bluuebunny/crossref_metadata_2025 huggingface dataset:
TREK-1 K+channels couple angiotensin II receptors to membrane depolarization and aldosterone secretion in bovine adrenal glomerulosa cells
Authors: Enyeart Judith A. , Danthi Sanjay J. , Enyeart John J.
Published: August 2004

Bovine adrenal glomerulosa (AZG) cells were shown to express bTREK-1 background K+channels that set the resting membrane potential and couple angiotensin II (ANG II) receptor activation to membrane depolarization and aldosterone secretion. Northern blot and in situ hybridization studies demonstrated that bTREK-1 mRNA is uniformly distributed in the bovine adrenal cortex, including zona fasciculata and zona glomerulosa, but is absent from the medulla. TASK-3 mRNA, which codes for the predominant background K+channel in rat AZG cells, is undetectable in the bovine adrenal cortex. In whole cell voltage clamp recordings, bovine AZG cells express a rapidly inactivating voltage-gated K+current and a noninactivating background K+current with properties that collectively identify it as bTREK-1. The outwardly rectifying K+current was activated by intracellular acidification, ATP, and superfusion of bTREK-1 openers, including arachidonic acid (AA) and cinnamyl 1–3,4-dihydroxy-α-cyanocinnamate (CDC). Bovine chromaffin cells did not express this current. In voltage and current clamp recordings, ANG II (10 nM) selectively inhibited the noninactivating K+current by 82.1 ± 6.1% and depolarized AZG cells by 31.6 ± 2.3 mV. CDC and AA overwhelmed ANG II-mediated inhibition of bTREK-1 and restored the resting membrane potential to its control value even in the continued presence of ANG II. Vasopressin (50 nM), which also physiologically stimulates aldosterone secretion, inhibited the background K+current by 73.8 ± 9.4%. In contrast to its potent inhibition of bTREK-1, ANG II failed to alter the T-type Ca2+current measured over a wide range of test potentials by using pipette solutions of identical nucleotide and Ca2+-buffering compositions. ANG II also failed to alter the voltage dependence of T channel activation under these same conditions. Overall, these results identify bTREK-1 K+channels as a pivotal control point where ANG II receptor activation is transduced to depolarization-dependent Ca2+entry and aldosterone secretion.
Postnatal intracerebroventricular exposure to leptin causes an altered adult female phenotype
Authors: Varma Amit , He Jing , Shin Bo-Chul , Weissfeld Lisa A. , Devaskar Sherin U.
Published: August 2004

We investigated the effect of daily intracerebroventricular (ICV) leptin administration (neonatal age 2–7 days) on hypothalamic neuropeptides (neuropeptide Y, α-melanocyte-stimulating hormone) that regulate food intake, body weight (BW) gain, and the metabolic/hormonal profile in suckling (8 and 21 days) and adult rat (35, 60, 90, and 120 days). ICV leptin (0.16 μg·g BW−1·dose−1; n = 70) led to a postnatal decline in BW ( P = 0.0002) that persisted only in the adult females ( P = 0.002). The postnatal decline in BW due to leptin was associated with a decline in food intake ( P = 0.01) and hypothalamic leptin receptor ( P = 0.008) and neuropeptide Y ( P = 0.008) immunoreactivities and an increase in α-melanocyte-stimulating hormone ( P = 0.008) immunoreactivity. In addition, hyperinsulinemia ( P = 0.01) with hypocorticosteronemia ( P = 0.007) occurred during the postnatal period with hypercorticosteronemia ( P = 0.007) and hypoleptinemia ( P = 0.008) and an increase in leutinizing hormone ( P = 0.01) in the adult male and female progeny. Persistent hyperinsulinemia ( P = 0.015) with hyperglycemia ( P = 0.008) and glucose intolerance ( P = 0.001) were observed only in the adult female. We conclude that postnatal leptin administration alters the adult female phenotype and speculate that this may relate to retention of leptin sensitivity resulting in a lipoatrophic state.
Placental uptake and transport of ACP, a neutral nonmetabolizable amino acid, in an ovine model of fetal growth restriction
Authors: de Vrijer Barbra , Regnault Timothy R. H. , Wilkening Randall B. , Meschia Giacomo , Battaglia Frederick C.
Published: August 2004

Reductions in fetal plasma concentrations of certain amino acids and reduced amino acid transport in vesicle studies suggest impaired placental amino acid transport in human fetal growth restriction (FGR). In the present study, we tested the hypothesis of an impairment in amino acid transport in the ovine model of hyperthermia-induced FGR by determining transplacental and placental retention and total placental clearance of a branched-chain amino acid (BCAA) analog, the nonmetabolizable neutral amino acid aminocyclopentane-1-carboxylic acid (ACP), in singleton control (C) and FGR pregnancies at 135 days gestation age (dGA; term 147 dGA). At study, based on the severity of the placental dysfunction, FGR fetuses were allocated to severe (sFGR, n = 6) and moderate FGR (mFGR, n = 4) groups. Fetal (C, 3,801.91 ± 156.83; mFGR, 2,911.33 ± 181.35; sFGR, 1,795.99 ± 238.85 g; P < 0.05) and placental weights (C, 414.38 ± 38.35; mFGR, 306.23 ± 32.41; sFGR, 165.64 ± 28.25 g; P < 0.05) were reduced. Transplacental and total placental clearances of ACP per 100 g placenta were significantly reduced in the sFGR but not in the mFGR group, whereas placental retention clearances were unaltered. These data indicate that both entry of ACP into the placenta and movement from the placenta into fetal circulation are impaired in severe ovine FGR and support the hypothesis of impaired placental BCAA transport in severe human FGR.
Manipulation of dietary carbohydrate and muscle glycogen affects glucose uptake during exercise when fat oxidation is impaired by β-adrenergic blockade
Authors: Zderic Theodore W. , Schenk Simon , Davidson Christopher J. , Byerley Lauri O. , Coyle Edward F.
Published: August 2004

