bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorophore‐assisted light inactivation produces both targeted and collateral effects on N‐type calcium channel modulation in rat sympathetic neurons
Authors: Guo Juan , Chen Huanmian , Puhl Henry L. , Ikeda* Stephen R.
Published: July 2006

Fluorophore‐assisted light inactivation (FALI) is a method to inactivate specific proteins on a time scale of seconds to minutes using either diffuse or coherent light. Here we examine a novel FALI modality that utilizes a fluorescein‐conjugated polypeptide, α‐bungarotoxin (BTX) and a 13 amino acid BTX‐binding site engineered into the N‐terminus of metabotropic glutamate receptor 8a (mGluR8a), a class C G‐protein‐coupled receptor (GPCR). The tagged mGluR8a was expressed in rat sympathetic neurons and labelled with fluorescein‐conjugated BTX (FL‐BTX). The efficacy of FALI was evaluated by monitoring mGluR8a‐mediated inhibition of calcium currents (ICa) using whole‐cell voltage‐clamp techniques. Following either wide‐field or laser illumination of FL‐BTX‐labelled neurons, mGluR8a‐mediated ICa inhibition was greatly attenuated whereas holding current and basal ICa, measures of non‐specific effects, were minimally affected. Sodium azide, a collision quencher of singlet oxygen, reduced the magnitude of FALI‐mediated effects supporting a role for reactive oxygen species in the process. Although these results were consistent with an acute inactivation of mGluR8a, the intended target, two findings confounded this interpretation. First, effects on a natively expressed signalling pathway, α2‐adrenergic receptor‐mediated ICa modulation, were observed following illumination of neurons expressing FL‐BTX‐labelled sodium channel β2 subunits or ionotropic 5‐HT3 receptors, proteins with no overt relationship to GPCR signalling pathways. Second, GPCR‐independent ICa modulation induced with intracellular guanylyl imidophosphate was also attenuated by FALI. These data challenge the assumption that the fluorophore‐tagged protein is the sole target of FALI and provide evidence that collateral damage to proximal proteins occurs following fluorophore illumination.
Resistance exercise increases AMPK activity and reduces 4E‐BP1 phosphorylation and protein synthesis in human skeletal muscle
Authors: Dreyer Hans C. , Fujita Satoshi , Cadenas Jerson G. , Chinkes David L. , Volpi Elena , Rasmussen Blake B.
Published: July 2006

Resistance exercise is a potent stimulator of muscle protein synthesis and muscle cell growth, with the increase in protein synthesis being detected within 2–3 h post‐exercise and remaining elevated for up to 48 h. However, during exercise, muscle protein synthesis is inhibited. An increase in AMP‐activated protein kinase (AMPK) activity has recently been shown to decrease mammalian target of rapamycin (mTOR) signalling to key regulators of translation initiation. We hypothesized that the cellular mechanism for the inhibition of muscle protein synthesis during an acute bout of resistance exercise in humans would be associated with an activation of AMPK and an inhibition of downstream components of the mTOR pathway (4E‐BP1 and S6K1). We studied 11 subjects (seven men, four women) before, during, and for 2 h following a bout of resistance exercise. Muscle biopsy specimens were collected at each time point from the vastus lateralis. We utilized immunoprecipitation and immunoblotting methods to measure muscle AMPKα2 activity, and mTOR‐associated upstream and downstream signalling proteins, and stable isotope techniques to measure muscle fractional protein synthetic rate (FSR). AMPKα2 activity (pmol min−1 (mg protein)−1) at baseline was 1.7 ± 0.3, increased immediately post‐exercise (3.0 ± 0.6), and remained elevated at 1 h post‐exercise (P < 0.05). Muscle FSR decreased during exercise and was significantly increased at 1 and 2 h post‐exercise (P < 0.05). Phosphorylation of 4E‐BP1 at Thr37/46 was significantly reduced immediately post‐exercise (P < 0.05). We conclude that AMPK activation and a reduced phosphorylation of 4E‐BP1 may contribute to the inhibition of muscle protein synthesis during resistance exercise. However, by 1–2 h post‐exercise, muscle protein synthesis increased in association with an activation of protein kinase B, mTOR, S6K1 and eEF2.
Prestin and the cochlear amplifier
Authors: Dallos Peter , Zheng Jing , Cheatham Mary Ann
Published: July 2006

In non‐mammalian, hair cell‐bearing sense organs amplification is associated with mechano‐electric transducer channels in the stereovilli (commonly called stereocilia). Because mammals possess differentiated outer hair cells (OHC), they also benefit from a novel electromotile process, powered by the motor protein, prestin. Here we consider new work pertaining to this protein and its potential role as the mammalian cochlear amplifier.
Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress
Authors: Alfieri Roberta R. , Bonelli Mara A. , Cavazzoni Andrea , Brigotti Maurizio , Fumarola Claudia , Sestili Piero , Mozzoni Paola , De Palma Giuseppe , Mutti Antonio , Carnicelli Domenica , Vacondio Federica , Silva Claudia , Borghetti Angelo F. , Wheeler Kenneth P. , Petronini Pier Giorgio
Published: July 2006

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H2O)−1. This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na+ dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in Vmax of the Na+‐dependent component, with no significant change in the Km value of about 75 μmol l−1. Quantitative real‐time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo‐inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l−1 to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.
CYK-4/GAP Provides a Localized Cue to Initiate Anteroposterior Polarity upon Fertilization
Authors: Jenkins Noah , Saam Jennifer R. , Mango Susan E.
Published: July 2006

