elderly individuals approximated 50% of the maximum oxygen uptake [Formula: see text] for men, and 60% of the maximum oxygen uptake [Formula: see text] for women. The findings demonstrate that TCQG is a low intensity exercise and can be prescribed as an alternative exercise program for cardiopulmonary rehabilitation.
Expression Profiling Identifies 147 Genes Contributing to a Unique Primate Neointimal Smooth Muscle Cell Phenotype
Authors: Geary Randolph L. , Wong James M. , Rossini Anthony , Schwartz Stephen M. , Adams Lawrence D.
Published: December 2002

Objective—

This study represents the first in an effort to systematically characterize different intimas by using expression array analysis.

Methods and Results—

We compared smooth muscle cells (SMCs) of the neointima formed 4 weeks after aortic grafting with those from normal aorta and vena cava from cynomolgus monkeys. Hybridization to cDNA arrays identified subsets of 147 and 45 genes differentially expressed in the neointima versus the aorta and vena cava, respectively. The expression pattern differentiating neointima from aortic SMCs was characterized largely by suppression. Only 13 genes were induced in the neointima: 7 encoded matrix proteins (6 collagens and 1 versican) and 2 encoded inducers of matrix synthesis (osteoblast-specific factor-2/Cbfa1 and connective tissue growth factor). The genes suppressed most in the neointima included the regulator of G-protein signaling-5, SPARClike-1/hevin, and nonmuscle myosin heavy chain-B. A smaller gene set differentiated the neointima from the vena cava. Most were induced (39 of 45 genes), and overlap with the neointima-aorta set was significant (10 of 13 genes). Array results were validated with Northern analysis, in situ hybridization, or immunohistochemistry.

Conclusions—

These data underscore the importance of matrix synthesis in neointimal maturation, and novel genes, newly associated with neointimal SMCs (regulator of G-protein signaling-5 and osteoblast-specific factor-2/Cbfa1), have raised new hypotheses regarding the pathogenesis of intimal hyperplasia.
In the Elderly, Interleukin-6 Plasma Levels and the −174G>C Polymorphism Are Associated With the Development of Cardiovascular Disease
Authors: Jenny Nancy S. , Tracy Russell P. , Ogg Malcolm S. , Luong Le Ahn , Kuller Lewis H. , Arnold Alice M. , Sharrett A. Richey , Humphries Steve E.
Published: December 2002

Objective—Interleukin (IL)-6–mediated inflammation is involved in cardiovascular disease (CVD). We assessed IL-6 levels and the −174G>C genotype in a case-control study of men and women (average age 73 years) within the Cardiovascular Health Study.

Methods and Results—Cases included incident angina, myocardial infarction (MI), and stroke (5-year follow-up), prevalent MI, and MRI-detectable infarcts. A control group and a group free of subclinical CVD were used for comparison. The −174C allele was associated with higher C-reactive protein (11% higher,P=0.02), fibrinogen (3% higher,P=0.02), and IL-6 (5% higher;P=0.16). IL-6 was associated with increased atherosclerosis when the control group was compared with the group free of subclinical CVD. No further association with CVD events was found when case groups were compared with the control group. Compared with its absence, presence of the −174C allele was associated with risk of MRI infarcts (odds ratio 1.5).

Conclusions—IL-6 levels differentiated those with subclinical CVD from those without. Although the −174C allele was not associated with incident events, associations of the genotype with inflammation and MRI infarcts, combined with the plasma IL-6 results, suggest that IL-6 may chronically predispose an individual to develop atherosclerosis.
Quantitative Trait Loci Influencing Blood and Liver Cholesterol Concentration in Rats
Authors: Bonné Anita C.M. , den Bieman Maria G. , Gillissen Gert F. , Lankhorst Ægidius , Kenyon Christopher J. , van Zutphen Bert F.M. , van Lith Hein A.
Published: December 2002

Objective—

The LEW/OlaHsd and BC/CpbU rat inbred strains differ markedly in blood and hepatic cholesterol levels before and after a cholesterol-rich diet. To define loci controlling these traits and related phenotypes, an F
2
population derived from these strains was genetically analyzed.

