bluuebunny/crossref_metadata_2025 huggingface dataset:
ON EXTENSIONS OF EMBEDDINGS INTO THE ENUMERATION DEGREES OF THE ${\Sigma_2^0}$-SETS
Authors: LEMPP STEFFEN , SLAMAN THEODORE A. , SORBI ANDREA
Published: January 2006

We give an algorithm for deciding whether an embedding of a finite partial order [Formula: see text] into the enumeration degrees of the [Formula: see text]-sets can always be extended to an embedding of a finite partial order [Formula: see text].
cGMP modulation of ACh-stimulated exocytosis in guinea pig antral mucous cells
Authors: Saad Adel H. , Shimamoto Chikao , Nakahari Takashi , Fujiwara Shoko , Katsu Ken-ichi , Marunaka Yoshinori
Published: January 2006

In guinea pig antral mucous cells, ACh stimulates the Ca2+-regulated exocytosis, which has a characteristics feature: an initial transient phase followed by a sustained phase. The effects of cGMP on ACh-stimulated exocytosis were studied in guinea pig antral mucous cells using video microscopy. cGMP enhanced the frequency of ACh-stimulated exocytotic events, whereas cGMP alone did not induce any exocytotic events under the ACh-unstimulated condition. cGMP did not stimulate either Ca2+mobilization or cAMP accumulation. The Ca2+dose-response studies demonstrated that cGMP shifted the dose-response curve upward with no shift to the lower concentration. This indicates that cGMP increased maximal responsiveness of the Ca2+-regulated exocytosis, but not the Ca2+sensitivity. Moreover, under a condition of ATP depletion by dinitrophenol (DNP) or anoxia (N2bubbling), ACh evoked only a sustained phase in exocytotic events with no initial transient phase. However, ACh evoked an initial transient phase followed by a sustained phase with addition of cGMP before ATP depletion, whereas only a sustained phase was evoked in a case of cGMP addition after ATP depletion. Thus cGMP-induced enhancement in ACh-stimulated exocytotic events requires ATP, suggesting that cGMP modulates ATP-dependent priming of Ca2+-regulated exocytosis in antral mucous cells. In conclusion, cGMP increases the number of primed granules via acceleration of the ATP-dependent priming, which enhances the Ca2+-regulated exocytosis stimulated by ACh.
Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTα-OSTβ in cholestasis in humans and rodents
Authors: Boyer James L. , Trauner Michael , Mennone Albert , Soroka Carol J. , Cai Shi-Ying , Moustafa Tarek , Zollner Gernot , Lee Jin Young , Ballatori Nazzareno
Published: January 2006

Organic solute transporter (OSTα-OSTβ) is a novel heteromeric bile acid and sterol transporter expressed at the basolateral membranes of epithelium in the ileum, kidney, and liver. To determine whether OSTα-OSTβ undergoes farnesoid X receptor (FXR)-dependent adaptive regulation following cholestatic liver injury, mRNA and protein expression levels were analyzed in patients with primary biliary cirrhosis (PBC) and following common bile duct ligation (CBDL) in rats and Fxr null and wild-type mice. Hepatic OSTα and OSTβ mRNA increased 3- and 32-fold, respectively, in patients with PBC compared with controls, whereas expression of Ostα and Ostβ also increased in the liver of rats and mice following CBDL. In contrast, expression of Ostα and Ostβ mRNA was generally lower in Fxr null mice, and CBDL failed to enhance expression of Ostα and Ostβ compared with wild-type mice. HepG2 cells treated for 24 h with chenodeoxycholic acid, a selective FXR ligand, had higher levels of OSTα and OSTβ mRNA and protein. Increases in OST protein were visualized by confocal microscopy at the plasma membrane. These results indicate that expression of Ostα and Ostβ are highly regulated in response to cholestasis and that this response is dependent on the FXR bile acid receptor.
Selective matrix metalloproteinase inhibition attenuates progression of left ventricular dysfunction and remodeling in dogs with chronic heart failure
Authors: Morita Hideaki , Khanal Sanjaya , Rastogi Sharad , Suzuki George , Imai Makoto , Todor Anastassia , Sharov Victor G. , Goldstein Sidney , O'Neill Timothy P. , Sabbah Hani N.
Published: January 2006

Matrix metalloproteinases (MMPs) contribute to the progression of left ventricular (LV) dysfunction and remodeling associated with heart failure (HF). The present study examined the long-term effects of a selective MMP inhibitor PG-530742 (PG) on the progression of LV dysfunction and remodeling in dogs with HF. Chronic HF [LV ejection fraction (LVEF), ≤36%] was produced by multiple sequential intracoronary microembolizations in 24 dogs. Two weeks after the last embolization, dogs were randomized to 3 mo of therapy with either high-dose (HD) PG (3.5 mg/kg, n = 8), low-dose (LD) PG (0.2 mg/kg, n = 8), or to a matched placebo (PL, n = 8). PG has been shown to produce complete inhibition of MMP-2, -3, -9, and -13, while sparing MMPs-1 and -7. Hemodynamic and echocardiographic measurements were made before and 3 mo after initiating therapy. In PL and LD dogs, LVEF decreased significantly, and LV end-systolic volume (ESV) and LV end-diastolic volume (EDV) increased significantly during the 3-mo follow-up period. Whereas in HD dogs ejection fraction increased from 36 ± 1 to 40 ± 1% ( P = 0.003), EDV and ESV decreased (59 ± 4 vs. 57 ± 4 ml, P = 0.02; and 38 ± 2 vs. 34 ± 2 ml, P = 0.00001, respectively). When compared with controls, HD-treated dogs showed 30% reduction in replacement fibrosis, 29% reduction in interstitial fibrosis, and 28% reduction in myocyte cross-sectional area. mRNA expression of selective MMPs was also reduced in LV tissue in HD- but not LD-treated dogs. In conclusion, in dogs with moderate HF, long-term monotherapy with HD selective MMP inhibitor PG prevents LV remodeling and the progression of global LV dysfunction.
Ankyrin repeat and SOCS box protein 15 regulates protein synthesis in skeletal muscle
Authors: McDaneld T. G. , Hannon K. , Moody D. E.
Published: January 2006