We have recently reported that, during moderate intensity exercise, low muscle glycogen concentration and utilization caused by a high-fat diet is associated with a marked increase in fat oxidation with no effect on plasma glucose uptake (Rdglucose). It is our hypothesis that this increase in fat oxidation compensates for low muscle glycogen, thus preventing an increase in Rdglucose. Therefore, the purpose of this study was to determine whether low muscle glycogen availability increases Rdglucose under conditions of impaired fat oxidation. Six cyclists exercised at 50% peak O2consumption (V̇o2 peak) for 1 h after 2 days on either a high-fat (HF, 60% fat, 24% carbohydrate) or control (CON, 22% fat, 65% carbohydrate) diet to manipulate muscle glycogen to low and normal levels, respectively. Two hours before the start of exercise, subjects ingested 80 mg of propanolol (βB), a nonselective β-adrenergic receptor blocker, to impair fat oxidation during exercise. HF significantly decreased calculated muscle glycogen oxidation ( P < 0.05), and this decrease was partly compensated for by an increase in fat oxidation ( P < 0.05), accompanied by an increase in whole body lipolysis ( P < 0.05), despite the presence of βB. Although HF increased fat oxidation, plasma glucose appearance rate, Rdglucose, and glucose clearance rate were also significantly increased by 13, 15, and 26%, respectively (all P < 0.05). In conclusion, when lipolysis and fat oxidation are impaired, in this case by βB, fat oxidation cannot completely compensate for a reduction in muscle glycogen utilization, and consequently plasma glucose turnover increases. These findings suggest that there is a hierarchy of substrate compensation for reduced muscle glycogen availability after a high-fat, low-carbohydrate diet, with fat being the primary and plasma glucose the secondary compensatory substrate. This apparent hierarchy likely serves to protect against hypoglycemia when endogenous glucose availability is low.
ANG II activates effectors of mTOR via PI3-K signaling in human coronary smooth muscle cells
Authors: Hafizi Sassan , Wang Xuemin , Chester Adrian H. , Yacoub Magdi H. , Proud Christopher G.
Published: August 2004

We have previously shown that the vasoconstrictive peptide angiotensin II (ANG II) is a hypertrophic agent for human coronary artery smooth muscle cells (cSMCs), which suggests that it plays a role in vascular wall thickening. The present study investigated the intracellular signal transduction pathways involved in the growth response of cSMCs to ANG II. The stimulation of protein synthesis by ANG II in cSMCs was blocked by the immunosuppressant rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway that includes the 70-kDa S6 kinase (p70S6k) and plays a key role in cell growth. The inhibitory effect of rapamycin was reversed by a molar excess of FK506; this indicates that both agents act through the common 12-kDa immunophilin FK506-binding protein. ANG II caused a rapid and sustained activation of p70S6k activity that paralleled its phosphorylation, and both processes were blocked by rapamycin. In addition, both of the phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002 abolished the ANG II-induced increase in protein synthesis, and wortmannin also blocked p70S6k phosphorylation. Furthermore, ANG II triggered dissociation of the translation initiation factor, eukaryotic initiation factor-4E, from its regulatory binding protein 4E-BP1, which was also inhibited by rapamycin and wortmannin. In conclusion, we have shown that ANG II activates components of the rapamycin-sensitive mTOR signaling pathway in human cSMCs and involves activation of phosphatidylinositol 3-kinase, p70S6k, and eukaryotic initiation factor-4E, which leads to activation of protein synthesis. These signaling mechanisms may mediate the growth-promoting effect of ANG II in human cSMCs.
Chronic hypoxia induces nonreversible right ventricle dysfunction and dysplasia in rats
Authors: Bonnet Pierre , Bonnet Sébastien , Boissière Julien , Net Jean-Loïc Le , Gautier Mathieu , Dumas de la Roque Eric , Eder Véronique
Published: August 2004