The
Caenorhabditis elegans
anteroposterior axis is established in response to fertilization by sperm. Here we present evidence that RhoA, the guanine nucleotide–exchange factor ECT-2, and the Rho guanosine triphosphatase–activating protein CYK-4 modulate myosin light-chain activity to create a gradient of actomyosin, which establishes the anterior domain. CYK-4 is enriched within sperm, and paternally donated CYK-4 is required for polarity. These data suggest that CYK-4 provides a molecular link between fertilization and polarity establishment in the one-cell embryo. Orthologs of CYK-4 are expressed in sperm of other species, which suggests that this cue may be evolutionarily conserved.
Superfluidity of Grain Boundaries and Supersolid Behavior
Authors: Sasaki S. , Ishiguro R. , Caupin F. , Maris H. J. , Balibar S.
Published: July 2006

When two communicating vessels are filled to a different height with liquid, the two levels equilibrate because the liquid can flow. We have looked for such equilibration with solid
4
He. For crystals with no grain boundaries, we see no flow of mass, whereas for crystals containing several grain boundaries, we detect a mass flow. Our results suggest that the transport of mass is due to the superfluidity of grain boundaries.
Production of a Heterologous Nonheme Catalase by
Lactobacillus casei
: an Efficient Tool for Removal of H
2
O
2
and Protection of
Lactobacillus bulgaricus
from Oxidative Stress in Milk
Authors: Rochat Tatiana , Gratadoux Jean-Jacques , Gruss Alexandra , Corthier Gérard , Maguin Emmanuelle , Langella Philippe , van de Guchte Maarten
Published: August 2006

Lactic acid bacteria (LAB) are generally sensitive to H
2
O
2
, a compound that they can paradoxically produce themselves, as is the case for
Lactobacillus bulgaricus. Lactobacillus plantarum
ATCC 14431 is one of the very few LAB strains able to degrade H
2
O
2
through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species:
L. bulgaricus
ATCC 11842,
Lactobacillus casei
BL23, and
Lactococcus lactis
MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in
L. casei
. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in
L. plantarum
and
L. casei
but at least 10- and 100-fold lower in
Lactococcus lactis
and
L. bulgaricus
, respectively. The expression of the MnKat gene in
L. casei
conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H
2
O
2
, and improved long-term survival in aerated cultures. In mixtures of
L. casei
producing MnKat and
L. bulgaricus
,
L. casei
can eliminate H
2
O
2
from the culture medium, thereby protecting both
L. casei
and
L. bulgaricus
from its deleterious effects.
Random Insertion of a TetR-Inducing Peptide Tag into
Escherichia coli
Proteins Allows Analysis of Protein Levels by Induction of Reporter Gene Expression
Authors: Schlicht Maximilian , Berens Christian , Daam Janko , Hillen Wolfgang
Published: August 2006

The insertion element InsTipα was constructed to generate protein expression data. It randomly fuses the TetR-inducing peptide Tip to the affected reading frame. Fusion protein expression is quantified by Tet-regulated reporter gene expression. The expression patterns of tagged
Escherichia coli
genes fully agree with published data from transcriptional fusions or microarrays, validating the Tip tag approach.
Skin adnexal neoplasms—part 1: An approach to tumours of the pilosebaceous unit
Authors: Alsaad K O , Obaidat N A , Ghazarian D
Published: August 2006

Skin adnexal neoplasms comprise a wide spectrum of benign and malignant tumours that exhibit morphological differentiation towards one or more types of adnexal structures found in normal skin. Most adnexal neoplasms are relatively uncommonly encountered in routine practice, and pathologists can recognise a limited number of frequently encountered tumours. In this review, the first of two, the normal histology of the skin adnexal structures is reviewed, and the histological features of selected but important benign and malignant tumours and tumour-like lesions of pilosebaceous origin discussed, with emphasis on the diagnostic approach and pitfalls in histological diagnosis.
What determines whether patients are willing to participate in resuscitation studies requiring exception from informed consent?
Authors: Abboud P-A , Heard K , Al-Marshad A A , Lowenstein S R
Published: July 2006

Objectives:To examine the willingness of patients to participate in a resuscitation study that requires exception from informed consent and to determine if willingness to participate is associated with demographic and other characteristics.

Methods:Adult patients in an emergency department and in a geriatric outpatient clinic were surveyed. Patients were asked to imagine that they presented to an emergency department with cardiac arrest and asked about their willingness to (1) receive a new drug outside of a study, (2) receive a new drug as part of a study and (3) participate in a randomised controlled trial (RCT) for a new drug. Patients were also asked about participation in studies of invasive procedures.

Results:213 patients from a geriatric clinic and 207 from an emergency department were surveyed. Two thirds of patients from the geriatric clinic and 83% from the emergency department were willing to receive an experimental drug outside of a study. Patients were less willing to participate in a study of the new drug and even less likely to participate in an RCT for the new drug (χ2test for trend, p<0.001 for both settings). Patients were less likely to participate in a study of thoracotomy than in a study that required placement of a femoral catheter (p = 0.008 for the geriatric clinic, p = 0.01 for the emergency department). Willingness to participate was not associated with trust in the doctors.

