bluuebunny/crossref_metadata_2025 huggingface dataset:
The fa leptin receptor mutation and the heritability of respiratory frequency in a Brown Norway and Zucker intercross
Authors: Iyengar Sudha K. , Stein Catherine M. , Russo Karlie , Erokwu Bernadette O. , Strohl Kingman P.
Published: March 2004

We sought to determine whether the fa (leptin receptor) mutation was a major determinant of the putative obesity effects on respiratory frequency in an intercross between the Brown Norway (low breathing frequency, nonobese strain) and the Zucker (moderately high breathing frequency, with the fa mutation) strains. The hypothesis was that rats bearing one (heterozygote) or two (homozygote) alleles of the Glu296Pro point mutation ( fa) would have a uniformly high respiratory frequency in the second filial (F2) generation, compared with wild-type animals. In addition to breathing frequency, tidal volume and minute ventilation were assessed during baseline, acute hypoxic (10% O2-0% CO2-balance nitrogen), hypercapnic (93% O2-7% CO2), hyperoxic (100% O2-0% CO2), and combined (10% O2-3% CO2-balance nitrogen) challenges in fa homozygote ( fa/fa; n = 24), fa heterozygote ( fa/wt; n = 33), and wild-type (wt/wt; n = 19) animals. Phenotypes were adjusted with stepwise regression analyses for the effects of age, sex, length, and litter size. Broad-sense heritability was estimated by examining the variance of the traits in first filial and F2 generations. ANOVAs were used to determine the mode of inheritance of the fa allele in the F2 generation. As anticipated, weight demonstrated the greatest overall broad-sense heritability (77%) and was the result of the recessive mutation. Breathing parameters during the hypoxic, hypercapnic, and combined challenges demonstrated a wide range of heritability from 5 to 96%, with a very nonuniform proportion of heritability explained by the leptin receptor. At best, for frequency 4.5 min into the hypercapnic hypoxic challenge, ∼20% of the total heritability (∼67%) could be attributed to an effect of the leptin receptor mutation. We conclude that, unlike its major effect on weight, the effect of the fa allele is not a major gene involved in the regulation of breathing frequency.
Chronic intermittent hypoxia and incremental cycling exercise independently depress muscle in vitro maximal Na+-K+-ATPase activity in well-trained athletes
Authors: Aughey R. J. , Gore C. J. , Hahn A. G. , Garnham A. P. , Clark S. A. , Petersen A. C. , Roberts A. D. , McKenna M. J.
Published: March 2004

Athletes commonly attempt to enhance performance by training in normoxia but sleeping in hypoxia [live high and train low (LHTL)]. However, chronic hypoxia reduces muscle Na+-K+-ATPase content, whereas fatiguing contractions reduce Na+-K+-ATPase activity, which each may impair performance. We examined whether LHTL and intense exercise would decrease muscle Na+-K+-ATPase activity and whether these effects would be additive and sufficient to impair performance or plasma K+regulation. Thirteen subjects were randomly assigned to two fitness-matched groups, LHTL ( n = 6) or control (Con, n = 7). LHTL slept at simulated moderate altitude (3,000 m, inspired O2fraction = 15.48%) for 23 nights and lived and trained by day under normoxic conditions in Canberra (altitude ∼600 m). Con lived, trained, and slept in normoxia. A standardized incremental exercise test was conducted before and after LHTL. A vastus lateralis muscle biopsy was taken at rest and after exercise, before and after LHTL or Con, and analyzed for maximal Na+-K+-ATPase activity [K+-stimulated 3- O-methylfluorescein phosphatase (3- O-MFPase)] and Na+-K+-ATPase content ([3H]ouabain binding sites). 3- O-MFPase activity was decreased by −2.9 ± 2.6% in LHTL ( P < 0.05) and was depressed immediately after exercise ( P < 0.05) similarly in Con and LHTL (−13.0 ± 3.2 and −11.8 ± 1.5%, respectively). Plasma K+concentration during exercise was unchanged by LHTL; [3H]ouabain binding was unchanged with LHTL or exercise. Peak oxygen consumption was reduced in LHTL ( P < 0.05) but not in Con, whereas exercise work was unchanged in either group. Thus LHTL had a minor effect on, and incremental exercise reduced, Na+-K+-ATPase activity. However, the small LHTL-induced depression of 3- O-MFPase activity was insufficient to adversely affect either K+regulation or total work performed.
Activity of Rostral Superior Colliculus Neurons During Passive and Active Viewing of Motion
Authors: Krauzlis Richard J.
Published: March 2004

The superior colliculus (SC) has long been known to be important for the control of saccades, and recent findings indicate that the rostral SC (rSC) plays some role in pursuit as well. The recent finding that the prelude activity of some SC neurons exhibits directional selectivity suggests that the rSC might process visual motion signals relevant for the control of pursuit. We have now tested the activity of buildup neurons in the rSC during the passive viewing of motion stimuli placed within their response field and also during the previewing of visual motion stimuli that were subsequently tracked with pursuit eye movements. We found that rSC buildup neurons typically responded well to motion stimuli, but that they exhibited essentially no selectivity for the direction or speed of visual motion, and that they also responded well to stationary flickering dots. However, during the previewing of visual motion prior to the onset of pursuit, many neurons did exhibit a buildup of activity similar to that exhibited before saccades. These results are inconsistent with the notion that the rSC mediates visual motion signals used to drive pursuit, but instead support the idea that visual motion signals can be used by rSC neurons as part of a mechanism for selecting targets for pursuit and saccades.
Linkage of myostatin pathway genes with knee strength in humans
Authors: Huygens W. , Thomis M. A. , Peeters M. W. , Aerssens J. , Janssen R. , Vlietinck R. F. , Beunen G.
Published: March 2004

