bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and characterization of delayed rectifying K+ channels in anterior rat taste buds
Authors: Liu Lidong , Hansen Dane R. , Kim Insook , Gilbertson Timothy A.
Published: June 2005

Delayed rectifying K+ (DRK) channels in taste cells have been implicated in the regulation of cell excitability and as potential targets for direct and indirect modulation by taste stimuli. In the present study, we have used patch-clamp recording to determine the biophysical properties and pharmacological sensitivity of DRK channels in isolated rat fungiform taste buds. Molecular biological assays at the taste bud and single-cell levels are consistent with the interpretation that taste cells express a variety of DRK channels, including members from each of the three major subfamilies: KCNA, KCNB, and KCNC. Real-time PCR assays were used to quantify expression of the nine DRK channel subtypes. While taste cells express a number of DRK channels, the electrophysiological and molecular biological assays indicate that the Shaker Kv1.5 channel (KCNA5) is the major functional DRK channel expressed in the anterior rat tongue.
Calcineurin-NFAT signaling, together with GABP and peroxisome PGC-1α, drives utrophin gene expression at the neuromuscular junction
Authors: Angus Lindsay M. , Chakkalakal Joe V. , Méjat Alexandre , Eibl Joe K. , Bélanger Guy , Megeney Lynn A. , Chin Eva R. , Schaeffer Laurent , Michel Robin N. , Jasmin Bernard J.
Published: June 2005

We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP- and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1α alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1α with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1α.
Direct association of RhoA with specific domains of PKC-α
Authors: Pang Haiyan , Bitar Khalil N.
Published: June 2005

Previous studies performed at our laboratory have shown that agonist-induced contraction of smooth muscle is associated with translocation of protein kinase C (PKC)-α and RhoA to the membrane and that this interaction is due to a direct protein-protein interaction. To determine the domains of PKC-α involved in direct interaction with RhoA, His-tagged PKC-α proteins of individual domains and different combinations of PKC-α domains were used to perform in vitro binding assays with the fusion protein glutathione- S-transferase (GST)-RhoA. Coimmunoprecipitation was also performed using smooth muscle cells transfected with truncated forms of PKC-α in this study. The data indicate that RhoA directly bound to full-length PKC-α, both in vitro (82.57 ± 15.26% above control) and in transfected cells. RhoA bound in vitro to the C1 domain of PKC-α [PKC-α (C1)] (70.48 ± 20.78% above control), PKC-α (C2) (72.26 ± 29.96% above control), and PKC-α (C4) (90.58 ± 26.79% above control), but not to PKC-α (C3) (0.64 ± 5.18% above control). RhoA bound in vitro and in transfected cells to truncated forms of PKC-α, PKC-α (C2, C3, and C4), and PKC-α (C3 and C4) (94.09 ± 12.13% and 85.10 ± 16.16% above control, respectively), but not to PKC-α (C1, C2, and C3) or to PKC-α (C2 and C3) (0.47 ± 1.26% and 7.45 ± 10.76% above control, respectively). RhoA bound to PKC-α (C1 and C2) (60.78 ± 13.78% above control) only in vitro, but not in transfected cells, and PKC-α (C2, C3, and C4) and PKC-α (C3 and C4) bound well to RhoA. These data suggest that RhoA bound to fragments that may mimic the active form of PKC-α. The studies using cells transfected with truncated forms of PKC-α indicate that PKC-α (C1 and C2), PKC-α (C1, C2, and C3), and PKC-α (C2 and C3) did not associate with RhoA. Only full-length PKC-α, PKC-α (C2, C3, and C4), and PKC-α (C3 and C4) associated with RhoA. The association increased upon stimulation with acetylcholine. These results suggest that the functional association of PKC-α with RhoA may require the C4 domain.
Stress inhibits nucleocytoplasmic shuttling of heat shock protein hsc70
Authors: Kodiha Mohamed , Chu Angel , Lazrak Omar , Stochaj Ursula
Published: June 2005

Heat shock proteins of the hsp/hsc70 family are essential chaperones, implicated in the stress response, aging, and a growing number of human diseases. At the molecular level, hsc70s are required for the proper folding and intracellular targeting of polypeptides as well as the regulation of apoptosis. Cytoplasmic members of the hsp/hsc70 family are believed to shuttle between nuclei and cytoplasm; they are found in both compartments of unstressed cells. Our experiments demonstrate that actin filament-destabilizing drugs trigger the nuclear accumulation of hsc70s in unstressed and heat-shocked cells recovering from stress. Using human-mouse heterokaryons, we show that stress inhibits shuttling and sequesters the chaperone in nuclei. The inhibition of hsc70 shuttling upon heat shock is only transient, and transport is reestablished when cells recover from stress. Hsc70 shuttling is controlled by hsc70 retention in the nucleus, a process that is mediated by two distinct mechanisms, ATP-sensitive binding of hsc70s to chaperone substrates and, furthermore, the association with nucleoli. The nucleolar protein fibrillarin and ribosomal protein rpS6 were identified as components that show an increased association with hsc70s in the nucleus upon stress exposure. Together, our data suggest that stress abolishes the exit of hsc70s from the nucleus to the cytoplasm, thereby limiting their function to the nuclear compartment. We propose that during recovery from stress hsc70s are released from nuclear and nucleolar anchors, which is a prerequisite to restore shuttling.
Intestinal renal metabolism ofl-citrulline andl-arginine following enteral or parenteral infusion ofl-alanyl-l-[2,15N]glutamine orl-[2,15N]glutamine in mice
Authors: Boelens Petra G. , van Leeuwen Paul A. M. , Dejong Cornelis H. C. , Deutz Nicolaas E. P.
Published: June 2005

