bluuebunny/crossref_metadata_2025 huggingface dataset:
Sed1p and Srl1p are required to compensate for cell wall instability in Saccharomyces cerevisiae mutants defective in multiple GPI‐anchored mannoproteins
Authors: Hagen Ilja , Ecker Margit , Lagorce Arnaud , Francois Jean M. , Sestak Sergej , Rachel Reinhard , Grossmann Guido , Hauser Nicole C. , Hoheisel Jörg D. , Tanner Widmar , Strahl Sabine
Published: May 2004

The covalently linked cell wall protein Ccw12p of Saccharomyces cerevisiae is a GPI‐anchored protein (V. Mrsa et al., 1999, J Bacteriol 181: 3076–3086). Although only 121 amino acids long, the haemagglutinin‐tagged protein released by laminarinase from the cell wall possesses an apparent molecular mass of > 300 kDa. A membrane‐bound form with an apparent molecular mass of 58 kDa is highly O‐ and N‐glycosylated and contains the GPI anchor. With a half‐life of 2 min, the membrane form is transformed to the > 300 kDa form. The deletion mutant ccw12Δ grows slower than the wild type, is highly sensitive to Calcofluor white and contains 2.5 times more chitin. Further, compared with wild‐type yeast, significantly more proteins are released from intact cells when treated with dithiothreitol. Interestingly, these defects become less pronounced when further GPI‐anchored cell wall proteins are deleted. Mutant ΔGPI (simultaneous deletion of CCW12, CCW13/DAN1, CCW14, TIP1 and CWP1) is similar in many respects to wild‐type yeast. To find out how the cell wall is stabilized in mutant ΔGPI, a genome‐wide transcription analysis was performed. Of 159 significantly regulated genes, 14 encode either known or suspected cell wall‐associated proteins. Analysis of genes affected in transcription revealed that SED1 and SRL1 in particular are required to reconstruct cell wall stability in the absence of multiple GPI‐anchored mannoproteins.
Activation of the redox sensor Pap1 by hydrogen peroxide requires modulation of the intracellular oxidant concentration
Authors: Vivancos Ana P. , Castillo Esther A. , Jones Nic , Ayté José , Hidalgo Elena
Published: May 2004

The transcription factor Pap1 and the MAP kinase Sty1 are key regulators of hydrogen peroxide‐induced responses in Schizosaccharomyces pombe. Pap1 can be activated quickly at low, but not high, hydrogen peroxide concentrations. The MAP kinase Sty1 has been reported to participate in Pap1 activation by the oxidant. Here, we provide biochemical and genetic evidence for the in vivo formation of a hydrogen peroxide‐induced disulphide bond in Pap1, which precedes the rapid and reversible nuclear accumulation of the transcription factor. We show that activation of the Sty1 cascade before the oxidative insult, or overexpression of the Sty1‐regulated genes ctt1 (encoding catalase) or gpx1 (encoding glutathione peroxidase), can accelerate Pap1 entry even at high doses of hydrogen peroxide. In fact, the lack of Sty1 impedes Pap1 nuclear localization, but only at high doses of the oxidant. We propose that, whereas low doses of hydrogen peroxide lead directly to Pap1 oxidation‐activation, high concentrations of the oxidant initially activate the Sty1 pathway, with the consequent increase in scavenging enzymes, which in turn helps to decompose the excess of hydrogen peroxide and achieve an appropriate concentration for the subsequent activation of Pap1. Our results also suggest that activation of Sty1 at high doses of hydrogen peroxide may also be required to trigger other antioxidant activities such as those reverting the overoxidation of cysteine residues at the Pap1 pathway.
The major structural components of two cell surface filaments of Porphyromonas gingivalis are matured through lipoprotein precursors
Authors: Shoji Mikio , Naito Mariko , Yukitake Hideharu , Sato Keiko , Sakai Eiko , Ohara Naoya , Nakayama Koji
Published: May 2004

Bacterial cell surface filaments play significant roles in adherence to and invasion of host cells. They are generated by the chaperone/usher pathway system (class I fimbriae), the type II secretion system (type IV pili) and the nucleation‐dependent polymerization system (Curli filaments) that are categorized by their modes of expression and assembly. In this study, we found that the periodontal pathogen Porphyromonas gingivalis expressed the major structural components of two cell surface filaments (fimbrilin and the 75 kDa protein) that had extremely long prosequences in their primary gene products. N‐terminal amino acid sequencing of the prosequences, treatment of P. gingivalis cells with globomycin, an inhibitor for lipoprotein‐specific signal peptidase, amino acid substitution of the cysteine residue of the prosequence of fimbrilin and [3H]‐palmitic acid labelling implied that fimbrilin and the 75 kDa protein were matured through their lipoprotein precursor forms. Accumulation of precursor forms of fimbrilin and the 75 kDa protein on the cell surface of the gingipain‐null mutant revealed that Arg‐gingipain processed these precursors on the surface to yield their mature forms, which subsequently assembled into the filamentous structures, suggesting that the transport and assembly of the major component proteins appear to be novel.
P–T conditions and kinematics of shear zones from the southern Adelaide Fold–Thrust Belt, South Australia: insights into the dynamics of a deeply eroded orogenic wedge
Authors: YASSAGHI A. , JAMES P. R. , FLÖTTMANN T. , WINSOR C. N.
Published: May 2004

