|
| ||
|
|
||
Cellular Mechanisms (cellular + mechanism)
Terms modified by Cellular Mechanisms Selected AbstractsCellular Mechanisms of Vagally Mediated Atrial Tachyarrhythmia in Isolated Arterially Perfused Canine Right AtriaJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 9 2002MASAMICHI HIROSE M.D. Mechanism of Vagally Mediated AT.Introduction: Increased vagal tone significantly enhances susceptibility to atrial fibrillation (AF); however, the cellular mechanisms responsible for vagally mediated AF are not completely understood. Methods and Results: In 12 isolated arterially perfused canine right atria, high-resolution optical mapping techniques were used to measure action potentials during control conditions, during intracardiac parasympathetic nerve stimulation (IPS; 30 to 50 Hz) as a surrogate for vagal stimulation, and during acetylcholine (ACh) infusion (10 to 30 ,M). During steady-state pacing, action potential duration was shorter during ACh infusion (43 ± 9 msec) than during IPS (78 ± 7 msec, P < 0.001) or control (129 ± 5 msec, P < 0.001). In contrast, repolarization gradients were larger during IPS (13 ± 3 msec/mm) than during ACh infusion (3 ± 1 msec/mm, P < 0.01) or control (5 ± 1 msec/mm, P < 0.01). Transmural repolarization gradients were relatively small for each intervention tested. During ACh infusion, atrial tachyarrhythmia (AT) was easily initiated with a single premature stimulus and was associated with a focal pattern of activation (84%). AT also was easily initiated by a single premature stimulus during IPS; however, when repolarization gradients were large, patterns of conduction block and incomplete macroreentry were often observed (64%). Importantly, AT initiation during IPS was associated with focal activity (36%) when repolarization gradients were small. Conclusion: In contrast to ACh infusion, IPS generally increased dispersion of repolarization and was often associated with patterns of conduction block and incomplete macroreentry, similar to that associated with in vivo cervical vagal stimulation. However, IPS also was associated with a focal pattern of initiation that was independent of local repolarization gradients. These results suggest that during vagal stimulation, AT initiation does not always depend on repolarization gradients. [source] Molecular and Cellular Mechanisms of Ectodomain SheddingTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 6 2010Kazutaka Hayashida Abstract The extracellular domain of several membrane-anchored proteins is released from the cell surface as soluble proteins through a regulated proteolytic mechanism called ectodomain shedding. Cells use ectodomain shedding to actively regulate the expression and function of surface molecules, and modulate a wide variety of cellular and physiological processes. Ectodomain shedding rapidly converts membrane-associated proteins into soluble effectors and, at the same time, rapidly reduces the level of cell surface expression. For some proteins, ectodomain shedding is also a prerequisite for intramembrane proteolysis, which liberates the cytoplasmic domain of the affected molecule and associated signaling factors to regulate transcription. Ectodomain shedding is a process that is highly regulated by specific agonists, antagonists, and intracellular signaling pathways. Moreover, only about 2% of cell surface proteins are released from the surface by ectodomain shedding, indicating that cells selectively shed their protein ectodomains. This review will describe the molecular and cellular mechanisms of ectodomain shedding, and discuss its major functions in lung development and disease. Anat Rec, 293:925,937, 2010. © 2010 Wiley,Liss, Inc. [source] Cellular mechanisms of cobalt-induced hippocampal epileptiform dischargesEPILEPSIA, Issue 1 2009Jiwei He Summary Purpose:, To explore the cellular mechanisms of cobalt-induced epileptiform discharges in mouse hippocampal slices. Methods:, Hippocampal slices were prepared from adult mice and briefly exposed to a CoCl2 -containing external solution. Population and single cell activities were examined via extracellular and whole-cell patch recordings. Results:, Brief cobalt exposure induced spontaneous, ictal-like discharges originating from the CA3 area. These discharges were suppressed by anticonvulsants, gap junction blockers, or by raising extracellular Ca2+, but their generation was not associated with overall hyperexcitability or impairment in GABAergic inhibition in the CA3 circuit. Electroencephalographic ictal discharges of similar waveforms were observed in behaving rats following intrahippocampal cobalt infusion. Discussion:, Mechanisms involving activity-dependent facilitation of gap junctional communication may play a major role in cobalt-induced epileptiform discharges. [source] Cellular mechanisms of the trigeminally evoked startle responseEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2003Susanne Schmid Abstract The startle response is an important mammalian model for studying the cellular mechanisms of emotions and of learning. It consists of contractions of facial and skeletal muscles in response to sudden acoustic, tactile or vestibular stimuli. Whereas the acoustic startle pathway is well described, only a few recent studies have investigated the tactile startle pathway. It was proposed that there is a direct projection from the principal sensory nucleus to the central sensorimotor interface of the startle response, which is formed by the giant neurons in the caudal pontine reticular formation. We explored this projection in greater detail in vitro. Anterograde tracing in rat brain slices confirmed projections with large axon terminals from the ventral part of the principal sensory nucleus to the lateral caudal pontine reticular formation. Electrophysiological studies revealed a monosynaptic glutamatergic connection between principal sensory nucleus neurons and caudal pontine reticular formation giant neurons. The synapses displayed paired-pulse facilitation at high-frequency stimulation, and homosynaptic depression at 1 Hz stimulation. The latter form of plasticity is thought to underlie habituation of the startle response. Furthermore, postsynaptic currents in caudal pontine reticular formation giant neurons evoked by principal sensory nucleus neuron stimulation summed in a linear way with signals evoked by stimulation of auditory afferents. Synaptic plasticity and summation of synaptic currents correspond well with in vivo data previously published by other groups. We thus presume that these synapses mediate trigeminal input to the startle pathway. [source] Cellular mechanisms of potassium transport in plantsPHYSIOLOGIA PLANTARUM, Issue 4 2008Dev T. Britto Potassium (K+) is the most abundant ion in the plant cell and is required for a wide array of functions, ranging from the maintenance of electrical potential gradients across cell membranes, to the generation of turgor, to the activation of numerous enzymes. The majority of these functions depend more or less directly upon the activities and regulation of membrane-bound K+ transport proteins, operating over a wide range of K+ concentrations. Here, we review the physiological aspects of potassium transport systems in the plasma membrane, re-examining fundamental problems in the field such as the distinctions between high- and low-affinity transport systems, the interactions between K+ and other ions such as NH4+ and Na+, the regulation of cellular K+ pools, the generation of electrical potentials and the problems involved in measurement of unidirectional K+ fluxes. We place these discussions in the context of recent discoveries in the molecular biology of K+ acquisition and produce an overview of gene families encoding K+ transporters. [source] Degeneration and regeneration of ultraviolet cone photoreceptors during development in rainbow troutTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2006W. Ted Allison Abstract Ultraviolet-sensitive (UVS) cones disappear from the retina of salmonid fishes during a metamorphosis that prepares them for deeper/marine waters. UVS cones subsequently reappear in the retina near sexual maturation and the return migration to natal streams. Cellular mechanisms of this UVS cone ontogeny were investigated using electroretinograms, in situ hybridization, and immunohistochemistry against opsins during and after thyroid hormone (TH) treatments of rainbow trout (Oncorhynchus mykiss). Increasing TH levels led to UVS cone degeneration. Labeling demonstrated that UVS cone degeneration occurs via programmed cell death and caspase inhibitors can inhibit this death. After the cessation of TH treatment, UVS cones regenerated in the retina. Bromodeoxyuridine (BrdU) was applied after the termination of TH treatment and was detected in the nuclei of cells expressing UVS opsin. BrdU was found in UVS cones but not other cone types. The most parsimonious explanation for the data is that UVS cones degenerated and UVS cones were regenerated from intrinsic retinal progenitor cells. Regenerating UVS cones were functionally integrated such that they were able to elicit electrical responses from second-order neurons. This is the first report of cones regenerating during natural development. Both the death and regeneration of cones in retinae represent novel mechanisms for tuning visual systems to new visual tasks or environments. J. Comp. Neurol. 