Methods and Results—

For each of the 192 F
2
animals, phenotypes were determined, and genomic DNA was screened for polymorphic microsatellite markers. Significant quantitative trait loci (QTLs) were detected for basal serum cholesterol level on chromosome 1 (
D1Rat335-D1Rat27
: total population, lod score 9.6; females, lod score 10.3) and chromosome 7 (
D7Rat69
: males, lod score 4.1), for postdietary serum cholesterol level on chromosome 2 (
D2Rat69
: total population, lod score 4.4) and chromosome 16 (
D16Rat6-D16Rat44
: total population, lod score 3.3), for postdietary serum phospholipid level on chromosome 11 (
D11Rat10
: total population, lod score 4.1; females, lod score 3.6), and for postdietary serum aldosterone level on chromosome 1 (
D1Rat14
: females, lod score 3.7) and chromosome 18 (
D18Rat55-D18Rat8
: females, lod score 2.9). In addition, QTLs with borderline significance were found on chromosomes 3, 5 to 11, 15, and 18.

Conclusions—

QTLs involved in blood and/or hepatic cholesterol concentrations (or related phenotypes) in the rat were identified. This contributes to the value of the rat as an animal model in studies researching the role of cholesterol in the pathogenesis of atherosclerosis and other cholesterol-related diseases.
Circulating Autoantibodies to Oxidized LDL Correlate With Impaired Coronary Endothelial Function After Cardiac Transplantation
Authors: Fang James C. , Kinlay Scott , Behrendt Dominik , Hikita Hiroyuki , Witztum Joseph L. , Selwyn Andrew P. , Ganz Peter
Published: December 2002

Objective—

The oxidative modification of low density lipoprotein (LDL) may play a role in the pathogenesis of transplant-associated arteriosclerosis. Oxidized LDL (OxLDL) is immunogenic as well as atherogenic, and the level of autoantibodies to OxLDL has been taken as an index of the oxidant state of LDL. Because endothelial dysfunction is key in the initiation of transplant-associated arteriosclerosis, we postulated that the level of OxLDL autoantibody is associated with the degree of impairment of coronary endothelial function.

Methods and Results—

Coronary endothelium-dependent dilation was assessed by using intracoronary acetylcholine and endothelium-independent dilation by nitroglycerin in 36 cardiac transplant recipients within 1 year of transplantation. The coronary responses to acetylcholine ranged from −37% (vasoconstriction) to 31% (vasodilation), and the responses to nitroglycerin ranged from 0% to 42% (vasodilation). The coronary vasomotor response to acetylcholine was significantly and inversely related to OxLDL autoantibody levels (
r
=−0.43,
P
<0.01) but not LDL levels (
r
=−0.04,
P
=0.83) or circulating OxLDL levels detected by monoclonal antibody EO6 (
r
=−0.27,
P
=0.11). The coronary artery response to nitroglycerin was not related to levels of OxLDL autoantibodies, LDL, or EO6 (all
P
=NS).

Conclusions—

Autoantibodies to OxLDL are increased in patients with coronary endothelial dysfunction in the first year after cardiac transplantation. The oxidative modification of LDL by inducing endothelial dysfunction in cardiac transplant recipients may be a critical step in the atherogenic effects of LDL and may provide a potential target for therapy.
Deficiency of Glutathione Peroxidase-1 Sensitizes Hyperhomocysteinemic Mice to Endothelial Dysfunction
Authors: Dayal Sanjana , Brown Kara L. , Weydert Christine J. , Oberley Larry W. , Arning Erland , Bottiglieri Teodoro , Faraci Frank M. , Lentz Steven R.
Published: December 2002

Objective—

We tested the hypothesis that deficiency of cellular glutathione peroxidase (GPx-1) enhances susceptibility to endothelial dysfunction in mice with moderate hyperhomocysteinemia.