Mechanisms are examined for the development of shear zones in the external portion of the southern Adelaide Fold–Thrust Belt, based on geometric and kinematic analyses, balanced cross‐sections, microstructure, pressure, and temperature of deformation estimates. Kinematic indicators reveal the zones were overthrust towards the west‐northwest. Shear‐zone quartz‐grain microstructures show mainly intracrystalline deformation and dynamic recrystallisation where there is greater grain dislocation. These features become less well developed in the shear zones closer to the hinterland, than in those on the foreland side. Primary fluid inclusions in deformed quartz veins reveal that average temperatures were in excess of 358°C and indicate an average pressure of about 300 MPa. Microprobe analyses of coexisting biotite–muscovite–quartz–feldspar reveal an average peak metamorphic pressure of 250 ± 100 MPa, compatible with the estimated pressure of 300 MPa from primary fluid inclusions. Modified fluid inclusions, however, suggest an average entrapment pressure of less than 150 MPa, which suggests that they suffered overpressuring during exhumation, implying that the amount of shear‐zone exhumation decreases towards the foreland. Based on this information, an orogenic wedge model is the preferred mechanism for shear‐zone development. The earliest generated and propagated shear zones in the hinterland were buried at greater depth and remained at higher temperatures and pressures before being exhumed. Later shear zones generated closer to the foreland propagated from shallower depths, implying that they experienced lower pressures and temperatures before uplift.
Miocene volcanic seamounts on northern Lord Howe Rise: lithology, age and origin
Authors: EXON N. F. , QUILTY P. G. , LAFOY Y. , CRAWFORD A. J. , AUZENDE J‐M.
Published: May 2004

Multibeam sonar swath‐mapping has revealed small submarine volcanic cones on the northeastern Lord Howe Rise, a submerged ribbon continent. Two such cones, aligned north‐northwest and 120 km apart, were dredged at 23–24°S. Water depth is about 1150 m nearby: the southern cone rises to 750 m and the northern to 900 m. Volcanic rocks dredged from the cones are predominantly highly altered hyaloclastites with minor basalt. The clasts are mostly intensely altered vesicular brownish glass with lesser basalt, set in zeolitic, clayey, micritic, or ferruginous cement. Lavas and hyaloclastites contain altered phenocrysts of olivine and plagioclase, and fresh clinopyroxene. The latter have compositions between acmite and Ti‐augite, matching clinopyroxene phenocrysts in undersaturated intraplate basanitic mafic lavas. Interbedded micrites in the volcaniclastics represent calcareous ooze that was deposited with (or later than) the volcanic pile. Foraminifers indicate that the micrite is dominantly Early Miocene (ca 16 Ma), and that the ooze was deposited in cool water. Upper Miocene to Pliocene micrites, presumed to be later infillings, all contain warm‐water forms. This evidence strongly suggests that both cones formed in pelagic depths in the Early Miocene. Ferromanganese crusts from the two cones are up to 7 cm thick and similar physically, but different chemically. The average growth rate is 4 mm/106 y. Copper, nickel, and cobalt content are relatively high in the north, but copper does not exceed 0.08 wt%, nickel 0.65%, and cobalt 0.25%. The Mn:Fe ratio is high in the south (average 13.7) suggesting strong hydrothermal influence. Such small volcanic cones related to intraplate hot‐spot‐type volcanism may occur in extensive fields on the Lord Howe Rise, like those off southern Tasmania. On the Lord Howe Rise, the known small volcanic cones coincide with broad gravity highs in areas of shallow continental basement. The highs are aligned parallel to the Lord Howe Rise margins, and it is probable that the Miocene volcanism is related to jostling on older faults. Other fields of small volcanic seamounts may occur on the Lord Howe Rise on such fractured gravity highs.
The Hazards of Starting and Quitting Smoking: Some Australian Evidence
Authors: Kidd Michael P. , Hopkins Sandra
Published: May 2004

The empirical analysis employs individual level data from the Australian Health Survey combined with retrospective data on tobacco price matched to the age at which the individual started and quit smoking. Split‐population hazard models are estimated for both starting and quitting smoking. The analysis suggests price plays a significant role in the decision to start smoking but not in the decision to quit. Further sensitivity analysis of different age groups and an alternative data source, questions the robustness of the significant role of price in the smoking initiation decision. From a policy perspective, the results indicate that increases in tobacco taxation can be an important instrument in reducing the incidence of smoking, but should be combined with other mechanisms such as mandating smoke‐free environments and antismoking education. Our results strongly support the targeting of antismoking campaigns towards teenagers.
Cultural structures and political life: The cultural matrix of democracy in Spain1
Authors: BENEDICTO JORGE
Published: May 2004

Abstract.  This article seeks to show empirically the importance of cultural aspects in the understanding of political events and the impact of cultural structures on the development of political life in contemporary societies. It uses as a case study the democratization process that has been taking place in Spain for the last 25 years. The Spanish case is especially interesting because of the powerful cultural framework established around the process of transition to democracy; a framework from which most political symbols and meanings of the new Spanish democracy emanate. After analyzing the basic categories of this cultural structure and the main consequences for the functioning of political life, the article goes on to argue that these cultural elements have shaped a special relationship between citizens and politics.
Unidirectional Atrio‐Atrial Conduction after Surgical Isolation of the Posterior Part of the Left Atrium and Pulmonary Veins for Atrial Fibrillation:
Authors: NOGAMI AKIHIKO , TADA HIROSHI , NAITO SHIGETO , KANEKO TATSUO
Published: May 2004