This study was the first to explore the potential role of the myostatin ( GDF8) pathway in relation to muscle strength and estimated muscle cross-sectional area in humans using linkage analysis with a candidate gene approach. In young male sibs ( n = 329) 11 polymorphic markers in or near 10 candidate genes from the myostatin pathway were genotyped. Muscle mass was estimated by anthropometric measurements, and maximal knee strength was evaluated using isokinetic dynamometers (Cybex NORM). Single-point nonparametric variance components and linear quantitative trait locus regression linkage analysis methods were used. Linkage patterns were observed between knee extension and flexion peak torque with markers D2S118 ( GDF8), D6S1051 ( CDKN1A), and D11S4138 ( MYOD1), and a maximum LOD score of 2.63 ( P = 0.0002) was observed with D2S118. The ratios of peak torque over muscle and bone area of the midthigh of the lower contraction velocity (60°/s) showed more frequently significant LOD scores than the torques at high velocity (240°/s). Although myostatin is physiologically more related to muscle mass through possible effects of hyperplasia and hypertrophy than it is to strength, only two estimated muscle cross-sectional areas were marginally linked (LOD 1.06 and 1.07, P = 0.01) with marker D2S118 near GDF8 (2q32.2). The present results gave suggestive evidence that the myostatin pathway might be important for strength phenotypes, and GDF8, CDKN1A, and MYOD1 are potential candidate regions for a further and denser mapping with respect to these phenotypes.
Large Artery Stiffness Is Not Related to Plasma Cholesterol in Older Subjects with Hypertension
Authors: Dart Anthony M. , Gatzka Christoph D. , Cameron James D. , Kingwell Bronwyn A. , Liang Yu-Lu , Berry Karen L. , Reid Christopher M. , Jennings Garry L.
Published: March 2004

Objective—

Previous studies have demonstrated a prognostic role of large artery stiffness in hypertensive subjects and increased stiffness in subjects with coronary artery disease. Although plasma cholesterol is an established risk factor for cardiovascular disease, its relationship with large artery properties in a hypertensive population is unclear.

Methods and Results—

Plasma cholesterol and large artery properties were measured at baseline in a subset of participants of a randomized controlled trial (ANBP2) evaluating hypertension treatment in older (65 to 84 years) subjects. Noninvasive measures of large artery behavior were central augmentation index (AI), systemic arterial compliance (SAC), and transverse expansion of the aortic arch (aortic distensibility). Arterial waveforms acceptable for analysis were obtained in ≈80% of cases yielding valid measurements of AI in 868, SAC in 846, and aortic distensibility in 680 subjects. Mean total and high-density lipoprotein (HDL) concentrations were 5.5±1.0 and 1.4±0.5 mmol L
−1
. Total and HDL cholesterol and AI were greater in females than males, whereas SAC and aortic distensibility were greater in males. In multiple regression analyses there were no significant associations between stiffness parameters and total or HDL cholesterol. Significant independent associations in such analyses were found for mean arterial blood pressure, gender, age, height, and heart rate, in keeping with previous findings.

Conclusions—

In the largest cohort of elderly hypertensive subjects studied to date, plasma cholesterol per se was not associated with large artery stiffness. Such independence from cholesterol increases the potential for artery stiffness measurements to additionally contribute to cardiovascular risk assessment in this population.
Prevention of Coronary Hyperreactivity in Preatherogenic Menopausal Rhesus Monkeys by Transdermal Progesterone
Authors: Hermsmeyer R. Kent , Mishra Rajesh G. , Pavcnik Dusan , Uchida Barry , Axthelm Michael K. , Stanczyk Frank Z. , Burry Kenneth A. , Illingworth D. Roger , Kaski Juan Carlos , Nordt Frank J.
Published: March 2004

Objective—

To test if transdermal progesterone (P) confers coronary vascular protection in surgically menopausal preatherosclerotic rhesus monkeys.

Methods and Results—

Ovariectomized rhesus monkeys fed an atherogenic diet (AD) for 19 months were treated with an investigational transdermal P cream (n=7) or identical placebo cream (n=5) for 4 weeks. Aorta and carotids showed fatty streaks and Oil Red O staining demonstrated lipid deposition. Serum P levels in P-treated rhesus monkeys (0.6 ng/mL) were significantly greater than placebo (0.2 ng/mL). Significant elevation of cholesterol, LDL cholesterol, and HDL cholesterol, was noted in all animals. Lp(a) was significantly attenuated in the AD-fed P-treated monkeys. Coronary angiographic experiments stimulating vasoconstriction by intracoronary injections of serotonin plus U46619 showed exaggerated prolonged actions amplified by AD, but significant protection against severe prolonged vasoconstriction in P-treated monkeys. Immunocytochemistry confirmed co-expression of P and thromboxane prostanoid (TP) receptors in coronaries and aorta. Western blotting demonstrated TP receptor attenuation in vascular muscle after P treatment.

Conclusions—

Coronary hyperreactivity, a putative component of coronary artery disease mediated via increased vascular muscle thromboxane prostanoid receptors, can be prevented by subphysiological levels of P, not only in nonatherosclerotic (previously shown) but also in preatherosclerotic primates.
Vascular Endothelial Zinc Finger 1 Is Involved in the Regulation of Angiogenesis: Possible Contribution of Stathmin/OP18 as a Downstream Target Gene
Authors: Miyashita Hiroki , Kanemura Masanori , Yamazaki Tohru , Abe Mayumi , Sato Yasufumi
Published: March 2004

Objective—

Vascular endothelial zinc finger 1 (Vezf1) is a recently identified zinc finger transcription factor that is expressed in endothelial cells (ECs) during vascular development in mouse embryo. Here, we present that Vezf1 was expressed in ECs at the site of postnatal angiogenesis. We therefore examined whether Vezf1 was involved in the regulation of angiogenesis.