Previously, we observed increased plasma arginine (ARG) concentrations after glutamine (GLN)-enriched diets, in combination with clinical benefits. GLN delivers nitrogen for ARG synthesis, and the present study was designed to quantify the interorgan relationship of exogenous l-GLN or GLN dipeptide, by enteral or parenteral route, contributing to intestinal citrulline (CIT) and renal de novo ARG synthesis in mice. To study this, we used a multicatheterized mouse model with Swiss mice ( n = 43) in the postabsorptive state. Stable isotopes were infused into the jugular vein or into the duodenum {per group either free l-[2,15N]GLN or dipeptide l-ALA-l-[2,15N]GLN, all with l-[ureido-13C-2H2]CIT and l-[guanidino-15N2-2H2]ARG} to establish renal and intestinal ARG and CIT metabolism. Blood flow was measured using14C-para-aminohippuric acid. Net intestinal CIT release, renal uptake of CIT, and net renal ARG efflux was found, as assessed by arteriovenous flux measurements. Quantitatively, more de novo l-[2,15N]CIT was produced when free l-[2,15N]GLN was given than when l-ALA-l-[2,15N]GLN was given, whereas renal de novo l-[2,15N]ARG was similar in all groups. In conclusion, the intestinal-renal axis is hereby proven in mice in that l-[2,15N]GLN or dipeptide were both converted into de novo renal l-[2,15N]ARG; however, not all was derived from intestinal l-[2,15N]CIT production. In this model, the feeding route and form of GLN did not influence de novo renal ARG production derived from GLN.
Role of renal nerves in development of hypertension in DOCA-salt model in rats: a telemetric approach
Authors: Jacob Frédéric , Clark Leah A. , Guzman Pilar Ariza , Osborn John W.
Published: June 2005

Centrally mediated hyperactivity of the autonomic nervous system contributes to DOCA hypertension; however, the targeted peripheral vascular bed(s) remain unclear. We propose that if renal sympathetic activity is a factor in the development of DOCA-salt hypertension, then renal denervation (RDNX) should attenuate the hypertensive response. In protocol 1, uninephrectomized RDNX ( n = 9) and sham-denervated ( n = 6) Sprague-Dawley rats were allowed free access to 0.9% NaCl solution and 0.1% NaCl diet. Mean arterial pressure (MAP) and heart rate were telemetrically recorded for 4 days before and 36 days after DOCA (100 mg/rat) implantation; sodium and water balances were recorded daily. Protocol 2 was similar except that saline intake in sham rats ( n = 7) was matched to that observed in RDNX rats of protocol 1 for 30 days; for the last 10 days, the rats were allowed free access to saline. Before DOCA in protocol 1, MAP was lower ( P < 0.05) in RDNX rats (99 ± 1 mmHg) compared with sham rats (111 ± 3 mmHg); however, heart rate and sodium and water balances were similar between groups. RDNX attenuated the MAP response to DOCA by ∼50% (ΔMAP = 22 ± 3 mmHg, where Δ is change in MAP) when compared with sham rats (ΔMAP = 38 ± 6). RDNX rats consumed significantly less saline than sham rats, and cumulative sodium and water balances were reduced by 33% and 23%, respectively. In protocol 2, a similar pattern in MAP elevation was observed in RDNX and saline-restricted, sham-denervated rats even when saline restriction was removed. These results indicate that the renal sympathetic nerves are important in hypertension development but that other factors are also involved.
Selective disruption of MMP-2 gene exacerbates myocardial inflammation and dysfunction in mice with cytokine-induced cardiomyopathy
Authors: Matsusaka Hidenori , Ikeuchi Masaki , Matsushima Shouji , Ide Tomomi , Kubota Toru , Feldman Arthur M. , Takeshita Akira , Sunagawa Kenji , Tsutsui Hiroyuki
Published: June 2005

Tumor necrosis factor-α (TNF-α) plays a pathophysiological role in the development and progression of heart failure. Matrix metalloproteinase (MMP)-2 is involved in extracellular matrix remodeling. Recent evidence suggests a protective role for this protease against tissue inflammation. Although MMP-2 is upregulated in the failing heart, little is known about its pathophysiological role. We thus hypothesized that ablation of the MMP-2 gene could affect cardiac remodeling and failure in TNF-α-induced cardiomyopathy. We crossed transgenic mice with cardiac-specific overexpression of TNF-α (TG) with MMP-2 knockout (KO) mice. Four groups of male and female mice were studied: wild-type (WT) with wild MMP-2 (WT/MMP+/+), WT with MMP-2 KO (WT/MMP−/−), TNF-α TG with wild MMP-2 (TG/MMP+/+), and TG with MMP-2 KO (TG/MMP−/−). The upregulation of MMP-2 zymographic activity in TG/MMP+/+ mice was completely abolished in TG/MMP−/− mice, and other MMPs and tissue inhibitors of metalloproteinase were comparable between groups. Survival was shorter for male TG/MMP−/− than TG/MMP+/+ mice. Female TG/MMP−/− mice were more severely affected than TG/MMP+/+ mice with diminished cardiac function. Myocardial TNF-α and other proinflammatory cytokines were increased in TG/MMP+/+ mice, and this increase was similarly observed in TG/MMP−/− mice. The extent of myocardial infiltrating cells including macrophages was greater in TG/MMP−/− than in TG/MMP+/+ mice. Selective ablation of the MMP-2 gene reduces survival and exacerbates cardiac failure in association with the increased level of myocardial inflammation. MMP-2 may play a cardioprotective role in the pathogenesis of cytokine-induced cardiomyopathy.
Prenatal programming of adult blood pressure: role of maternal corticosteroids
Authors: Woods Lori L. , Weeks Douglas A.
Published: June 2005