A 66‐year‐old woman underwent mitral valve replacement and surgical isolation of the posterior part of the left atrium for persistent AF. During sinus rhythm an isolated atrial rhythm was recorded from the isolated posterior left atrium using an esophageal electrode catheter. After the intravenous administration of adenosine triphosphate sinus rhythm was suppressed and 1:1 conduction from the posterior left atrium to the rest of the atrium was recorded. Unidirectional atrio‐atrial conduction was revealed by a simple electrophysiological evaluation.(PACE 2004; 27[Pt. I]:812–814)
Atrial Septal Pacing:
Authors: PADELETTI LUIGI , MICHELUCCI ANTONIO , PIERAGNOLI PAOLO , COLELLA ANDREA , MUSILLI NICOLA
Published: May 2004

Atrial pacing may prevent the onset of atrial fibrillation (AF) because of: (1) prevention of the relative bradycardia that triggers paroxysmal AF; (2) prevention of the bradycardia induced dispersion of refractoriness; (3)suppression or reduction of premature atrial contractions that initiate reentry and predispose to AF; (4) preservation of AV synchrony, which might prevent switch induced changes in atrial repolarization predisposing to AF. Atrial pacing locations that decrease atrial activation and dispersion of refractoriness may be preferable in patients with a history of AF. Two different interatrial septum sites have been proposed: the Bachmann's bundle and the coronary sinus ostium. The results of two prospective randomized studies indicate that septal pacing, when compared to the traditional right atrial appendage pacing, significantly reduces : (1) paroxysmal AF recurrences and burden; and (2) progression to chronic AF. (PACE 2004; 27[Pt. II]:850–854)
Randomized Trials for Selective Site Pacing:
Authors: GAMMAGE MICHAEL D. , MARSH ANNA‐MARIE
Published: May 2004

Clinical evidence is accumulating that pacing from traditional right‐sided sites (right ventricular apex and right atrial appendage) may be associated with long‐term detriment to left ventricular function and promotion of atrial arrhythmias. Large numbers of small studies, some randomized, have addressed the effects of pacing from nontraditional or alternate sites, but the studies have varied in design such that comparison of results is often difficult. Many studies show data from acute studies only, although more recently longer‐term data have started to become available; definition and description of pacing site also varies, adding further to the difficulties in comparing data. This article attempts to examine the randomized studies available to date, and to consider how future studies may contribute to our understanding of the effects of pacing from traditional and selected, specific sites on the right side of the heart. (PACE 2004; 27[Pt. II]:878–882)
Selective Site Pacing:
Authors: LIEBERMAN RANDY , GRENZ DAVID , MOND HARRY G. , GAMMAGE MICHAEL D.
Published: May 2004

Selective site right ventricular pacing has been suggested as an approach to reduce the incidence of ventricular dysfunction and hopefully influence the morbidity resulting from traditional right ventricular apical pacing. Pacing from the right ventricular apex allows a stable ventricular rate, and together with atrial pacing and sensing, helps maintain atrioventricular synchrony but does not allow physiological activation of the left ventricle. Traditional atrial pacing sites like the right atrial appendage may encourage atrial tachyarrhythmias, whereas lead placement in right atrial septal sites may reduce the frequency of symptomatic atrial tachyarrhythmia episodes, especially when combined with prevention algorithms. Researchers attempting to pace the heart from these selective sites have been hindered by the lack of uniform definitions of where these sites actually lie and the inadequacy of tools to consistently reach these locations and verify correct placement. This lack of definition consensus may have contributed to the apparent conflict of data, particularly in the right ventricle. There is an urgent need for a standardization of terms and identifying measures for selective pacing sites. (PACE 2004; 27[Pt. II]:883–886)
Selective Site Pacing:
Authors: YEE RAYMOND , KLEIN GEORGE J. , KRAHN ANDREW C. , SKANES ALLAN C.
Published: May 2004

Current tools and techniques for pacemaker and ICD lead insertion have been optimized for implantation of leads at traditional sites but may be ill‐suited for achieving alternate or selective site placement. If clinical studies confirm that optimal pacing is achieved from locations other than the right atrial appendage and right ventricular apex, new tools for reliably, easily and safely implanting leads at these sites will need to be created and methods for identifying these optimal pacing site(s) in each patient will be needed. Implant centers will need to understand the benefits of selective site pacing and make the transition to using these new tools. The various factors affecting the change from traditional to selective site pacing lead placement are discussed. (PACE 2004; 27[Pt. II]:894–896)
In vitro prediction of canine urolith mineral composition using computed tomographic mean beam attenuation measurements
Authors: Pressler Barrak M. , Mohammadian Lenore A. , Li Erning , Vaden Shelly L. , Levine Jay F. , Mathews Kyle G. , Robertson Ian D.
Published: May 2004