The purpose of this study was to evaluate the reversibility of right ventricular (RV) remodelling after pulmonary artery hypertension (PAHT) secondary to 3 wk of hypobaric hypoxia. A group of 10 adult male Wistar rats were studied and were the following: control normoxic (C), after 3 wk of chronic hypoxia (CH), and after 3 wk of exposure to hypoxia followed by 3 wk of normoxia recovery (N-RE). Mean pulmonary artery pressure was 11 ± 2 mmHg in the C group, 35 ± 2 mmHg in the CH group, and 14 ± 3 mmHg in the N-RE group. RV function was assessed by echocardiography. In the CH group, the pulmonary flow measured in Doppler mode depicted a midsystolic notch and a decrease of the pulmonary acceleration time compared with control [17 ± 1 vs. 34 ± 1 ms ( n = 10), respectively; P < 0.05]. RV thickening measured in M-mode was apparent in the CH group compared with the control group [2.84 ± 0.40 vs. 1.73 ± 0.26 mm ( n = 10), P < 0.05]. In the N-RE group, the RV wall was significantly thinner compared with the CH group [1.56 ± 0.08 vs. 1.73 ± 0.26 mm ( n = 10), P < 0.05]. The calculated RV diameter shortness fraction was not different between the CH group and C group (34 ± 4.2% vs. 36 ± 2.8%) but decreased in the N-RE group [20 ± 2.4% ( n = 10), P < 0.01]. The E-to-A wave ratio on the tricuspid Doppler inflow was significantly lower in the CH group and N-RE group compared with the C group [0.70 ± 0.8 and 0.72 ± 0.1 vs. 0.88 ± 0.2 ( n = 10), respectively; P < 0.05]. In the isolated perfused heart using the Langendorff method, RV compliance was increased in the CH group and decreased in the N-RE group. In the N-RE group, fibrous bands with metaplasia were observed on histological sections of the RV free wall. We conclude that PAHT induces nonreversible RV dysfunction with dysplasia.
Dietary supplementation with 2-deoxy-d-glucose improves cardiovascular and neuroendocrine stress adaptation in rats
Authors: Wan Ruiqian , Camandola Simonetta , Mattson Mark P.
Published: August 2004

Dietary restriction and physical exercise can enhance stress resistance and reduce the risk of cardiovascular disease. We investigated the effects of dietary supplementation with 2-deoxy-d-glucose (2-DG), a glucose analog that limits glucose availability at the cellular level, on cardiovascular and neuroendocrine responses to stress in rats. Young adult male Sprague-Dawley rats were implanted with telemetry probes to monitor blood pressure (BP), heart rate, body temperature, and body movements. These variables were measured at designated times during a 6-mo period in rats fed control and 2-DG-supplemented (0.4% 2-DG, fed ad libitum on a schedule of 2 days on the diet and 1 day off the diet) diets during unperturbed conditions and during and after immobilization stress or cold-water swim stress. Rats fed the 2-DG diet exhibited significant reductions in resting BP, attenuated BP responses during stress, and accelerated recovery to baseline after stress. Plasma concentrations of ACTH and corticosterone were elevated under nonstress conditions in rats fed the 2-DG diet and exhibited differential responses to single (enhanced response) and multiple (reduced response) stress sessions compared with rats fed control rat chow ad libitum. The 2-DG diet improved glucose metabolism, as indicated by decreased concentrations of blood glucose and insulin under nonstress conditions, but glucose and insulin responses to stress were maintained. We conclude that improvements in some cardiovascular risk factors and stress adaptation in rats maintained on a 2-DG-supplemented diet are associated with reduced neuroendocrine responses to the stressors.
Hypoxic induction ofCtgfis directly mediated by Hif-1
Authors: Higgins Debra F. , Biju Mangatt P. , Akai Yasuhiro , Wutz Anton , Johnson Randall S. , Haase Volker H.
Published: August 2004

CTGF plays a significant role in the development of renal fibrosis by mediating the fibrotic effects of transforming growth factor (TGF)-β1and has been shown to be hypoxia inducible in human breast cancer cells. It has been suggested that hypoxia is an important underlying cause for the development of renal fibrosis through the modulation of profibrotic genes. One of the key mediators of the cell's response to lowered oxygen environments is hypoxia-inducible-factor-1 (HIF-1), a basic helix-loop-helix transcription factor, which enables cells to adapt to hypoxia by regulating the expression of genes involved in increasing oxygen availability ( VEGF, erythropoietin) and enhancing glucose uptake and metabolism ( Glut-1, PGK). In this paper, we have used primary tubular epithelial cell cultures from a tetracycline-inducible- Hif- 1α knockout murine model to further elucidate the role of Hif-1 in the hypoxic-induction of Ctgf expression. We show that hypoxia response elements present upstream of Ctgf enable direct interaction of Hif-1 transcription factor with the Ctgf promoter, resulting in increased transcription of Ctgf mRNA. Cells deficient in Hif- 1α were incapable of inducing Ctgf mRNA in response to hypoxia, suggesting an absolute requirement of Hif-1. Furthermore, the observed Hif-1-mediated hypoxic stimulation of Ctgf expression was found to occur independently of TGF-β1signaling. Our findings have important implications for a number of fibrotic disorders in which hypoxia, CTGF, and TGF-β1are involved, including renal, dermal, hepatic, and pulmonary fibrosis.
Role of p53 in cisplatin-induced tubular cell apoptosis: dependence on p53 transcriptional activity
Authors: Jiang Man , Yi Xiaolan , Hsu Stephen , Wang Cong-Yi , Dong Zheng
Published: August 2004

