bluuebunny/crossref_metadata_2025 huggingface dataset:
An Optimized CD8+T-Cell Response Controls Productive and Latent Gammaherpesvirus Infection
Authors: Braaten Douglas C. , Sparks-Thissen Rebecca L. , Kreher Scott , Speck Samuel H. , Virgin Herbert W.
Published: January 2005

Strategies to prime CD8+T cells againstMurine gammaherpesvirus 68(γHV68; MHV68) latency have, to date, resulted in only limited effects. While early forms of latency (<21 days) were significantly reduced, effects were not seen at later times, indicating loss of control by the primed CD8+T cells. In the present study, we evaluated CD8+T cells in an optimized system, consisting of OTI T-cell-receptor (TCR) transgenic mice, which generate clonal CD8+T cells specific for Kb-SIINFEKL of OVA, and a recombinant γHV68 that expresses OVA (γHV68.OVA). Our aim was to test whether this optimized system would result in more effective control not only of acute infection but also of later forms of latent infection than was seen with previous strategies. First, we show that OTI CD8+T cells effectively controlled acute replication of γHV68.OVA in liver, lung, and spleen at 8 and 16 days after infection of OTI/RAG mice, which lack expression of B and CD4+T cells. However, we found that, despite eliminating detectable acute replication, the OTI CD8+T cells did not prevent the establishment of latency in the OTI/RAG mice. We next evaluated the effectiveness of OTI T cells in OTI/B6 animals, which express B cells—a major site of latency in wild-type mice—and CD4+T cells. In OTI/B6 mice OTI CD8+T cells not only reduced the frequency of cells that reactivate from latency and the frequency of cells bearing the viral genome at 16 days after infection (similar to what has been reported before) but also were effective at reducing latency at 42 days after infection. Together, these data show that CD8+T cells are sufficient, in the absence of B cells and CD4+T cells, for effective control of acute replication. The data also demonstrate for the first time that a strong CD8+T-cell response can limit long-term latent infection.
Role of Viral Factor E3L in Modified Vaccinia Virus Ankara Infection of Human HeLa Cells: Regulation of the Virus Life Cycle and Identification of Differentially Expressed Host Genes
Authors: Ludwig Holger , Mages Jörg , Staib Caroline , Lehmann Michael H. , Lang Roland , Sutter Gerd
Published: January 2005

Modified vaccinia virus Ankara (MVA) is a highly attenuated virus strain being developed as a vaccine for delivery of viral and recombinant antigens. The MVA genome lacks functional copies of numerous genes interfering with host response to infection. The interferon resistance gene E3L encodes one important viral immune defense factor still made by MVA. Here we demonstrate an essential role of E3L to allow for completion of the MVA molecular life cycle upon infection of human HeLa cells. A deletion mutant virus, MVA-ΔE3L, was found defective in late protein synthesis, viral late transcription, and viral DNA replication in infected HeLa cells. Moreover, we detected viral early and continuing intermediate transcription associated with degradation of rRNA, indicating rapid activation of 2′-5′-oligoadenylate synthetase/RNase L in the absence of E3L. Further molecular monitoring of E3L function by microarray analysis of host cell transcription in MVA- or MVA-ΔE3L-infected HeLa cells revealed an overall significant down regulation of more than 50% of cellular transcripts expressed under mock conditions already at 5 h after infection, with a more prominent shutoff following MVA-ΔE3L infection. Interestingly, a cluster of genes up regulated exclusively in MVA-ΔE3L-infected cells could be identified, including transcripts for interleukin 6, growth arrest and DNA damage-inducible protein β, and dual-specificity protein phosphatases. Our data indicate that lack of E3L inhibits MVA antigen production in human HeLa cells at the level of viral late gene expression and suggest that E3L can prevent activation of additional host factors possibly affecting the MVA molecular life cycle.
Inhibition of Cell Division by the Human Cytomegalovirus IE86 Protein: Role of the p53 Pathway or Cyclin-Dependent Kinase 1/Cyclin B1
Authors: Song Yoon-Jae , Stinski Mark F.
Published: January 2005

The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G
0
/G
1
to G
1
/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser
15
p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G
2
/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53
+/+
and p53
−/−
cells.
Early Steps of Clathrin-Mediated Endocytosis Involved in Phagosomal Escape of Fcγ Receptor-Targeted Adenovirus
Authors: Meier Oliver , Gastaldelli Michele , Boucke Karin , Hemmi Silvio , Greber Urs F.
Published: January 2005

Adenovirus type 2 (Ad2) and Ad5 enter epithelial cells via the coxsackievirus B Ad receptor (CAR) and αvintegrin coreceptors. In the absence of CAR, they can be directed to the Fcγ receptor 1 of hematopoietic cells by an adaptor comprising the extracellular CAR domain and the Fc portion of a human immunoglobulin G (CARex-Fc). This gives rise to Ad aggregates and single particles which together enhance gene delivery up to 250-fold compared to adaptor-less viruses. A small interfering RNA knockdown of the clathrin heavy chain and quantitative electron microscopy of hematopoietic leukemia cells showed that the majority of Ads were phagocytosed as clusters of 1 to 3 μm in diameter and that about 10% of the particles entered cells by clathrin-mediated endocytosis. The clathrin knockdown did not affect phagocytosis but, surprisingly, inhibited viral escape from phagosomes. Similarly, blocking an early stage of clathrin-coated pit assembly inhibited phagosomal escape and infection but not aggregate uptake, unlike blocking of a late stage of clathrin-coated pit formation. We propose a cooperative interaction of clathrin-mediated endocytosis and phagocytosis triggering phagosomal lysis and infection.
Civets Are Equally Susceptible to Experimental Infection by Two Different Severe Acute Respiratory Syndrome Coronavirus Isolates
Authors: Wu Donglai , Tu Changchun , Xin Chaoan , Xuan Hua , Meng Qingwen , Liu Yonggang , Yu Yedong , Guan Yuntao , Jiang Yu , Yin Xunnan , Crameri Gary , Wang Muping , Li Changwen , Liu Shengwang , Liao Ming , Feng Li , Xiang Hua , Sun Jinfu , Chen Jinding , Sun Yanwei , Gu Shoulin , Liu Nihong , Fu Dexia , Eaton Bryan T. , Wang Lin-Fa , Kong Xiangang
Published: January 2005