Methods and Results—

The specific downregulation of Vezf1 by antisense oligodeoxynucleotide (AS-ODN) significantly inhibited the proliferation, migration, and network formation of cultured ECs as well as angiogenesis in vivo. Vezf1 AS-ODN downregulated the expression of stathmin/oncoprotein18 (OP18), a microtubule-destabilizing protein, in ECs, whereas transient transfection of Vezf1 cDNA increased the expression of stathmin/OP18 in ECs. To explore the relationship between Vezf1 and stathmin/OP18, we specifically downregulated stathmin/OP18. We found that stathmin/OP18 AS-ODN inhibited the proliferation, migration, and network formation of ECs as Vezf1 AS-ODN did. Moreover, Vezf1 AS-ODN decreased G2/M population of ECs and increased apoptosis, which reproduced the characteristic feature of stathmin/OP18 inhibition.

Conclusion—

These results suggest that Vezf1 is involved in the regulation of angiogenesis, at least in part, through the expression of stathmin/OP18 in ECs.
Myocyte Nitric Oxide Synthase 2 Contributes to Blunted β-Adrenergic Response in Failing Human Hearts by Decreasing Ca
2+
Transients
Authors: Ziolo Mark T. , Maier Lars S. , Piacentino Valentino , Bossuyt Julie , Houser Steven R. , Bers Donald M.
Published: March 2004

Background—

Human heart failure (HF) usually exhibits blunted response to β-adrenergic receptor (AR) stimulation. Here, we examined whether expression of nitric oxide synthase-2 (NOS2, or inducible NOS) contributes to this loss of inotropic reserve in human HF.

Methods and Results—

Failing human hearts were obtained at transplantation. Contraction and [Ca
2+
]
i
measurements were performed in isolated cardiac myocytes and trabeculae. In HF myocytes and muscle, isoproterenol (ISO), a β-AR agonist, led to small inotropic and lusitropic responses. Specific inhibition of NOS2 by aminoguanidine (AG) or L-NIL dramatically increased the ISO-induced inotropy and lusitropy, such that the ISO+AG response in HF approached that seen with ISO alone in nonfailing human myocytes or muscles. Ca
2+
transient data directly paralleled these results, indicating that altered cellular Ca
2+
handling is responsible. In nonfailing human hearts, NOS2 inhibition had no effects. In addition, NOS2 inhibition also had no effect in 30% of failing hearts, but in these myocytes and muscles, the ISO response alone was similar to that of nonfailing hearts. In line with these functional findings, NOS2 protein expression measured by Western blotting was induced in HF when AG/L-NIL had a functional effect but not when AG/L-NIL had no effect on contractility and Ca
2+
transients.

Conclusions—

NOS2 expression strongly limited ISO-induced increases in contraction, twitch Δ[Ca
2+
]
i
, and lusitropy in trabeculae and isolated myocytes from failing human hearts. Thus, the β-AR hyporesponsiveness in human HF is mediated in large part by NO (or related congeners) produced within cardiac myocytes via NOS2.
Early Predictors of In-Hospital Death in Infective Endocarditis
Authors: Chu Vivian H. , Cabell Christopher H. , Benjamin Daniel K. , Kuniholm Erin F. , Fowler Vance G. , Engemann John , Sexton Daniel J. , Corey G. Ralph , Wang Andrew
Published: March 2004

Background—

Data on early determinants of outcome in infective endocarditis (IE) are limited. We evaluated the prognostic significance of early clinical characteristics in a large, prospective cohort of patients with IE.

Methods and Results—

Two hundred sixty-seven consecutive patients with definite or possible IE by modified Duke criteria and echocardiography performed within 7 days of presentation were evaluated. Acute physiology was assessed by the Acute Physiology, Age, Chronic Health Evaluation II (APACHE II) score at the time of presentation, and early heart failure was diagnosed by Framingham criteria. In-hospital mortality rate in the cohort was 19% and similar for patients with definite or possible IE (20% versus 16%, respectively;
P
=0.464). Independent predictors of death determined by logistic regression modeling were diabetes mellitus (OR 2.48; 95% CI, 1.24 to 4.96),
Staphylococcus aureus
as causative organism (OR, 2.06; 95% CI, 1.01 to 4.20), APACHE II score (OR, 1.07; 95% CI, 1.01 to 1.12), and embolic event (OR, 2.79; 95% CI, 1.15 to 6.80). Early echocardiographic findings of the Duke criteria were not predictive of death.

Conclusions—

Early in the course of IE, readily available clinical characteristics that reflect the host-pathogen interaction are predictive of in-hospital death. These factors may identify those patients with IE for more aggressive treatment.
Glucose-Cholesterol Interaction Magnifies Coronary Heart Disease Risk for Hypertensive Patients
Authors: Cohen Hillel W. , Hailpern Susan M. , Alderman Michael H.
Published: March 2004

Elevated cholesterol and glucose are known independent risk factors for coronary heart disease. This study examines whether an adverse synergistic interaction of cholesterol and glucose magnifies coronary heart disease risk among treated hypertensive patients. Subjects were hypertensive patients (n=6672) in a worksite treatment program, with entry fasting glucose <6.99 mmol/L (126 mg/dL) and total cholesterol <6.72 mmol/L (260 mg/dL) observed for mean 5.6±4.5 years follow-up (range 0.5 to 21.7 years). Outcome events were incident hospitalization or death due to coronary heart disease. Cox proportional hazard models were constructed for the whole sample to assess interaction and then stratified by fasting glucose categories with thresholds defined either at impaired fasting glucose (≥6.11 mmol/L [110 mg/dL]) or upper quartile (≥5.72 mmol/L [103 mg/dL]). An interaction product term of total cholesterol and fasting glucose as continuous variables significantly (
P
=0.009) improved a Cox proportional hazards model, adjusting for total cholesterol, fasting glucose, and other coronary heart disease risk factors. Adjusted hazard ratios for 3 upper total cholesterol categories (with total cholesterol <5.17mmol/L [200 mg/dL] as reference) in the higher fasting glucose stratum were more than double the corresponding hazard ratios in the lower stratum, whether using impaired fasting glucose or upper quartile fasting glucose as the cut point. These results suggest that an adverse synergistic interaction between glucose and cholesterol magnifies coronary heart disease risk associated with total cholesterol among hypertensive patients, raising the possibility that coronary heart disease prevention might be enhanced if cholesterol intervention criteria were modified by glucose status.
G Protein-Coupled Receptor Internalization Signaling Is Required for Cardioprotection in Ischemic Preconditioning
Authors: Tong Haiyan , Rockman Howard A. , Koch Walter J. , Steenbergen Charles , Murphy Elizabeth
Published: March 2004