Both maternal glucocorticoid administration and maternal dietary protein or food restriction in pregnancy cause fewer nephrons and hypertension in the adult offspring. The purpose of these studies was to determine the extent to which nutritional factors contribute to programming of offspring hypertension by maternal glucocorticoids. Pregnant rats were treated with dexamethasone (100 μg·kg−1·d−1sc) on days 1–10 (ED) or days 15–20 (LD) of pregnancy. Additional groups of pregnant animals were pair fed to the early (EDPF) and late (LDPF) dexamethasone-treated groups, and another group was untreated or given vehicle (C). The dams treated with dexamethasone reduced their food intake and lost or failed to gain a normal amount of weight during treatment; body weights of ED dams caught up to normal after the treatment period, whereas those of LD dams did not. In adulthood (∼21 wks), chronically instrumented male offspring of ED had normal blood pressures (125 ± 2 mmHg vs. 126 ± 1 mmHg in C), whereas LD offspring were hypertensive (136 ± 3 mmHg). However, LDPF offspring were equally hypertensive (134 ± 2 mmHg). Glomerular filtration rates normalized to body weight were not significantly different among groups. Qualitatively similar results were found in female offspring. Thus the long-term effects of maternal glucocorticoid administration at this dose on offspring’s blood pressure may, in large part, be accounted for by the reduction in maternal food intake. These data suggest that maternal glucocorticoids and maternal food or protein restriction may, at least in part, share a common mechanism in programming offspring for hypertension. The window of sensitivity of future offspring blood pressure to either maternal insult coincides with nephrogenesis in the rat, suggesting that impaired renal development could play an important role in this programming.
CRF-related peptides contribute to stress response and regulation of appetite in hypoxic rainbow trout
Authors: Bernier Nicholas J. , Craig Paul M.
Published: June 2005

Hypoxia stress suppresses appetite in a variety of fish species, but the mechanisms mediating this response are not known. Therefore, given their anorexigenic and hypophysiotropic properties, we investigated the contribution of forebrain corticotropin-releasing factor (CRF) and urotensin I (UI) to the regulation of food intake and the hypothalamic-pituitary-interrenal (HPI) stress axis in hypoxic rainbow trout. Exposure to 50 and 35% O2 saturation for 24 h decreased food intake by 28 and 48%, respectively. The 35% O2 treatment also increased forebrain CRF and UI mRNA levels, plasma cortisol, and lactate. Exposure for 72 h to the same conditions resulted in similar reductions in food intake, increases in plasma cortisol proportional to the hypoxia severity, and increases in forebrain CRF and UI mRNA levels in the 50% O2 treatment. Relative to saline-infused fish, chronic intracranial infusion of the CRF receptor antagonist α-helical CRF(9–41) reduced the appetite-suppressing effects of 24-h exposure to 35% O2 and blocked the hypoxia-induced increase in plasma cortisol. Finally, forebrain microdissection revealed that 50 and 35% O2 exposure for 24 h specifically increases preoptic area CRF and UI mRNA levels in proportion to the severity of the hypoxic challenge and either has no effect or elicits small decreases in other forebrain regions. These results show that CRF-related peptides play a physiological role in regulating the HPI axis and in mediating at least a portion of the reduction in food intake under hypoxic conditions in rainbow trout and demonstrate that the response of forebrain CRF and UI neurons to this stressor is region specific.
Genetic and environmental determination of tracking in static strength during adolescence
Authors: Peeters Maarten W. , Thomis Martine A. , Maes Hermine H. M. , Beunen Gaston P. , Loos Ruth J. F. , Claessens Albrecht L. , Vlietinck Robert
Published: June 2005

The purpose of this study was to determine whether the observed phenotypic stability in static strength during adolescence, as measured by interage correlations in arm pull, is mainly caused by genetic and/or environmental factors. Subjects were from the Leuven Longitudinal Twin Study ( n = 105 pairs, equally divided over 5 zygosity groups). Arm-pull data were aligned on age at peak height velocity to attenuate the temporal fluctuations in interage correlations caused by differences in timing of the adolescent growth spurt. Developmental genetic models were fitted using structural equation modeling. After the data were aligned on age at peak height velocity, the annual interage correlations conformed to a quasi-simplex structure over a 4-yr interval. The best-fitting models included additive genetic and unique environmental sources of variation. Additive genetic factors that already explained a significant amount of variation at previous measurement occasions explained 44.3 and 22.5% of the total variation at the last measurement occasion in boys and girls, respectively. Corresponding values for unique environmental sources of variance are 31.2 and 44.5%, respectively. In conclusion, the observed stability of static strength during adolescence is caused by both stable genetic influences and stable unique environmental influences in boys and girls. Additive genetic factors seem to be the most important source of stability in boys, whereas unique environmental factors appear to be more predominant in girls.
Vascular adaptation to deconditioning and the effect of an exercise countermeasure: results of the Berlin Bed Rest study
Authors: Bleeker Michiel W. P. , De Groot Patricia C. E. , Rongen Gerard A. , Rittweger Jörn , Felsenberg Dieter , Smits Paul , Hopman Maria T. E.
Published: June 2005