499:702,715, 2006. © 2006 Wiley-Liss, Inc. [source] Excitotoxicity-induced endocytosis confers drug targeting in cerebral ischemia,ANNALS OF NEUROLOGY, Issue 3 2009Anne Vaslin MSc Objective Targeting neuroprotectants specifically to the cells that need them is a major goal in biomedical research. Many peptidic protectants contain an active sequence linked to a carrier such as the transactivator of transcription (TAT) transduction sequence, and here we test the hypothesis that TAT-linked peptides are selectively endocytosed into neurons stressed by excitotoxicity and focal cerebral ischemia. Methods In vivo experiments involved intracerebroventricular injection of TAT peptides or conventional tracers (peroxidase, fluorescein isothiocyanate-dextran) in young rats exposed to occlusion of the middle cerebral artery at postnatal day 12. Cellular mechanisms of uptake were analyzed in dissociated cortical neuronal cultures. Results In both models, all tracers were taken up selectively into stressed neurons by endocytosis. In the in vivo model, this was neuron specific and limited to the ischemic area, where the neurons displayed enhanced immunolabeling for early endosomal antigen-1 and clathrin. The highly efficient uptake of TAT peptides occurred by the same selective mechanism as for conventional tracers. All tracers were targeted to the nucleus and cytoplasm of neurons that appeared viable, although ultimately destined to die. In dissociated cortical neuronal cultures, an excitotoxic dose of N -methyl- D -aspartate induced a similar endocytosis. It was 100 times more efficient with TAT peptides than with dextran, because the former bound to heparan sulfate proteoglycans at the cell surface, but it depended on dynamin and clathrin in both cases. Interpretation Excitotoxicity-induced endocytosis is the main entry route for protective TAT peptides and targets selectively the neurons that need to be protected. Ann Neurol 2009;65:337,347 [source] Cellular mechanisms of injury after major traumaBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 10 2009I. H. Chaudry This is the Fourth article in the Journal's series on major trauma. Chaudry and Bland, leading experts in the field, consider the cellular implications of injury. Copyright © 2009 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. [source] Calcium-sensing receptor mediates phenylalanine-induced cholecystokinin secretion in enteroendocrine STC-1 cellsFEBS JOURNAL, Issue 18 2008Tohru Hira Intraluminal l -phenylalanine (Phe) stimulates cholecystokinin (CCK) secretion in vivo and in vitro. However, the cellular mechanism by which CCK-producing enteroendocrine cells sense Phe is unknown. The calcium-sensing receptor (CaR) can sense amino acids, and is expressed in the gastrointestinal tract. In the present study, we examined whether CaR functions as a receptor for Phe in CCK-producing enteroendocrine cells. CCK secretion and intracellular Ca2+ concentration in response to Phe were measured in the murine CCK-producing enteroendocrine cell line STC-1 at various extracellular Ca2+ concentrations or after treatment with a CaR antagonist. At more than 20 mm, Phe induced dose-dependent CCK secretion and intracellular Ca2+ mobilization in STC-1 cells. In the presence of 3.0 mm extracellular Ca2+, 10 and 20 mm Phe induced significantly higher CCK secretion than under normal conditions (1.2 mm extracellular Ca2+). Intracellular Ca2+ mobilization, induced by 10 or 20 mm Phe, was also enhanced by increasing extracellular Ca2+ concentrations. In addition, intracellular Ca2+ mobilization induced by addition of extracellular Ca2+ was augmented by the presence of Phe. These results closely match the known CaR properties. Treatment with a specific CaR antagonist (NPS2143) completely inhibited Phe-induced CCK secretion and the latter phase of intracellular Ca2+ mobilization. CaR mRNA expression was demonstrated by RT-PCR in STC-1 cells, as well as in other mouse tissues including the kidney, thyroid, stomach and intestine. In conclusion, CaR functions as a receptor for Phe, stimulating CCK secretion in enteroendocrine STC-1 cells. [source] Human remyelination promoting antibody inhibits apoptotic signaling and differentiation through Lyn kinase in primary rat oligodendrocytesGLIA, Issue 15 2010J. Watzlawik Abstract Purpose: Human remyelination promoting IgM mAbs target oligodendrocytes (OLs) and function in animal models of multiple sclerosis (MS). However, their mechanism of action is unknown. This study seeks to identify the cellular mechanism of action of a recombinant human IgM on OL survival. Methods: Binding of rHIgM22 to the surface of rat OLs was studied by co-localization with various markers. RHIgM22-mediated effects on apoptotic signaling in OLs, differentiation markers, and signaling molecules were detected by Western blotting and immunoprecipitation. Results: RHIgM22 co-localized with integrin ,3 but not other integrin ,-chains in OLs. Downstream of integrin ,3 we identified Src family kinase (SFK) Lyn as a key player of rHIgM22-mediated actions in OLs. Lyn immunoprecipitated in a complex together with integrin ,v,3 and PDGF,R. Lyn expression was 