Tubular damage by cisplatin leads to acute renal failure, which limits its use in cancer therapy. In tubular cells, a primary target for cisplatin is presumably the genomic DNA. However, the pathway relaying the signals of DNA damage to tubular cell death is unclear. In response to DNA damage, the tumor suppressor gene p53 is induced and is implicated in subsequent DNA repair and cell death by apoptosis. The current study was designed to examine the role of p53 in cisplatin-induced apoptosis in cultured rat kidney proximal tubular cells. Cisplatin at 20 μM induced apoptosis in ∼70% of cells, which was partially suppressed by carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone (VAD), a general caspase inhibitor. Of interest, cisplatin-induced apoptosis was also suppressed by pifithrin-α, a pharmacological inhibitor of p53. Cisplatin-induced caspase activation was completely inhibited by VAD, but only partially by pifithrin-α. Early during cisplatin treatment, p53 was phosphorylated and upregulated. The p53 activation was blocked by pifithrin-α, but not by VAD. Bcl-2 expression abolished cisplatin-induced apoptosis without blocking p53 phosphorylation or induction. The results suggest that p53 activation might be an early signal for apoptosis during cisplatin treatment. To further determine the role of p53, tubular cells were stably transfected with a dominant-negative mutant of p53 with diminished transcriptional activity. Expression of the mutant attenuated cisplatin-induced apoptosis and caspase activation. In conclusion, the results support an important role for p53 in cisplatin-induced apoptosis in renal tubular cells. p53 May regulate apoptosis through the transcription of apoptotic genes.
Forward and Backward Arm Cycling Are Regulated by Equivalent Neural Mechanisms
Authors: Zehr E. Paul , Hundza Sandra R.
Published: August 2004

It was shown some time ago that cutaneous reflexes were phase-reversed when comparing forward and backward treadmill walking. Activity of central-pattern-generating networks (CPG) regulating neural activity for locomotion was suggested as a mechanism involved in this “program reversal.” We have been investigating the neural control of arm movements and the role for CPG mechanisms in regulating rhythmic arm cycling. The purpose of this study was to evaluate the pattern of muscle activity and reflex modulation when comparing forward and backward arm cycling. During rhythmic arm cycling (forward and backward), cutaneous reflexes were evoked with trains (5 × 1.0 ms pulses at 300 Hz) of electrical stimulation delivered to the superficial radial (SR) nerve at the wrist. Electromyographic (EMG) recordings were made bilaterally from muscles acting at the shoulder, elbow, and wrist. Analysis was conducted on specific sections of the movement cycle after phase-averaging contingent on the timing of stimulation in the movement cycle. EMG patterns for rhythmic arm cycling are similar during both forward and backward motion. Cutaneous reflex amplitudes were similarly modulated at both early and middle latency irrespective of arm cycling direction. That is, at similar phases in the movement cycle, responses of corresponding sign and amplitude were seen regardless of movement direction. The results are generally parallel to the observations seen in leg muscles after stimulation of cutaneous nerves in the foot during forward and backward walking and provide further evidence for CPG activity contributing to neural activation and reflex modulation during rhythmic arm movement.
Modeling of Smooth Pursuit-Related Neuronal Responses in the DLPN and NRTP of the Rhesus Macaque
Authors: Ono Seiji , Das Vallabh E. , Economides John R. , Mustari Michael J.
Published: August 2004

The dorsolateral pontine nucleus (DLPN) and nucleus reticularis tegmenti pontis (NRTP) comprise obligatory links in the cortico-ponto-cerebellar system supporting smooth pursuit eye movements. We examined the response properties of DLPN and rNRTP neurons during step-ramp smooth pursuit of a small target moving across a dark background. Our neurophysiological studies were conducted in awake, behaving juvenile macaques ( Macaca mulatta). We used multiple linear-regression modeling to estimate the relative sensitivities of neurons to eye parameters (position, velocity, and acceleration) and retinal-error parameters (position, velocity, and acceleration). We found that a large proportion of pursuit-related DLPN neurons primarily code eye-velocity information, whereas a large proportion of rNRTP neurons primarily code eye-acceleration information. We calculated the relative decrease in variance found when using a six-component model that included both eye- and retinal-error parameters compared with three-component models that include either eye or retinal error. These comparisons show that a majority of DLPN (14/20) and rNRTP (17/19) neurons have larger contributions from eye compared with retinal-error parameters ( P < 0.001, paired t-test). Even though eye-motion parameters provide the strongest contributions in a given model, a significant contribution from retinal error was often present (i.e., >20% reduction in variance in 6-component model compared with 3-component models). Thus our results indicate that the DLPN plays a larger role in maintaining steady-state smooth pursuit eye velocity, whereas rNRTP contributes to both the initiation and maintenance of smooth pursuit.
Lack of Pharmacokinetic Interactions Between Cilomilast and Theophylline or Smoking in Healthy Volunteers
Authors: Murdoch Robert D. , Zussman Barry , Schofield J. Paul , Webber Dawn M.
Published: August 2004