The present study is designed to explore the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC). We used TG mice with cardiac-specific overexpression of a Gβγ-sequestering peptide, βARKct (TG βARKct mice), to test whether the protection of PC is Gβγ-dependent. To test the role of G
i
protein, we used wild-type mice pretreated with the G
i
inhibitor pertussis toxin. Recovery of left ventricular developed pressure and infarct size were measured as indices of protection. PC induced protection in wild-type mice, but this protection was blocked by pertussis toxin treatment and was also blocked in TG βARKct mice. To determine the mechanism of Gβγ-induced protection in PC, we investigated one of the downstream targets of Gβγ, the PI3K/p70S6K pathway. PC-induced phosphorylation of p70S6K was not blocked in TG βARKct hearts; therefore, we investigated other targets of Gβγ. Recent studies suggest a role for Gβγ in GPCR internalization. We found that βARKct, a specific PI3K inhibitor wortmannin, and bafilomycin A
1
, which all block receptor recycling, all blocked the protective effect of PC. To additionally test whether PI3K is involved in PC-activated receptor internalization and endosomal signaling, we used TG mice with cardiac-specific overexpression of a catalytically inactive mutant PI3Kγ, which disrupts the recruitment of functional PI3K to agonist-activated GPCRs in vivo. We found that the catalytically inactive mutant of PI3Kγ blocks the protection of PC. In summary, these data suggest the novel finding that the cardioprotective effect of PC requires receptor internalization.
Antiremodeling Effects of Iloprost and the Dual-Selective Phosphodiesterase 3/4 Inhibitor Tolafentrine in Chronic Experimental Pulmonary Hypertension
Authors: Schermuly Ralph Theo , Kreisselmeier Klaus Peter , Ghofrani Hossein Ardeschir , Samidurai Arun , Pullamsetti Soni , Weissmann Norbert , Schudt Christian , Ermert Leander , Seeger Werner , Grimminger Friedrich
Published: March 2004

Severe pulmonary hypertension is a disabling disease with high mortality. We investigated acute and chronic effects of iloprost, a long-acting prostacyclin analogue, and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in monocrotaline-induced pulmonary hypertension in rats. Twenty-eight and 42 days after administration of the alkaloid, right ventricular systolic pressure increased from 25.8±2.0 to 62.9±3.4 and 70.5±7.4 mm Hg, with concomitant decline in cardiac index, central venous oxygen saturation, and arterial oxygenation. Marked right heart hypertrophy was demonstrated by the strongly elevated ratio of right ventricle/left ventricle plus septum weight, and massive thickening of the precapillary artery smooth muscle layer was shown histologically. Western blot analysis demonstrated increased levels of matrix metalloproteinases (MMPs) -2 and -9 and increased gelatinolytic activities in isolated pulmonary arteries. In these animals, both intravenous iloprost and tolafentrine displayed characteristic features of pulmonary vasodilators. When chronically infused from days 14 to 28, both agents significantly attenuated all monocrotaline-induced hemodynamic and gas exchange abnormalities as well as right heart hypertrophy. Full normalization of all variables including right ventricle size was achieved on combined administration of both agents during this period. This was also true for MMP-2 and MMP-9 expression and activity. Moreover, when iloprost plus tolafentrine was used for late therapeutic intervention, with infusion from days 28 to 42 after full establishment of severe pulmonary hypertension and cor pulmonale, hemodynamic, gas exchange, and cardiac and pulmonary vascular remodeling changes were significantly reversed. We conclude that the combined administration of iloprost and a dual-selective phosphodiesterase 3/4 inhibitor prevents and reverses the development of pulmonary hypertension and cor pulmonale in response to monocrotaline in rats. This regimen may therefore offer a possible antiremodeling therapy in severe pulmonary hypertension.
Historical Analogies and the Use of Force
Authors: Garofano John
Published: March 2004

Using a recent book by Jeffrey Record as a point of departure, this essay considers the role of historical analogies in decisions by U.S. leaders to use force during the Cold War. The analogies considered by Record those of Munich and Vietnam may have had a bearing on some decisions, but it is often difficult to assess their relative weight compared to other critical variables. Moreover, several analogies not considered by Record Pearl Harbor, for example may have been far more salient during certain crises than the analogies he examines. In any case, we need a more systematic analysis of historical analogies than Record provides if we are to gauge the real influence and impact of historical analogies on the Cold War.
p73, the “Assistant” Guardian of the Genome?
Authors: MELINO GERRY
Published: March 2004

Abstract: Although p53 is clearly involved in the salvage pathway to DNA damage, its frequent mutations do not explain the efficacy of radiotherapy and chemotherapy. Indeed, around 50% of all human cancers show mutations in p53, and a further fraction show a functional inactivation of the protein. Nevertheless, patients seem to respond to therapy that would otherwise require a functional p53. At least in part, these responses could be explained by the pathway mediated by p73. This mechanism is parallel to, but independent of the p53 pathway. Several pieces of evidence show a significant interaction between these two proteins. Therefore, while p53 can be rightly defined as the guardian of the genome, we could think of p73 as the “assistant” guardian of the genome!
Signal Transduction of Cerulein‐Induced Cytokine Expression and Apoptosis in Pancreatic Acinar Cells
Authors: LEE JANGWON , SEO JEONGHUN , KIM HYEYOUNG , CHUNG JAE BOCK , KIM KYUNG HWAN
Published: March 2004