Ankyrin repeat and SOCS box protein 15 (ASB15) is an Asb family member expressed predominantly in skeletal muscle. We have previously reported that ASB15 mRNA abundance decreases after administration of β-adrenergic receptor agonists. Because β-adrenergic receptor agonists are known to stimulate muscle hypertrophy, the objective of this study was to determine whether ASB15 regulates cellular processes that contribute to muscle growth. Stable myoblast C2C12 cells expressing full-length ASB15 (ASB15-FL) and ASB15 lacking the ankyrin repeat (ASB15-Ank) or SOCS box (ASB15-SOCS) motifs were evaluated for changes in proliferation, differentiation, protein synthesis, and protein degradation. Expression of ASB15-FL caused a delay in differentiation, followed by an increase in protein synthesis of ∼34% ( P < 0.05). A consistent effect of ASB15 overexpression was observed in vivo, where ectopic expression of ASB15 increased skeletal muscle fiber area ( P < 0.0001) after 9 days. Expression of ASB15-SOCS altered differentiation of myoblasts, resulting in detachment of cells from culture plates. Expression of ASB15-Ank increased protein degradation by 84 h of differentiation ( P < 0.05), and in vivo ectopic expression of an ASB15 construct lacking both the ankyrin repeat and SOCS box motifs decreased skeletal muscle fiber area ( P < 0.0001). Together, these results suggest ASB15 participates in the regulation of protein turnover and muscle cell development by stimulating protein synthesis and regulating differentiation of muscle cells. This is the first study to demonstrate a role for an Asb family member in skeletal muscle growth.
Macrophages and skeletal muscle regeneration: a clodronate-containing liposome depletion study
Authors: Summan Mukesh , Warren Gordon L. , Mercer Robert R. , Chapman Rebecca , Hulderman Tracy , Van Rooijen Nico , Simeonova Petia P.
Published: January 2006

The study evaluates the influence of monocytes/macrophages in the mechanisms of skeletal muscle injury using a mouse model and selective depletion of peripheral monocyte with systemic injections of liposomal clodronate (dichloromethylene bisphosphonate). This pharmacological treatment has been demonstrated to induce specific apoptotic death in monocytes and phagocytic macrophages. In the current studies, the liposomal clodronate injections resulted in a marked attenuation of the peak inflammatory response in the freeze-injured muscle in the first three days after injury. The effect was accompanied by a transient reduction (at day 1 or 3 postinjury) of the expression of several genes coding for inflammatory, as well as growth-related mediators, including TNF, monocyte chemoattractant protein (MCP)-1, thioredoxin, high-mobility group AT-hook 1, insulin-like growth factor-binding protein (IGFBP), and IGF-1. In contrast, the expression of major myogenic factors (i.e., MyoD and myogenin) directly involved in the activation/proliferation and differentiation of muscle precursor cells was not altered by the clodronate liposome treatment. The repair process in the injured muscle of clodronate liposome-treated mice was characterized by prolonged clearance of necrotic myofibers and a tendency for increased muscle fat accumulation at day 9 and 14 postinjury, respectively. In conclusion, a significant reduction of the initial monocyte/macrophage influx into the injured muscle is associated with not improved, but moderately impaired, repair processes after skeletal muscle injury.
Measurement of activation energy and oxidative phosphorylation onset kinetics in isolated muscle fibers in the absence of cross-bridge cycling
Authors: Walsh B. , Howlett R. A. , Stary C. M. , Kindig C. A. , Hogan M. C.
Published: January 2006

This study utilized N-benzyl- p-toluene sulfonamide (BTS), a potent inhibitor of cross-bridge cycling, to measure 1) the relative metabolic costs of cross-bridge cycling and activation energy during contraction, and 2) oxygen uptake kinetics in the presence and absence of myosin ATPase activity, in isolated Xenopus laevis muscle fibers. Isometric tension development and either cytosolic Ca2+concentration ([Ca2+]c) or intracellular Po2(P[Formula: see text]) were measured during contractions at 20°C in control conditions (Con) and after exposure to 12.5 μM BTS. BTS attenuated tension development to 5 ± 0.4% of Con but did not affect either resting or peak [Ca2+]cduring repeated isometric contractions. To determine the relative metabolic cost of cross-bridge cycling, we measured the fall in P[Formula: see text] (ΔP[Formula: see text]; a proxy for V̇o2) during contractions in Con and BTS groups. BTS attenuated ΔP[Formula: see text] by 55 ± 6%, reflecting the relative ATP cost of cross-bridge cycling. Thus, extrapolating ΔP[Formula: see text] to a value that would occur at 0% tension suggests that actomyosin ATP requirement is ∼58% of overall ATP consumption during isometric contractions in mixed fiber types. BTS also slowed the fall in P[Formula: see text] (time to 63% of overall ΔP[Formula: see text]) from 75 ± 9 s (Con) to 101 ± 9 s (BTS) ( P < 0.05), suggesting an important role of the products of ATP hydrolysis in determining the V̇o2onset kinetics. These results demonstrate in isolated skeletal muscle fibers that 1) activation energy accounts for a substantial proportion (∼42%) of total ATP cost during isometric contractions, and 2) despite unchanged [Ca2+]ctransients, a reduced rate of ATP consumption results in slower V̇o2onset kinetics.
Oxytocin actions within the supraoptic and paraventricular nuclei: differential effects on peripheral and intranuclear vasopressin release
Authors: Neumann Inga D. , Torner Luz , Toschi Nicola , Veenema Alexa H.
Published: January 2006