The pharmacokinetic profile of cilomilast (Ariflo®), a selective phosphodiesterase 4 (PDE4) inhibitor, was investigated in three separate studies. Two of these studies explored the drug interaction potential of cilomilast with the nonselective PDE inhibitor, theophylline, and a third study compared the pharmacokinetic profile of cilomilast in smokers and nonsmokers. Repeated administration of cilomilast had no effect on the steady‐state pharmacokinetics of theophylline in either a pilot dose‐ranging or definitive therapeutic study. At therapeutic doses, the point estimate and 90% confidence interval for theophylline AUC0–12 and Cmax were completely contained within the range (0.8, 1.25). Similarly, repeated administration of theophylline had little clinically relevant effect on the steady‐state pharmacokinetics of cilomilast when compared to placebo, as only slight average increases in cilomilast AUC0–12 and Cmax (6% and 3%, respectively) were observed. In addition, mean cilomilast exposure (AUC0–∞) was found to be similar in both smokers and nonsmokers (8.47±2.20 μg•h/mL and 7.70 ± 2.25 μg•h/mL, respectively). Throughout all three studies, cilomilast was well tolerated, and concomitant use of these selective and nonselective inhibitors, although unlikely in the clinic, is hypothetically feasible. Taken together, these studies clearly differentiate cilomilast from theophylline for drug‐drugliability issues in a smoker and nonsmoker population, as well as highlight the potential to switch from one drug to another without undue clinical concern.
Pharmacokinetics and Pharmacodynamics of Argatroban in Combination With a Platelet Glycoprotein IIB/IIIA Receptor Antagonist in Patients Undergoing Percutaneous Coronary Intervention
Authors: Cox Donna S. , Kleiman Neal S. , Boyle Duane A. , Aluri Jagadeesh , Parchman L. Gerald , Holdbrook Frederick , Fossler Michael J.
Published: August 2004

The pharmacokinetic‐pharmacodynamic (PK‐PD) relationship of argatroban, administered in combination with a platelet glycoprotein IIb/IIIa receptor antagonist, was characterized in patients undergoing percutaneous coronary intervention (PCI). Plasma argatroban and activated clotting times (ACTs) were assessed periprocedurally in 152 patients administered argatroban (250‐ or 300‐μg/kg bolus, then 15‐μg/ kg/min infusion) in combination with abciximab or eptifibatide during PCI. The PK and PK‐PD models were developed utilizing a sequential population approach in NONMEM. Population PK estimates for clearance, central volume, and peripheral volume were 22.0 L/h, 11.0 L, and 13.0 L, respectively (coefficients of variation [CVs] ≤ 10%). By covariate analysis, clearance increased linearly with body weight. Plasma argatroban and ACT effect were well described using a sigmoidal Emax model. For argatroban in combination with platelet glycoprotein IIb/IIIa receptor blockade in patients undergoing PCI, population PK parameters are consistent with values reported for argatroban in healthy subjects. A predictable relationship exists between argatroban concentration and effect in this setting.
Pharmacokinetic and Pharmacodynamic Modeling of Recombinant Human Erythropoietin After Single and Multiple Doses in Healthy Volunteers
Authors: Ramakrishnan Rohini , Cheung Wing K. , Wacholtz Mary C. , Minton Neil , Jusko William J.
Published: August 2004

This study describes a pharmacokinetic (PK) model to account for serum recombinant human erythropoietin (rHuEpo) concentrations in healthy volunteers following intravenous (IV) and subcutaneous (SC) dosing; it also characterizes the pharmacodynamics (PD) of SC rHuEpo effects on reticulocytes, red blood cells (RBC), and hemoglobin (Hb) in blood. Data were obtained from 4 clinical studies carried out in healthy volunteers. Epoetin alfa (rHuEpo) was administered as 5 single IV doses ranging from 10 to 500 IU/kg, as 8 single SC doses ranging from 300 to 2400 IU/kg, and as 2 multiple SC dosage regimens (150 IU/kg/3 times a week [tiw] and 600 IU/kg/wk). A dual‐absorption rate model (fast zero‐order and slow first‐order inputs) with nonlinear disposition characterized the PK of SC rHuEpo. A high Km value was obtained indicating that clearance was mildly nonlinear. Absorption was slow (tmax ∼24 hours), and the bioavailability of SC rHuEpo increased with dose (ranging from 46%‐100%). A catenary cell production and loss model with a feedback down regulation component was used to fit the reticulocyte data yielding estimates of the stimulatory capacity (Smax), sensitivity (SC50), and life span parameters. These parameters were used for simulations of RBC and Hb profiles. An SC50 of 27 to 61 IU/L was estimated indicating that low physiological plasma rHuEpo concentrations were sufficient to produce pharmacological effects. No marked sex‐dependent differences in clinical responses to rHuEpo therapy were found despite baseline differences. Realistic pharmacokinetic and physiological models accounted for clinical responses from a wide array of dosing conditions with rHuEpo. The rationale for greater efficacy of SC administration of rHuEpo compared to IV was ascertained.
Active transport of imatinib into and out of cells: implications for drug resistance
Authors: Thomas Julia , Wang Lihui , Clark Richard E. , Pirmohamed Munir
Published: August 2004