Abstract: The signaling pathways mediating cytokine production and apoptosis in pancreatic acinar cells have not been fully understood. We investigated the signal transduction of cerulein‐induced cytokine IL‐6 expression and apoptosis in pancreatic acinar AR42J cells. The wild‐type cells and the cells transfected with mutant genes for signaling molecules (such as transcription factor NF‐κB and AP‐1) and ras, the upstream signal for mitogen‐activated protein kinase (MAPK), were used. IL‐6 level was determined in the medium by enzyme‐linked immunosorbent assay (ELISA). Apoptosis was determined by viable cell counts and DNA fragmentation. Wild‐type cells and transfected cells with control vector (pcDNA), IκB mutant gene (IκB mt), H‐ras mutant gene (Ras N17), or c‐jun dominant negative gene (TAM‐67) were treated with cerulein for 24 hours. As a result, cerulein induced IL‐6 expression time‐dependently at 10‐8 M, and apoptosis dose‐dependently at 24 h in the wild‐type cells. Cerulein‐induced IL‐6 expression (at 10‐8 M) and apoptosis (at 10‐7 M) were inhibited in the transfected cells with mutant gene (IκB mt, Ras N17, TAM‐67) as compared to pcDNA cells and the wild‐type cells. In conclusion, cerulein induces cytokine expression and apoptotic cell death, which may be regulated by NF‐κB, AP‐1, and possibly MAPK in pancreatic acinar cells.
Involvement of TNF‐Related Apoptosis‐Inducing Ligand (TRAIL) Induction in Interferon γ‐Mediated Apoptosis in Ewing Tumor Cells
Authors: ABADIE ANNIE , WIETZERBIN JUANA
Published: March 2004

Abstract: We investigated the effect of IFN γ or IFN γ/IFN α and IFN γ/TNF treatment on the apoptosis of Ewing tumor cells (SK‐N‐MC). The results show that treatment of cells with IFN γ resulted in 17% of cell death while no effect was observed when cells were treated with IFN α or TNF alone. Percentage cell death increased markedly in IFN γ/IFN α‐ and IFN γ/TNF‐treated cells (42% and 67%, respectively) as compared to IFN γ‐treated cells. These results suggest that IFN α and TNF amplified the apoptotic signal(s) induced by IFN γ. It was shown recently that Ewing cells undergo apoptosis upon treatment with recombinant TRAIL (TNF‐related apoptosis inducing ligand), a member of the TNF family. Thus, since cytokines were reported to be able to induce TRAIL in other cell systems, we were prompted to investigate whether TRAIL induction was involved in the mechanism responsible for IFN γ‐mediated cell death in Ewing cells. The results reported here are consistent with the notion that cell‐associated and secreted TRAIL contribute in an autocrine or paracrine manner to the triggering of Ewing cell apoptosis induced by IFN γ alone or combined with IFN α or TNF. The observations reported here might contribute to the development of alternative new approaches to the treatment of Ewing tumors resistant to conventional therapy.
Antagonism between Apoptotic (Bax/Bcl‐2) and Anti‐Apoptotic (IAP) Signals in Human Osteoblastic Cells under Vector‐Averaged Gravity Condition
Authors: NAKAMURA HIROSHI , KUMEI YASUHIRO , MORITA SADAO , SHIMOKAWA HITOYATA , OHYA KEIICHI , SHINOMIYA KENICHI
Published: March 2004

Abstract: A functional disorder associated with weightlessness is well documented in osteoblasts. The apototic features of this disorder are poorly understood. Harmful stress induces apoptosis in cells via mitochondria and/or Fas. The Bax triggers cytochrome c release from mitochondria, which can be blocked by the Bcl‐2. Released cytochrome c then activates the initiator caspase, caspase‐9, which can be blocked by the anti‐apototic (IAP) family of molecules. The effector caspase, caspase‐3, finally exerts DNA fragmentation. We conducted this study to examine the apoptotic effects of vector‐averaged gravity on normal human osteoblastic cells. Cell culture flasks were incubated on the clinostat, which generated vector‐averaged gravity condition (simulated microgravity) for 12, 24, 48, and 96 hours. Upon termination of clinostat cultures, the cell number and cell viability were assessed. DNA fragmentation was analyzed on the agarose‐gel electrophoresis. The mRNA levels for Bax, Bcl‐2, XIAP, and caspase‐3 genes were analyzed by semi‐quantitative RT‐PCR. Twenty‐four hours after starting clinostat rotation, the ratios of Bax/Bcl‐2 mRNA levels (indicator of apoptosis) were significantly increased to 136% of the 1G static controls. However, the XIAP mRNA levels (anti‐apoptotic molecule) were increased concomitantly to 138% of the 1G static controls. Thus, cell proliferation or cell viability was not affected by vector‐averaged gravity. DNA fragmentation was not observed in clinostat group as well as in control group. Finally, the caspase‐3 mRNA levels were not affected by vector‐averaged gravity. Simulated microgravity might modulate some apoptotic signals upstream the mitochondrial pathway.
Apoptotic Mitochondrial Dysfunction Induced by Benzo(a)pyrene in Liver Epithelial Cells
Authors: HUC LAURENCE , GILOT DAVID , GARDYN CLAIRE , RISSEL MARY , DIMANCHE‐BOITREL MARIE‐THÉRÈSE , GUILLOUZO ANDRÉ , FARDEL OLIVIER , LAGADIC‐GOSSMANN DOMINIQUE
Published: March 2004