In response to forced swimming (FS), AVP is released somato-dendritically within the supraoptic nucleus (SON) and paraventricular nucleus (PVN), but not from neurohypophyseal terminals into blood. Together with AVP, oxytocin (OXT) is released within the SON and PVN. Here, we studied the role of intra-SON and intra-PVN OXT in the regulation of local AVP release and into the blood in male rats. Within the SON, bilateral retrodialysis of an OXT receptor antagonist (OXT-A) increased local AVP release in response to FS [60 s, 21°C, vehicle twofold, not significant (ns); OXT-A: 15-fold increase, P < 0.05] without significantly affecting basal AVP release. In addition, local OXT-A elevated plasma AVP secretion under basal conditions (twofold increase, P < 0.05) without further elevation after FS. Within the PVN, exposure to FS elevated local AVP release, reaching significance only in the OXT-A group (vehicle: 1.4-fold, ns; OXT-A: 1.6-fold increase, P = 0.050). Bilateral OXT-A into the PVN did not affect peripheral AVP secretion either under basal or stress conditions. Basal ACTH concentrations tended to be elevated by local OXT-A within the PVN (1.7-fold increase, P = 0.076). In contrast, the swim-induced ACTH secretion was attenuated after retrodialysis of OXT-A within both the SON (at 5 min) and PVN (at 15 min) ( P < 0.05 both) compared with vehicle. The results demonstrate a receptor-mediated effect of OXT within the SON and PVN on local and neurohypophyseal AVP release, which depends upon the activity conditions. Further, while exerting an inhibitory effect on hypothalamo-pituitary-adrenal axis activity under basal conditions, hypothalamic OXT is essential for an adequate acute ACTH response.
Lateral ventricular ghrelin and fourth ventricular ghrelin induce similar increases in food intake and patterns of hypothalamic gene expression
Authors: Kinzig Kimberly P. , Scott Karen A. , Hyun Jayson , Bi Sheng , Moran Timothy H.
Published: January 2006

The gut peptide ghrelin has been shown to stimulate food intake after both peripheral and central administration, and the hypothalamic arcuate nucleus has been proposed to be the major site for mediating this feeding stimulatory action. Ghrelin receptors are widely distributed in the brain, and hindbrain ghrelin administration has been shown to potently stimulate feeding, suggesting that there may be other sites for ghrelin action. In the present study, we have further assessed potential sites for ghrelin action by comparing the ability of lateral and fourth ventricular ghrelin administration to stimulate food intake and alter patterns of hypothalamic gene expression. Ghrelin (0.32, 1, or 3.2 nmol) in the lateral or fourth ventricle significantly increased food intake in the first 4 h after injection, with no ventricle-dependent differences in degree or time course of hyperphagia. One nanomole of ghrelin into either the lateral or fourth ventricle resulted in similar increases in arcuate nucleus neuropeptide Y mRNA expression. Expression levels of agouti-related peptide or proopiomelanocortin mRNA were not affected by ghrelin administration. These data demonstrate that ghrelin can affect food intake and hypothalamic gene expression through interactions at multiple brain sites.
Direct and indirect effects of leptin on preadipocyte proliferation and differentiation
Authors: Wagoner Blair , Hausman Dorothy B. , Harris Ruth B. S.
Published: January 2006

Leptin has been shown to reduce body fat in vivo. Adipocytes express the leptin receptor; therefore, it is realistic to expect a direct effect of leptin on adipocyte growth and metabolism. In vitro studies examining the effect of leptin on adipocyte metabolism require supraphysiological doses of the protein to see a decrease in lipogenesis or stimulation of lipolysis, implying an indirect action of leptin. It also is possible that leptin reduces adipose mass by inhibiting preadipocyte proliferation (increase in cell number) and/or differentiation (lipid filling). Thus we determined direct and indirect effects of leptin on preadipocyte proliferation and differentiation in vitro. We tested the effect of leptin (0–500 ng/ml), serum from leptin-infused rats (0.25% by volume), and adipose tissue-conditioned medium from leptin-infused rats (0–30% by volume) on preadipocyte proliferation and differentiation in a primary culture of cells from male Sprague-Dawley rat adipose tissue. Leptin (50 ng/ml) stimulated proliferation of preadipocytes ( P < 0.05), but 250 and 500 ng leptin/ml inhibited proliferation of both preadipocyte and stromal vascular cell fractions ( P < 0.01), as measured by [3H]thymidine incorporation. Serum from leptin-infused rats inhibited proliferation of the adipose and stromal vascular fractions ( P = 0.01), but adipose tissue-conditioned medium had no effect on proliferation of either cell fraction. None of the treatments changed preadipocyte differentiation as measured by sn-glycerophosphate dehydrogenase activity. These results suggest that leptin could inhibit preadipocyte proliferation by modifying release of a factor from tissue other than adipose tissue.
Persistence of a behavioral food-anticipatory circadian rhythm following dorsomedial hypothalamic ablation in rats
Authors: Landry G. J. , Simon M. M. , Webb I. C. , Mistlberger R. E.
Published: January 2006