Determination of urolith mineral composition is critical for management of urolithiasis in dogs and cats. Using computed tomography, urolith physical density, and hence chemical composition, can be quantified using mean beam attenuation measurements (Hounsfield units; HU). This study was designed to establish in vitro reference ranges for three types of compositionally pure uroliths retrieved from dogs. Sixty‐six canine uroliths (22 uric acid, 21 calcium oxalate, 14 struvite, nine mixed or compound) were placed in a phantom array. Uroliths were scanned at 120 kVp, 200 mA, and 80 kVp, 200 mA. The region of interest (ROI) for mean HU calculation was determined using two techniques, and reference ranges were calculated for each kVp using either ROI technique. HU for urolith types of pure composition were statistically different (Wilcoxon's two‐sample test,P<0.0083 [Bonferonni correction with six comparisons for totalP<0.05]) using both ROI techniques at either kVp. Struvite uroliths were not statistically different from mixed or compound uroliths. The accuracy for determination of composition of pure uroliths ranged from 86% to 93%; the prediction accuracy for each urolith mineral type and for all uroliths in general was highest when the ROI was hand‐drawn just within the visible urolith border at 80 kVp. Technique of ROI determination and kVp that yielded the highest sensitivity, specificity, and positive and negative predictive values varied for each urolith type. Therefore, in this study, HU could be used to differentiate three types of uroliths of pure mineral composition in vitro. Further studies are needed to determine the predictive value of HU in vivo.
Use Of Traction During Magnetic Resonance Imaging Of Caudal Cervical Spondylomyelopathy (“Wobbler Syndrome”) In The Dog
Authors: Penderis Jacques , Dennis Ruth
Published: May 2004

Cervical spondylomyelopathy or “wobbler syndrome” is a well‐defined disorder of large‐breed dogs, characterized by a compressive lesion affecting the cervical spinal cord that in many dogs may have a dynamic component. Determination of whether this dynamic component is present is important in the decision‐making process as regards therapeutic intervention. Despite a significant risk of neurologic deterioration following myelography in some large dogs affected by wobbler syndrome, myelography is considered an essential part of the assessment, primarily as it allows assessment of whether a dynamic compression exists. This same neurologic deterioration is not apparent following magnetic resonance (MR) imaging; however, the use of MR imaging in the investigation of wobbler syndrome has thus far been limited by the inability to perform the traction studies required to ascertain whether a dynamic component to the spinal cord compression exists. This paper presents a technique of applying traction during MR imaging of the cervical spine to reduce a dynamic wobbler syndrome lesion.
Echocardiographic changes induced by moderate to marked hypobaric hypoxia in dogs
Authors: Glaus Tony M. , Tomsa Kamil , Hässig Michael , Reusch Claudia
Published: May 2004

Hypobaric (high‐altitude) hypoxia is a physiologic cause of pulmonary hypertension, and alters left ventricular (LV) systolic and diastolic function. In the presence of tricuspid regurgitation, systolic pulmonary artery pressure can be measured noninvasively using the peak tricuspid regurgitation velocity and the Bernoulli equation. In the absence of measurable tricuspid regurgitation, severity of pulmonary hypertension may be estimated using two‐dimensional, M‐mode, and Doppler‐derived parameters. To evaluate the usefulness of echocardiographic parameters for detecting mild‐to‐moderate pulmonary hypertension caused by moderate‐to‐marked hypoxia and to study the effect of high‐altitude hypoxia on systolic and diastolic LV function in dogs, 19 Greenland dogs were examined at moderate altitude (2300 m) and high altitude (3500 m), and 10 Greenland control dogs were examined at 700–900 m. Evaluated parameters were pulmonary flow profile (shape, right ventricular acceleration time (RVAT), ejection time (RVET), RVAT/ET), peak mitral inflow velocities (LVE, LVA, LVE/A‐ratio), LV % fractional shortening (FS), systolic time intervals (LVPEP, LVPEP/ET), and stroke volume index (SVI). Notching during deceleration was common in dogs at high altitude and in the control dogs, but not in dogs at moderate altitude. RVAT was shorter in dogs at high altitude compared with moderate altitude, but not compared with control dogs. Peak A‐velocity was higher and E/A‐ratio was lower in dogs at high altitude compared with moderate altitude and control dogs. FS was increased in dogs at high altitude compared with moderate altitude, and LVPEP and LVPEP/ET were shorter in the dogs at high altitude compared with moderate altitude and control dogs. In conclusion, significant differences in pulmonary flow profiles and systolic and diastolic parameters can be observed echocardiographically in dogs at different degrees of hypobaric hypoxia. However, overlap between the groups compromises their usefulness for diagnosing and estimating the degree of mild‐to‐moderate pulmonary hypertension in individual dogs.
Technique for equine cervical articular process joint injection
Authors: Mattoon John S. , Drost Wm Tod , Grguric Michael R. , Auld Danelle M. , Reed Stephen M.
Published: May 2004

Degenerative changes and osteochondrosis of articular processes are common sources of stiffness or pain in the equine cervical spine. Temporary relief of the clinical signs related to these maladies may be achieved by injecting corticosteroids into the joint. This is routinely done by ultrasound‐guided needle placement. The cervical articular processes and joint form an easily identifiable sonographic landmark, a step‐like echogenic surface described as having the appearance of a “chair.”
Expression of cyclooxygenase‐2 in canine epithelial nasal tumors
Authors: Kleiter Miriam , Malarkey David E. , Ruslander David E. , Thrall Donald E.
Published: May 2004