Methods and Results—

Mice that were wild type (
Gpx1
+/+
), heterozygous (
Gpx1
+/−
), or homozygous (
Gpx1
−/−
) for the mutated
Gpx1
allele were fed a control diet or a high-methionine diet for 17 weeks. Plasma total homocysteine was elevated in mice on the high-methionine diet compared with mice on the control diet (23±3 versus 6±0.3 μmol/L, respectively;
P
<0.001) and was not influenced by
Gpx1
genotype. In mice fed the control diet, maximal relaxation of the aorta in response to the endothelium-dependent dilator acetylcholine (10
−5
mol/L) was similar in
Gpx1
+/+
,
Gpx1
+/−
, and
Gpx1
−/−
mice, but relaxation to lower concentrations of acetylcholine was selectively impaired in
Gpx1
−/−
mice (
P
<0.05 versus
Gpx1
+/+
mice). In mice fed the high-methionine diet, relaxation to low and high concentrations of acetylcholine was impaired in
Gpx1
−/−
mice (maximal relaxation 73±6% in
Gpx1
−/−
mice versus 90±2% in
Gpx1
+/+
mice,
P
<0.05). No differences in vasorelaxation to nitroprusside or papaverine were observed between
Gpx1
+/+
and
Gpx1
−/−
mice fed either diet. Dihydroethidium fluorescence, a marker of superoxide, was elevated in
Gpx1

−/−

mice fed the high-methionine diet (
P
<0.05 versus
Gpx1
+/+
mice fed the control diet).

Conclusions—

These findings demonstrate that deficiency of GPx-1 exacerbates endothelial dysfunction in hyperhomocysteinemic mice and provide support for the hypothesis that hyperhomocysteinemia contributes to endothelial dysfunction through a peroxide-dependent oxidative mechanism.
Plasminogen Activator Inhibitor 1 and Vitronectin Protect Against Stenosis in a Murine Carotid Artery Ligation Model
Authors: de Waard Vivian , Arkenbout E. Karin , Carmeliet Peter , Lindner Volkhard , Pannekoek Hans
Published: December 2002

Objective—

We previously reported that plasminogen activator inhibitor 1 (PAI-1), in the presence of vitronectin (VN), inhibits thrombin activity in vitro. Furthermore, we demonstrated in human atherosclerotic plaques the colocalization of thrombin, PAI-1, and VN, as well as activity of thrombin and PAI-1. Here, we show that PAI-1 is a local thrombin inhibitor in vivo.

Methods and Results—

We used the murine carotid artery ligation model to assess the role of PAI-1 and VN in stenosis by using PAI-1–deficient (PAI-1
−/−
) and VN
−/−
mice. Ligation resulted in a smooth muscle cell (SMC)-rich intima without infiltrating cells. We show that PAI-1
−/−
and VN
−/−
mice generate a larger intima than wild-type mice as the result of more extensive SMC proliferation, as evidenced by cell counting and staining for proliferating cell-nuclear antigen.

Conclusions—

In PAI-1
−/−
mice, excessive intima formation is prevented by the thrombin-specific inhibitor hirudin. Finally, immunohistochemical analysis revealed PAI-1, VN, and (pro)thrombin antigen in intimal lesions. Our observations are compatible with inhibition of thrombin-mediated SMC proliferation by PAI-1/VN complexes.
Oxidized LDL-Induced Smooth Muscle Cell Proliferation Involves the EGF Receptor/PI-3 Kinase/Akt and the Sphingolipid Signaling Pathways
Authors: Auge Nathalie , Garcia Virginie , Maupas-Schwalm Françoise , Levade Thierry , Salvayre Robert , Negre-Salvayre Anne
Published: December 2002

Objective—

Oxidized low-density lipoprotein (oxLDL)-induced smooth muscle cell (SMC) proliferation requires the coactivation of various signaling pathways, namely sphingomyelin/ceramide/sphingosine-1-phosphate, epithelial growth factor receptor (EGFR), and phosphoinositide 3-kinase (PI-3K) pathways. This study aimed to clarify the respective role and the hypothetical cross-talk between sphingomyelin/ceramide/sphingosine-1-phosphate, EGFR, and PI-3K/Akt pathways in the balance between mitogenic and cytotoxic effects elicited by oxLDL.