Circadian rhythms of behavior in rodents are regulated by a system of circadian oscillators, including a master light-entrainable pacemaker in the suprachiasmatic nucleus that mediates synchrony to the day-night cycle, and food-entrainable oscillators located elsewhere that generate rhythms of food-anticipatory activity (FAA) synchronized to daily feeding schedules. Despite progress in elucidating neural and molecular mechanisms of circadian oscillators, localization of food-entrainable oscillators driving FAA remains an enduring problem. Recent evidence suggests that the dorsomedial hypothalamic nucleus (DMH) may function as a final common output for behavioral rhythms and may be critical for the expression of FAA (Gooley JJ, Schomer A, and Saper CB. Nat Neurosci 9: 398–407, 2006). To determine whether the reported loss of FAA by DMH lesions is specific to one behavioral measure or generalizes to other measures, rats received large radiofrequency lesions aimed at the DMH and were recorded in cages with movement sensors. Total and partial DMH ablation was associated with a significant attenuation of light-dark-entrained activity rhythms during ad libitum food access, because of a selective reduction in nocturnal activity. When food was restricted to a single 3-h daily meal in the middle of the lights-on period, all DMH and intact rats exhibited significant FAA. The rhythm of FAA persisted during a 48-h food deprivation test and reappeared during a 72-h deprivation test after ad libitum food access. The DMH is not the site of oscillators or entrainment pathways necessary for all manifestations of FAA, but may participate on the output side of this circadian function.
Nitric oxide in B6 mouse and nitric oxide-sensitive soluble guanylate cyclase in cat modulate acetylcholine release in pontine reticular formation
Authors: Lydic Ralph , Garza-Grande Ricardo , Struthers Richard , Baghdoyan Helen A.
Published: January 2006

ACh regulates arousal, and the present study was designed to provide insight into the neurochemical mechanisms modulating ACh release in the pontine reticular formation. Nitric oxide (NO)-releasing beads microinjected into the pontine reticular formation of C57BL/6J (B6) mice significantly ( P < 0.0001) increased ACh release. Microdialysis delivery of the NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12) to the mouse pontine reticular formation also caused a concentration-dependent increase in ACh release ( P < 0.001). These are the first neurochemical data showing that ACh release in the pontine reticular formation of the B6 mouse is modulated by NO. The signal transduction cascade through which NO modulates ACh release in the pontine reticular formation has not previously been characterized. Therefore, an additional series of studies quantified the effects of a soluble guanylate cyclase (sGC) inhibitor, 1 H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), on ACh release in the cat medial pontine reticular formation. During naturally occurring states of sleep and wakefulness, but not anesthesia, ODQ caused a significant ( P < 0.001) decrease in ACh release. These results show for the first time that NO modulates ACh in the medial pontine reticular formation of the cat via an NO-sensitive sGC signal transduction cascade. Isoflurane and halothane anesthesia have been shown to decrease ACh release in the medial pontine reticular formation. The finding that ODQ did not alter ACh release during isoflurane or halothane anesthesia demonstrates that these anesthetics disrupt the NO-sensitive sGC-cGMP pathway. Considered together, results from the mouse and cat indicate that NO modulates ACh release in arousal-promoting regions of the pontine reticular formation via an NO-sensitive sGC-cGMP pathway.
Central excitability does not limit postfatigue voluntary activation of quadriceps femoris
Authors: Kalmar J. M. , Cafarelli E.
Published: January 2006

After fatigue, motor evoked potentials (MEP) elicited by transcranial magnetic stimulation and cervicomedullary evoked potentials elicited by stimulation of the corticospinal tract are depressed. These reductions in corticomotor excitability and corticospinal transmission are accompanied by voluntary activation failure, but this may not reflect a causal relationship. Our purpose was to determine whether a decline in central excitability contributes to central fatigue. We hypothesized that, if central excitability limits voluntary activation, then a caffeine-induced increase in central excitability should offset voluntary activation failure. In this repeated-measures study, eight men each attended two sessions. Baseline measures of knee extension torque, maximal voluntary activation, peripheral transmission, contractile properties, and central excitability were made before administration of caffeine (6 mg/kg) or placebo. The amplitude of vastus lateralis MEPs elicited during minimal muscle activation provided a measure of central excitability. After a 1-h rest, baseline measures were repeated before, during, and after a fatigue protocol that ended when maximal voluntary torque declined by 35% (Tlim). Increased prefatigue MEP amplitude ( P = 0.055) and cortically evoked twitch ( P < 0.05) in the caffeine trial indicate that the drug increased central excitability. In the caffeine trial, increased MEP amplitude was correlated with time to task failure ( r = 0.74, P < 0.05). Caffeine potentiated the MEP early in the fatigue protocol ( P < 0.05) and offset the 40% decline in placebo MEP ( P < 0.05) at Tlim. However, this was not associated with enhanced maximal voluntary activation during fatigue or recovery, demonstrating that voluntary activation is not limited by central excitability.
Experimental determination of net protein charge, [A]tot, andKaof nonvolatile buffers in bird plasma
Authors: Stämpfli Henry , Taylor Michael , McNicoll Carl , Gancz Ady Y. , Constable Peter D.
Published: January 2006