Conclusions:Study design and invasiveness of the intervention were associated with the willingness of patients to participate in resuscitation studies that require exception from informed consent.
Is it in the best interests of an intellectually disabled infant to die?
Authors: Wilkinson D
Published: July 2006

One of the most contentious ethical issues in the neonatal intensive care unit is the withdrawal of life-sustaining treatment from infants who may otherwise survive. In practice, one of the most important factors influencing this decision is the prediction that the infant will be severely intellectually disabled. Most professional guidelines suggest that decisions should be made on the basis of the best interests of the infant. It is, however, not clear how intellectual disability affects those interests. Why should intellectual disability be more important than physical disability to the future interests of an infant? Is it discriminatory to base decisions on this? This paper will try to unravel the above questions. It seems that if intellectual disability does affect the best interests of the child it must do so in one of three ways. These possibilities will be discussed as well as the major challenges to the notion that intellectual disability should have a role in such decisions. The best interests of the child can be affected by severe or profound intellectual disability. It is, though, not as clear-cut as some might expect.
Research ethics committees in Europe: implementing the directive, respecting diversity
Authors: Hedgecoe A , Carvalho F , Lobmayer P , Raka F
Published: July 2006

With the recent Clinical Trials Directive, a degree of harmonisation into research ethics committees (RECs) across Europe, including the time taken to assess a trial proposal and the kinds of issues a committee should take into account, has been introduced by the European Union (EU). How four different member states—Hungary, Portugal, Sweden and the UK—have chosen to implement the directive is shown. Although this has resulted in four very different ways of structuring RECs, similar themes are present in all four cases, such as centralisation of control over RECs within member states, harmonisation of REC procedures across the EU and increased role of political decision making with regard to such committees.
Perceptions of patients on the utility or futility of end-of-life treatment
Authors: Rodriguez K L , Young A J
Published: July 2006

Background and objectives:Definitions of medical futility, offered by healthcare professionals, bioethicists and other experts, have been rigorously debated by many investigators, but the perceptions of patients of futility have been explored only by a few. Patients were allowed to discuss their concerns about end-of-life care, so that their ideas about treatment futility or utility could be extrapolated by us.

Methods:In this cross-sectional study, in-depth, semistructured interviews were conducted with 30 elderly people who were receiving outpatient care in a large, urban Veterans Affairs medical centre in the US. Each of their healthcare providers was also interviewed. Participants were asked to consider four terms commonly used in advance directive forms (ie, life-sustaining treatment, terminal condition, state of permanent unconsciousness and decision-making capacity) and to discuss what these terms meant to them. Audiotapes of the open-ended interviews were transcribed and responses were coded and categorised by constant comparison, a commonly used qualitative method.

Results:The following four factors were taken into account by the participants when discussing end-of-life interventions and outcomes: (1) expected quality of life; (2) emotional and financial costs of treatment; (3) likelihood of treatment success; and (4) expected effect on longevity.

Conclusions:Although the terms “utility” or “futility” were not generally used by the participants, segments of speech indicating their perceptions of these terms were identified. Treatment was not always discussed in the same way by patients and providers, but seemed to reflect the same four concerns. Therefore, it may be fruitful for providers to focus on these concerns when discussing end-of-life treatment options with their patients.
Prevention of Cerebral Oxidative Injury by Post-ischemic Intravenous Administration of Shengmai San
Authors: Ichikawa Haruyo , Wang Lei , Konishi Tetsuya
Published: August 2006

Shengmai San (SMS) is a traditional Chinese medicine (TCM) comprising three different herbal components, Panax ginseng, Ohiopogon japonicus and Fructus schisandrae and has been used for treating coronary heart diseases (Bensky and Barolet, 1990). It was shown that SMS effectively prevented cerebral oxidative injury in rats when it administered into the duodenum before cerebral ischemia-reperfusion.

In the present study, we examined whether post-ischemic administration of SMS can ameliorate cerebral ischemia-reperfusion injury in rats as well. Results showed that SMS injected immediately after ischemia also prevented the ischemia-reperfusion injury, when the effect was evaluated by the formation of protein carbonyl and thiobarbituric acid reactive substance (TBARS), and the loss of glutathione peroxidase (GPX). The preventative potential of SMS was decreased rapidly dependent on the time lag until SMS was injected after ischemia. However, it was noted that intravenously administered SMS protected the oxidative injury approximately 30% even after 60 min of reperfusion in terms of protein carbonyl formation. It is thus suggested that SMS injection might be useful for preventing the progression of injury in cerebral infarction after stroke.
Endothelium-Dependent Induction of Vasorelaxation by the Butanol Extract of Phellinus igniarius in Isolated Rat Aorta
Authors: Kang Dae Gill , Cao Li Hua , Lee Jun Kyoung , Choi Deok Ho , Kim Seung Ju , Lee Hyuck , Kim Jin Sook , Lee Ho Sub
Published: August 2006