Methods and Results—

Coimmunoprecipitation experiments and the use of inhibitors and dominant-negative mutant showed that oxLDL-induced PI-3K activation is dependent on EGFR. PI-3K activation is independent of the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, because PI-3K inhibition by LY294002 or dominant-negative Δp85 mutant does not abrogate sphingomyelin hydrolysis, and, conversely, the use of permeant C2-ceramide and of
N,N
-dimethyl-sphingosine, a sphingosine kinase inhibitor, does not alter PI-3K activity. Activation of Akt/PKB by oxLDL requires PI-3K, as shown by the inhibition by LY204002 and in Δp85 SMC. The inhibition of Akt/PKB by PI-3K inhibitor LY204002 or by overexpression of kinase-dead Akt shifted the mitogenic effect of oxLDL toward apoptosis, thus suggesting that the PI-3K/Akt pathway acts as a survival pathway.

Conclusions—

SMC proliferation elicited by moderate concentrations of oxLDL involves the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, which leads to extracellular regulated kinase 1/2 activation and DNA synthesis, and the EGFR/PI-3K/Akt pathway, which prevents the apoptotic effect of oxLDL.
Depolarization of Endothelial Cells Enhances Platelet Aggregation Through Oxidative Inactivation of Endothelial NTPDase
Authors: Krötz Florian , Sohn Hae Young , Keller Matthias , Gloe Torsten , Bolz Steffen Sebastian , Becker Bernhard F. , Pohl Ulrich
Published: December 2002

Objective—

The objective of this study was to investigate whether depolarization of cultured endothelial cells (human umbilical vein endothelial cells, HUVECs) affects their ectonucleotidase activity through superoxide (O
2
−
) production.

Methods and Results—

Depolarization by the cation channel gramicidin (100 nmol/L) or tetrabutylammonium chloride (1 mmol/L) induced O
2
−
release from HUVECs (n=4), which was decreased by superoxide dismutase (SOD, 500 U/mL). The activity of endothelial ectonucleotidases was assessed by the production of inorganic phosphate from ADP, which was decreased by chronic depolarization by 25% (n=6,
P
<0.05) and the amount of AMP derived from ADP in the presence of the 5′-nucleotidase inhibitor α,β-methylene-5′-diphosphate (100 μmol/L). AMP was decreased by chronic depolarization from 0.54±0.16 to 0.39±0.11 μmol/min/mg protein (n=6,
P
<0.05). This was abolished in the continuous presence of SOD (n=6). NTPDase protein was predominantly expressed in HUVECs (n=4). Protein abundance, viability of cells, and apoptosis rates were not altered by depolarization (n=10). Only in the presence of depolarized HUVECs, but not with control cells or with HUVECs depolarized in the presence of SOD, did 5 μmol/L of ADP cause irreversible platelet aggregation. Increases in transmural pressure induced endothelial depolarization in intact hamster small arterioles.

Conclusions—

Depolarization causes the endothelial production of O
2
−
, which inhibits the activity of endothelial ectonucleotidases. Increases in transmural pressure induce endothelial depolarization. In chronically hypertensive diseases, depolarization might favor platelet aggregation.
Thrombospondin-2 Polymorphism Is Associated With a Reduced Risk of Premature Myocardial Infarction
Authors: Boekholdt S. Matthijs , Trip Mieke D. , Peters Ron J.G. , Engelen Marc , Boer Jolanda M.A. , Feskens Edith J.M. , Zwinderman Aeilko H. , Kastelein John J.P. , Reitsma Pieter H.
Published: December 2002

Objective—

Recently, polymorphisms in thrombospondin (THBS) genes coding for THBS-1 (N700S), THBS-2 (T>G substitution in 3′-untranslated region), and THBS-4 (A387P) genes were proposed to modulate the risk of premature coronary artery disease (CAD) or myocardial infarction (MI). It was our objective to verify this hypothesis in an independent cohort.