The quantitative mechanistic acid-base approach to clinical assessment of acid-base status requires species-specific values for [A]tot(the total concentration of nonvolatile buffers in plasma) and Ka(the effective dissociation constant for weak acids in plasma). The aim of this study was to determine [A]totand Kavalues for plasma in domestic pigeons. Plasma from 12 healthy commercial domestic pigeons was tonometered with 20% CO2at 37°C. Plasma pH, Pco2, and plasma concentrations of strong cations (Na, K, Ca), strong anions (Cl, l-lactate), and nonvolatile buffer ions (total protein, albumin, phosphate) were measured over a pH range of 6.8–7.7. Strong ion difference (SID) (SID5= Na + K + Ca − Cl − lactate) was used to calculate [A]totand Kafrom the measured pH and Pco2and SID5. Mean (±SD) values for bird plasma were as follows: [A]tot= 7.76 ± 2.15 mmol/l (equivalent to 0.32 mmol/g of total protein, 0.51 mmol/g of albumin, 0.23 mmol/g of total solids); Ka= 2.15 ± 1.15 × 10−7; and p Ka= 6.67. The net protein charge at normal pH (7.43) was estimated to be 6 meq/l; this value indicates that pigeon plasma has a much lower anion gap value than mammals after adjusting for high mean l-lactate concentrations induced by restraint during blood sampling. This finding indicates that plasma proteins in pigeons have a much lower net anion charge than mammalian plasma protein. An incidental finding was that total protein concentration measured by a multianalyzer system was consistently lower than the value for total solids measured by refractometer.
Exercise-induced abdominal muscle fatigue in healthy humans
Authors: Taylor Bryan J. , How Stephen C. , Romer Lee M.
Published: January 2006

The abdominal muscles have been shown to fatigue in response to voluntary isocapnic hyperpnea using direct nerve stimulation techniques. We investigated whether the abdominal muscles fatigue in response to dynamic lower limb exercise using such techniques. Eleven male subjects [peak oxygen uptake (V̇o2 peak) = 50.0 ± 1.9 (SE) ml·kg−1·min−1] cycled at >90% V̇o2 peakto exhaustion (14.2 ± 4.2 min). Abdominal muscle function was assessed before and up to 30 min after exercise by measuring the changes in gastric pressure (Pga) after the nerve roots supplying the abdominal muscles were magnetically stimulated at 1–25 Hz. Immediately after exercise there was a decrease in Pga at all stimulation frequencies (mean −25 ± 4%; P < 0.001) that persisted up to 30 min postexercise (−12 ± 4%; P = 0.001). These reductions were unlikely due to changes in membrane excitability because amplitude, duration, and area of the rectus abdominis M wave were unaffected. Declines in the Pga response to maximal voluntary expiratory efforts occurred after exercise (158 ± 13 before vs. 145 ± 10 cmH2O after exercise; P = 0.005). Voluntary activation, assessed using twitch interpolation, did not change (67 ± 6 before vs. 64 ± 2% after exercise; P = 0.20), and electromyographic activity of the rectus abdominis and external oblique increased during these volitional maneuvers. These data provide new evidence that the abdominal muscles fatigue after sustained, high-intensity exercise and that the fatigue is primarily due to peripheral mechanisms.
FDG-PET imaging of pulmonary inflammation in healthy volunteers after airway instillation of endotoxin
Authors: Chen Delphine L. , Rosenbluth Daniel B. , Mintun Mark A. , Schuster Daniel P.
Published: January 2006

Recent studies indicate that a focal, limited, inflammatory response can be safely elicited after direct bronchial instillation of small doses of endotoxin into a single lung segment. Because the radiotracer [18F]fluorodeoxyglucose ([18F]FDG) is taken up at accelerated rates within inflamed tissues, we hypothesized that we could detect and quantify this regional inflammatory response with positron emission tomography (PET). We imaged 18 normal volunteers in a dose-escalation study with 3 endotoxin dosing groups ( n = 6 in each group): 1 ng/kg, 2 ng/kg, and 4 ng/kg. Endotoxin was instilled by bronchoscopy into a segment of the right middle lobe, with imaging performed ∼24 h later, followed by bronchoalveolar lavage (BAL). A “subtraction imaging analysis” was performed in the highest dose cohort to identify the area of inflammation, using the preendotoxin scan as a baseline. BAL neutrophil counts were significantly higher in the highest dose group compared with the other two groups (1,413 ± 625 vs. 511 ± 396 and 395 ± 400 cells/mm3; P < 0.05). Autoradiography performed on cells harvested by BAL showed specific [3H]deoxyglucose ([3H]DG) uptake limited to neutrophils. In vitro [3H]DG uptake in BAL neutrophils in the 4 ng/kg dose group (but not in the 2 ng/kg group) was statistically greater than in peripheral blood neutrophils obtained before endotoxin instillation. The rate of [18F]FDG uptake was greatest in the 4 ng/kg group, with a consistent, statistically significant increase in the rate of uptake after endotoxin instillation compared with baseline. We conclude that the inflammatory response to low-dose endotoxin in a single lung segment can be visualized and quantified by imaging with FDG-PET.
Ubiquitination of p53 at Multiple Sites in the DNA-Binding Domain
Authors: Chan Wan Mui , Mak Man Chi , Fung Tsz Kan , Lau Anita , Siu Wai Yi , Poon Randy Y.C.
Published: January 2006

The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain. (Mol Cancer Res 2006;4(1)15–25)
Calcium and Vitamin D Metabolism in Hypocalcemic Vitamin D-Resistant Rickets Carriers
Authors: Nicolaidou Polyxeni , Papadopoulou Anna , Georgouli Helen , Matsinos Y.G. , Tsapra Helen , Fretzayas Andreas , Giannoulia-Karantana Aglaia , Kitsiou Sophia , Douros Konstantinos , Papassotiriou Ioannis , Chrousos Georges P.
Published: January 2006