Severe acute respiratory syndrome (SARS) was caused by a novel virus now known as SARS coronavirus (SARS-CoV). The discovery of SARS-CoV-like viruses in masked palm civets (
Paguma larvata
) raises the possibility that civets play a role in SARS-CoV transmission. To test the susceptibility of civets to experimental infection by different SARS-CoV isolates, 10 civets were inoculated with two human isolates of SARS-CoV, BJ01 (with a 29-nucleotide deletion) and GZ01 (without the 29-nucleotide deletion). All inoculated animals displayed clinical symptoms, such as fever, lethargy, and loss of aggressiveness, and the infection was confirmed by virus isolation, detection of viral genomic RNA, and serum-neutralizing antibodies. Our data show that civets were equally susceptible to SARS-CoV isolates GZ01 and BJ01.
Characterization of the Minimal Replicator of Kaposi's Sarcoma-Associated Herpesvirus Latent Origin
Authors: Hu Jianhong , Renne Rolf
Published: January 2005

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) binds to two sites within the 801-bp-long terminal repeat (TR) and is the only viral protein required for episomal maintenance. While two or more copies of TR are required for long-term maintenance, a single TR confers LANA-dependent origin activity on plasmid DNA. Deletion mapping revealed a 71-bp-long minimal replicator containing two distinctive sequence elements: LANA binding sites (LBS1/2) and an adjacent 29- to 32-bp-long GC-rich sequence which we termed the replication element. Furthermore, the transcription factor Sp1 can bind to TR outside the minimal replicator and contributes to TR's previously reported enhancer activity.
Neutrophil-mediated accumulation of 2-ClHDA during myocardial infarction: 2-ClHDA-mediated myocardial injury
Authors: Thukkani Arun K. , Martinson Bradley D. , Albert Carolyn J. , Vogler George A. , Ford David A.
Published: January 2005

The pathophysiological sequelae of myocardial infarction include neutrophil infiltration into the infarct zone that contributes to additional damage to viable tissue and removal of cellular debris from necrosed tissue. Reactive chlorinating species produced by myeloperoxidase amplify the oxidant capacity of activated neutrophils. Plasmalogens are a major phospholipid subclass of both endothelial cells and cardiac myocytes. Recent studies have shown that plasmalogens are targeted by neutrophil-derived reactive chlorinating species that lead to the production of α-chloro fatty aldehydes. Results herein demonstrate that the α-chloro fatty aldehyde 2-chlorohexadecanal (2-ClHDA) accumulates in rat hearts subjected to left anterior descending coronary artery occlusion. Myocardia from rats subjected to surgical infarction had increased 2-ClHDA and neutrophil infiltration levels compared with myocardia from rats subjected to sham surgery. Additionally, infarcted myocardia from rats rendered neutropenic by treatments with an anti-neutrophil antibody had diminished 2-ClHDA and neutrophil infiltration levels compared with infarcted myocardia from normopenic rats; 2-ClHDA was shown to elicit myocardial damage as determined by lactate dehydrogenase release in isolated perfused rat hearts. Additionally, 2-ClHDA treatment of hearts resulted in decreased heart rate and ventricular performance. Taken together, the present results demonstrate a novel neutrophil-dependent myeloperoxidase-based mechanism that results in 2-ClHDA production in response to regional myocardial infarction that may also contribute to cardiac dysfunction.
Biomechanical stress induces IL-6 expression in smooth muscle cells via Ras/Rac1-p38 MAPK-NF-κB signaling pathways
Authors: Zampetaki Anna , Zhang Zhongyi , Hu Yanhua , Xu Qingbo
Published: January 2005

IL-6, a proinflammatory cytokine, has been implicated in the development of vascular diseases. We previously demonstrated that mechanical stress can initiate signaling pathways leading to smooth muscle cell (SMC) proliferation and apoptosis, but little is known concerning cyclic stress-induced inflammatory response. To explore the role of stretch in the upregulation of cytokine expression in SMCs we performed RNase protection assay for a panel of cytokines and found that mechanical stress resulted in a time-dependent induction of IL-6 mRNA but not other cytokines, e.g., IL-1α, IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-18, IFN-γ, and macrophage migration inhibitory factor (MIF). This induction also correlated with elevated IL-6 protein levels in the supernatant. Pretreatment of the cells with NF-κB inhibitors inhibited NF-κB activity and resulted in marked inhibition (50%) of IL-6 protein. Moreover, SMC lines stably expressing dominant-negative Ras (RasN17) or Rac (RacN17) exhibited a remarkable decrease in p38 MAPK activity and IL-6 mRNA induction by mechanical stress. Furthermore, a significant inhibition of 30 and 40% in IL-6 protein was observed in SMCs pretreated with inhibitors of p38 MAPK and ERK1/2, respectively, but not JNK. Interestingly, SMCs isolated from PKC-δ-deficient mice exhibited higher levels of IL-6 compared with wild-type cells. Finally, high levels of IL-6 expression were observed in atherosclerotic lesions of vein bypass grafts, which are related to altered biomechanical stress. Our findings demonstrate that biomechanical stress-induced IL-6 expression occurs via a mechanism that involves Ras/Rac/p38 MAPK/NF-κB/NF-IL6 signaling pathways, which is downregulated by PKC-δ, and suggest that modulation of this event contributes to the pathogenesis of atherosclerosis.
Extracellular acidosis induces heme oxygenase-1 expression in vascular smooth muscle cells
Authors: Christou Helen , Bailey Nicole , Kluger Morgan S. , Mitsialis S. Alex , Kourembanas Stella
Published: January 2005