Deconditioning is a risk factor for cardiovascular disease. The physiology of vascular adaptation to deconditioning has not been elucidated. The purpose of the present study was to assess the effects of bed rest deconditioning on vascular dimension and function of leg conduit arteries. In addition, the effectiveness of resistive vibration exercise as a countermeasure for vascular deconditioning during bed rest was evaluated. Sixteen healthy men were randomly assigned to bed rest (BR-Ctrl) or to bed rest with resistive vibration exercise (BR-RVE). Before and after 25 and 52 days of strict horizontal bed rest, arterial diameter, blood flow, flow-mediated dilatation (FMD), and nitroglycerin-mediated dilatation were measured by echo Doppler ultrasound. In the BR-Ctrl group, the diameter of the common femoral artery decreased by 13 ± 3% after 25 and 17 ± 1% after 52 days of bed rest ( P < 0.001). In the BR-RVE group this decrease in diameter was significantly attenuated (5 ± 2% after 25 days and 6 ± 2% after 52 days, P < 0.01 vs. BR-Ctrl). Baseline blood flow did not change after bed rest in either group. After 52 days of bed rest, FMD and nitroglycerin-mediated dilatation of the superficial femoral artery were increased in both groups, possibly by increased nitric oxide sensitivity. In conclusion, bed rest deconditioning is accompanied by a reduction in the diameter of the conduit arteries and by an increased reactivity to nitric oxide. Resistive vibration exercise effectively attenuates the diameter decrease of leg conduit arteries after bed rest.
Effect of inflation on the interaction between the left and right hemidiaphragms
Authors: De Troyer André , Cappello Matteo , Scillia Pierre
Published: June 2005

At resting end expiration [functional residual capacity (FRC)], the actions of the left and right hemidiaphragms on the lung are synergistic. However, the synergism decreases in magnitude as muscle tension decreases. Therefore, the hypothesis was tested in anesthetized dogs that the degree of synergism between the two hemidiaphragms also decreases with increasing lung volume. In a first experiment, the changes in airway opening pressure (ΔPao) and abdominal pressure (ΔPab) obtained during simultaneous stimulation of the left and right phrenic nerves (measured changes in pressure) at different lung volumes were compared with the sum of the pressure changes produced by their separate stimulation (predicted changes in pressure). Although the pressure changes decreased markedly with increasing lung volume, the measured ΔPao and ΔPab were substantially greater than the predicted values at all lung volumes. The ratio of the measured to the predicted ΔPao, in fact, remained constant. In a second experiment, radiographic measurements showed that the fractional shortening of the muscle during bilateral contraction at high lung volumes was similar to that during unilateral contraction. During unilateral contraction at high lung volumes, however, the passive hemidiaphragm moved in the cranial direction, whereas, during unilateral contraction at FRC, it moved in the caudal direction. These observations indicate that 1) for a given muscle tension, the synergism between the two halves of the diaphragm is greater at high lung volumes than at FRC; and 2) this difference is primarily related to the greater distortion of the muscle configuration.
Effect of carbohydrate ingestion on exercise-induced alterations in metabolic gene expression
Authors: Cluberton Laura J. , McGee Sean L. , Murphy Robyn M. , Hargreaves Mark
Published: June 2005

Skeletal muscle possesses a high degree of plasticity and can adapt to both the physical and metabolic challenges that it faces. An acute bout of exercise is sufficient to induce the expression of a variety of metabolic genes, such as GLUT4, pyruvate dehydrogenase kinase 4 (PDK-4), uncoupling protein-3 (UCP3), and peroxisome proliferator-activated receptor-γ coactivator 1 (PGC-1). Reducing muscle glycogen levels before exercise potentiates the effect of exercise on many genes. Similarly, altered substrate availability induces transcription of many of these genes. The purpose of this study was to determine whether glucose ingestion attenuates the exercise-induced increase in a variety of exercise-responsive genes. Six male subjects (28 ± 7 yr; 83 ± 3 kg; peak pulmonary oxygen uptake = 46 ± 6 ml·kg−1·min−1) performed 60 min of cycling at 74 ± 2% of peak pulmonary oxygen uptake on two separate occasions. On one occasion, subjects ingested a 6% carbohydrate drink. On the other occasion, subjects ingested an equal volume of a sweet placebo. Muscle samples were obtained from vastus lateralis at rest, immediately after exercise, and 3 h after exercise. PDK-4, UCP3, PGC-1, and GLUT4 mRNA levels were measured on these samples using real-time RT-PCR. Glucose ingestion attenuated ( P < 0.05) the exercise-induced increase in PDK-4 and UCP3 mRNA. A similar trend ( P = 0.09) was observed for GLUT4 mRNA. In contrast, PGC-1 mRNA increased following exercise to the same extent in both conditions. These data suggest that glucose availability can modulate the effect of exercise on metabolic gene expression.
Skeletal muscle mitochondrial DNA content in exercising humans
Authors: Marcuello A. , González-Alonso J. , Calbet J. A. L. , Damsgaard R. , López-Pérez M. J. , Díez-Sánchez C.
Published: June 2005