Background/Aims: Hypocalcemic vitamin D-resistant rickets (HVDRR) is a rare monogenic autosomal recessive disorder associated with mutations in the gene of the vitamin D receptor (VDR), the mediator of 1,25(OH)2D3 action. Although many investigations have discussed the clinical manifestations and molecular etiology of this disease, only a few have investigated the biochemical and hormonal status of heterozygous HVDRR. The aim of the current work was to investigate the profile of selected biochemical and hormonal parameters related to the vitamin D endocrine system in a large number of HVDRR heterozygotes. Methods: 67 relatives of 2 HVDRR patients, all members of an extended Greek kindred of five generations with a common ancestor, were included in the study. Direct sequencing was used to identify VDR gene mutations. Serum Ca, P, 25(OH)D, iPTH, and 1,25(OH)2D levels were determined in all members of the kindred. Results: DNA analysis of the participants led to the design of two study groups: the HVDRR carriers (24) and the control subjects (43). Our results showed elevated circulating serum levels of 1,25(OH)2D3 and lower levels of PTH than their age- and sex-matched controls. No hypocalcemia or hypophosphatemia were detected in HVDRR carriers. Conclusions: Our findings suggest that HVDRR carriers may have compensatory elevated serum levels of 1,25(OH)2D3 through which they restrain PTH secretion. The study of HVDRR carriers could be a useful tool for the investigation of the vitamin D endocrine system.
Topical Application of Mitomycin C before versus after Myringotomy: An Experimental Study
Authors: Kaftan Holger , Hosemann Werner
Published: January 2006

The objective of this animal study was to compare topical mitomycin C (MMC) before and after nonlaser myringotomy. Bilateral myringotomies were performed in 12 rats. On the left side, MMC was applied to the intact tympanic membrane for 10 min before myringotomy (preapplication group). On the right side, MMC was applied to the tympanic membrane following myringotomy. Tympanic membranes were observed for a total of 8 weeks. By day 35, all myringotomies with application of MMC after perforation of the tympanic membrane were closed, whereas 42% of the myringotomies with application of MMC prior to perforation remained patent. One tympanic membrane in the preapplication group showed an enlargement of the perforation with partial destruction of the malleus. The present study showed that the application of MMC to the intact tympanic membrane prolongs the patency of nonlaser myringotomies in rats. Specific side effects of the MMC application must be kept in mind.
Modulation of Hepatic Inflammatory Risk Markers of Cardiovascular Diseases by PPAR–α Activators
Authors: Zambon Alberto , Gervois Philippe , Pauletto Paolo , Fruchart Jean-Charles , Staels Bart
Published: January 2006

Atherosclerosis is a long-term chronic inflammatory disease associated with increased concentrations of inflammatory hepatic markers, such as CRP and fibrinogen, and of peripheral origin, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6. Peroxisome proliferator-activated receptor (PPAR-)-α is a ligand-activated transcription factor that regulates expression of key genes involved in lipid homeostasis and modulates the inflammatory response both in the vascular wall and the liver. PPAR-α is activated by natural ligands, such as fatty acids, as well as the lipid-lowering fibrates. PPAR-α agonists impact on different steps of atherogenesis: (1) early markers of atherosclerosis, such as vascular wall reactivity, are improved, (2) however, reduced expression of adhesion molecules on the surface of endothelial cells, accompanied by decreased levels of inflammatory cytokines, such as TNF-α, IL-1, and IL-6, leads to a decreased leukocyte recruitment into the arterial wall; (3) in later stages of the atherosclerotic process, PPAR-α agonists may promote plaque stabilization and reduce cardiovascular events, via effects on metalloproteinases, such as MMP9. Moreover, PPAR-α activation by fibrates also impairs proinflammatory cytokine-signaling pathways in the liver resulting in the modulation of the acute phase response reaction via mechanisms independent of changes in lipoprotein levels. Effective coronary artery disease (CAD) prevention requires the use of agents that act beyond low-density lipoprotein cholesterol-lowering. PPAR-α agonists appear to comprehensively address some of the abnormalities of the most common clinical phenotypes of the high CAD risk patient of the 21st century such as in the metabolic syndrome and type 2 diabetes: low high-density lipoprotein cholesterol, high triglycerides, small, dense low-density lipoprotein, and a proinflammatory, procoagulant state.
Enhanced T-Cell Expression of RANK Ligand in Acute Coronary Syndrome
Authors: Sandberg Wiggo J. , Yndestad Arne , Øie Erik , Smith Camilla , Ueland Thor , Ovchinnikova Olga , Robertson Anna-Karin L. , Müller Fredrik , Semb Anne G. , Scholz Hanne , Andreassen Arne K. , Gullestad Lars , Damås Jan Kristian , Frøland Stig S. , Hansson Göran K. , Halvorsen Bente , Aukrust Pål
Published: January 2006

Objective—Based on its role in inflammation and matrix degradation, we hypothesized a role for osteoprotegerin (OPG), RANK, and RANK ligand (RANKL) in coronary artery disease.

Methods and Results—We examined the expression of various members of the OPG/RANKL/RANK axis in patients with stable and unstable angina and in the atherosclerotic lesions of apolipoprotein E–deficient (apoE−/−) mice. Our findings were: (1) Serum levels of OPG were raised in patients with unstable angina (n=40), but not in those with stable angina (n=40), comparing controls (n=20); (2) mRNA levels of RANKL were increased in T-cells in unstable angina patients accompanied by increased expression of RANK in monocytes; (3) strong immunostaining of OPG/RANKL/RANK was seen within thrombus material obtained at the site of plaque rupture during acute myocardial infarction; (4) OPG/RANKL/RANK was expressed in the atherosclerotic plaques of apoE−/−mice, with RANKL located specifically to the plaques; and (5) RANKL enhanced the release of monocyte chemoattractant peptide-1 in mononuclear cells from unstable angina patients, and promoted matrix metalloproteinase (MMP) activity in vascular smooth muscle cells.