Extracellular acidosis (EA) has profound effects on vascular homeostasis, including vascular bed-specific alterations in vascular tone. Regulation of gene expression by EA has been observed in a variety of cells including vascular endothelial cells. Whether EA regulates gene expression in vascular smooth muscle cells (VSMCs) is not known. Heme oxygenase (HO)-1 is expressed in vascular cells, and its expression is regulated by cellular stressors such as heat, radiation, and hypoxia. Increased HO-1 expression in VSMCs leads to increased production of CO and its second messenger cGMP, which are important regulators of vascular tone and paracrine interactions in the vasculature. We examined whether EA regulates the expression of HO-1 in VSMCs. Exposure of VSMCs to acidic medium (pH 6.8) significantly increased HO-1 mRNA and protein compared with exposure to medium of physiological pH (pH 7.4). The acidic induction of HO-1 expression was time dependent and involved both transcriptional activation of the HO-1 gene and enhanced stability of HO-1 mRNA. Nitric oxide did not appear to mediate this response. We conclude that HO-1 is transcriptionally and posttranscriptionally upregulated by EA in VSMCs. This induction is time dependent and reversible. We speculate that EA, as an important tissue and cellular stressor for VSMCs, may elicit changes in gene expression patterns that contribute to the maintenance or disruption of vascular homeostasis.
Antagonistic regulation of swelling-activated Cl−current in rabbit ventricle by Src and EGFR protein tyrosine kinases
Authors: Ren Zuojun , Baumgarten Clive M.
Published: January 2005

Regulation of swelling-activated Cl−current ( ICl,swell) is complex, and multiple signaling cascades are implicated. To determine whether protein tyrosine kinase (PTK) modulates ICl,swelland to identify the PTK involved, we studied the effects of a broad-spectrum PTK inhibitor (genistein), selective inhibitors of Src (PP2, a pyrazolopyrimidine) and epidermal growth factor receptor (EGFR) kinase (PD-153035), and a protein tyrosine phosphatase (PTP) inhibitor (orthovanadate). ICl,swellevoked by hyposmotic swelling was increased 181 ± 17% by 100 μM genistein, and the genistein-induced current was blocked by the selective ICl,swellblocker tamoxifen (10 μM). Block of Src with PP2 (10 μM) stimulated tamoxifen-sensitive ICl,swellby 234 ± 27%, mimicking genistein, whereas the inactive analog of PP2, PP3 (10 μM), had no effect. Moreover, block of PTP by orthovanadate (1 mM) inhibited ICl,swelland prevented its stimulation by PP2. In contrast with block of Src, block of EGFR kinase with PD-153035 (20 nM) inhibited ICl,swell. Several lines of evidence argue that the PP2-stimulated current was ICl,swell: 1) the stimulation was volume dependent, 2) the current was blocked by tamoxifen, 3) the current outwardly rectified with both symmetrical and physiological Cl−gradients, and 4) the current reversed near the Cl−equilibrium potential. To rule out contributions of other currents, Cd2+(0.2 mM) and Ba2+(1 mM) were added to the bath. Surprisingly, Cd2+suppressed the decay of ICl,swell, and Cd2+plus Ba2+eliminated time-dependent currents between −100 and +100 mV. Nevertheless, these divalent ions did not eliminate ICl,swellor prevent its stimulation by PP2. The results indicate that tyrosine phosphorylation controls ICl,swell, and regulation of ICl,swellby the Src and EGFR kinase families of PTK is antagonistic.
Protease-activated receptor and endothelial-myocyte uncoupling in chronic heart failure
Authors: Moshal Karni S. , Tyagi Neetu , Henderson Brooke , Ovechkin Alexander V. , Tyagi Suresh C.
Published: January 2005