The butanol extract of Phellinus igniarius (BPI) induced relaxation of the phenylephrin e-precontracted rat aorta in a dose-dependent manner, and its effect was abolished by the removal of functional endothelium. Pretreatment of the aortic tissues with NG -nitro-L-arginine methyl ester (L-NAME), methylene blue, or 1H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin1-one (ODQ) inhibited the vascular relaxation induced by BPI. BPI-induced vascular relaxations were also markedly attenuated by the addition of verapamil or diltiazem, while the relaxant effect of BPI was not blocked by pretreatment with indomethacine, glibenclamide, tetraethylammonium (TEA), atropine, or propranolol. Incubation of endothelium-intact rat aorta with BPI increased the production of cGMP in a dose-dependent manner. These results suggest that BPI dilates vascular smooth muscle via endothelium-dependent nitric oxide-cGMP signaling pathway, with the possible involvement of L-type Ca 2+ channels.
Samul Extract Protects Against theH2O2-Induced Apoptosis of H9c2 Cardiomyoblasts via Activation of Extracellular Regulated Kinases (Erk) 1/2
Authors: Park Channy , So Hong Seob , Kim Se Jin , Youn Myung Ja , Moon Byung-Soon , Shin Sun-Ho , Lee In , Moon Seong-Keun , Park Raekil
Published: August 2006

Samul extract, containing Radix Rehmanniae, Radix Angelicae Gigantis, Radix Paeoniae, and Rhizoma Cnidii, has been traditionally used for treatment of ischemic heart and brain damages in Oriental medicine. However, little is known about the mechanism by which Samul rescues cells from cytotoxic damage. This study was designed to investigate the protective mechanisms of Samul on H2O2-induced death of H9c2 cells. Treatment with H2O2markedly decreased the viability of H9c2 cells in a dose- and time-dependent manner, which was significantly prevented by pre-treatment with Samul. The nature of death of H9c2 cells by H2O2was demonstrated by apoptotic features, including ladder-pattern fragmentation of genomic DNA and chromatin condensation, which were markedly abolished by pretreatment of Samul in H2O2-treated cells. We further demonstrated that MEK inhibitor, PD98059, dose-dependently attenuated the protective effects of Samul against H2O2, whereas inhibitors of Jnk and p38 did not. Consistently, Samul induced the early phosphorylation of Erk, p44, in H2O2-treated cells. In addition, treatment with Samul also resulted in an increase of expression of anti-apotogenic Bcl2 protein, which was decreased by H2O2. However, it inhibited the expression of apotogenic Bax protein in H2O2-treated cells. Taken together, these results suggest that the protective effects of Samul against oxidative damage may be achieved via activation of MAP kinase, Erk as well as Bcl2 family proteins.
APPLICATION OF ADOMIAN'S METHOD TO NONLINEAR ABSORPTION
Authors: SANCHEZ F. , LEBLOND H. , BRUNEL M. , AMEUR K. AÏT
Published: August 2006

We use the method of Adomian to investigate the solution of the nonlinear equation giving the evolution of a plane wave in a medium exhibiting linear, two- and three-photon absorption. The second-order solution is in good agreement with numerical simulations. An effective two-photon absorption coefficient is conveniently defined to fit experimental data.
Transcriptional alterations in the left ventricle of three hypertensive rat models
Authors: Cerutti Catherine , Kurdi Mazen , Bricca Giampiero , Hodroj Wassim , Paultre Christian , Randon Jacques , Gustin Marie-Paule
Published: August 2006

Left ventricular hypertrophy (LVH) is commonly associated with hypertension and represents an independent cardiovascular risk factor. The aim of this study was to test the hypothesis that the cardiac overload related to hypertension is associated to a specific gene expression pattern independently of genetic background. Gene expression levels were obtained with microarrays for 15,866 transcripts from RNA of left ventricles from 12-wk-old rats of three hypertensive models [spontaneously hypertensive rat (SHR), Lyon hypertensive rat (LH), and heterozygous TGR(mRen2)27 rat] and their respective controls. More than 60% of the detected transcripts displayed significant changes between the three groups of normotensive rats, showing large interstrain variability. Expression data were analyzed with respect to hypertension, LVH, and chromosomal distribution. Only four genes had significantly modified expression in the three hypertensive models among which a single gene, coding for sialyltransferase 7A, was consistently overexpressed. Correlation analysis between expression data and left ventricular mass index (LVMI) over all rats identified a larger set of genes whose expression was continuously related with LVMI, including known genes associated with cardiac remodeling. Positioning the detected transcripts along the chromosomes pointed out high-density regions mostly located within blood pressure and cardiac mass quantitative trait loci. Although our study could not detect a unique reprogramming of cardiac cells involving specific genes at early stage of LVH, it allowed the identification of some genes associated with LVH regardless of genetic background. This study thus provides a set of potentially important genes contained within restricted chromosomal regions involved in cardiovascular diseases.
Strongly irresolvable spaces
Authors: Rose David , Sizemore Kari , Thurston Ben
Published: August 2006

Several new characterizations of strongly irresolvable topological
spaces are found and precise relationships are noted between
strong irresolvability, hereditary irresolvability, and
submaximality. It is noted that strong irresolvablity is a faint
topological property, while neither hereditary irresolvablity nor
submaximality are semitopological.
A Photon Counting Technique for Quantitatively Evaluating Progression of Peritoneal Tumor Dissemination
Authors: Yanagihara Kazuyoshi , Takigahira Misato , Takeshita Fumitaka , Komatsu Teruo , Nishio Kazuto , Hasegawa Fumio , Ochiya Takahiro
Published: August 2006