9-fold up-regulated and Lyn activation was 3-fold higher inrHIgM22-treated OL cultures compared with controls. RHIgM22 inhibited apoptotic signaling by greater than 10-fold reduction of caspase-3 and capsase-9 cleavage and reduced by 4-fold expression of differentiation markers MBP and MOG in OLs. SFK inhibitors PP2 and SU6656 inhibited Lyn activity and restored caspase-cleavage in OLs. A human IgM that did not promote remyelination and medium wereused as controls. Conclusions: rHIgM22 prevented apoptotic signaling andinhibited OL differentiation by Lyn implying thatIgM-mediated remyelination is due toprotection of OPC and OLs rather than promotion of OPC differentiation. © 2010 Wiley-Liss, Inc. [source] Autophagy activation by rapamycin eliminates mouse Mallory-Denk bodies and blocks their proteasome inhibitor-mediated formation,HEPATOLOGY, Issue 6 2008Masaru Harada The proteasomal and lysosomal/autophagy pathways in the liver and other tissues are involved in several biological processes including the degradation of misfolded proteins. Exposure of hepatocyte cell lines to proteasome inhibitors (PIs) results in the formation of inclusions that resemble Mallory-Denk bodies (MDBs). Keratins are essential for MDB formation and keratin 8 (K8)-overexpressing transgenic mice are predisposed to MDB formation. We tested the hypothesis that PIs induce MDBs in vivo and that autophagy participates in MDB turnover. The effect of the PI bortezomib (which is used to treat some malignancies) on MDB formation was tested in K8-overexpressing mice and in cultured cells. Inclusion formation was examined using immune and conventional electron microscopy (EM). Bortezomib induced MDB-like inclusions composed of keratins, ubiquitin, and p62 in cultured cells. Short-term exposure to bortezomib induced similar inclusions in K8-overexpressing but not in nontransgenic mice, without causing liver injury. In bortezomib-treated mice, autophagy was activated in hepatocytes as determined by EM and biochemical analysis. Further activation of autophagy by rapamycin (Rap) decreased the number of inclusions in bortezomib-treated K8 transgenic mice significantly. Rap also led to resorption of spontaneously formed MDBs in aging K8-overexpressing mice. Immune EM demonstrated K8-positive and ubiquitin-positive structures in autophagic vacuoles in the mouse liver. Conclusion: PIs alone are sufficient to induce MDBs in susceptible animals, while Rap-mediated activation of autophagy prevents MDB formation and causes MDB resorption. These findings suggest that some patients treated with PIs may become predisposed to MDB formation. Autophagy provides a potential cellular mechanism for the resorption of cytoplasmic inclusions. (HEPATOLOGY 2008.) [source] A mixture of seven antiandrogens induces reproductive malformations in ratsINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2008Cynthia V. Rider Summary To date, regulatory agencies have not considered conducting cumulative risk assessments for mixtures of chemicals with diverse mechanisms of toxicity because it is assumed that the chemicals will act independently and the individual chemical doses are not additive. However, this assumption is not supported by new research addressing the joint effects of chemicals that disrupt reproductive tract development in the male rat by disrupting the androgen signalling pathway via diverse mechanisms of toxicity [i.e. androgen receptor (AR) antagonism in the reproductive tract vs. inhibition of androgen synthesis in the foetal testis]. In this study, pregnant rats were exposed to four dilutions of a mixture containing vinclozolin, procymidone, linuron, prochloraz, benzyl butyl phthalate, dibutyl phthalate and diethylhexyl phthalate during the period of sexual differentiation and male offspring were assessed for effects on hormone sensitive endpoints including: anogenital distance, infant areolae retention and reproductive tract tissue weights and malformations. The ratio of the chemicals in the mixture was based upon each chemical's ED50 for inducing reproductive tract malformations (hypospadias or epididymal agenesis). The observed responses from the mixture were compared with predicted responses generated with a toxic equivalency approach and models of dose addition, response addition or integrated addition. As hypothesized, we found that the mixture of chemicals that alter the androgen signalling pathway via diverse mechanisms disrupted male rat reproductive tract differentiation and induced malformations in a cumulative, dose-additive manner. The toxic equivalency and dose addition models provided the best fit to observed responses even though the chemicals do not act via a common cellular mechanism of action. The current regulatory framework for conducting cumulative risk assessments needs to consider the results, including those presented