Imatinib is a tyrosine kinase inhibitor that is effective in the treatment of chronic myeloid leukemia (CML). Not all patients achieve cytogenetic response. Some patients even lose the initial cytogenetic response. In this study, we investigated the active cellular transport of imatinib to gain a better understanding of the possible mechanisms of imatinib resistance. We used the leukemic cell line CCRFCEM and its drug-resistant subline VBL100 to measure the uptake of carbon 14 (14C)-labeled imatinib. Imatinib uptake was temperature dependent, indicative of an active uptake process. Additionally, incubations with transport inhibitors showed that verapamil, amantadine, and procainamide, inhibitors of the human organic cation transporter 1 (hOCT1), significantly decreased imatinib uptake into CEM cells, whereas the inhibition of hOCT2 or hOCT3 had no effect, indicating that influx into the cells is an active process likely to be mediated by hOCT1. Studies using transfected MDCK cell lines revealed an active efflux component attributable to MDR1 (ABCB1). Both hOCT1 and MDR1 were expressed in CML primary cells and cell lines. The results indicate that active transport processes mediate the influx and efflux of imatinib. Differential expression of influx (hOCT1) and efflux (MDR1) transporters may be a critical determinant of intracellular drug levels and, hence, resistance to imatinib.
Antithrombotic activity of dermatan sulfate in heparin cofactor II-deficient mice
Authors: Vicente Cristina P. , He Li , Pavão Mauro S. G. , Tollefsen Douglas M.
Published: August 2004

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of dermatan sulfate in wild-type and HCII-deficient mice. Intravenous administration of porcine skin dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII+/+ mice that was not observed in HCII-/- animals. Pharmacokinetic studies suggested that porcine skin dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate dermatan sulfates, which have higher charge densities than mammalian dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII-/- mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged dermatan sulfates possess weak antithrombotic activity independent of HCII. (Blood. 2004;104:3965-3970)
RNAi-mediated silencing of CD40 prevents leukocyte adhesion on CD154-activated endothelial cells
Authors: Pluvinet Raquel , Pétriz Jordi , Torras Joan , Herrero-Fresneda Inmaculada , Cruzado Josep M. , Grinyó Josep M. , Aran Josep M.
Published: August 2004

The CD40-CD154 dyad has a central role in the development of immune-inflammatory processes. Therefore, disruption of CD40 signaling has the potential to be therapeutically useful in a number of disease indications, including autoimmune syndromes, atherosclerosis, and allograft rejection. Blocking antibodies to CD154 have been successfully employed in experimental animal models, and recently in clinical trials, to prevent or treat these immunologically induced diseases. However, the thrombotic events observed in some of these studies raise important issues regarding future use of anti-CD154 antibodies in humans. In this study, we demonstrate that a small interfering RNA (siRNA) can effectively reduce the surface expression of the human CD40 costimulatory receptor. Moreover, by rendering endothelial cells unresponsive to CD154+ Jurkat cell–mediated activation through RNA interference, induction of endothelial cell-adhesion molecule expression and leukocyte adhesion is prevented in vitro. Thus, anti-CD40 siRNA may become a safe and effective therapeutic option for interfering with CD40-CD154–mediated acute or chronic immune-inflammatory conditions.
Interactions between the megakaryocyte/platelet-specific β1 tubulin and the secretory leukocyte protease inhibitor SLPI suggest a role for regulated proteolysis in platelet functions
Authors: Schulze Harald , Korpal Manav , Bergmeier Wolfgang , Italiano Joseph E. , Wahl Sharon M. , Shivdasani Ramesh A.
Published: August 2004

Platelet-restricted β1 tubulin is required for optimal thrombopoiesis and discoid cell shape. To identify interacting factors, we used the divergent β1-tubulin C-terminus as the bait in a yeast 2-hybrid screen of megakaryocyte (MK) cDNAs. We isolated secretory leukocyte protease inhibitor (SLPI), a serine protease antagonist characterized principally as a secreted factor with multiple roles in inflammation. SLPI is expressed in MKs and platelets in 2 discrete compartments. One pool resides in punctate cytoplasmic structures, whereas a significant fraction localizes along peripheral microtubules (MTs) and is lost with cold-induced MT disruption or in β1 tubulin-/- platelets. These findings reveal unexpected interaction between a prominent cytoskeletal protein and an inhibitor of proteolysis. SLPI-/- mice show intact proplatelet formation, platelet numbers and shape, and marginal MT bands; thus, SLPI is not essential for thrombopoiesis. However, SLPI is released upon platelet activation, which also reverses its association with the resting marginal band. Platelet SLPI inhibits neutrophil elastase, an activity that is reduced when β1 tubulin is absent. We conclude that SLPI localizes in part along the MK and platelet MT cytoskeleton by virtue of specific interactions with β1 tubulin. SLPI may thus have unanticipated roles in MK and platelet functions, including regulated proteolysis after activation. (Blood. 2004;104:3949-3957)
The adaptor protein SH2D1A regulates signaling through CD150 (SLAM) in B cells
Authors: Mikhalap Svitlana V. , Shlapatska Larysa M. , Yurchenko Olga V. , Yurchenko Maria Y. , Berdova Ganna G. , Nichols Kim E. , Clark Edward A. , Sidorenko Svetlana P.
Published: August 2004