We recently established a mouse model of peritoneal dissemination of human gastric carcinoma, including the formation of ascites, by orthotopic transplantation of cultured gastric carcinoma cells. To clarify the processes of expansion of the tumors in this model, nude mice were sacrificed and autopsied at different points of time after the orthotopic transplantation of the cancer cells for macroscopic and histopathologic examination of the tumors. The cancer cells grew actively in the gastric submucosa and invaded the deeper layers to reach the serosal plane. The tumor cells then underwent exfoliation and became free followed by the formation of metastatic lesions initially in the greater omentum and subsequent colonization and proliferation of the tumors on the peritoneum. Although this model allowed the detection of even minute metastases, it was not satisfactory from the viewpoint of quantitative and objective evaluation. To resolve these problems, we introduced a luciferase gene into this tumor cell line with a high metastasizing potential and carried out in vivo photon counting analysis. This photon counting technique was found to allow objective and quantitative evaluation of the progression of peritoneal dissemination on a real-time basis. This animal metastatic model is useful for monitoring the responses of tumors to anticancer agents. (Cancer Res 2006; 66(15): 7532-9)
Divergent Routes to Oral Cancer
Authors: Hunter Keith D. , Thurlow Johanna K. , Fleming Janis , Drake Paul J.H. , Vass J. Keith , Kalna Gabriela , Higham Des J. , Herzyk Pawel , MacDonald D. Gordon , Parkinson E. Ken , Harrison Paul R.
Published: August 2006

Most head and neck squamous cell carcinoma (HNSCC) patients present with late-stage cancers, which are difficult to treat. Therefore, early diagnosis of high-risk premalignant lesions and incipient cancers is important. HNSCC is currently perceived as a single progression mechanism, resulting in immortal invasive cancers. However, we have found that ∼40% of primary oral SCCs are mortal in culture, and these have a better prognosis. About 60% of oral premalignancies (dysplasias) are also mortal. The mortal and immortal tumors are generated in vivo as judged by p53 mutations and loss of p16INK4A expression being found only in the original tumors from which the immortal cultures were derived. To investigate the relationships of dysplasias to SCCs, we did microarray analysis of primary cultures of 4 normal oral mucosa biopsies, 19 dysplasias, and 16 SCCs. Spectral clustering using the singular value decomposition and other bioinformatic techniques showed that development of mortal and immortal SCCs involves distinct transcriptional changes. Both SCC classes share most of the transcriptional changes found in their respective dysplasias but have additional changes. Moreover, high-risk dysplasias that subsequently progress to SCCs more closely resemble SCCs than nonprogressing dysplasias. This indicates for the first time that there are divergent mortal and immortal pathways for oral SCC development via intermediate dysplasias. We believe that this new information may lead to new ways of classifying HNSCC in relation to prognosis. (Cancer Res 2006; 66(15): 7405-13)
Determinants of Sensitivity and Resistance to Rapamycin-Chemotherapy Drug Combinations In vivo
Authors: Wendel Hans-Guido , Malina Abba , Zhao Zhen , Zender Lars , Kogan Scott C. , Cordon-Cardo Carlos , Pelletier Jerry , Lowe Scott W.
Published: August 2006

The phosphatidylinositol-3-OH kinase [PI(3)K] pathway is frequently activated in human cancers and represents a rational target for therapeutic intervention. We have previously shown that enforced expression of Akt, which is a downstream effector of PI(3)K, could promote tumorigenesis and drug resistance in the Eμ-myc mouse lymphoma model, and that these tumors were particularly sensitive to inhibition of mammalian target of rapamycin (mTOR) with rapamycin when combined with conventional chemotherapy. We now show that reduced dosage of PTEN, a negative regulator of PI(3)K signaling, is sufficient to activate Akt, but has only a modest effect on lymphomagenesis in the same model. Nonetheless, loss of even one PTEN allele resulted in lymphomas that were resistant to conventional chemotherapy yet sensitive to rapamycin/chemotherapy combinations. These effects could be recapitulated by using RNA interference to suppress PTEN expression in lymphomas, which were previously established in the absence of PI(3)K lesions. Finally, the introduction of lesions that act downstream of mTOR (eIF4E) or disable apoptosis (Bcl-2 and loss of p53) into PTEN+/− lymphomas promoted resistance to rapamycin/chemotherapy combinations. Thus, whether activation of the PI(3)K pathway confers sensitivity or resistance to therapy depends on the therapy used as well as secondary genetic events. Understanding these genotype-response relationships in human tumors will be important for the effective use of rapamycin or other compounds targeting the PI(3)K pathway in the clinic. (Cancer Res 2006; 66(15): 7639-46)
Immunosurveillance ofErbb2Carcinogenesis in Transgenic Mice Is Concealed by a Dominant Regulatory T-Cell Self-Tolerance
Authors: Ambrosino Elena , Spadaro Michela , Iezzi Manuela , Curcio Claudia , Forni Guido , Musiani Piero , Wei Wei-Zen , Cavallo Federica
Published: August 2006

To assess the role of CD4+CD25+Foxp3+ regulatory T (Treg) cells in overcoming immunosurveillance of Erbb2 (HER-2/neu) mammary lesions, we studied the effects of their sustained removal in BALB/c female mice made transgenic for the rat Erbb2 (r-Erbb2) oncogene (BALB-neuT mice), which develop multiple mammary carcinomas. During the progression of these lesions, Treg cells expand in the spleen, tumor draining lymph nodes, and tumors. Repeated administration of anti-CD25 antibodies extends tumor-free survival, reduces carcinoma multiplicity, and leads to the manifestation of a natural antibody and CTL-mediated reactivity against r-Erbb2. Loss of Foxp3+ Treg cells during anti-CD25 treatment remarkably caused the disappearance of Gr1+ immature myeloid cells, suggesting a cross-talk between these two inhibitory immune cell types. Treg cell expansion associated with r-Erbb2 overexpression may be seen as a physiologic response to dampen the immune reaction elicited by local anomalous overexpression of a self-antigen. (Cancer Res 2006; 66(15): 7734-40)
Inhalation of Low-Level Formaldehyde Increases the Bcl-2/Bax Expression Ratio in the Hippocampus of Immunologically Sensitized Mice
Authors: Tsukahara Shinji , Yamamoto Shoji , Shwe Tin-Tin-Win , Ahmed Sohel , Kunugita Naoki , Arashidani Keiichi , Fujimaki Hidekazu
Published: August 2006