Cyclooxygenase‐2 (COX‐2) is an enzyme upregulated in some human and animal tumors. Enzymatic products are associated with tumorigenic activities. Given the poor response of canine nasal tumors to radiation, we considered the possibility that some of this resistance may be associated with COX‐2 expression. To test this, 21 formalin‐fixed, paraffin‐embedded, and archived biopsy samples from canine epithelial nasal tumors were analyzed for COX‐2 expression using immunohistochemistry. The biopsies were collected from dogs prior to radiation therapy. COX‐2 expression was present in 17 of 21 (81%) tumors. The expression was observed in several different tumor types, including nasal carcinomas, adenocarcinomas, and squamous cell carcinomas. Samples from five control dogs without nasal neoplasia were also analyzed for COX‐2 staining. These specimens were characterized by varying degrees of lymphoplasmacytic rhinitis with scattered regions of COX‐2 positive respiratory epithelial and stromal cells. Whether the intensity and distribution of COX‐2 expression in nasal tumors can be used as a prognostic marker requires further investigation. A combination therapy of irradiation and a selective COX‐2 inhibitor appears worthy of clinical investigation in the treatment of canine epithelial nasal tumors.
Hsp104 Catalyzes Formation and Elimination of Self-Replicating Sup35 Prion Conformers
Authors: Shorter James , Lindquist Susan
Published: May 2004

The protein-remodeling factor Hsp104 governs inheritance of [
PSI
+
], a yeast prion formed by self-perpetuating amyloid conformers of the translation termination factor Sup35. Perplexingly, either excess or insufficient Hsp104 eliminates [
PSI
+
]. In vitro, at low concentrations, Hsp104 catalyzed the formation of oligomeric intermediates that proved critical for the nucleation of Sup 35 fibrillization de novo and displayed a conformation common among amyloidogenic polypeptides. At higher Hsp104 concentrations, amyloidogenic oligomerization and contingent fibrillization were abolished. Hsp104 also disassembled mature fibers in a manner that initially exposed new surfaces for conformational replication but eventually exterminated prion conformers. These Hsp104 activities differed in their reaction mechanism and can explain [
PSI
+
] inheritance patterns.
AUTOMATED METHODS IN CLUSTERED MICROCALCIFICATIONS DETECTION MODULE OF A CAD SYSTEM
Authors: DIYANA WAN MIMI , BESAR ROSLI
Published: May 2004

Defining region of interests (ROIs) containing abnormal lesions on digital mammograms is the first step in many Computer-Aided-Diagnosis (CAD) systems for the classification of early signs of breast cancer as malignant or benign. The motivation of this paper is to study the efficiency of automated methods used in clustered microcalcifications (MCCs) detection module of a proposed CAD system. The proposed methods are based on several image processing concepts, such as morphological processing, fractal analysis, adaptive wavelet transform, local maxima detection and high-order statistics (HOS) tests. We applied these methods on a set of MIAS database mammograms. The mammograms consisted of two groups, which were cancerous (clustered MCCs) and non-cancerous (normal) and they were digitized at a size of 1024 by 1024 with 256 gray levels. The results showed that the efficiency of HOS test, fractal analysis and morphological approach were 99%, 92% and 74%, respectively. It was proven that the HOS test was the most efficient, and gave reliable results for every mammogram tested.
THE EFFECT OF FREEZE STORAGE ON THE TENSILE PROPERTIES OF TENDONS
Authors: NG BOON-HO , CHOU SIAW-MENG
Published: May 2004

The objective of the current study is to elucidate the influence of freeze storage on the tensile properties of tendons by testing specimens at numerous adjacent durations to eliminate the influence of biological variation and experimental errors. Chicken flexor digitorum profundus tendons were stored intact in the digits at -20°C and specimens were tensile tested at 33 durations over 360 days. Scanning electron microscopy was done on fresh specimens and specimens freeze-stored for 30, 233 and 427 days to investigate microstructure change after freeze storage. The tensile properties of tendons, as represented by ultimate tensile strength, strained at ultimate tensile strength. The elastic modulus did not deviate significantly (p>0.01) after freeze storage while the collagen fiber density of specimens stored for 233 and 427 days decreased with porosity growth. In contrast, no obvious microstructure difference was observed between fresh tendons and tendons stored for 30 days. These findings suggest that the postmortem degradation incurred a minor effect on the structural integrity of the tendons and therefore did not affect the overall tensile properties. We conclude that freeze storage at -20°C does not alter the tensile properties of tendons for as many as 360 days.
Involvement of G protein βγ-subunits in diverse signaling induced by Gi/o-coupled receptors: study using the Xenopus oocyte expression system
Authors: Uezono Yasuhito , Kaibara Muneshige , Murasaki Osamu , Taniyama Kohtaro
Published: May 2004