herein, which indicate that chemicals that disrupt foetal tissues during sexual differentiation act in a cumulative, dose-additive manner irrespective of the specific cellular mechanism of toxicity. [source] Integrin signaling through FAK in the regulation of mammary stem cells and breast cancerIUBMB LIFE, Issue 4 2010Jun-Lin Guan Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase identified as a key mediator of intracellular signaling by integrins, a major family of cell surface receptors for extracellular matrix, in the regulation of different cellular functions in a variety of cells. Upon activation by integrins through disruption of an autoinhibitory mechanism, FAK undergoes autophosphorylation and forms a complex with Src and other cellular proteins to trigger downstream signaling through its kinase activity or scaffolding function. A number of integrins are identified as surface markers for mammary stem cells (MaSCs), and both integrins and FAK are found to play crucial roles in the maintenance of MaSCs in studies using mouse models, suggesting that integrin signaling through FAK may serve as a functional marker for MaSCs. Consistent with previous studies linking increased expression and activation of FAK to human breast cancer, these findings suggest a novel cellular mechanism of FAK promotion of mammary tumorigenesis by maintaining the pools of MaSCs as targets of oncogenic transformation. Furthermore, FAK inactivation in mouse models of breast cancer also reduced the pool of mammary cancer stem cells (MaCSCs), decreased their self-renewal in vitro, and compromised their tumorigenicity and maintenance in vivo, suggesting a potential role of integrin signaling through FAK in breast cancer growth and progression through its functions in MaCSCs. This review discusses these recent advances and future studies into the mechanism of integrin signaling through FAK in breast cancer through regulation of MaCSCs that may lead to development of novel therapies for this deadly disease. © 2010 IUBMB IUBMB Life, 62(4): 268,276, 2010 [source] The Percentage of Pituitary Gonadotropes with Immunoreactive Oestradiol Receptors Increases in the Follicular Phase of the Ovine Oestrous CycleJOURNAL OF NEUROENDOCRINOLOGY, Issue 10 2001V. A. Tobin Abstract During the oestrous cycle, there is an alteration in gonadotrope responsiveness to gonadotropin releasing hormone (GnRH). One cellular mechanism that may be involved in these changes at the pituitary level is the hormonal regulation of oestrogen receptor (ER) expression. Using double-label immunohistochemistry, we examined the proportion of gonadotropes, lactotropes and somatotropes with immunoreactive (ir) oestrogen receptor alpha (ER,) in pituitary sections from ewes at three stages of the ovine oestrous cycle (n = 8 per group). The percentage of ER, positive cells that also stained positive for luteinizing hormone (LH) increased in the transition from the luteal phase to the follicular phase (n = 8), with no further increase at the time of oestrus (n = 8). In the pituitaries from the luteal phase sheep, only a small number (15%) of lactotropes and 4% of somatotropes were found to contain ir-ER, and there were no alterations across the oestrous cycle. When we examined pituitaries from ovariectomized (OVX) ewes treated (i.m.) with either oestradiol benzoate (50 µg) or oil vehicle for 2, 4, 6 or 16 h (n = 4 per group), there was no effect of treatment. In fact, the percentage of gonadotropes that were ER,-positive in OVX ewes was similar to that observed in the pituitaries from the follicular phase ewes, both of which display a high frequency of pulsatile GnRH secretion. We conclude that the number of gonadotropes that contain ir-ER, increases in the follicular phase of the oestrous cycle and this may enhance the responsiveness of these cells to oestrogen and GnRH. We suggest that this may be due to increased pulsatile GnRH input rather than rising oestrogen levels. [source] Behavioral and electrophysiological effects of 5-HT in globus pallidus of 6-hydroxydopamine lesioned ratsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2010Shu-Jing Zhang Abstract Anatomical studies have shown that the globus pallidus receives abundant 5-hydroxytryptamine (5-HT) innervations from raphe nuclei. 