We examined the hypothesis that oxidants generated nitroso derivatives, activated latent matrix metalloproteinase (MMP), and induced proteinase-activated receptor 1 (PAR-1), leading to disconnection between the endothelium and myocytes. Administration of cardiospecific tissue inhibitor of metalloproteinase-4 (TIMP-4/CIMP) ameliorated the oxidative-proteolytic stress and endothelial-myocyte uncoupling in chronic heart failure (CHF) in mice. Aortic-vena cava fistula (AVF) was created in 30 male mice (C57BL/6J) and studied at 0-, 2-, and 8-wk AVF. To reverse cardiac remodeling, as measured by MMP activation, purified CIMP was administered by an osmotic minipump subcutaneously after 8-wk AVF, and groups of mice ( n = 6 mice/group) were examined after 12 and 16 wk. Levels of PAR-1 in the left ventricle (LV) were increased at 2 and 8 wk (compared with 0 wk of no CIMP treatment) but were normal at 12 and 16 wk after CIMP treatment, as measured by Western blot analysis. Similar results were obtained for LV levels of nitrotyrosine, MMP-2 and -9 activities, and TIMP-1 and -3. However, the levels of TIMP-4, endothelial cell density, and responses of cardiac rings to acetylcholine and bradykinin were attenuated at 2 and 8 wk and normalized after CIMP administration in AVF mice. CIMP induced nitric oxide in microvascular endocardial endothelial cells. The results suggest that nitro generation activated MMP and PAR-1, leading to endothelial-myocyte uncoupling. CIMP treatment normalized PAR-1 expression and ameliorated endothelial-myocyte uncoupling by decreasing oxidant-mediated proteolytic stress in CHF.
Lysophosphatidic acid-mediated augmentation of cardiomyocyte lipoprotein lipase involves actin cytoskeleton reorganization
Authors: Pulinilkunnil Thomas , An Ding , Ghosh Sanjoy , Qi Dake , Kewalramani Girish , Yuen Gloria , Virk Naureen , Abrahani Ashraf , Rodrigues Brian
Published: January 2005

The lipoprotein lipase (LPL)-augmenting property of lysophosphatidylcholine requires the formation of lysophosphatidic acid (LPA) ( J Mol Cell Cardiol 37: 931–938, 2004). Given that the actin cytoskeleton has been implicated in regulating cardiomyocyte LPL, we examined whether LPL secretion after LPA involves actin cytoskeleton reassembly. Incubation of myocytes with LPA (1–100 nM) increased basal and heparin-releasable LPL (HR-LPL), an effect that was independent of shifts in LPL mRNA. The influence of LPA on myocyte LPL was reflected at the coronary lumen, with substantial increases of the enzyme at this location. Incubation of myocytes with cytochalasin D not only blocked LPA-induced augmentation of HR-LPL but also abrogated filamentous actin formation. These effects of LPA were likely receptor mediated. Exposure of myocytes to LPA facilitated significant membrane translocation of RhoA and its downstream effector Rho kinase I (ROCK I), and blocking this effect with Y-27632 appreciably reduced basal and HR-LPL activity. Incubation of adipose tissue with LPA also significantly enhanced basal and HR-LPL activity, suggesting that sarcomeric actin likely has a limited role in influencing the LPL secretory function of LPA in the myocyte. Comparable to LPA, hyperglycemia also caused significant membrane translocation of RhoA and ROCK I in hearts isolated from diazoxide-treated animals, effects that were abrogated using insulin. Overall, our data suggest that comparable to hyperglycemia, LPA-induced increases in cardiac LPL occurred via posttranscriptional mechanisms and processes that likely required RhoA activation and actin polymerization. Whether this increase in LPL augments triglyceride deposition in the heart leading to eventual impairment in contractile function is currently unknown.
Endothelin isoforms and the response to myocardial stretch
Authors: Ennis Irene L. , Garciarena Carolina D. , Pérez Néstor G. , Dulce Raúl A. , Camilión de Hurtado María C. , Cingolani Horacio E.
Published: January 2005

Myocardial stretch elicits a biphasic increase in developed force with a first rapid force response and a second slow force response (SFR). The rapid phase is due to an increase in myofilament Ca2+ responsiveness; the SFR, analyzed here, is ascribed to a progressive increase in Ca2+ transients. Experiments were performed in cat papillary muscles to further elucidate the signaling pathway underlying the SFR. Although the SFR was diminished by BQ-123, a similar endothelin (ET)-1-induced increase in force was not affected: 23 ± 2 vs. 23 ± 3% (not significant). Instead, BQ-123 suppressed the contractile effects of ET-2 or ET-3 (21 ± 2 and 25 ± 3% vs. −1 ± 1 and −7 ± 3% respectively, P < 0.05), suggesting that ET-2 or ET-3, but not ET-1, was involved in the SFR. Each isoform activated the Na+/H+ exchanger (NHE-1), increasing intracellular Na+ concentration by 2.0 ± 0.1, 2.3 ± 0.1, and 2.1 ± 0.4 mmol/l for ET-1, ET-2, and ET-3, respectively ( P < 0.05). The NHE-1 inhibitor HOE-642 prevented the increases in force and intracellular Na+ concentration induced by all the ET isoforms, but only ET-2 and ET-3 effects were sensitive to BQ-123. Real-time RT-PCR measurements of prepro-ET-1, -ET-2, and -ET-3 were performed before and 5, 15, and 30 min after stretch. No changes in ET-1 or ET-2, but an increase of ∼60% in ET-3, mRNA after 15 min of stretch were detected. Stretch-induced ET-3 mRNA upregulation and its mechanical counterpart were suppressed by AT1 receptor blockade with losartan. These data suggest a role for AT1-mediated ET-3 released in the early activation of NHE-1 that follows myocardial stretch.
Ginsenosides block HIV protease inhibitor ritonavir-induced vascular dysfunction of porcine coronary arteries
Authors: Chai Hong , Zhou Wei , Lin Peter , Lumsden Alan , Yao Qizhi , Chen Changyi
Published: January 2005