We studied the functions of βγ-subunits of Gi/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl− currents in oocytes expressing β2-adrenoceptor and the protein kinase A-dependent Cl− channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [d-Ala2, d-Leu5]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing β2-adrenoceptor-CFTR and 5-HT1A receptor (5-HT1AR), δ-opioid receptor, or GABAB receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT1AR. The 5-HT-induced enhancement of Gs-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin α (Gtα). The 5-HT-induced enhancement was further augmented by coexpression of the Gβγ-activated form of adenylate cyclase (AC) type II but not AC type III. Thus βγ-subunits of Gi/o protein contribute to the enhancement of Gs-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT1AR or δ-opioid receptor alone. They elicited Ca2+-activated Cl− currents in oocytes coexpressing these receptors with the Gβγ-activated form of phospholipase C (PLC)-β2 but not with PLC-β1. These currents were inhibited by pretreatment with PTX and coexpression of Gtα, suggesting that βγ-subunits of Gi/o protein activate PLC-β2 and then cause intracellular Ca2+ mobilization. Our results indicate that βγ-subunits of Gi/o protein participate in diverse intracellular signals, enhancement of Gs-coupled receptor-mediated responses, and intracellular Ca2+ mobilization.
Insulin secretion in lipodystrophic HIV-infected patients is associated with high levels of nonglucose secretagogues and insulin resistance of β-cells
Authors: Haugaard Steen B. , Andersen Ove , Storgaard Heidi , Dela Flemming , Holst Jens Juul , Iversen Johan , Nielsen Jens Ole , Madsbad Sten
Published: May 2004

We examined whether plasma concentrations of nonglucose insulin secretagogues are associated with prehepatic insulin secretion rates (ISR) in nondiabetic, insulin-resistant, human immunodeficiency virus (HIV)-infected, lipodystrophic patients (LIPO). Additionally, the negative feedback of insulin on ISR was evaluated. ISR were estimated by deconvolution of plasma C-peptide concentrations during fasting (basal) and during the last 30 min of a 120-min euglycemic insulin clamp (40 mU·m−2·min−1). Eighteen normoglycemic LIPO were compared with 25 normoglycemic HIV-infected patients without lipodystrophy (controls). Thirty minutes before start of the clamp, a bolus of glucose was injected intravenously to stimulate endogenous insulin secretion. Insulin sensitivity index (SiRd) was estimated from glucose tracer analysis. LIPO displayed increased basal ISR (69%), clamp ISR (114%), basal insulin (130%), and clamp insulin (32%), all P ≤ 0.001, whereas SiRdwas decreased (57%, P < 0.001). In LIPO, ISRbasalcorrelated significantly with basal insulin, alanine, and glucagon (all r > 0.65, P < 0.01), but not with glucose. In control subjects, ISRbasalcorrelated significantly with insulin, glucagon, and glucose (all r > 0.41, P < 0.05), but not with alanine. In LIPO, ISRclampcorrelated significantly with clamp free fatty acids (FFA), alanine, triglyceride, and glucagon (all r > 0.51, P < 0.05). In control subjects, ISRclampcorrelated with clamp triglyceride ( r = 0.45, P < 0.05). Paradoxically, in LIPO, ISRclampcorrelated positively with clamp insulin ( r = 0.68, P < 0.01), which suggests an absent negative feedback of insulin on ISR. Our data support evidence that lipodystrophic, nondiabetic, HIV-infected patients exhibit increased ISR, which can be partially explained by an impaired negative feedback of insulin on β-cells and an increased stimulation of ISR by FFA, alanine, triglyceride, and glucagon.
Differential sensitivity of intranuclear and systemic oxytocin release to central noradrenergic receptor stimulation during mid- and late gestation in rats
Authors: Lipschitz David L. , Crowley William R. , Bealer Steven L.
Published: May 2004

A number of changes occur in the oxytocin (OT) system during gestation, such as increases in hypothalamic OT mRNA, increased neural lobe and systemic OT, and morphological and electrophysiological changes in OT-containing magnocellular neurons, suggestive of altered neuronal sensitivity, which may be mediated by ovarian steroids. Because central norepinephrine (NE) and histamine (HA) are potent stimulators of OT release during parturition and lactation, the present study investigated the effects of central noradrenergic and histaminergic receptor activation on systemic (NE, HA) and intranuclear (NE) OT release in pregnant rats and in ovariectomized rats treated with ovarian steroids. Plasma OT levels in late gestation were significantly higher compared with all other groups, and neither adrenergic nor histaminergic receptor blockade decreased these elevated levels. Furthermore, the α-adrenergic agonist phenylephrine, but not histamine, stimulated systemic OT release to a significantly greater extent in late gestation than in midpregnant, ovariectomized, or steroid-treated females. Although basal extracellular OT levels in the paraventricular nucleus, as measured with microdialysis, were unchanged during pregnancy or steroid treatment, noradrenergic receptor stimulation of intranuclear OT release was significantly elevated in midgestation females compared with all other groups. These studies indicate that sensitivity of intranuclear and systemic OT release to noradrenergic receptor activation differentially varies during the course of gestation.
Therapeutic gene transfer to dystrophic diaphragm by an adenoviral vector deleted of all viral genes
Authors: Matecki Stefan , Dudley Roy W. R. , Divangahi Maziar , Gilbert Renald , Nalbantoglu Josephine , Karpati George , Petrof Basil J.
Published: May 2004