Objective: A recent study from our research group showed that repeated exposure to low-level formaldehyde (FA) increases the production of nerve growth factor, involving the survival and maintenance of neurons, in the hippocampus of immunized mice. In the present study, we examined the effects of FA on apoptotic mechanisms regulating survival and death of cells and on N-methyl-D-aspartate (NMDA) receptors related to hippocampal functions in the mouse hippocampus. Methods: Western blot analyses were performed for Bcl-2, Bax and NMDA receptor subtypes 2A and 2B of the hippocampus taken from C3H mice exposed to 0 or 400 ppb of FA with or without ovalbumin (OVA) immunization. Immunohistochemical analysis for active caspase-3 was also carried out for these mice. Results: The ratio of Bcl-2 to Bax expression levels significantly increased with 400-ppb FA exposure in OVA-immunized mice but not in mice without OVA immunization, although differences in each protein level were not significant among groups. Active caspase- 3-immunoreactive cells were found in the hippocampus. However, the number was only a few and not significantly affected by FA exposure and OVA immunization. NMDA receptor type 2A and 2B expression levels of FA-exposed mice were sustained at comparative levels with those for the control mice with or without OVA immunization. Conclusions: These results indicate that changes in the Bcl-2/Bax expression ratio, which occurs with low-level FA exposure and immunization and may follow enhancement of nerve growth factor production, exerts a protective effect against cell death by apoptosis.
Nuclear Receptors Nur77, Nurr1, and NOR-1 Expressed in Atherosclerotic Lesion Macrophages Reduce Lipid Loading and Inflammatory Responses
Authors: Bonta Peter I. , van Tiel Claudia M. , Vos Mariska , Pols Thijs W.H. , van Thienen Johannes V. , Ferreira Valérie , Arkenbout E. Karin , Seppen Jurgen , Spek C. Arnold , van der Poll Tom , Pannekoek Hans , de Vries Carlie J.M.
Published: July 2006

Objective—

Atherosclerosis is an inflammatory disease in which macrophage activation and lipid loading play a crucial role. In this study, we investigated expression and function of the NR4A nuclear receptor family, comprising Nur77 (NR4A1, TR3), Nurr1 (NR4A2), and NOR-1 (NR4A3) in human macrophages.

Methods and Results—

Nur77, Nurr1, and NOR-1 are expressed in early and advanced human atherosclerotic lesion macrophages primarily in areas of plaque activation/progression as detected by in situ-hybridization and immunohistochemistry. Protein expression localizes to the nucleus. Primary and THP-1 macrophages transiently express NR4A-factors in response to lipopolysaccharide and tumor necrosis factor α. Lentiviral overexpression of Nur77, Nurr1, or NOR-1 reduces expression and production of interleukin (IL)-1β and IL-6 proinflammatory cytokines and IL-8, macrophage inflammatory protein-1α and -1β and monocyte chemoattractant protein-1 chemokines. In addition, NR4A-factors reduce oxidized–low-density lipoprotein uptake, consistent with downregulation of scavenger receptor-A, CD36, and CD11b macrophage marker genes. Knockdown of Nur77 or NOR-1 with gene-specific lentiviral short-hairpin RNAs resulted in enhanced cytokine and chemokine synthesis, increased lipid loading, and augmented CD11b expression, demonstrating endogenous NR4A-factors to inhibit macrophage activation, foam-cell formation, and differentiation.

Conclusion—

NR4A-factors are expressed in human atherosclerotic lesion macrophages and reduce human macrophage lipid loading and inflammatory responses, providing further evidence for a protective role of NR4A-factors in atherogenesis.
Fenofibrate Reduces Atherogenesis in ApoE*3Leiden Mice
Authors: Kooistra T. , Verschuren L. , de Vries-van der Weij J. , Koenig W. , Toet K. , Princen H.M.G. , Kleemann R.
Published: July 2006

Objective—

To demonstrate, quantify, and mechanistically dissect antiatherosclerotic effects of fenofibrate besides lowering plasma cholesterol per se.

Methods and Results—

ApoE*3Leiden transgenic mice received either a high-cholesterol diet (HC) or HC containing fenofibrate (HC+FF) resulting in 52% plasma cholesterol-lowering. In a separate low-cholesterol diet (LC) control group, plasma cholesterol was adjusted to the level achieved in the HC+FF group. Low plasma cholesterol alone (assessed in LC) resulted in reduced atherosclerosis (lesion area, number and severity) and moderately decreased plasma serum amyloid-A (SAA) concentrations. Compared with LC, fenofibrate additively reduced lesion area, number and severity, and the total aortic plaque load. This additional effect in HC+FF was paralleled by an extra reduction of aortic inflammation (macrophage content; monocyte adhesion; intercellular adhesion molecule-1 [ICAM-1], soluble vascular cell adhesion molecule-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), MCP-1, and NF-κB expression), systemic inflammation (plasma SAA and fibrinogen levels), and by an upregulation of plasma apoE levels. Also, enhanced expression of ABC-A1 and SR-B1 in aortic macrophages may contribute to the antiatherosclerotic effect of fenofibrate by promoting cholesterol efflux.