The CD150 receptor is expressed on activated T and B lymphocytes, dendritic cells, and monocytes. A TxYxxV/I motif in the CD150 cytoplasmic tail can bind different SH2-containing molecules, including tyrosine and inositol phosphatases, Src family kinases, and adaptor molecules. To analyze CD150-initiated signal transduction pathways, we used DT40 B-cell sublines deficient in these molecules. CD150 ligation on DT40 transfectants induced the extracellular signal-regulated kinase (ERK) pathway, which required SH2-containing inositol phosphatase (SHIP) but not SH2 domain protein 1A (SH2D1A). CD150-mediated Akt phosphorylation required Syk and SH2D1A, was negatively regulated by Lyn and Btk, but was SHIP independent. Lyn directly phosphorylated Y327 in CD150, but the Akt pathway did not depend on CD150 tyrosine phosphorylation and CD150-SHP-2 association. Analysis of CD150 and SH2D1A expression in non-Hodgkin and Hodgkin lymphomas revealed stages of B-cell differentiation where these molecules are expressed alone or coexpressed. Signaling studies in Hodgkin disease cell lines showed that CD150 is linked to the ERK and Akt pathways in neoplastic B cells. Our data support the hypothesis that CD150 and SH2D1A are coexpressed during a narrow window of B-cell maturation and SH2D1A may be involved in regulation of B-cell differentiation via switching of CD150-mediated signaling pathways. (Blood. 2004;104:4063-4070)
Results of a prospective randomized clinical trial of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) followed by radiation therapy (RT) versus ABVD alone for stages I, II, and IIIA nonbulky Hodgkin disease
Authors: Straus David J. , Portlock Carol S. , Qin Jing , Myers Jane , Zelenetz Andrew D. , Moskowitz Craig , Noy Ariela , Goy André , Yahalom Joachim
Published: August 2004

To determine whether combined modality therapy (CMT) is superior to chemotherapy (CT) alone, 152 untreated Hodgkin disease patients with clinical stages (CSs) IA, IB, IIA, IIB, and IIIA without bulk disease were prospectively randomized to 6 cycles of doxorubicin, bleomycin, vinblastine, dacarbazine (ABVD) alone or 6 cycles of ABVD followed by radiation therapy (RT) (3600 cGy: involved field for 11 patients, modified extended field for the rest). Of 76 patients randomized to receive RT, 65 actually received it, and 11 did not (4 progressed, 1 had bleomycin toxicity, 6 refused). For ABVD + RT, the complete remission (CR) percentage was 94% and no major response, 6%. For ABVD alone, 94% achieved a CR; 1.5%, a partial response (PR); and 4.5%, no major response. At 60 months CR duration, freedom from progression (FFP), and overall survival (OS) for ABVD + RT versus ABVD alone are 91% versus 87% (P = .61), 86% versus 81% (P = .61), and 97% versus 90% (P = .08), respectively (log-rank). The 95% confidence intervals for CR duration, FFP, and OS differences at 5 years were –8% to 15%, –8% to 18%, and –4% to 12%, respectively. Although significant differences were not seen, it is possible that a benefit in outcome of less than 20% for CMT might be seen in a larger trial.
Platelets synthesize large amounts of active plasminogen activator inhibitor 1
Authors: Brogren Helén , Karlsson Lena , Andersson Maria , Wang Lingwei , Erlinge David , Jern Sverker
Published: August 2004

Previous studies have suggested that plasminogen activator inhibitor 1 (PAI-1) released from platelets convey resistance of platelet-rich blood clots to thrombolysis. However, the majority of PAI-1 in platelets is inactive and therefore its role in clot stabilization is unclear. Because platelets retain mRNA and capacity for synthesis of some proteins, we investigated if platelets can de novo synthesize PAI-1 with an active configuration. PAI-1 mRNA was quantified with real-time polymerase chain reaction and considerable amounts of PAI-1 mRNA were detected in all platelet samples. Over 24 hours, the amount of PAI-1 protein as determined by an enzyme-linked immunosorbent assay increased by 25% (P = .001). Metabolic radiolabeling with 35S-methionine followed by immunoprecipitation confirmed an ongoing PAI-1 synthesis, which could be further stimulated by thrombin and inhibited by puromycin. The activity of the newly formed PAI-1 was investigated by incubating platelets in the presence of tissue-type plasminogen activator (tPA). This functional assay showed that the majority of the new protein was in an active configuration and could complexbind tPA. Thus, there is a continuous production of large amounts of active PAI-1 in platelets, which could be a mechanism by which platelets contribute to stabilization of blood clots. (Blood. 2004;104:3943-3948)
Alternative mapping of probes to genes for Affymetrix chips
Authors: Gautier Laurent , Møller Morten , Friis-Hansen Lennart , Knudsen Steen
Published: August 2004

Background

Short oligonucleotide arrays have several probes measuring the expression level of each target transcript. Therefore the selection of probes is a key component for the quality of measurements. However, once probes have been selected and synthesized on an array, it is still possible to re-evaluate the results using an updated mapping of probes to genes, taking into account the latest biological knowledge available.

Methods

We investigated how probes found on recent commercial microarrays for human genes (Affymetrix HG-U133A) were matching a recent curated collection of human transcripts: the NCBI RefSeq database. We also built mappings and used them in place of the original probe to genes associations provided by the manufacturer of the arrays.

Results

In a large number of cases, 36%, the probes matching a reference sequence were consistent with the grouping of probes by the manufacturer of the chips. For the remaining cases there were discrepancies and we show how that can affect the analysis of data.