Human immunodeficiency virus (HIV) protease inhibitor ritonavir (RTV) may induce vascular dysfunction through oxidative stress. Ginsenosides have been shown to have potential benefits on the cardiovascular system through diverse mechanisms, including antioxidative property. The objective of this study was to determine whether ginsenosides could prevent coronary arteries from RTV-induced dysfunction. Porcine coronary artery rings were incubated with RTV and ginsenosides Rb1, Rc, and Re for 24 h. Vasomotor function was recorded by a myograph tension system. In response to the thromboxane A2 analog U-46619, the contraction of the vessel rings was significantly reduced. When cocultured with Rb1, Rc, and Re, the contractility significantly increased. In response to bradykinin at 10−5 M, the endothelium-dependent relaxation of vessel rings was significantly reduced by 59% for RTV compared with controls ( P < 0.05). When cocultured with Rb1, Rc, and Re, the relaxation significantly increased 100%, 90%, and 134%, respectively, compared with the RTV-alone groups ( P > 0.05). In response to sodium nitroprusside, RTV significantly reduced vasorelaxation. In addition, the endothelial nitric oxide synthase (eNOS) mRNA levels were significantly reduced by 78% for RTV group ( P < 0.05) by real-time PCR analysis. The eNOS protein levels measured by Western blot analysis and nitrite concentrations measured by Griess assay were also decreased, whereas O2− production by lucigenin-enhanced chemiluminescence was significantly increased in the RTV-treated group. These effects of RTV were effectively blocked by ginsenosides. Thus HIV protease inhibitor RTV significantly impaired the vasomotor function of porcine coronary arteries. This effect may be mediated by the downregulation of eNOS and overproduction of O2−. These results suggest that ginsenosides can effectively block RTV-induced vascular dysfunction.
Chloride channel activity in human lung fibroblasts and myofibroblasts
Authors: Yin Zhaohong , Watsky Mitchell A.
Published: January 2005

It is well established that transforming growth factor (TGF)-β stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl−channel current ( ICl-LPA) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of ICl-LPAon fibroblast-to-myofibroblast differentiation. We recorded ICl-LPAusing the amphotericin perforated-patch technique. We activated ICl-LPAusing LPA or sphingosine-1-phosphate. We determined phenotype by Western blotting and immunohistochemistry using an anti-α-smooth muscle actin (SMA) antibody. RT-PCR was performed to determine which phospholipid growth factor receptors are present in HLF. We found that HLF cultured in TGF-β (myofibroblasts) had significantly elevated α-SMA levels and ICl-LPAcurrent density compared with control fibroblasts. ICl-LPAactivation was blocked by DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (1 μM). DIDS and NPPB, in a dose-dependent manner, significantly reduced α-SMA levels in HLF stimulated with TGF-β. These results demonstrate the receptor-mediated activation of ICl-LPAby LPA and sphingosine-1-phosphate in cultured human lung myofibroblasts, with only minimal ICl-LPAactivity in fibroblasts. This Cl−channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.
Transcription of macrophage IGF-I exon 1 is positively regulated by the 5′-untranslated region and negatively regulated by the 5′-flanking region
Authors: Wynes Murry W. , Riches David W. H.
Published: January 2005

Idiopathic pulmonary fibrosis (IPF) is an insidious lung disease with no known cure or effective therapy. Macrophage-derived insulin-like growth factor-I (IGF-I) is thought to play a role in the pathogenesis of IPF; however, little is known about the control of IGF-I expression in macrophages. In this report we investigated the cis-regulatory elements that control basal expression using luciferase reporter constructs in RAW 264.7 macrophages. We show that the +95 to +329 region contains elements necessary to direct maximal promoter activity, whereas the +251 to +329 region contains the minimal promoter. Mapping transcriptional start sites for endogenous IGF-I in primary macrophages revealed that the major transcriptional start site is centered at +150, whereas the most 3′-transcriptional start site is centered at +255. Nuclear proteins from primary and RAW 264.7 macrophages bind specifically to the region required for maximal promoter activity (+134 to +173) and to the region required for minimal promoter activity (+267 to +299). Antibody supershift assays indicate that Sp3 bound to the +267 to +299 region. Moreover, mutation of the putative binding site reduced Sp3 binding in EMSAs and increased promoter activity in luciferase reporter gene assays. We also found that the regions from −1711 to −855 and −855 to −337 contain putative macrophage-specific suppressor elements that do not function in HeLa or COS-7 epithelial cell lines. These data support the view that macrophage IGF-I expression is positively regulated by elements located in the 5′-untranslated region and negatively regulated by elements in the 5′-flanking region of the IGF-I gene.
Pulmonary vascular effects of sildenafil on the development of chronic pulmonary hypertension in the ovine fetus
Authors: Larrue B. , Jaillard S. , Lorthioir M. , Roubliova X. , Butrous G. , Rakza T. , Warembourg H. , Storme L.
Published: January 2005

We investigated the pulmonary vascular effects of prophylactic use of sildenafil, a specific phosphodiesterase-5 inhibitor, in late-gestation fetal lambs with chronic pulmonary hypertension. Fetal lambs were operated on at 129 ± 1 days gestation (term = 147 days). Ductus arteriosus (DA) was compressed for 8 days to cause chronic pulmonary hypertension. Fetuses were treated with sildenafil (24 mg/day) or saline. Pulmonary vascular responses to increase in shear stress and in fetal PaO2were studied at, respectively, day 4 and 6. Percent wall thickness of small pulmonary arteries (%WT) and the right ventricle-to-left ventricle plus septum ratio (RVH) were measured after completion of the study. In the control group, DA compression increased PA pressure (48 ± 5 to 72 ± 8 mmHg, P < 0.01) and pulmonary vascular resistance (PVR) (0.62 ± 0.08 to 1.15 ± 0.11 mmHg·ml−1·min−1, P < 0.05). Similar increase in PAP was observed in the sildenafil group, but PVR did not change significantly (0.54 ± 0.06 to 0.64 ± 0.09 mmHg·ml−1·min−1). Acute DA compression, after brief decompression, elevated PVR 25% in controls and decreased PVR 35% in the sildenafil group. Increased fetal PaO2did not change PVR in controls but decreased PVR 60% in the sildenafil group. %WT and RVH were not different between groups. Prophylactic sildenafil treatment prevents the rise in pulmonary vascular tone and altered vasoreactivity caused by DA compression in fetal lambs. These results support the hypothesis that elevated PDE5 activity is involved in the consequences of chronic pulmonary hypertension in the perinatal lung.
Dopamine D2 receptor modulation of carotid body type 1 cell intracellular calcium in developing rats
Authors: Carroll J. L. , Boyle K. M. , Wasicko M. J. , Sterni L. M.
Published: January 2005