Several weeks of intense endurance training enhances mitochondrial biogenesis in humans. Whether a single bout of exercise alters skeletal muscle mitochondrial DNA (mtDNA) content remains unexplored. Double-stranded mtDNA, estimated by slot-blot hybridization and real time PCR and expressed as mtDNA-to-nuclear DNA ratio (mtDNA/nDNA) was obtained from the vastus lateralis muscle of healthy human subjects to investigate whether skeletal muscle mtDNA changes during fatiguing and nonfatiguing prolonged moderate intensity [2.0–2.5 h; ∼60% maximal oxygen consumption (V̇o2 max)] and short repeated high-intensity exercise (5–8 min; ∼110% V̇o2 max). In control resting and light exercise (2 h; ∼25% V̇o2 max) studies, mtDNA/nDNA did not change. Conversely, mtDNA/nDNA declined after prolonged fatiguing exercise (0.863 ± 0.061 vs. 1.101 ± 0.067 at baseline; n = 14; P = 0.005), remained lower after 24 h of recovery, and was restored after 1 wk. After nonfatiguing prolonged exercise, mtDNA/nDNA tended to decline ( n = 10; P = 0.083) but was reduced after three repeated high-intensity exercise bouts (0.900 ± 0.049 vs. 1.067 ± 0.071 at baseline; n = 7; P = 0.013). Our findings indicate that prolonged and short repeated intense exercise can lead to significant reductions in human skeletal muscle mtDNA content, which might function as a signal stimulating mitochondrial biogenesis with exercise training.
In vivo assessment of human vaginal oxygen and carbon dioxide levels during and post menses
Authors: Hill Donna R. , Brunner Marianne E. , Schmitz Deborah C. , Davis Catherine C. , Flood Janine A. , Schlievert Patrick M. , Wang-Weigand Sherry Z. , Osborn Thomas W.
Published: June 2005

Previous in vitro and in vivo animal studies showed that O2and CO2concentrations can affect virulence of pathogenic bacteria such as Staphylococcus aureus. The objective of this work was to measure O2and CO2levels in the vaginal environment during tampon wear using newly available sensor technology. Measurements by two vaginal sensors showed a decrease in vaginal O2levels after tampon insertion. These decreases were independent of the type of tampons used and the time of measurement (mid-cycle or during menstruation). These results are not in agreement with a previous study that concluded that oxygenation of the vaginal environment during tampon use occurred via delivery of a bolus of O2during the insertion process. Our measurements of gas levels in menses showed the presence of both O2and CO2in menses. The tampons inserted into the vagina contained O2and CO2levels consistent with atmospheric conditions. Over time during tampon use, levels of O2in the tampon decreased and levels of CO2increased. Tampon absorbent capacity, menses loading, and wear time influenced the kinetics of these changes. Colonization with S. aureus had no effect on the gas profiles during menstruation. Taken collectively, these findings have important implications on the current understanding of gaseous changes in the vaginal environment during menstruation and the potential role(s) they may play in affecting bacterial virulence factor production.
TRL, IDL, and LDL Apolipoprotein B-100 and HDL Apolipoprotein A-I Kinetics as a Function of Age and Menopausal Status
Authors: Matthan Nirupa R. , Jalbert Susan M. , Lamon-Fava Stefania , Dolnikowski Gregory G. , Welty Francine K. , Barrett Hugh R. , Schaefer Ernst J. , Lichtenstein Alice H.
Published: June 2005

Objective—

To determine mechanisms contributing to the altered lipoprotein profile associated with aging and menopause, apolipoprotein B-100 (apoB-100) and apoA-I kinetic behavior was assessed.

Methods and Results—

Eight premenopausal (25±3 years) and 16 postmenopausal (65±6 years) women consumed for 6 weeks a standardized Western diet, at the end of which a primed-constant infusion of deuterated leucine was administered in the fed state to determine the kinetic behavior of triglyceride-rich lipoprotein (TRL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) apoB-100, and high-density lipoprotein (HDL) apoA-I. Data were fit to a multicompartmental model using SAAM II to calculate fractional catabolic rate (FCR) and production rate (PR). Total cholesterol, LDL cholesterol (LDL-C), TRL-C, and triglyceride levels were higher (50%, 55%, 130%, and 232%, respectively) in the postmenopausal compared with the premenopausal women, whereas HDL-C levels were similar. Plasma TRL, IDL, and LDL-apoB-100 levels and pool sizes (PS) were significantly higher in the postmenopausal than premenopausal women. These differences were accounted for by lower TRL, IDL, and LDL apoB-100 FCR (
P
<0.05), with no difference in PR. There was no significant difference between groups in HDL-C levels or apoA-I kinetic parameters. Plasma TRL-C concentrations were negatively correlated with TRL apoB-100 FCR (
r
=−0.46;
P
<0.05) and positively correlated with PR (
r
=0.62;
P
<0.01). Plasma LDL-C concentrations were negatively correlated with LDL apoB-100 FCR (
r
=−0.70;
P
<0.001) but not PR.

Conclusions—

The mechanism for the increase in TRL and LDL apoB-100 PS observed in the postmenopausal women was determined predominantly by decreased TRL and LDL catabolism rather than increased production. No differences were observed in HDL apoA-I kinetics between groups.
Association Between 5-Lipoxygenase Expression and Plaque Instability in Humans
Authors: Cipollone Francesco , Mezzetti Andrea , Fazia Maria Luigia , Cuccurullo Chiara , Iezzi Annalisa , Ucchino Sante , Spigonardo Francesco , Bucci Marco , Cuccurullo Franco , Prescott Stephen M. , Stafforini Diana M.
Published: June 2005

Objective—

The participation of 5-lipoxygenase (5-LO) in the development of atherosclerosis has been suggested by recent studies. However, a role for 5-LO as a modulator of atherosclerotic plaque instability has not been previously reported in humans. Thus, the aims of this study was to analyze the expression of 5-LO in human carotid plaques and to investigate the mechanism by which this enzyme could lead to plaque instability and rupture.