Methods and Results—

We performed a case-control study among patients (n=503) referred to our institution for symptomatic CAD that occurred before the age of 50 years and a group of age- and sex-matched population-based controls free of CAD (n=1071). The THBS-1 variant allele was not associated with an altered risk of premature CAD or MI. Homozygosity for the THBS-2 variant allele and the THBS-4 variant (387P) allele was significantly associated with a reduced risk of premature MI compared with wild-type individuals (OR=0.44, 0.24 to 0.84 and OR=0.43, 0.22 to 0.85, respectively). The latter observation is in contrast with a previous report, although confidence intervals overlap.

Conclusions—

We conclude that a relationship between the THBS-1 N700S polymorphism and premature CAD is unlikely. For the THBS-4 A387P polymorphism, additional studies are required to elucidate its role in premature CAD. Finally, we conclude that the THBS-2 polymorphism is associated with a reduced risk of premature MI.
Nonendothelial Source of Nitric Oxide in Arterioles But Not in Venules
Authors: Kashiwagi Satoshi , Kajimura Mayumi , Yoshimura Yasunori , Suematsu Makoto
Published: December 2002

This study aimed to examine topographic distribution of microvascular NO generation in vivo. To this end, nitrosonium ion (NO
+
)–sensitive diaminofluorescein diacetate was superfused continuously on the rat mesentery and the fluorescence was visualized in the microvessels through laser confocal microfluorography. Two major sites exhibited a time-dependent elevation of the fluorescence: microvascular endothelia and mast cells. As judged by the fluorescence sensitivity to local application of different inhibitors of NO synthase (NOS), NO availability in arteriolar endothelium and mast cells appeared to be maintained mainly by NOS1, whereas that in venular endothelium greatly depends on NOS3. In venules, the magnitude of inhibitory responses elicited by the inhibitors was positively correlated with the density of leukocyte adhesion. NOS inhibitors significantly reduced, but did not eliminate, the NO
+
-associated fluorescence in arterioles, capillaries, and venules, suggesting alternative sources of NO in circulation for these microvessels. Immunohistochemistry for NOS isozymes revealed that NOS1 occurred not only in nerve fibers innervated to arterioles but also abundantly in mast cells. Laser flow cytometry of peritoneal cells in vitro revealed abundant expression of NOS1 in mast cells. Interestingly, NOS3 occurred in endothelia of capillaries and venules but not in those of distal arterioles with comparable diameters. These results suggest that the arterioles receive NO from nonendothelial origins involving NOS1 present in nerve terminals and mast cells, whereas venules depend on the endothelial NOS as a major source. Furthermore, nonenzymatic sources of NO from circulating reservoirs constitute a notable fraction throughout different classes of microvessels. The full text of this article is available at http://www.circresaha.org.
Neuronal Nitric Oxide Synthase Gene Transfer Promotes Cardiac Vagal Gain of Function
Authors: Mohan R.M. , Heaton D.A. , Danson E.J.F. , Krishnan S.P.R. , Cai S. , Channon K.M. , Paterson D.J.
Published: December 2002

Nitric oxide (NO) generated from neuronal nitric oxide synthase (NOS-1) in intrinsic cardiac ganglia has been implicated in parasympathetic-induced bradycardia. We provide direct evidence that NOS-1 acts in a site-specific manner to promote cardiac vagal neurotransmission and bradycardia. NOS-1 gene transfer to the guinea pig right atrium increased protein expression and NOS-1 immunolocalization in cholinergic ganglia. It also increased the release of acetylcholine and enhanced the heart rate (HR) response to vagal nerve stimulation (VNS) in vitro and in vivo. NOS inhibition normalized the HR response to VNS in the NOS-1–treated group compared with the control groups (enhanced green fluorescent protein and sham) in vitro. In contrast, an acetylcholine analogue reduced HR to the same extent in all groups before and during NOS inhibition. These results demonstrate that NOS-1–derived NO acts presynaptically to facilitate vagally induced bradycardia and that upregulation of NOS-1 via gene transfer may provide a novel method for increasing cardiac vagal function.
Lipopolysaccharides fromLegionellaandRhizobiumstimulate mouse bone marrow granulocytes via Toll-like receptor 2
Authors: Girard Robert , Pedron Thierry , Uematsu Satoshi , Balloy Viviane , Chignard Michel , Akira Shizuo , Chaby Richard
Published: December 2002