Carotid chemoreceptor type 1 cells release dopamine, which inhibits carotid chemoreceptor activity via dopamine D2 autoreceptors on type 1 cells. Postnatal changes in dopaminergic modulation may be involved in postnatal chemoreceptor development. The present study explores dopaminergic modulation of the intracellular calcium ([Ca2+]i) response to hypoxia in type 1 cells from 1, 3, and 11- to 16-day-old rats. Using fura-2, we studied the effects of quinpirole, a D2 receptor agonist, on type 1 cell [Ca2+]iresponse to 90-s hypoxia challenges (Po2∼1–2 mmHg). Cells were sequentially exposed to the following challenges: 1) hypoxia control, 2) hypoxia plus quinpirole, and 3) hypoxia plus quinpirole plus sulpiride (D2 receptor antagonist). In the 11- to 16-day-old group, type 1 cell [Ca2+]iincreased ∼3 to 4-fold over resting [Ca2+]iin response to hypoxia. Quinpirole (10 μM) significantly blunted the peak [Ca2+]iresponse to hypoxia. Repeat challenge with hypoxia plus 10 μM quinpirole in the presence of 10 μM sulpiride partially restored the hypoxia [Ca2+]iresponse. In sharp contrast to the older aged group, 10 μM quinpirole had minimal effect on hypoxia response of type 1 cells from 1-day-olds and a small but significant effect at 3 days of age. We conclude that stimulation of dopamine D2 receptors inhibits type 1 cell [Ca2+]iresponse to hypoxia, consistent with an inhibitory autoreceptor role. These findings suggest dopamine-mediated inhibition and oxygen sensitivity increase with age on a similar time course and do not support a role for dopamine as a major mediator of carotid chemoreceptor resetting.
The role of tourist icons for sustainable tourism
Authors: Becken Susanne
Published: January 2005

This paper provides a discussion of tourist icons as pull factors of a destination and problems associated with icons. According to interviews held with major wholesalers in Australia and inbound tour operators in New Zealand, tourist icons vary in importance to different segments of the market, with coach tourists placing more importance on icons than free independent travellers. Moreover, the need to base overseas marketing on icons depends on the maturity of the targeted market. A tourist survey in New Zealand revealed that Milford Sound and Rotorua were indeed the most commonly known attractions, but that in addition to these two key icons, tourists were attracted to natural features in general, such as mountains and glaciers.

This paper argues that an overemphasis on tourist icons potentially leads to unsustainable tourism development, for the following reasons: tourists travel large distances to visit the spatially very dispersed tourist icons, and this results in considerable amounts of greenhouse gas emissions, even if they visit New Zealand for a short period of time; the high visitation levels at popular sites result in infrastructural bottlenecks, reaching carrying capacity, which results in potentially significant environmental and social impacts; and the uneven geographical distribution of tourism spending is a missed opportunity for much-needed regional development. The current ‘100% Pure’ campaign by Tourism New Zealand potentially contributes to a more holistic promotion of New Zealand as an attractive ‘natural landscape’ destination.
Staying in the spa marketing game: Trends, challenges, strategies and techniques
Authors: McNeil Kimberly R. , Ragins Edna J.
Published: January 2005

The spa industry in the USA has shown robust growth in sales volume and in the number of facilities. This growth is driven by baby-boomers who want a place where they can get spa treatments that improve their physical and mental well-being, and which offers rest, relaxation, exercise and pampering. Spas are providing a myriad of opportunities for improving the mind and body. Despite the growth currently taking place in the spa industry, there are still challenges to consider. The authors highlight current issues and trends in the US spa industry. Strategies and techniques for spa marketers to combat potential challenges are suggested under the headings of target markets, product, place, promotion and price.
The ski chalet party: An under-researched and highly successful industry sector
Authors: Major Bridget
Published: January 2005

This paper aims to explore the origins of the concept of the ski chalet party, an under-researched and yet extremely successful British initiative. The chalet party product is examined in the context of the post-war development of the European winter sports industry, and qualitative research is undertaken which places this type of service and accommodation provision within a European resort development perspective. Primary research based on an interview with the pioneer of the European ski chalet party outlines the origins and unique nature and characteristics of this product. Statistics in the format of data from two of the UK market leaders are provided demonstrating the importance and significance of this type of accommodation to winter sports tourism. The exploration of the concept of the chalet party is important in evaluating how this model has led to the product becoming such an integral part of the winter sports industry, particularly pertinent given its unique characteristics which have implications for its management. The author explores the nature of the product and details its main features and characteristics. The exploration of the emergence of the concept and the positioning of the format leads to the identification of further areas for research and consideration of the fact that the model has also been successfully adapted and utilised in the provision of non-winter sports holidays, but not to the same degree.
The detailed structure and origin of clay minerals at the Cretaceous/Tertiary boundary, Stevns Klint (Denmark)
Authors: Drits V. A. , Lindgreen H. , Sakharov B. A. , Jakobsen H. J. , Zviagina B. B.
Published: January 2005