Methods and Results—

We obtained atherosclerotic plaques from 60 patients undergoing carotid endarterectomy. We divided the plaques into symptomatic and symptomatic according to clinical evidence of plaque instability. Clinical evidence of plaque instability was provided by the assessment of recent ischemic symptoms attributable to the stenosis and by the presence of ipsilateral cerebral lesion(s) determined by computed tomography. Plaques were analyzed for CD68+ macrophages, CD3+ T cells, α-actin+ smooth muscle cells, 5-LO, cyclooxygenase 2, matrix metalloproteinase (MMP)-2, and MMP-9 by immunohistochemical, immunoblotting, and densitometric analyses. MMP activity was assessed by zymography. Leukotriene (LT) B
4
and collagen were quantified by ELISA and Sirius red polarization, respectively. The percentage of macrophage-rich and T-cell–rich areas was larger in symptomatic compared with asymptomatic patients (25±6% versus 8±4%,
P
<0.0001, and 74±17 versus 18±4 cell/mm
2
,
P
<0.003). 5-LO expression was higher in symptomatic compared with asymptomatic plaques (24±4% versus 6±3%,
P
<0.0001) and was associated with increased MMP-2 and MMP-9 expression (27±4% versus 7±3%,
P
<0.0001, and 29±5% versus 8±2%,
P
<0.0001) and activity and with decreased collagen content (6.9±2.4% versus 17.8±3.1%,
P
<0.01). Immunofluorescence showed that 5-LO and MMPs colocalize in activated macrophages. Notably, higher 5-LO in symptomatic plaques correlated with increased LTB
4
production (18.15±3.56 versus 11.27±3.04 ng/g tissue,
P
<0.0001).

Conclusions—

The expression of 5-LO is elevated in symptomatic compared with asymptomatic plaques and is associated with acute ischemic syndromes, possibly through the generation of LTB
4
, subsequent MMP biosynthesis, and plaque rupture.
Retroviral Infection and Selection of Culture-Derived Platelets Allows Study of the Effect of Transgenes on Platelet Physiology Ex Vivo and on Thrombus Formation In Vivo
Authors: Gillitzer Angelika , Peluso Mario , Laugwitz Karl-Ludwig , Münch Götz , Massberg Steffen , Konrad Ildiko , Gawaz Meinrad , Ungerer Martin
Published: June 2005

Background—

We recently reported the development of culture-derived (CD) platelets with the aim to express any protein of interest in these platelets.

1

We now report a specific protocol of retroviral infection into the progenitor cells and subsequent selection, which allows to generate large amounts of highly homogenous transgene-expressing CD platelets and to study transgene function rapidly and reliably at large-scale ex vivo and in vivo settings.

Methods and Results—

After retroviral infection and selection, the activation-dependent expression profile of surface markers, aggregation, and granule release were investigated. The function of transgene-expressing CD platelets, the precursor cells of which had been retrovirally infected, compared well to noninfected CD platelets or freshly isolated platelets. Hence, the retroviral infection protocol did not alter platelet physiology. In contrast, adenoviral infection of precursors to CD platelets resulted in marked functional alterations that obviated their use in analytic experiments. Additionally, sufficient amounts of selected CD platelets were generated to warrant intravenous injections into living mice. This approach permitted study of their adhesive profile at endothelial lesions and their effect on thrombus formation in vivo by intravital videofluorescence microscopy.

Conclusion—

The novel selection method allowed us to produce recombinant transgene-expressing platelets in sufficient amounts to study genetically modified platelets in vitro and in vivo.
Reduced Immunoregulatory CD31
+
T Cells in the Blood of Atherosclerotic Mice With Plaque Thrombosis
Authors: Caligiuri Giuseppina , Groyer Emilie , Khallou-Laschet Jamila , Zen Ayman Al Haj , Sainz Julie , Urbain Dominique , Gaston Anh-Thu , Lemitre Mathilde , Nicoletti Antonino , Lafont Antoine
Published: June 2005

Objective—

Lymphocyte activation is thought to play a major role in the pathogenesis of atherosclerotic complications such as plaque thrombosis. Circulating CD31
+
T cells have been shown to regulate human T cell activation. Aim of this study was to evaluate whether the proportion of circulating immunoregulatory CD31
+
T cells is correlated to the occurrence of plaque thrombosis in aged apolipoprotein (apo) E knockout (KO) mice.

Methods and Results—

CD31
+
T cell depletion of spleen T cells enhanced proliferation (
P
<0.05) and interferon-γ production (
P
<0.01) while reducing interleukin (IL)-4 (
P
<0.001) and IL-10 (
P
=0.001) secretion in response to minimally modified low-density lipoprotein. CD31
+
T cells were counted in 65 apoE KO mice (46-week-old) by flow cytometry. Organizing thrombi could be documented in 28 of 195 (14%) lesions and in at least one of the aorta root lesions in 23 of 65 mice (35%). CD31
+
T cell count was significantly reduced in mice showing plaque thrombosis (72.3±1.5% versus 84.1±1.2%;
P
<0.0001), but such reduction did not follow induced plaque rupture or experimentally controlled thrombosis.