Lipopolysaccharide (LPS) derived from enterobacteria elicit in several cell types cellular responses that are restricted in the use of Toll-like receptor 4 (TLR4) as the principal signal-transducing molecule. A tendency to consider enterobacterial LPS as a prototypic LPS led some authors to present this mechanism as a paradigm accounting for all LPSs in all cell types. However, the structural diversity of LPS does not allow such a general statement. By using LPSs from bacteria that do not belong to the Enterobacteriaceae, we show that in bone marrow cells (BMCs) the LPS of Rhizobium species Sin-1 and of three strains of Legionella pneumophila require TLR2 rather than TLR4 to elicit the expression of CD14. In addition, exposure of BMCs from TLR4-deficient (C3H/HeJ) mice to the lipid A fragment of the Bordetella pertussis LPS inhibits their activation by the Legionella lipid A. The data show selective action of different LPSs via different TLRs, and suggest that TLR2 can interact with many lipid A structures, leading to either agonistic or specific antagonistic effects.
Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis
Authors: Martin-Verdeaux Sophie , Pombo Isabel , Iannascoli Bruno , Roa Michèle , Varin-Blank Nadine , Rivera Juan , Blank Ulrich
Published: December 2002

Compound exocytosis of inflammatory mediators from mast cells requires SNARE and a series of accessory proteins. However, the molecular steps that regulate secretory granule movement and membrane fusion as well as the role of the cytoskeleton are still poorly understood. Here, we report on our investigation of the role of syntaxin-binding Munc18 isoforms and the microtubule network in this process. We found that mast cells express Munc18-2, which interacts with target SNAREs syntaxin 2 or 3, as well as Munc18-3, which interacts with syntaxin 4. Munc18-2 was localised to secretory granules, whereas Munc18-3 was found on the plasma membrane. Increased expression of Munc18-2 and derived peptides containing an interfering effector loop inhibited IgE-triggered exocytosis, while increased expression of Munc18-3 showed no effect. Munc18-2 localisation on granules is polarised; however, upon stimulation Munc18-2 redistributed into forming lamellipodia and persisted on granules that were aligned along microtubules, but was excluded from F-actin ruffles. Disruption of the microtubule network with nocodazole provoked Munc18-2 redistribution and affected mediator release. These findings suggest a role for Munc18-2 and the microtubule network in the regulation of secretory granule dynamics in mast cells.
α5β1 integrin mediates strong tissue cohesion
Authors: Robinson Elizabeth E. , Zazzali Kathleen M. , Corbett Siobhan A. , Foty Ramsey A.
Published: December 2002

Integrins and cadherins are considered to have distinct and opposing functions. Integrins are traditionally cited for their role in cell-substratum interactions, whereas cadherins are thought to mediate strong intercellular cohesion. Together, these adhesion systems play crucial roles in a wide variety of cellular and developmental processes including cell migration, morphology, differentiation and proliferation. In this manuscript we present evidence that integrins possess the ability to mediate strong intercellular cohesion when cells are grown as 3D aggregates.