Conclusions—We show enhanced expression of the OPG/RANKL/RANK system both in clinical and experimental atherosclerosis, with enhanced T-cell expression of RANKL as an important feature of unstable disease.
Missense Mutations in the PCSK9 Gene Are Associated With Hypocholesterolemia and Possibly Increased Response to Statin Therapy
Authors: Berge Knut Erik , Ose Leiv , Leren Trond P.
Published: January 2006

Objective—

The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene encodes a proprotein convertase that causes degradation of cell surface low-density lipoprotein receptors (LDLRs). Mutations in the PCSK9 gene that disrupt the normal function of PCSK9 could therefore result in increased number of LDLRs and hypocholesterolemia. Also, the cholesterol-lowering effect of statins could be increased in subjects carrying mutations in the PCSK9 gene.

Methods and Results—

We have screened 38 unrelated hypocholesterolemic subjects as well as 25 unrelated familial hypercholesterolemia (FH) heterozygotes who responded particularly well to statin therapy for mutations in the 12 exons of the PCSK9 gene by DNA sequencing. Six of the 38 (15.8%) hypocholesterolemic subjects were heterozygous for 1 of the 3 mutations R46L, G106R, or R237W in the PCSK9 gene. In the group of 25 FH heterozygotes who responded particularly well to statin therapy, 3 (8.8%) were heterozygous for mutations R46L or N157K in the PCSK9 gene. None of 441 hypercholesterolemic subjects without mutations in the LDLR gene or in the apolipoprotein B-100 gene possessed any of the 4 mutations.

Conclusion—

The 4 missense mutations R46L, G106R, N157K, and R237W are associated with hypocholesterolemia and possibly increased response to statin therapy.
Vascular Responses to α
1
-Adrenergic Receptors in Small Rat Mesenteric Arteries Depend on Mitochondrial Reactive Oxygen Species
Authors: Hao Li , Nishimura Tamiko , Wo Hua , Fernandez-Patron Carlos
Published: January 2006

Background—

Agonists of G-protein–coupled receptors (eg, adrenoceptors and angiotensin receptors) signal, at least in part, through matrix metalloproteinases (such as matrix metalloproteinase [MMP]-7) that transactivate the epidermal growth factor receptor (EGFR). Focusing on adrenoceptors, we examined whether the MMP-dependent signaling pathway depends on reactive oxygen species (ROS).

Methods and Results—

In isolated rat mesenteric arteries, selective stimulation of α
1
-adrenoceptors with phenylephrine induced MMP transactivation of the EGFR, mitochondrial ROS production (detected by MitoTrackerRed-CM-H
2
XRos-fluorescence and dihydroethidium-fluorescence and high-performance liquid chromatography [HPLC]/MS assay) and vasoconstriction. Inhibition of the synthesis of either MMP-7 or EGFR with anti-sense or siRNA oligonucleotides, respectively, decreased mitochondrial ROS production in response to phenylephrine. Targeted mitochondrial ROS scavenging with MitoTrackerRed-CM-H
2
XRos inhibited adrenergic vasoconstriction. Adrenoceptor-induced ROS increased mitochondrial membrane potential (Δψm), which was prevented by blockers of MMPs (GM6001, doxycycline), EGFR (AG1478), or complex I, all of which also prevented ROS production as well as vasoconstriction.

Conclusions—

Production of mitochondrial ROS is a new event in the pathway by which vasoactive agonists that induce MMP transactivation of the EGFR modulate vascular tone. Moreover, our findings suggest a connection between agonist-induced activity of MMPs, the promotion of oxidative stress, enhanced vascular tone, and hypertrophy, which are all implicated in the development and progression of vascular disease.
Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics
Authors: Kim-Shapiro Daniel B. , Schechter Alan N. , Gladwin Mark T.
Published: January 2006

The ability of oxyhemoglobin to inhibit nitric oxide (NO)-dependent activation of soluble guanylate cyclase and vasodilation provided some of the earliest experimental evidence that NO was the endothelium-derived relaxing factor (EDRF). The chemical behavior of this dioxygenation reaction, producing nearly diffusion limited and irreversible NO scavenging, presents a major paradox in vascular biology: The proximity of large amounts of oxyhemoglobin (10 mmol/L) to the endothelium should severely limit paracrine NO diffusion from endothelium to smooth muscle. However, several physical factors are now known to mitigate NO scavenging by red blood cell encapsulated hemoglobin. These include diffusional boundaries around the erythrocyte and a red blood cell free zone along the endothelium in laminar flowing blood, which reduce reaction rates between NO and red cell hemoglobin by 100- to 600-fold. Beyond these mechanisms that reduce NO scavenging by hemoglobin within the red cell, 2 additional mechanisms have been proposed suggesting that NO can be stored in the red blood cell either as nitrite or as an S-nitrosothiol (S-nitroso-hemoglobin). The latter controversial hypothesis contends that NO is stabilized, transported, and delivered by intra-molecular NO group transfers between the heme iron and β-93 cysteine to form S-nitroso-hemoglobin (SNO-Hb), followed by hypoxia-dependent delivery of the S-nitrosothiol in a process that links regional oxygen deficits with S-nitrosothiol–mediated vasodilation. Although this model has generated a field of research examining the potential endocrine properties of intravascular NO molecules, including S-nitrosothiols, nitrite, and nitrated lipids, a number of mechanistic elements of the theory have been challenged. Recent data from several groups suggest that the nitrite anion (NO
2
−
) may represent the major intravascular NO storage molecule whose transduction to NO is made possible through an allosterically controlled nitrite reductase reaction with the heme moiety of hemoglobin. As subsequently understood, the hypoxic generation of NO from nitrite is likely to prove important in many aspects of physiology, pathophysiology, and therapeutics.
Influence of Folate on Arterial Permeability and Stiffness in the Absence or Presence of Hyperhomocysteinemia
Authors: Symons J. David , Zaid Ussama B. , Athanassious Christian N. , Mullick Adam E. , Lentz Steven R. , Rutledge John C.
Published: January 2006

Objective—

Elevated plasma total homocysteine (tHcy) is associated with risk for cardiovascular disease. A common cause of mild hyperhomocysteinemia (HHcy) is folate deficiency. We sought to determine whether folate deficiency per se increases arterial permeability (quantitative fluorescence microscopy) and stiffness (vessel elastigraph), and whether the effects of folate deficiency are more severe in the presence of mild HHcy.