Conclusions

While the probes on Affymetrix arrays remain the same for several years, the biological knowledge concerning the genomic sequences evolves rapidly. Using up-to-date knowledge can apparently change the outcome of an analysis.
Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex
Authors: Kluge Christoph , Seidel Thorsten , Bolte Susanne , Sharma Shanti S , Hanitzsch Miriam , Satiat-Jeunemaitre Beatrice , Roß Joachim , Sauer Markus , Golldack Dortje , Dietz Karl-Josef
Published: August 2004

Background

Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed.

Results

To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells.

Conclusions

All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.
Cluster randomised trials in the medical literature: two bibliometric surveys
Authors: Bland J Martin
Published: August 2004

Background

Several reviews of published cluster randomised trials have reported that about half did not take clustering into account in the analysis, which was thus incorrect and potentially misleading. In this paper I ask whether cluster randomised trials are increasing in both number and quality of reporting.

Methods

Computer search for papers on cluster randomised trials since 1980, hand search of trial reports published in selected volumes of the British Medical Journal over 20 years.

Results

There has been a large increase in the numbers of methodological papers and of trial reports using the term 'cluster random' in recent years, with about equal numbers of each type of paper. The British Medical Journal contained more such reports than any other journal. In this journal there was a corresponding increase over time in the number of trials where subjects were randomised in clusters. In 2003 all reports showed awareness of the need to allow for clustering in the analysis. In 1993 and before clustering was ignored in most such trials.

Conclusion

Cluster trials are becoming more frequent and reporting is of higher quality. Perhaps statistician pressure works.
Is drug-induced toxicity a good predictor of response to neo-adjuvant chemotherapy in patients with breast cancer? -A prospective clinical study
Authors: Chintamani  , Singhal Vinay , Singh JP , Lyall Ashima , Saxena Sunita , Bansal Anju
Published: August 2004

Background

Neo-adjuvant chemotherapy is an integral part of multi-modality approach in the management of locally advanced breast cancer and it is vital to predict the response in order to tailor the regime for a patient. The common final pathway in the tumor cell death is believed to be apoptosis or programmed cell death and chemotherapeutic drugs like other DNA-damaging agents act on rapidly multiplying cells including both the tumor and the normal cells by following the same common final pathway. This could account for both the toxic effects and the response. Absence or decreased apoptosis has been found to be associated with chemo resistance. The change in expression of apoptotic markers (Bcl-2 and Bax proteins) brought about by various chemotherapeutic regimens is being used to identify drug resistance in the tumor cells. A prospective clinical study was conducted to assess whether chemotherapy induced toxic effects could serve as reliable predictors of apoptosis or response to neo-adjuvant chemotherapy in patients with locally advanced breast cancer.

Methods

50 cases of locally advanced breast cancer after complete routine and metastatic work up were subjected to trucut biopsy and the tissue evaluated immunohistochemically for apoptotic markers (bcl-2/bax ratio). Three cycles of Neoadjuvant Chemotherapy using FAC regime (5-fluorouracil, adriamycin, cyclophosphamide) were given at three weekly intervals and patients assessed for clinical response as well as toxicity after each cycle. Modified radical mastectomy was performed in all patients three weeks after the last cycle and the specimen were re-evaluated for any change in the bcl-2/bax ratio. The clinical response, immunohistochemical response and the drug-induced toxicity were correlated and compared.

Descriptive studies were performed with SPSS version 10 and the significance of response was assessed using paired t-test. Significance of correlation between various variables was assessed using chi-square test and coefficient of correlation calculated by Pearson correlation coefficient.

Results

There was a statistically significant correlation observed between clinical, immunohistochemical response (bcl-2/bax ratio) and the drug-induced toxicity.

Conclusion

Responders also had significant toxicity while non-responders did not show significant toxicity following neoadjuvant chemotherapy. The chemotherapy-induced toxicity was observed to be a cost effective and reliable predictor of response to neo-adjuvant chemotherapy.
Factors associated with psychotropic drug use among community-dwelling older persons: A review of empirical studies
Authors: Voyer Philippe , Cohen David , Lauzon Sylvie , Collin Johanne
Published: August 2004

Background

In the many descriptive studies on prescribed psychotropic drug use by community-dwelling older persons, several sociodemographic and other factors associated with drug use receive inconsistent support.

Method

Empirical reports with data on at least benzodiazepine or antidepressant drug use in samples of older persons published between 1990 and 2001 (n = 32) were identified from major databases and analyzed to determine which factors are most frequently associated with psychotropic drug use in multivariate analyses. Methodological aspects were also examined.

Results

Most reports used probability samples of users and non-users and employed cross-sectional designs. Among variables considered in 5 or more reports, race, proximity to health centers, medical consultations, sleep complaints, and health perception were virtually always associated to drug use. Gender, mental health, and physical health status were associated in about two-thirds of reports. Associations with age, marital status, medication coverage, socioeconomic status, and social support were usually not observed.

Conclusions

The large variety of methods to operationalize drug use, mental health status, and social support probably affected the magnitude of observed relationships. Employing longitudinal designs and distinguishing short-term from long-term use, focusing on samples of drug users exclusively, defining drug use and drug classes more uniformly, and utilizing measures of psychological well-being rather than only of distress, might clarify the nature of observed associations and the direction of causality. Few studies tested specific hypotheses. Most studies focused on individual characteristics of respondents, neglecting the potential contribution of health care professionals to the phenomenon of psychotropic drug use among seniors.