Duchenne muscular dystrophy is caused by defects in the dystrophin gene, and the mdx mouse is the most frequently employed genetic model of this disease. It is well known that different muscle groups do not respond in the same way to dystrophin deficiency. In particular, the mdx mouse diaphragm exhibits severe morphological and functional changes not found in other mdx muscles. Use of early generation adenoviral vectors to deliver genes to the diaphragm in immunocompetent mdx mice has been associated with substantial functional toxicity and a rapid loss of transgene expression. Here we determined the response to dystrophin gene replacement in the mdx diaphragm using a “gutted” adenoviral vector that contains the coding sequence of two full-length dystrophin genes and is deleted of most viral DNA sequences. At 1 wk postdelivery of the vector, 23.6 ± 4% of total fibers in the injected diaphragm bundle expressed dystrophin at the sarcolemma, which remained stable over the study duration of 30 days without the need for continuous immunosuppression. Treated diaphragms showed a significantly improved resistance to the abnormal force deficits induced by high-stress muscle contractions, the latter being a functional hallmark of dystrophin-deficient muscle. This functional amelioration was achieved despite the presence of mildly increased inflammation (CD4+ and CD8+ lymphocytes) within the vector-treated diaphragms. To our knowledge, this is the first demonstration that a viral vector can achieve reversal of functional abnormalities in the dystrophic diaphragm via therapeutic dystrophin gene transfer without the need for sustained immunosuppressive therapy.
Sildenafil alters calcium signaling and vascular tone in pulmonary arteries from chronically hypoxic rats
Authors: Pauvert Olivier , Bonnet Sébastien , Rousseau Eric , Marthan Roger , Savineau Jean-Pierre
Published: May 2004

Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been recently proposed as a therapeutic tool to treat or prevent pulmonary artery hypertension (PAHT). We thus studied the effect of sildenafil on both the calcium signaling of isolated pulmonary artery smooth muscle cells (PASMCs) and the reactivity of pulmonary artery (PA) obtained from chronic hypoxia (CH)-induced pulmonary hypertensive rats compared with control (normoxic) rats. CH rats were maintained in an hypobaric chamber (50.5 kPa) for 3 wk leading to full development of PAHT. Intracellular calcium concentration ([Ca2+]i) was measured in PASMCs loaded with the calcium fluorophore indo 1. Unlike in control rats, sildenafil (10–100 nM) decreased the resting [Ca2+]ivalue in PASMCs obtained from CH rats. In PASMCs from both control and CH rats, sildenafil concentration dependently inhibited the [Ca2+]iresponse induced by G-coupled membrane receptor agonists such as angiotensin II and phenylephrine but had no effect on the amplitude of the [Ca2+]iresponse induced by caffeine. Sildenafil (0.1 nM–1 μM) concentration dependently reduced basal PA tone that is present in CH rats and relaxed PA rings precontracted with phenylephrine in both control and CH rats. These data show that sildenafil is a potent pulmonary artery relaxant in CH rats and that it normalizes CH-induced increases in resting [Ca2+]iand basal tone. Consequently, pharmacological inhibition of sildenafil-sensitive PDE5 downregulates the Ca2+signaling pathway involved in this model of pulmonary hypertension.
The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells
Authors: Kelsen Steven G. , Aksoy Mark O. , Yang Yi , Shahabuddin Syed , Litvin Judith , Safadi Fayez , Rogers Thomas J.
Published: May 2004

Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were ∼35% of β-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% β-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was ∼78,000 receptors/cell when assessed by competitive displacement of125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.
Antisense oligodeoxynucleotides directed against a novel angiotensinogen mRNA-stabilizing protein reduce blood pressure in spontaneously hypertensive rats
Authors: Klett Christoph P. R. , Anderson Dock , Sholook Myssara , Granger Joey P.
Published: May 2004

We have previously reported that hypertension in the young spontaneously hypertensive rat (SHR) is associated with an elevation in tissue angiotensinogen and a novel polysomal protein known to stabilize angiotensinogen mRNA. In our current study we determined the role of the mRNA-stabilizing protein in the regulation of tissue angiotensinogen expression and mean arterial pressure (MAP) in the SHR utilizing antisense oligodeoxynucleotide (AON) inhibition. Three AONs (RNASTAAS1, position 31–50; RNASTAAS2, position 21–40; RNASTAAS3, position 143–162 of the cDNA coding for the polysomal protein) were administered intravenously (dose 450, 900, and 1,800 μg/kg; 1 dosage/day over 3 days) in conscious, chronically instrumented male SHRs at the age of 7 wk. Control SHRs received corresponding scrambled oligodeoxynucleotide sequences (SCR1, SCR2, SCR3). Each animal received the increasing dose schedule. RNASTAAS2 resulted in a reduced expression of the polysomal protein to 21% (liver), 12% (brain), 27% (heart), 18% (renal cortex), and 22% (renal medulla) of control. Angiotensinogen expression was inhibited to 54% (liver), 41% (brain), 68% (heart), 52% (renal cortex), and 74% (renal medulla) compared with control SHRs. Decreases in plasma concentrations of angiotensinogen and plasma renin activities were associated with a significant decrease in MAP from 147 ± 6 mmHg (after SCR2) to 106 ± 4 mmHg after RNASTAAS2. The effects of the two other AONs on MAP were less (RNASTAAS1, −31 mmHg; RNASTAAS3, −16 mmHg) with corresponding decreases in mRNAs coding for angiotensinogen and the polysomal protein. A significant decrease in intracellular concentrations of the polysomal protein accompanied AON inhibition. The magnitude of effects (−15 to −41 mmHg) was comparable to the effects of captopril (100 mg·kg−1·day−1for 3 days: −32 mmHg) and an AT1receptor antagonist (L-158809, 1.5 mg·kg−1·day−1for 3 days: −36 mmHg). These data suggest an important role of the mRNA-stabilizing protein for hepatic and extrahepatic angiotensinogen expression and MAP in the SHR.
Cisplatin-induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells
Authors: Arany Istvan , Megyesi Judit K. , Kaneto Hideaki , Price Peter M. , Safirstein Robert L.
Published: May 2004