Much of the data elucidating the role of integrins as mediators of cell-extracellular matrix (ECM) interactions have been generated using conventional cell culture techniques in which cells are plated onto ECM-coated 2D surfaces. In vivo, cells are embedded in a 3D meshwork of ECM proteins. We hypothesized that, within this meshwork, integrin-ECM interactions may impart cohesivity to an aggregate of cells by linking adjacent cells together. To test this hypothesis, we transfected Chinese hamster ovary (CHO-B2) cells to express α5β1 integrin and found that these cells formed compact, spherical aggregates. We measured aggregate cohesivity using tissue surface tensiometry, a novel technique that quantifies cell-cell cohesivity of spheroids under physiological conditions. We determined that α5β1 integrin is capable of conferring strong cohesivity (σ=8.22±0.68 dynes/cm) to aggregates of α5-integrin-transfected cells. This cohesion was found to be independent of cadherin expression and was significantly greater than the cohesivity conferred onto CHO-B2 cells transfected with N-cadherin (σ=3.14±0.20 dynes/cm, P≤0.0001), a more traditional cell-cell cohesion system.

Fibronectin-null CHO cells that express α5β1 integrin but do not secrete endogenous fibronectin do not form aggregates in fibronectin-depleted medium. Addition of increasing amounts of exogenous dimeric fibronectin to these cells resulted in a dose-dependent compaction. However, compaction failed to occur in the presence of fibronectin monomers. These data indicate that fibronectin is required for α5β1-mediated compaction and that the dimeric structure of fibronectin is essential for this process. Additionally, aggregate formation of the α5 integrin transfectants was inhibited by an RGD peptide thus confirming α5β1 integrin specificity. Collectively, these data confirm our hypothesis that α5β1 integrin acts through fibronectin to link adjacent cells together, thus promoting strong intercellular cohesion in 3D cellular aggregates.
Analysis of the interactions between BP180, BP230, plectin and the integrin α6β4 important for hemidesmosome assembly
Authors: Koster Jan , Geerts Dirk , Favre Bertrand , Borradori Luca , Sonnenberg Arnoud
Published: December 2002

Hemidesmosomes (HDs) are multi-protein complexes that promote stable adhesion of epithelial cells to the underlying extracellular matrix. We assessed the interactions between different hemidesmosomal components with each other, mapped the binding sites and studied the importance of these interactions for HD assembly in yeast two-hybrid and cell-transfection assays. The results show that: (1) bullous pemphigoid antigen (BP) 180 binds not only to BP230, but also to plectin. The interactions between these proteins are facilitated by the Y subdomain in the N-terminal plakin domain of BP230 and plectin, and residues 145-230 of the cytoplasmic domain of BP180; (2) different, but overlapping, sequences on BP180 mediate binding to β4, which, in turn associates with BP180 via its third fibronectin type III repeat; (3) sequences in the N-terminal extremity of BP230 mediate its binding to β4, which requires the C-terminal end of the connecting segment up to the fourth FNIII repeat of the β4 subunit. (4) Finally, cell-transfection studies showed that the localization of BP230 into hemidesmosome-like structures depends on its Z-Y subdomains as well as on the availability of BP180. By having further uncovered interactions between various hemidesmosomal components, mapped the involved binding sites and dissected a hierarchy of interactions relevant for their topogenic fate, our findings give novel insights into the molecular organization of hemidesmosomes.
Isotope Effects of Water Molecules on the Hydration of Alkali Metal and Halide Ions
Authors: Kakiuchi Masahisa
Published: December 2002

Based on the results of hydrogen isotope fractionation in aqueous alkali halide solutions at 25 °C, reported in previous papers, the hydrogen isotope effect between water molecules hydrated to a cation or an anion and bulk water molecules is estimated with the assumption that the equilibrium constant of the hydrogen isotope exchange reaction between the hydration water of sodium ion and bulk water is unity. The orders of the hydrogen isotope effects of alkali metal cations and halide anions are found to be Cs+ > K+ > Na+ > Li+, and I− > Br− > Cl− for a constant hydration number, respectively, and the hydrogen isotope effect is mainly influenced in anions rather than in cations. The oxygen isotope effect for alkali and halide ions is evaluated from the literature. The hydrogen and oxygen isotopic effects for the cations and anions show the same trend, but the magnitude of the hydrogen isotope effect for anions is significantly larger than that of the oxygen isotope effect.