Methods and Results—

Heterozygous cystathionine β-synthase (CBS)-deficient mice (CBS
+/−
) and their wild-type littermates (CBS
+/+
) were fed chow containing either standard (Con) or relatively low amounts of folate (LF) for 18±3 weeks. Liver folate (μg folate/g liver) and tHcy (μM), respectively, were 12±1 and 8±1 in CBS
+/+
Con mice (n=12), and 8±1 and 8±1 in CBS
+/+
LF animals (n=5). Carotid arterial permeability was &38% greater (
P
<0.05) in CBS
+/+
LF versus Con mice, but vascular stiffening was unaltered. Liver folate and tHcy, respectively, were 13±1 and 11±1 in CBS
+/−
Con mice (n=16), and 8±1 and 16±3 in CBS
+/−
LF animals (n=6). Carotid arterial dextran accumulation was &31% greater, and maximal strain in aortae was &20% lower (both
P
<0.05) in CBS
+/−
LF versus Con mice.

Conclusion—

Taken together, low folate (
P
<0.05) combined with mild HHcy (
P
<0.05) in CBS
+/−
mice produced more arterial dysfunction compared with low folate alone (ie, CBS
+/+
mice). These findings may be particularly relevant to elderly individuals because tHcy and deficiencies of folate metabolism increase with age.
PPARγ Gene Transfer Sustains Apoptosis, Inhibits Vascular Smooth Muscle Cell Proliferation, and Reduces Neointima Formation After Balloon Injury in Rats
Authors: Lim Soo , Jin Cheng Ji , Kim Min , Chung Sung Soo , Park Ho Seon , Lee In Kyu , Lee Choon Taek , Cho Young Min , Lee Hong Kyu , Park Kyong Soo
Published: January 2006

Objective—

There is still debate as to whether antiatherosclerotic effect of PPARγ ligands is dependant on PPARγ gene itself or some other pathway.

Methods and Results—

To investigate the effect of PPARγ gene modulation on neointima formation after balloon injury, we delivered adenoviral vectors expressing the wild-type (WT) dominant negative (DN) PPARγ, or a control gene (β-galactosidase [BG]) into carotid artery after balloon injury in rosiglitazone (a PPARγ ligand)-treated (R+) (3 mg/kg/d) and nontreated (R−) rats. Two weeks after gene delivery, in both R+ and R− animals, the PPARγ-WT gene transfer showed a significantly lower intima-media ratio (IMR) than control group. Moreover, the delivery of a PPARγ-DN form showed the highest IMR (in R+WT, 0.51±0.15; R+BG, 0.89±0.14; R+DN, 1.20±0.18,
P
<0.05 and in R−WT, 0.91±0.21; R−BG, 1.44±0.23; R−DN, 1.74±0.29,
P
<0.05). Proliferation and migration showed same result pattern as IMR. In addition, apoptotic indices were significantly higher in the PPARγ-WT gene transferred group than in the PPARγ-DN group.

Conclusions—

In vivo transfer of the PPARγ-WT gene was found to inhibit smooth muscle proliferation, sustain apoptosis, and reduce neointima formation after balloon injury irrespective of rosiglitazone treatment. These results indicate that PPARγ overexpression itself has a protective role against restenosis after balloon injury.
A Possible Association Between Coronary Plaque Instability and Complex Plaques in Abdominal Aorta
Authors: Momiyama Yukihiko , Kato Ryuichi , Fayad Zahi A. , Tanaka Nobukiyo , Taniguchi Hiroaki , Ohmori Reiko , Kihara Teruyoshi , Kameyama Akira , Miyazaki Koji , Kimura Kazuo , Arakawa Koh , Kusuhara Masatoshi , Nagata Masayoshi , Nakamura Haruo , Ohsuzu Fumitaka
Published: January 2006

Objective—

Coronary plaque instability causes myocardial infarction (MI). Angiographic lesions with such instability are complex lesions. Complex carotid plaques were reported to be prevalent in unstable angina. We investigated associations between coronary plaque instability, such as MI and angiographic complex coronary lesions, and aortic plaques.

Methods and Results—

Aortic MRI was performed in 146 patients undergoing coronary angiography, of whom 108 had coronary artery disease (CAD) and 44 also had MI. Prevalence of plaques in thoracic and abdominal aortas was higher in patients with than without CAD (73% and 94% versus 32% and 79%), but it was similar in CAD patients with and without MI. Notably, complex plaques in abdominal aorta were more prevalent in CAD patients with than without MI (36% versus 14%;
P
<0.025). In multivariate analysis, abdominal complex plaques were associated with MI (odds ratio [OR], 4.5; 95% CI, 1.5 to 13.8). Among patients without MI, thoracic and abdominal complex plaques were more prevalent in patients with than without complex coronary lesions (22% and 33% versus 2% and 7%;
P
<0.05). Abdominal complex plaques were also associated with complex coronary lesions (OR, 9.8; 95% CI, 1.1 to 85.9).

Conclusion—

Complex plaques in abdominal aorta were associated with MI and complex coronary lesions, suggesting a link between coronary and aortic plaque instability.