Conclusion—

Fenofibrate reduces atherosclerosis more than can be explained by lowering total plasma cholesterol per se. Impaired recruitment of monocytes/macrophages, reduced vascular and systemic inflammation, and stimulation of cholesterol efflux may all contribute to these beneficial effect of fenofibrate.
Microcirculatory Hemodynamics and Endothelial Dysfunction in Systemic Lupus Erythematosus
Authors: Wright Stephen A. , O’Prey Fiona M. , Rea Derrick J. , Plumb Rick D. , Gamble Andrew J. , Leahey William J. , Devine Adrian B. , McGivern R. Canice , Johnston Dennis G. , Finch Michael B. , Bell Aubrey L. , McVeigh Gary E.
Published: July 2006

Objective—

Impaired flow-mediated dilation (FMD) occurs in disease states associated with atherosclerosis, including SLE. The primary hemodynamic determinant of FMD is wall shear stress, which is critically dependent on the forearm microcirculation. We explored the relationship between FMD, diastolic shear stress (DSS), and the forearm microcirculation in 32 patients with SLE and 19 controls.

Methods and Results—

DSS was calculated using (mean diastolic velocity×8×blood viscosity)/baseline brachial artery diameter. Doppler velocity envelopes from the first 15 seconds of reactive hyperemia were analyzed for resistive index (RI), and interrogated in the frequency domain to assess forearm microvascular hemodynamics. FMD was significantly impaired in SLE patients (median, 2.4%; range, −2.1% to 10.7% versus median 5.8%; range, 1.9% to 14%;
P
<0.001). DSS (dyne/cm
2
) was significantly reduced in SLE patients (median, 18.5; range, 3.9 to 34.0 versus median 21.8; range, 14.1 to 58.7;
P
=0.037). A strong correlation between FMD and DSS, r
s
=0.65,
P
=0.01 was found. Postischemic RI was not significantly different between the 2 groups; however, there were significant differences in the power-frequency spectrums of the Doppler velocity envelopes (
P
<0.05).

Conclusions—

These data suggest that in SLE, altered structure and function of the forearm microcirculation contributes to impaired FMD through a reduction in shear stress stimulus.
Association Between Birth Weight and Blood Pressure Is Robust, Amplifies With Age, and May Be Underestimated
Authors: Davies Anna A. , Smith George Davey , May Margaret T. , Ben-Shlomo Yoav
Published: August 2006

Data on the early life origins of adult hypertension have been widely reported: however, recent research shows that the strength of association between small size at birth and higher blood pressure weakens as study size increases. In this article, we retest the association between birth weight and systolic blood pressure in a large cohort, examine whether age interacts with birth weight to predict blood pressure, and explore reasons why birth weight-blood pressure associations tend to weaken with increasing study size. Measurements from 25874 employees of a large United Kingdom company (mean [SD] age: 38.0 [7.9] years), undertaking voluntary occupational health screening, were available. Using linear regression analysis, we observed that systolic blood pressure changed −0.8 (95% CI: −1.1 to −0.5) mmHg per 1-kg increase in birth weight (
P
<0.001) adjusted for age and sex and −1.1 (95% CI: −1.3 to −0.8) mmHg/kg (
P
<0.001) after further adjustment for body size. This inverse association amplified with age (age/birth weight interaction term
P
<0.001). In participants reporting birth weight from hospital records (n=744), systolic blood pressure changed −1.4 (95% CI: −3.1 to 0.2) mmHg/kg compared with −0.8 (95% CI: −1.0 to −0.5) mmHg/kg in all of the other participants. Finally, the data show evidence of “fixed-category blood pressure allocation,” where participants are allocated certain blood pressure values, such as 120/80 mmHg, independent of actual blood pressure. Although the association between birth weight and systolic blood pressure was weaker than observed in smaller studies, recalled birth weight and fixed blood pressure measurement error may generate a trend toward weaker associations in larger studies.
Recombinant Adeno-Associated Virus Serotype 9 Leads to Preferential Cardiac Transduction In Vivo
Authors: Pacak Christina A. , Mah Cathryn S. , Thattaliyath Bijoy D. , Conlon Thomas J. , Lewis Melissa A. , Cloutier Denise E. , Zolotukhin Irene , Tarantal Alice F. , Byrne Barry J.
Published: July 2006