Abstract: How pHi changes, more specifically alkalinization, affect the apoptotic cascade has yet to be determined. The aim of the present work was to test the involvement of mitochondria in the apoptotic cascade triggered by benzo(a)pyrene [B(a)P] and to determine the role of pHi changes and p53 relative to mitochondria. Our results indicate that B(a)P‐induced apoptosis might rely upon a p53‐dependent and a pH‐sensitive mitochondrial dysfunction.
Early Activation and Induction of Apoptosis in T Cells Is Independent of c‐Fos
Authors: BIERBAUM HANNA , BAUMANN SVEN , HERR INGRID , TUCKERMANN JAN P. , HESS JOCHEN , SCHORPP‐KISTNER MARINA , ANGEL PETER
Published: March 2004

Abstract: We used c‐Fos‐deficient activated T cells from the spleen and c‐Fos‐deficient thymocytes to address the capacity of these cells to undergo apoptosis in response to various stimuli. To determine the role of c‐Fos in apoptosis regulation in thymocytes, we challenged thymocytes from wild‐type and c‐Fos‐deficient mice with either TPA or the glucocorticoid dexamethasone. After various time points cells were stained according to the Nicoletti method and analyzed by FACS. Thymocytes from both genotypes exhibited similar efficiency of apoptosis in response to treatment with TPA or dexamethasone. Our data provide clear evidence that c‐Fos is not required for apoptosis regulation in activated T cells as well as in thymocytes.
NF‐κB Inhibition Restores Sensitivity to Fas‐Mediated Apoptosis in Lymphoma Cell Lines
Authors: MELI MARIA , D'ALESSANDRO NATALE , TOLOMEO MANLIO , RAUSA LUCIANO , NOTARBARTOLO MONICA , DUSONCHET LUISA
Published: March 2004

Abstract: Failure to perform the Fas‐related apoptosis pathway can account for tumor resistance both to chemotherapeutic agents and to immunological effectors. We studied the role of NK‐κB in Fas‐resistance, employing the Fas‐sensitive human T‐lymphoma HuT78 cell line and its Fas‐resistant variants HuT78B1 and HuT78G9. All these cell lines expressed high levels of constitutively activated NF‐κB. Pretreatment of cells with NF‐κB inhibitors (PDTC, MG132, or SN50) strongly enhanced CH11‐induced apoptosis in HuT78 and Hut78G9 cells, while only MG132 showed a similar potentiating effect in HuT78B1. The described synergism was significantly inhibited by pretreatment with the anti‐Fas‐blocking antibody ZB4 or with the pancapsase inhibitor Z‐VAD‐FMK, but not by capsase‐8 or ‐9 inhibitors. Overall, these data suggest that NF‐κB inhibition may restore the Fas‐pathway in Fas‐resistant NF‐κB‐overexpressing tumors.
Role of NF‐κB and DNA Repair Protein Ku on Apoptosis in Pancreatic Acinar Cells
Authors: SONG JI YEON , LIM JOO WEON , KIM HYEYOUNG , KIM KYUNG HWAN
Published: March 2004

Abstract: Reactive oxygen species have been known to cause DNA damage and induce apoptosis. During DNA damage, DNA repair proteins Ku70 and Ku80 prevent cell death, but severe DNA damage beyond the repair capacity of the DNA repair proteins triggers necrosis or apoptosis. Recent reports have shown that NF‐κB plays a critical role in protecting the cells from apoptosis. We investigated whether glucose oxidase acting on β‐D‐glucose (G/GO), which continuously produces H2O2, induces apoptosis, and whether NF‐κB and Ku are involved in G/GO‐induced apoptosis in pancreatic acinar AR42J cells. Electron microscopic observation showed that apoptotic cells with characteristic nuclear condensation and shrinkage as well as large vacuoles were detected after G/GO treatment. G/GO treatment induced apoptotic cell death, as determined by viable cell count and DNA fragmentation. G/GO‐induced apoptosis was increased in the cells transfected with the Ku‐dominant negative mutant (Ku D/N) and mutated IκBα gene (IκB mt) as compared to the wild‐type cells (Wild) and the cells transfected with the control pcDNA3 vector (pcN‐3). G/GO treatment caused nuclear loss of both Ku70 and Ku80 in Wild cells and pcN‐3 cells. Even without G/GO treatment, nuclear loss of Ku proteins was observed in IκB mt cells. These results suggest that oxidative stress‐induced reduction of nuclear Ku proteins may cause loss of defense against DNA damage and thus induce apoptosis in pancreatic acinar cells. The novel finding is that nuclear translocation of Ku proteins may be mediated by NF‐κB.
The Selective Estrogen Receptor Modulator 4‐Hydroxy Tamoxifen Induces G1 Arrest and Apoptosis of Multiple Myeloma Cell Lines
Authors: GAUDUCHON JULIETTE , GOUILLEUX FABRICE , MAILLARD SÉBASTIEN , MARSAUD VÉRONIQUE , RENOIR MICHEL J. , SOLA BRIGITTE
Published: March 2004

Abstract: Multiple myeloma (MM) is an incurable hematological malignancy for which new therapeutic strategies should be envisaged. The selective estrogen receptor modulator (SERM), 4‐hydroxy tamoxifen (4‐OHTam), in the range of 1 to 10 μM, was able to impair the cell proliferation of MM cell lines. This was achieved by blocking cells at the G1 phase of the cell cycle and by inducing apoptosis. This cellular response was observed in five out of six tested cell lines, all five expressing both α and β estrogen receptor forms. No modifications of Bcl‐2, Bcl‐X, and Bax levels were observed, as well as no changes in Pi3K/Akt and JAK/STAT pathways that are often constitutively active in these cells. The signalization of 4‐OHTam‐induced cell death needs further investigation.
Gene Expression Induced by X‐ray Irradiation in Human Blood Cell Lines
Authors: MORI M , BENOTMANE M A. , VERHEYDE J , VAN HUMMELEN P , HOOGHE‐PETERS E L. , DESAINTES C
Published: March 2004