The free energy of transfer of alkali metal and halide ions from H2O to HDO is calculated from the hydrogen isotope effect for the individual ions. The calculated values are compared with the free energy of transfer from H2O to D2O obtained by electromotive force measurements, and are compared with the free energy of transfer from H216O to H218O calculated from the oxygen isotope effect.
Thermodynamic Parameters for the Complexation of the Pyridinio Derivatives of Cyclodextrins with Some Inorganic Anions in D2O Solution
Authors: Yamamoto Tatsuyuki , Nagata Takuya , Ohtsuki Hideki , Ono Masaharu , Matsui Yoshihisa
Published: December 2002

Substitution of the pyridinio groups for the C(6)-OH’s of cyclodextrins (CD’s) brought about a remarkable increase in the binding constants (Ka) for 1 : 1 complexes of CD’s with inorganic anions, such as I−, SCN−, and ClO4−. The thermodynamic parameters for the complexation of native α-CD and its pyridinio derivatives showed that the increase in Ka is mainly driven by an enthalpy term (ΔH), and that the contribution of an entropy term (TΔS) is minor. In contrast, the increase in Ka is mainly driven by TΔS, and the contribution of ΔH is minor, for the systems of β- and γ-CD’s with the larger interior cavities. These results were explained in terms of the combined action of van der Waals and electrostatic interactions between the CD’s and anions, together with desolvation upon complexation.
Formation of Electronically Excited Fragments Resulting from Collisions of He(23S) Atoms with CCl4 and Related Chloromethanes
Authors: Tokue Ikuo , Kobayashi Masaaki , Kudo Takeyuki , Yamasaki Katsuyoshi
Published: December 2002

The photoemissions from fragments produced by collisions of He(23S) atoms with CHCl3, CCl2F2, CCl3F, CCl4, and CBrCl3 have been studied in the collision energy range of 120–210 meV using a crossed beam. The Cl2(D′–A′) band is predominant for CCl4 and CBrCl3, while the A–X and B–X bands of CF are intense for CCl2F2 and CCl3F. The sum of the emission cross sections (σem) for several systems of neutral fragments in the 190–600 nm region was estimated to be in the (0.3–1.2) × 10−20 m2 range for these chloromethanes at a collision energy of 150 meV. The dependences of σem’s for the Cl2(D′–A′) band from CHCl3 and CCl4 on the collision energy are either nearly zero or slightly negative. The sum of σem’s for Cl2 and other ion-pair bands reveals a fairly good correlation with the electron affinity of the targets. These results support the harpoon mechanism for the formation of Cl2(D′) from the He(23S) + chloromethanes reactions.
Multielement Determination and Distributions of Major-to-Ultratrace Elements in Industrial Waste Incineration Bottom Ash as Studied by ICP-AES and ICP-MS
Authors: Fujimori Eiji , Ichikawa Kenji , Shiozawa Ryuji , Asai Katsuichi , Chiba Koichi , Haraguchi Hiroki
Published: December 2002

The multielement determination of major-to-ultratrace elements in industrial waste incineration bottom ash was carried out by inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS) after alkali fusion using LiBO2. The concentrations of 50 elements in bottom ash could be determined over the concentration range from 192 mg g−1 of Si to 0.072 μg g−1 of In. Silicon, Al, Fe, Ca, Na, and Ti, which except for Fe are lithophile elements, were major elements of industrial waste incineration bottom ash. The distributions of major-to-ultratrace elements in incineration bottom ash were evaluated by the enrichment factors. The enrichment factors of major elements in incineration bottom ash indicated that their relative contents were quite similar to those in the continental crust. It was also found that most minor and trace elements in the lithophile element group provided almost the same enrichment factors as those of major elements. On the other hand, Cu, Zn, Cd, and Pb in the chalcophile element group as well as some of the siderophile ones, such as Co and Ni, provided the significantly larger enrichment factors, although they were more enriched in fly ash than in bottom ash. Such large abundances of many transition elements in industrial waste incineration ashes may reflect the large amounts of metal use in industry as well as in modern society.