The structure of an illite-smectite (I-S) sample (HSI) from the Tertiary–Cretaceous boundary layer at Stevns, Denmark, i.e. the so-called Fish Clay, is investigated in order to discuss the origin of this clay and its similarity to clays from Maastrichtian-Danian chalk of Denmark and the North Sea. The phase compositions and layer sequences have been determined by X-ray diffraction. The structural formulae are determined from chemical analysis and solid-state 27Al magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. The octahedral vacancy pattern has been determined by thermal analysis and the particle shape by atomic force microscopy. The HSI sample I-S consists of two phases, a high-smectitic (HS) phase (70%) having 95% smectite and 5% illite (leucophyllite) layers, and a low-smectitic (LS) phase (30%) having 50% smectite and 50% illite (leucophyllite) layers. Both phases have trans-vacant dioctahedral sheets and contain only traces of IVAl, the charge of the illite (leucophyllite) layers being provided predominantly by octahedral Mg for Al substitution. The structure of the tetrahedral and octahedral sheets for the HSI are compared to the structure of an I-S sample (GRI) from the Maastrichtian chalk at Stevns below the Tertiary-Cretaceous boundary and to the standard smectite SAz-1 from Arizona. For all three samples the 27Al MAS NMR spectra show the presence of two resolved IVAl resonances, which indicate the presence of two different IVAl sites. The NMR, infrared spectroscopy and chemical data show that both HSI and GRI have highly ordered Mg for Al substitution in the octahedral sheet, each Al having one Mg and two Al neighbours, whereas substitution of Mg for Al in SAz-1 is random. For the HSI sample, both the HS and LS phases are probably formed from volcanic ash. The structural similarity of the phases in HSI and GRI shows that GRI formed from a material similar to HSI by illitization of the HS phase and chloritization of the LS phase.
In situ transformation of amorphous gels into spherical aggregates of kaolinite: A HRTEM study
Authors: Huertas F. J. , Fiore S. , Linares J.
Published: January 2005

Initial stages of the gel-to-kaolinite transformation were studied using HRTEM. Spherical aggregates of kaolinite crystals were produced during the synthesis of kaolinite by hydrothermal treatment of Si-Al amorphous gels. Prior to sphere formation, gels transform into pseudospherical domains that have a Si/Al ratio of one and display no SAED pattern. The spherical particles consist of radially arranged sectors of stacks of planar crystallites. Crystals display nonbasal spacings of 4.5, 4.2 and 3.8 Å and a basal spacing of 7.1 Å , the c* axis following the radius of the sphere.

Interpretation of the 3D nanostructure of the spheres is difficult. The c* axis exhibits a radial disposition, but the relative orientation of the a* and b* axes in neighbouring crystallites may produce bent layers or incoherent contacts. In addition, curved layers with a d spacing of 7.4 Å may be attributed to halloysite layers collapsed under microscopy conditions. The disappearance of the spheres during the hydrothermal treatment is probably due to preferential dissolution of either highstress areas near bent layers or non-crystalline material filling crystal boundaries. Dissolution leads to sphere disaggregation and allows the component columnar crystals to continue to grow. Observations of the gel matrix suggest that under our experimental conditions kaolinite crystallizes via an in situ transformation of the gel.
Experimental study of swelling in unsaturated compacted clays
Authors: Saiyouri N. , Tessier D. , Hicher P. Y.
Published: January 2005

This paper describes the swelling properties of two highly compacted clays, natural, untreated Wyoming montmorillonite (MX80) and Fourges smectite (FoCa7), saturated with Na and Ca, respectively.

The initially compacted samples were hydrated by subjecting them to different suction pressures in a range between 100 MPa and 1 kPa. For each equilibrium state, the volume change (swelling) and water content (hydration) were measured. The samples were then studied by X-ray diffraction using a transmission device to determine interlayer distance and particle size, in order to clarify both the swelling and hydration mechanisms. The distances between clay layers ranged between 10 and 21.6 Å , i.e. corresponding to between 0 and 4 water layers. Upon hydration, the particle size decreased from 350 and 100 clay layers per particle to 10 layers per particle when the suction pressure decreased from 100 MPa to 1 kPa for MX80 and FoCa7, respectively. The first swelling stage is described as being an insertion of water molecules between the layers. Then a division of the initial particles into particles of smaller size with increasingly large inter-particle distances was observed. Observations by transmission electronic microscopy confirmed these results.
Diagenetic to low-grade metamorphic evolution of clay mineral assemblages in Palaeozoic to early Mesozoic rocks of the Eastern Taurides, Turkey
Authors: Bozkaya Ö. , Yalçin H.
Published: January 2005