Abstract: The response to X‐ray irradiation of three different human hematopoietic cell lines originating from T (Jurkat), B (Raji), and promyelocytic (HL60) leukemia was analyzed. The survival after irradiation differed among the three cell lines, with Jurkat cells being the most vulnerable and HL60 being the least sensitive. The profile of gene expression was studied with the microarray technique in both Jurkat and HL60 cell lines. Out of the 13,800 different genes spotted on microarrays, very few genes (<0.5%) appeared to be induced more than 2‐fold or repressed more than 2.5‐fold in both cell lines.
Staurosporine Induces Apoptotic Volume Decrease (AVD) in ECV304 Cells
Authors: PORCELLI A M. , GHELLI A , ZANNA C , VALENTE P , FERRONI S , RUGOLO M
Published: March 2004

Abstract: Incubation of ECV304 cells with 1 μM staurosporine (STS) causes apoptotic cell death. In the present study, we investigate whether a significant apoptotic volume decrease (AVD) was apparent during the very early times (1 h) of the apoptotic process. Our data suggest that upregulation of Cl‐ (and possibly K+) channels by STS may be a very early primary event required for the subsequent onset of AVD, which results in apoptosis.
The Developing Mouse Dentition
Authors: PETERKOVÁ RENATA , PETERKA MIROSLAV , LESOT HERVÉ
Published: March 2004

Abstract: Developing limb or differentiating neural and blood cells are traditional models used to study programmed cell death in mammals. The developing mouse dentition can also be an attractive model for studying apoptosis regulation. Apoptosis is most extant during early odontogenesis in mice. The embryonic tooth pattern is comprised not only of anlagen of functional teeth (incisor, molars), but also of vestiges of ancestral tooth primordia that must be suppressed. Apoptosis is involved in (a) the elimination of vestigial tooth primordia in the prospective toothless gap (diastema) between the incisor and molars and (b) the shaping of germs in functional teeth. This type of apoptosis occurs in the dental epithelium according to a characteristic temporo‐spatial pattern. Where apoptosis concentrates, specific signaling is also found. We proposed a hypothesis to explain the stimulation of apoptosis in the dental epithelium by integrating two concepts: (1) The regulation of epithelial budding by positional information generated from interactions between growth‐activating and growth‐inhibiting signals, and (2) apoptosis stimulation by the failure of death‐suppressing signals. During the budding of the dental epithelium, local excess in growth inhibitors (e.g., Bmps) might lead to the epithelial cells' failure to receive adequate growth‐activating (apoptosis‐suppressing) signals (e.g., Fgfs). The resulting signal imbalance leads to cell “suicide” by apoptosis. Understanding of apoptosis regulation in the vestigial tooth primordia can help to elucidate the mechanism of their suppression during evolution and to identify factors essential for tooth survival. The latter knowledge will be important for developing a technology of tooth engineering.
Surfactant Protein D Binds Genomic DNA and Apoptotic Cells, and Enhances Their Clearance, in Vivo
Authors: PALANIYAR NADES , CLARK HOWARD , NADESALINGAM JEYA , HAWGOOD SAMUEL , REID KENNETH B.M.
Published: March 2004

Abstract: Collectins, such as surfactant protein D (SP‐D), bind apoptotic cells; however, the ligands that they recognize on these cells are unknown. We hypothesized that SP‐D binds to the DNA present on these cells. We show that SP‐D binds and aggregates mouse alveolar macrophage DNA effectively. Alveolar macrophages of SP‐D(−/−) mice contained more nicked DNA than those of SP‐A(−/−) and wild type mice. Our results also suggest that carbohydrate recognition domains of SP‐D may recognize DNA present on the apoptotic cells. Therefore, cell‐surface DNA could be a ligand for recognition of apoptotic cells by collectins.
Apoptosis in Cultured Cells Infected with Feline Calicivirus
Authors: ROBERTS LISA O. , AL‐MOLAWI NAEMA , CARTER MICHAEL J. , KASS GEORGE E. N.
Published: March 2004

Abstract: Caliciviruses are important pathogens of man and animals; feline calicivirus (FCV) is responsible for an acute upper respiratory tract disease in cats. To date, little is known about the mechanism of cell damage induced by these viruses. We set out to determine if apoptosis played any role in cell death in FCV infection of cultured cells. We demonstrate that caspase‐2, ‐3, and ‐7 were activated during FCV infection, as evidenced by pro‐form processing and an increase in acetyl‐Asp‐Glu‐Val‐Asp‐7‐amido‐4‐trifluoromethylcoumarin cleavage activity, as well as cleavage of poly(ADP‐ribose)polymerase. Caspase activation coincided with the condensation of chromatin. At about 8 h post infection we also detected cleavage of the FCV capsid protein; this was prevented by caspase inhibitors. Taken together these results suggest that FCV triggers apoptosis within infected cells and that caspases are involved in the cleavage of the capsid protein.
Increased Apoptotic Cell Death in Sporadic and Genetic Alzheimer's Disease
Authors: ECKERT ANNE , MARQUES CELIO A. , KEIL UTA , SCHÜSSEL KATRIN , MÜLLER WALTER E.
Published: March 2004