Conclusions—

Reduced CD31
+
T cells in circulating blood is a hallmark of atherosclerotic plaque thrombosis. Our data suggest that CD31
+
T cells may play an important regulatory role in the development of plaque thrombosis.
Induced Spreading Depression Activates Persistent Neurogenesis in the Subventricular Zone, Generating Cells With Markers for Divided and Early Committed Neurons in the Caudate Putamen and Cortex
Authors: Yanamoto Hiroji , Miyamoto Susumu , Tohnai Norimitsu , Nagata Izumi , Xue Jing-Hui , Nakano Yoshikazu , Nakajo Yukako , Kikuchi Haruhiko
Published: June 2005

Background and Purpose—

Status epilepticus and cerebral ischemia stimulate persistent neurogenesis in the adult brain, but both conditions cause neuronal damage. We determined whether spreading depression, a common epiphenomenon of these conditions, stimulates persistent neurogenesis.

Methods—

We analyzed the effect of KCl-induced spreading depression on persistent neurogenesis and the spatio-temporal distribution of cells exhibiting immunohistochemical markers for divided and early committed neurons (new neurons) in the adult rat brain.

Results—

After induction of spreading depression for 48 hours, the density of mitotic cells, divided cells, and new neurons in the subventricular zone increased at days 1 to 3, days 3 to 6, and day 6, respectively (
P
<0.05). The divided cell density in the rostral migratory stream and the stream size increased at day 12 (
P
<0.001). Vehicle (saline) infusion or induction of spreading depression for 4 hours only did not increase the divided cell density, but the latter increased new neuron density in the subventricular zone (
P
<0.001). Double-labeled new neuron-like cells also appeared in the caudate putamen or cortex in ectopic fashion at day 3, with dramatic increases at days 6 and 12. Administration of the NMDA receptor antagonist, MK-801, which inhibits the propagation of spreading depression, abolished the increase in new neurons in the subventricular zone and the appearance of ectopic new neuron-like cells after 48-hour KCl infusion. There was no neuronal damage, as evidenced by mature neuron density, neurite density, and apoptotic cell appearance after spreading depression for 48 hours.

Conclusions—

Spreading depression has the potential to stimulate persistent neurogenesis or to produce ectopic new neuron-like cells.
Familial Hypercholesterolemia Patients Treated With Statins at No Increased Risk for Intracranial Vascular Lesions Despite Increased Cholesterol Burden and Extracranial Atherosclerosis
Authors: Soljanlahti Sami , Autti Taina , Lauerma Kirsi , Raininko Raili , Keto Pekka , Turtola Hannu , Vuorio Alpo F.
Published: June 2005

Background and Purpose—

To correlate known vascular disease risk factors and the signs of extracranial and intracranial changes of vascular origin in young patients with heterozygous familial hypercholesterolemia (FH).

Methods—

39 DNA test-verified heterozygous FH North Karelian patients (FH-NK), aged 6 to 48, 28 of them treated with statins, and 25 healthy controls underwent brain magnetic resonance imaging (MRI) and carotid ultrasound.

Results—

Common carotid intima-media thickness was significantly greater in the patients (
P
=0.005). MR angiography showed no pathological changes, other than 1 incidental aneurysm. The number and size of white matter hyperintensities on T2-weighted MR images, considered as markers of microvascular alterations, did not differ between patients and controls.

Conclusions—

FH-NK patients treated with statins seem to be at no increased risk for brain infarcts or other brain lesions of vascular origin when younger than age 50.
Association of a Functional Polymorphism in the Clopidogrel Target Receptor Gene,
P2Y12
, and the Risk for Ischemic Cerebrovascular Events in Patients With Peripheral Artery Disease
Authors: Ziegler Sophie , Schillinger Martin , Funk Marion , Felber Katharina , Exner Markus , Mlekusch Wolfgang , Sabeti Schila , Amighi Jasmin , Minar Erich , Brunner Martin , Müller Markus , Mannhalter Christine
Published: June 2005

Background and Purpose—

There is considerable variability in the antiplatelet effects of the thienopyridine agent “clopidogrel.” We tested for an association of gene sequence variations in
P2Y12
and occurrence of neurological adverse events in patients with symptomatic peripheral artery disease (PAD) during clopidogrel treatment.

Methods—

We studied 137 patients undergoing antiplatelet therapy with clopidogrel and 336 patients with aspirin for the occurrence of neurological events (ischemic stroke and/or carotid revascularization). Prevalence of 2 previously described exonic polymorphisms of the
P2Y12
gene, 34C>T and 52G>T, was determined by polymerase chain reaction.

Results—

Genotype frequencies for mutated, heterozygous, and wild-type alleles for the 34C>T and the 52G>T polymorphisms were 9% (n=40), 44% (n=210), and 47% (n=223), and 4% (n=17), 27% (n=127), and 70% (n=329), respectively. During the median follow-up of 21 months, neurological events occurred in 8% of patients. In patients with aspirin therapy, neither polymorphism was associated with neurological events. However, in clopidogrel patients, carriers of at least one 34T allele had a 4.02-fold increased adjusted risk for neurological events compared with carriers of only 34C alleles (95% confidence interval, 1.08 to 14.9). Neither polymorphism was associated with all-cause mortality.