Palaeozoic to early Mesozoic rocks of both autochthonous and allochthonous units of the Eastern Tauride Belt have different textural and mineralogical features related to their varying deposition regimes and thermal histories. The Devonian and Carboniferous formations in the allocthonous units have anchizonal to epizonal grades of metamorphism, whereas those of the autochthon experienced only early diagenetic conditions. A well-developed penetrative slaty cleavage is also found in Devonian formations of the allocthonous units, which is not observed in equivalent rocks of the autochthon. Phyllosilicates of the uncleaved diagenetic assemblages consist mainly of illite, chlorite, kaolinite, mixed-layer illite-smectite, chlorite-vermiculite and chloritesmectite, whereas in addition to these, metamorphic allochthonous units contain some index minerals such as dickite, paragonite, Na and K mica, pyrophyllite, stilpnomelane and chloritoid. These latter assemblages show a higher degree of textural and mineralogical maturation and lower b cell dimensions than the autochthonous rocks, suggesting that they originated in a relatively high heat flow basin such as an extensional setting.
Effectiveness of different databases in identifying studies for systematic reviews: experience from the WHO systematic review of maternal morbidity and mortality
Authors: Betrán Ana P , Say Lale , Gülmezoglu A Metin , Allen Tomas , Hampson Lynn
Published: January 2005

Background

Failure to be comprehensive can distort the results of a systematic review. Conversely, extensive searches may yield unmanageable number of citations of which only few may be relevant. Knowledge of usefulness of each source of information may help to tailor search strategies in systematic reviews.

Methods

We conducted a systematic review of prevalence/incidence of maternal mortality and morbidities from 1997 to 2002. The search strategy included electronic databases, hand searching, screening of reference lists, congress abstract books, contacting experts active in the field and web sites from less developed countries. We evaluated the effectiveness of each source of data and discuss limitations and implications for future research on this topic.

Results

Electronic databases identified 64098 different citations of which 2093 were included. Additionally 487 citations were included from other sources. MEDLINE had the highest yield identifying about 62% of the included citations. BIOSIS was the most precise with 13.2% of screened citations included. Considering electronic citations alone (2093), almost 20% were identified uniquely by MEDLINE (400), 7.4% uniquely by EMBASE (154), and 5.6% uniquely by LILACS (117). About 60% of the electronic citations included were identified by two or more databases.

Conclusions

This analysis confirms the need for extending the search to other sources beyond well-known electronic databases in systematic reviews of maternal mortality and morbidity prevalence/incidence. These include regional databases such as LILACS and other topic specific sources such as hand searching of relevant journals not indexed in electronic databases. Guidelines for search strategies for prevalence/incidence studies need to be developed.
Injury morbidity in an urban and a rural area in Tanzania: an epidemiological survey
Authors: Moshiro Candida , Heuch Ivar , Åstrøm Anne Nordrehaug , Setel Philip , Hemed Yusuf , Kvåle Gunnar
Published: January 2005

Background

Injuries are becoming a major health problem in developing countries. Few population based studies have been carried out in African countries. We examined the pattern of nonfatal injuries and associated risk factors in an urban and rural setting of Tanzania.

Methods

A population-based household survey was conducted in 2002. Participants were selected by cluster sampling. A total of 8,188 urban and 7,035 rural residents of all ages participated in the survey. All injuries reported among all household members in the year preceding the interview and resulting in one or more days of restricted activity were included in the analyis.

Results

A total of 206 (2.5%) and 303 (4.3%) persons reported to have been injured in the urban and rural area respectively. Although the overall incidence was higher in the rural area, the incidence of major injuries (≥ 30 disability days) was similar in both areas. Males were at a higher risk of having an injury than females. Rural residents were more likely to experience injuries due to falls (OR = 1.6; 95% CI = 1.1 – 2.3) and cuts (OR = 4.3; 95% CI = 3.0 – 6.2) but had a lower risk of transport injuries. The most common causes of injury in the urban area were transport injuries and falls. In the rural area, cuts and stabs, of which two thirds were related to agriculture, formed the most common cause. Age was an important risk factor for certain types of injuries. Poverty levels were not significantly associated with experiencing a nonfatal injury.

Conclusion

The patterns of injury differ in urban and rural areas partly as a reflection of livelihoods and infrastructure. Rural residents are at a higher overall injury risk than urban residents. This may be important in the development of injury prevention strategies.
The changes of CD4+CD25+/CD4+ proportion in spleen of tumor-bearing BALB/c mice
Authors: Liu Ji-Yan , Zhang Xiao-Shi , Ding Ya , Peng Rui-Qing , Cheng Xia , Zhang Nian-Hua , Xia Jian-Chuan , Zeng Yi-Xin
Published: January 2005

CD4+CD25+ regulatory T lymphocytes (TR) constitute 5–10% of peripheral CD4+ T cells in naive mice and humans, and play an important role in controlling immune responses. Accumulating evidences show that TR cells are involved in some physiological processes and pathologic conditions such as autoimmune diseases, transplantation tolerance and cancer, and might be a promising therapeutic target for these diseases.

To evaluate the change of CD4+CD25+ TR cells in mouse tumor models, CD4+CD25+ subset in peripheral blood and spleen lymphocytes from normal or C26 colon-carcinoma-bearing BABL/c mice were analyzed by flow cytometry using double staining with CD4 and CD25 antibodies.

The proportion of CD4+CD25+/CD4+ in spleen lymphocytes was found to be higher than that in peripheral blood lymphocytes in normal mice. No difference was observed in the proportion in peripheral blood lymphocytes between tumor bearing mice and normal mice, while there was a significant increase in the proportion in spleen lymphocytes in tumor bearing mice as compared with normal mice. Moreover, the proportion increased in accordance with the increase in the tumor sizes. The increase in the proportion was due to the decrease in CD4+ in lymphocytes, which is resulted from decreased CD4+CD25- subset in lymphocytes. Our observation suggests the CD4+CD25+/CD4+ proportion in spleen lymphocytes might be a sensitive index to evaluate the TR in tumor mouse models, and our results provide some information on strategies of antitumor immunotherapy targeting CD4+CD25+ regulatory T lymphocytes.