Abstract: Mounting evidence indicates increased susceptibility to cell death and increased oxidative damage as common features in neurons from sporadic Alzheimer's disease (AD) patients but also from familial AD (FAD) cases. Autosomal dominant forms of FAD are caused by mutations of the amyloid precursor protein (APP) gene and by mutations of the genes encoding for presenilin 1 or presenilin 2 (PS1/2). We investigated the effect of the Swedish APP double mutation (APPsw) on oxidative stress‐induced cell death mechanisms in PC12 cells. This mutation results in from three‐ to sixfold increased β‐amyloid (Aβ) production compared with wild‐type APP (APPwt). Because APPsw cells secrete low Aβ levels similar to the situation in FAD brains, our cell model represents a very suitable approach to elucidate the AD‐specific cell death pathways under more likely physiological conditions. We found that APPsw‐bearing cells show decreased mitochondrial membrane potential after exposure to hydrogen peroxide. In addition, activity of the executor caspase 3 after treatment with hydrogen peroxide was elevated in APPsw cells, which seems to be the result of an enhanced activation of both intrinsic and extrinsic apoptosis pathways. Our findings provide evidence that the massive neurodegeneration in early age of FAD patients could be a consequence of an increased vulnerability of neurons by mitochondrial abnormalities resulting in activation of different apoptotic pathways as a consequence to elevated oxidative stress levels. Finally, we propose a hypothetical sequence of the pathogenic steps linking sporadic AD, FAD, Aβ production, mitochondrial dysfunction with caspase pathway, and neuronal loss.
Stable‐Tau Overexpression in Human Neuroblastoma Cells
Authors: DELOBEL PATRICE , MAILLIOT CHRISTEL , HAMDANE MALIKA , SAMBO ANNE‐VÉRONIQUE , BÉGARD SÉVERINE , VIOLLEAU ANNE , DELACOURTE ANDRÉ , BUÉE LUC
Published: March 2004

Abstract: Many neurodegenerative disorders referred to as “tauopathies” are characterized by the accumulation and aggregation of Tau proteins into filaments. In these pathologies, Tau proteins are hyperphosphorylated and also abnormally phosphorylated. Moreover, they differ from each other by the preferential aggregation of isoforms exhibiting either three microtubule‐binding repeats (3R) or four repeats (4R) Tau. To investigate the effects of an intracellular accumulation of Tau, we stably transfected neuroblastoma cell line SY5Y with either 3R or 4R Tau. Our data showed that an increase in intracellular Tau expression has led to their hyperphosphorylation. Conversely, an abnormal Tau phosphorylation and/or aggregation were never observed. Furthermore, SY5Y cells transfected with 4R Tau showed an increased susceptibility to cell death. Finally, in apoptotic conditions, Tau proteins were degraded at their carboxy terminus by caspase, leading to an apparent decrease in Tau phosphorylation in this region. Because truncated Tau generated during apoptosis are not commonly found in Tau aggregates, apoptotic processes may not be of interest in neurofibrillary degeneration.
Transient Leukocytosis, Granulocyte Colony‐Stimulating Factor Plasma Concentrations, and Apoptosis Determined by Binding of Annexin V by Peripheral Leukocytes in Patients with Severe Sepsis
Authors: WEISS MANFRED , ELSHARKAWI MOHAMMED , WELT KATRIN , SCHNEIDER ELISABETH‐MARION
Published: March 2004

Abstract: This study was undertaken to clarify the relation between transient increases in the numbers of leukocytes and granulocyte colony‐stimulating factor (G‐CSF) plasma concentrations as well as the degree of apoptosis, as determined by binding of annexin V by these cells in patients with severe sepsis and septic shock. Over a 6‐month period, annexin V binding by leukocytes was determined daily using flow cytometry and FITC‐labeled annexin V in 33 postoperative patients with severe sepsis or septic shock during their intensive care unit stay. The percentage of annexin V binding neutrophils, monocytes, and lymphocytes was significantly lower, and G‐CSF plasma concentrations were higher in patients than in controls on most days. Nine, 19, and 18 peaks in neutrophil, monocyte, and lymphocyte counts (increase of ≥30% within 2 days, followed by a decrease of ≥30% within 2 days) occurred in 6, 11, and 16 patients. During leukocyte peaks, the absolute numbers of annexin V binding neutrophils, monocytes, and lymphocytes paralleled those of neutrophil, monocyte, and lymphocyte numbers. However, the percentage of annexin V binding neutrophils, monocytes, and lymphocytes did not differ significantly from one day to another. Increased G‐CSF serum concentrations preceded neutrophil and monocyte peaks. In conclusion, apoptosis of leukocytes is lowered during severe sepsis and septic shock in critically ill patients. Moreover, the degree of apoptosis does not increase during transient leukocytosis. G‐CSF might contribute to the low degree of apoptosis of neutrophils and monocytes in those patients.
Troponin I is required for myofibrillogenesis and sarcomere formation inDrosophilaflight muscle
Authors: Nongthomba Upendra , Clark Sam , Cummins Mark , Ansari Maqsood , Stark Meg , Sparrow John C.
Published: March 2004

Myofibrillar proteins assemble to form the highly ordered repetitive contractile structural unit known as a sarcomere. Studies of myogenesis in vertebrate cell culture and embryonic developmental systems have identified some of the processes involved during sarcomere formation. However, isoform changes during vertebrate muscle development and a lack of mutants have made it difficult to determine how these proteins assemble to form sarcomeres. The indirect flight muscles (IFMs) of Drosophila provide a unique genetic system with which to study myofibrillogenesis in vivo. We show in this paper that neither sarcomeric myosin nor actin are required for myoblast fusion or the subsequent morphogenesis of muscle fibres, i.e. fibre morphogenesis does not depend on myofibrillogenesis. However, fibre formation and myofibrillogenesis are very sensitive to the interactions between the sarcomeric proteins. A troponin I (TnI) mutation, hdp3, leads to an absence of TnI in the IFMs and tergal depressor of trochanter (TDT) muscles due to a transcript-splicing defect. Sarcomeres do not form and the muscles degenerate. TnI is part of the thin filament troponin complex which regulates muscle contraction. The effects of the hdp3 mutation are probably caused by unregulated acto-myosin interactions between the thin and thick filaments as they assemble. We have tested this proposal by using a transgenic myosin construct to remove the force-producing myosin heads. The defects in sarcomeric organisation and fibre degeneration in hdp3 IFMs are suppressed, although not completely, indicating the need for inhibition of muscle contraction during muscle development. We show that mRNA and translated protein products of all the major thin filament proteins are reduced in hdp3 muscles and discuss how this and previous studies of thin filament protein mutants indicate a common co-ordinated control mechanism that may be the primary cause of the muscle defects.