Cisplatin treatment induces extensive death of the proximal tubules in mice. We also demonstrated that treatment of immortalized mouse proximal tubule cells (TKPTS) with 25 μM cisplatin induces apoptotic death in vitro. Here, we demonstrate that members of the MAPKs such as ERK, JNK, and p38 are all activated after cisplatin treatment both in vivo and in vitro. Because MAPKs mediate cell survival and death, we studied their role in cisplatin-induced cell death in vitro. Apoptosis was confirmed by cell morphology, fluorescence-activated cell-sorting analysis, annexin V/propidium iodide binding, and caspase-3 activation in TKPTS cells. Inhibition of ERK, but not JNK or p38, abolished caspase-3 activation and apoptotic death, suggesting a prodeath role of ERK in cisplatin-induced injury. We also determined that cisplatin-induced ERK as well as caspase-3 activation are epidermal growth factor receptor (EGFR) and c- src dependent because inhibition of these genes inhibited ERK and caspase-3 activation and attenuated apoptotic death. These results suggest that caspase-3 mediates cisplatin-induced cell death in TKPTS cells via an EGFR/src/ERK-dependent pathway. We also suggest that the prodeath effect of ERK is injury type dependent because during oxidant injury, ERK supports survival rather than death in the same cells. We propose that injury-specific outcome diverges downstream from ERK in cisplatin- or H2O2-mediated cell survival and death.
Estrogen receptor α-mediated events promote sex-specific diabetic glomerular hypertrophy
Authors: Lovegrove Alecia S. , Sun Jianhong , Gould Karen A. , Lubahn Dennis B. , Korach Kenneth S. , Lane Pascale H.
Published: May 2004

Sex differences in the incidence and progression of renal diseases suggest a protective role for estrogen. This study examined the role of estrogen receptor α (ERα)-mediated events in normal and diabetic renal and glomerular growth. Wild-type and ERα-null mice (ERKO) were observed over 2 wk of streptozocin-induced diabetes. Blood glucose was monitored, and insulin was given daily to maintain levels of 250–350 mg/dl. Body weight, kidney weight, glucose, insulin, renal transforming growth factor-β1, and glomerular area were examined for effects of sex, genotype, and diabetes. Genotype had no effect on glomerular or renal size in male mice regardless of metabolic state. Nondiabetic female ERKO mice had kidney weights approaching those of wild-type males and much greater than those of wild-type females (0.15 ± 0.04 vs. 0.11 ± 0.04 g; P < 0.001). When only diabetic mice were studied, sex and/or genotype showed no effect on renal weight. Diabetic female ERKO mice had smaller glomerular areas than wild types (2,799 ± 159 vs. 3,409 ± 187 μm2; P = 0.01). Glomerular areas were similar in diabetic wild-type and ERKO males (3,020 ± 199 vs. 3,406 ± 176 μm2). Transforming growth factor-β1 levels, expressed as picograms per milligram total protein, were similar in diabetic wild-type and ERKO males (1.0 ± 0.6 vs. 0.9 ± 0.6). In diabetic females, wild types had significantly higher levels of this growth factor than ERKO mice (3.8 ± 0.7 vs. 1.1 ± 0.6; P = 0.005). ERα-mediated processes influence normal and diabetic renal and glomerular size, but only in female mice. These data do not support a protective role for ERα-mediated events in diabetic nephropathy.
Stress-related genomic responses during the course of heat acclimation and its association with ischemic-reperfusion cross-tolerance
Authors: Horowitz Michal , Eli-Berchoer Luba , Wapinski Ilan , Friedman Nir , Kodesh Einat
Published: May 2004

Acclimation to heat is a biphasic process involving a transient perturbed phase followed by a long lasting period during which acclimatory homeostasis is developed. In this investigation, we used cDNA stress microarray (Clontech Laboratory) to characterize the stress-related genomic response during the course of heat acclimation and to test the hypotheses that 1) heat acclimation influences the threshold of activation of protective molecular signaling, and 2) heat-acclimation-mediated ischemic-reperfusion (I/R) protection is coupled with reprogrammed gene expression leading to altered capacity or responsiveness of protective-signaling pathways shared by heat and I/R cytoprotective systems. Rats were acclimated at 34°C for 0, 2, and 30 days.32P-labeled RNA samples prepared from the left ventricles of rats before and after subjection to heat stress (HS; 2 h, 41°C) or after I/R insult (ischemia: 75%, 45 min; reperfusion: 30 min) were hybridized onto the array membranes. Confirmatory RT-PCR of selected genes conducted on samples taken at 0, 30, and 60 min after HS or total ischemia was used to assess the promptness of the transcriptional response. Cluster analysis of the expressed genes indicated that acclimation involves a “two-tier” defense strategy: an immediate transient response peaking at the initial acclimating phase to maintain DNA and cellular integrity, and a sustained response, correlated with slowly developed adaptive, long-lasting cytoprotective signaling networks involving genes encoding proteins that are essential for the heat-shock response, antiapoptosis, and antioxidation. Gene activation was stress specific. Faster activation and suppression of signaling pathways shared by HS and I/R stressors probably contribute to heat-acclimation I/R cross-tolerance.