Heart disease is often the end result of inherited genetic defects, which may potentially be treatable using a gene-transfer approach. Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a realistic method for the treatment of such disorders. Here, we demonstrate and compare the natural affinity of specific AAV serotype capsids for transduction of cardiac tissue. We compared the previously accepted optimal rAAV serotype for transduction of skeletal muscle, rAAV2/1, with rAAV2/8 and the newer rAAV2/9 vectors carrying the CMV-
lacZ
construct in their respective abilities to transcend vasculature and transduce myocardium following intravenous delivery of 1×10
11
vector genomes in neonatal mice. We found that both rAAV2/8 and rAAV2/9 are able to transduce myocardium at ≈20- and 200-fold (respectively) higher levels than rAAV2/1. Biodistribution analysis revealed that rAAV2/9 and rAAV2/8 demonstrate similar behavior in extracardiac tissue. Vector genome quantification showed an increase in genome copy numbers in cardiac tissue for several weeks following administration, which corresponds to expression data. In addition, we intravenously administered 1×10
11
vector genomes of rAAV2/9-CMV-
lacZ
into adult mice and achieved an expression biodistribution profile similar to that found following delivery to newborns. Although higher doses of virus will be necessary to approach those levels observed following neonatal injections, adult myocardium is also readily transduced by rAAV2/9. Finally, we have demonstrated physiological disease correction by AAV9 gene transfer in a mouse model of Pompe disease via ECG tracings and that intravenous delivery of the same vector preferentially transduces cardiac tissue in nonhuman primates.
Distribution and Functional Characterization of Equilibrative Nucleoside Transporter-4, a Novel Cardiac Adenosine Transporter Activated at Acidic pH
Authors: Barnes Kay , Dobrzynski Halina , Foppolo Sophie , Beal Paul R. , Ismat Fouzia , Scullion Elspeth R. , Sun Lijie , Tellez James , Ritzel Mabel W.L. , Claycomb William C. , Cass Carol E. , Young James D. , Billeter-Clark Rudi , Boyett Mark R. , Baldwin Stephen A.
Published: July 2006

Adenosine plays multiple roles in the efficient functioning of the heart by regulating coronary blood flow, cardiac pacemaking, and contractility. Previous studies have implicated the equilibrative nucleoside transporter family member equilibrative nucleoside transporter-1 (ENT1) in the regulation of cardiac adenosine levels. We report here that a second member of this family, ENT4, is also abundant in the heart, in particular in the plasma membranes of ventricular myocytes and vascular endothelial cells but, unlike ENT1, is virtually absent from the sinoatrial and atrioventricular nodes. Originally described as a monoamine/organic cation transporter, we found that both human and mouse ENT4 exhibited a novel, pH-dependent adenosine transport activity optimal at acidic pH (apparent
K
m
values 0.78 and 0.13 mmol/L, respectively, at pH 5.5) and absent at pH 7.4. In contrast, serotonin transport by ENT4 was relatively insensitive to pH. ENT4-mediated nucleoside transport was adenosine selective, sodium independent and only weakly inhibited by the classical inhibitors of equilibrative nucleoside transport, dipyridamole, dilazep, and nitrobenzylthioinosine. We hypothesize that ENT4, in addition to playing roles in cardiac serotonin transport, contributes to the regulation of extracellular adenosine concentrations, in particular under the acidotic conditions associated with ischemia.
Two Distinct Pools of Mesenchyme Contribute to the Development of the Atrial Septum
Authors: Mommersteeg Mathilda T.M. , Soufan Alexandre T. , de Lange Frederik J. , van den Hoff Maurice J.B. , Anderson Robert H. , Christoffels Vincent M. , Moorman Antoon F.M.
Published: July 2006

Closure of the primary atrial foramen is achieved by fusion of the atrioventricular cushions with the mesenchymal cap on the leading edge of the muscular primary atrial septum. A fourth component involved is the vestibular spine, originally described by His in 1880 as an intra-cardiac continuation of the extra-cardiac mesenchyme of the dorsal mesocardium. The morphogenesis of this area is of great clinical interest, because of the high incidence of atrial and atrioventricular septal defects. Nonetheless, the origin of the participating components is largely unknown. Here we report that the primary atrial foramen is surrounded in its entirety by mesenchyme derived from endocardium. A second population of mesenchyme not derived from endocardium was observed at the caudal margin of the mesenchymal atrial cap, entirely embedded within the mesenchyme derived from endocardium and contiguous with the mesenchyme of the dorsal mesocardium. Our reconstructions show this second population does indeed take the form of a short spine, albeit that it is the right pulmonary ridge, rather than this spine, that protrudes into the atrial lumen. From the stance of morphological description, therefore, there is little thus far to substantiate the existence of an atrial spine.
Activation of Estrogen Receptor-α Reduces Aortic Smooth Muscle Differentiation
Authors: Montague Christine R. , Hunter Melissa G. , Gavrilin Mikhail A. , Phillips Gary S. , Goldschmidt-Clermont Pascal J. , Marsh Clay B.
Published: July 2006

Women are at high risk of dying from unrecognized cardiovascular disease. Many differences in cardiovascular disease between men and women appear to be mediated by vascular smooth muscle cells (SMC). Because estrogen reduces the proliferation of SMC, we hypothesized that activation of estrogen receptor-α (ERα) by agonists or by growth factors altered SMC function. To determine the effect of growth factors, estrogen, and ERα expression on SMC differentiation, human aortic SMC were cultured in serum-free conditions for 10 days. SMC from men had lower spontaneous expression of ERα and higher levels of the differentiation markers calponin and smooth muscle α-actin than SMC from women. When SMC containing low expression of ERα were transduced with a lentivirus containing ERα, activation of the receptor by ligands or growth factors reduced differentiation markers. Conversely, inhibiting ERα expression by small interfering RNA (siRNA) in cells expressing high levels of ERα enhanced the expression of differentiation markers. ERα expression and activation reduced the phosphorylation of Smad2, a signaling molecule important in differentiation of SMC and initiated cell death through cleavage of caspase-3. We conclude that ERα activation switched SMC to a dedifferentiated phenotype and may contribute to plaque instability.