Conclusions—

In PAD patients, clopidogrel response variability exists, which may result in increased risk for cerebrovascular events. Sequence alterations of the target receptor gene represent one possible mechanism for clopidogrel failure. Whether identification of the 34C>T polymorphism as a contributor to this process could serve as risk stratification tool, an indicator for higher clopidogrel doses, or the use of alternate agents warrants further investigation.
Possible Role for K
+
in Endothelium-Derived Hyperpolarizing Factor–Linked Dilatation in Rat Middle Cerebral Artery
Authors: McNeish Alister J. , Dora Kim A. , Garland Christopher J.
Published: June 2005

Background and Purpose—

Endothelium-derived hyperpolarizing factor (EDHF) and K
+
are vasodilators in the cerebral circulation. Recently, K
+
has been suggested to contribute to EDHF-mediated responses in peripheral vessels. The EDHF response to the protease-activated receptor 2 ligand SLIGRL was characterized in cerebral arteries and used to assess whether K
+
contributes as an EDHF.

Methods—

Rat middle cerebral arteries were mounted in either a wire or pressure myograph. Concentration-response curves to SLIGRL and K
+
were constructed in the presence and absence of a variety of blocking agents. In some experiments, changes in tension and smooth muscle cell membrane potential were recorded simultaneously.

Results—

SLIGRL (0.02 to 20 μmol/L) stimulated concentration and endothelium-dependent relaxation. In the presence of
N
G
-nitro-
l
-arginine methyl ester, relaxation to SLIGRL was associated with hyperpolarization and sensitivity to a specific inhibitor of IK
Ca
, 1-[(2-chlorophenyl)diphenylmethyl]-1
H
-pyrazole (1μmol/L), reflecting activation of EDHF. Combined inhibition of K
IR
with Ba
2+
(30μmol/L) and Na
+
/K
+
-ATPase with ouabain (1 μmol/L) markedly attenuated the relaxation to EDHF. Raising extracellular [K
+
] to 15 mmol/L also stimulated smooth muscle relaxation and hyperpolarization, which was also attenuated by combined application of Ba
2+
and ouabain.

Conclusions—

SLIGRL evokes EDHF-mediated relaxation in the rat middle cerebral artery, underpinned by hyperpolarization of the smooth muscle. The profile of blockade of EDHF-mediated hyperpolarization and relaxation supports a pivotal role for IK
Ca
channels. Furthermore, similar inhibition of responses to EDHF and exogenous K
+
with Ba
2+
and ouabain suggests that K
+
may contribute as an EDHF in the middle cerebral artery.
Transformation of Diffuse β-Amyloid Precursor Protein and β-Amyloid Deposits to Plaques in the Thalamus After Transient Occlusion of the Middle Cerebral Artery in Rats
Authors: van Groen Thomas , Puurunen Kirsi , Mäki Hanna-Mari , Sivenius Juhani , Jolkkonen Jukka
Published: June 2005

Background and Purpose—

The present study examined the long-term presence of β-amyloid precursor protein (APP) and β-amyloid (Aβ) accumulation in the rat thalamus after focal cerebral ischemia.

Methods—

Male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 2 hours. Sensorimotor outcome was assessed using a tapered/ledged beam-walking task after operation. The distribution of APP and Aβ was examined immunohistochemically at 1 week, 1 month, and 9 months after MCAO.

Results—

MCAO caused a long-lasting deficit in forelimb and hind limb function assessed using the beam-walking test. Histologic examination revealed a transient increase in APP and Aβ staining in axons in the corpus callosum and in neurons at the border of the ischemic region. APP and Aβ deposits persisted in the thalamic nuclei (ventroposterior lateral and ventroposterior medial nuclei), eventually leading to dense plaque-like deposits by the end of the 9-month follow-up. The deposits were surrounded by an astroglial scar. The deposits were positive for Aβ and N-terminal APP, but not for C-terminal APP. Antibodies against the C-terminal of Aβ, ie, Aβ42 and Aβ40, showed a preferential staining for Aβ42. Congo red or thioflavine S did not stain the deposits.

Conclusions—

The present results demonstrated the persistent presence and aggregation of APP and Aβ, or their fragments, to dense plaque-like deposits in the ventroposterior lateral and ventroposterior medial nuclei of rats subjected to focal cerebral ischemia.
Family History in Ischemic Stroke Before 70 Years of Age
Authors: Jood Katarina , Ladenvall Claes , Rosengren Annika , Blomstrand Christian , Jern Christina
Published: June 2005

Background and Purpose—

Results from twin and family history studies of ischemic stroke suggest that future molecular genetic studies should focus on strictly defined stroke subtypes and younger cases. Accordingly, we investigated stroke subtypes, vascular risk factors, and family history in a large study of patients with ischemic stroke onset before age 70 years.

Methods—

Six hundred consecutive white participants with ischemic stroke (18 to 69 years) and 600 age- and sex-matched controls were examined for vascular risk factors and family history of stroke and myocardial infarction (MI). Stroke subtype was defined using Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria.

Results—

Family history of stroke was associated with overall ischemic stroke (multivariate odds ratio [OR], 1.75; 95% confidence interval [CI], 1.26 to 2.43), large-vessel disease (LVD) (OR, 1.88; 95% CI, 1.02 to 3.44), small-vessel disease (SVD, OR, 1.79; 95% CI, 1.13 to 2.84), and cryptogenic stroke (OR, 1.70; 95% CI, 1.13 to 2.56), but not with cardioembolic stroke. Family history of MI was strongly associated with LVD (OR, 3.25; 95% CI, 1.74 to 6.07), whereas no significant association were observed for other subtypes. We also found an independent association between family history of stroke and a favorable outcome after 3 months.

Conclusion—

Family history of stroke is an independent risk factor for ischemic stroke with onset before age 70 years. For the first time to our knowledge, we report this association not only for LVD and SVD but also for cryptogenic stroke, implying that future studies of the genetics of ischemic stroke should target these 3 subtypes.