5-HT may occupy an important position in the modulation of motor function through its affect on the activity of globus pallidus. In the present study, intrapallidal microinjection of 5-HT (0.1 mM) alone did not induce any motor behavior or postural asymmetry in the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats. However, when infused concomitantly with a low dose of 3, 4-dihydroxyphenylalanine (L-DOPA, 3 mg/kg i.p.), which itself can induce modest contralateral rotational behavior, 5-HT significantly potentiated the number of contralateral rotations. To elucidate the cellular mechanism, in vivo extracellular recordings were performed to examine the effects of 5-HT on globus pallidus neurons. In normal rats, the predominant effect of micropressure ejection of 5-HT on pallidal neurons was excitation. In 6-OHDA-lesioned rats, although 5-HT increased the firing rate in most pallidal neurons, 5-HT-induced inhibitory effects was stronger than that on the unlesioned side as well as normal rats. Furthermore, 5-HT1B receptors are mainly involved in 5-HT-induced excitation while 5-HT1A receptors are involved in 5-HT-induced inhibition. The results suggest that 5-HT may potentiate the antiparkinsonian effect of L-DOPA through modulating the activity of globus pallidus. © 2009 Wiley-Liss, Inc. [source] Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD-14,Toll-like receptor,nuclear factor ,B pathwayJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2003Jun-ichi Kido Objectives:, Calprotectin is a cytosolic protein with antibacterial action in leukocytes and its level increases in some inflammatory diseases, including periodontal diseases, rheumatoid arthritis and ulcerative colitis. Recently, we found that the lipopolysaccharide of Porphyromonas gingivalis (P-LPS) induced calprotectin release from human neutrophils. P-LPS, a major virulence factor of periodontal pathogens, is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor (TLR) and nuclear factor ,B (NF-,B). In the present study, we investigated whether calprotectin release by P-LPS is induced via the CD14,TLR,NF-,B pathway and the cellular mechanism of calprotectin release in human neutrophils. Material and methods:, Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, TLR2 and TLR4, or several inhibitors of NF-,B, microtubules and microfilaments, and then incubated with P-LPS. The calprotectin amount in the culture medium was determined using ELISA, and the nuclear extracts from cells were used for the examination of NF-,B binding activity using electrophoretic mobility shift assays. Results:, P-LPS increased calprotectin release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR2 antibodies, but not by two anti-TLR4 antibodies. NF-,B inhibitors suppressed P-LPS-induced NF-,B binding activity and calprotectin release. The inhibitors of microtubule and microfilament polymerization significantly decreased P-LPS-induced calprotectin release. Conclusion:, These results suggest that calprotectin release is induced by P-LPS via the CD14,TLR2,NF-,B signal pathway in human neutrophils and may be dependent on microtubule and microfilament systems. [source] Facilitation of Myocardial PI3K/Akt/nNOS Signaling Contributes to Ethanol-Evoked Hypotension in Female RatsALCOHOLISM, Issue 7 2009Mahmoud M. El-Mas Background:, The mechanism by which ethanol reduces cardiac output (CO) and blood pressure (BP) in female rats remains unclear. We tested the hypothesis that enhancement of myocardial phosphatidylinositol 3-kinase (PI3K)/Akt signaling and related neuronal nitric oxide synthase (nNOS) and/or endothelial nitric oxide synthase (eNOS) activity constitutes a cellular mechanism for the hemodynamic effects of ethanol. Methods:, We measured the level of phosphorylated eNOS (p-eNOS) and p-nNOS in the myocardium of ethanol (1 g/kg intragastric, i.g.) treated female rats along with hemodynamic responses [BP, CO, stroke volume, (SV), total peripheral resistance, (TPR)], and myocardial nitrate/nitrite levels (NOx) levels. Further, we investigated the effect of selective pharmacological inhibition of nNOS with N, -propyl- l -arginine (NPLA) or eNOS with N5 -(1-iminoethyl)- l -ornithine (l -NIO) on cellular, hemodynamic, and biochemical effects of ethanol. The effects of PI3K inhibition by wortmannin on the cardiovascular actions of ethanol and myocardial Akt phosphorylation were also investigated. Results:, The hemodynamic effects of ethanol (reductions in BP, CO, and SV) were associated with significant increases in myocardial NOx and myocardial p-nNOS and p-Akt expressions while myocardial p-eNOS remained unchanged. Prior nNOS inhibition by NPLA (2.5 or 12.5 ,g/kg) attenuated hemodynamic effects of ethanol and abrogated associated increases in myocardial NOx and cardiac p-nNOS contents. The hemodynamic effects of ethanol and increases in myocardial p-Akt phosphorylation were reduced by wortmannin (15 ,g/kg). On the other hand, although eNOS inhibition by l -NIO (4 or 20 mg/kg) in a dose-dependent manner attenuated ethanol-evoked hypotension, the concomitant reductions in CO and SV remained unaltered. Also, selective eNOS inhibition uncovered dramatic increases in TPR in response to ethanol, which appeared to have offset the reduction in CO. Neither NPLA nor l -NIO altered plasma ethanol levels. Conclusions:, These findings implicate the myocardial PI3K/Akt/nNOS signaling in the reductions in BP and CO produced by ethanol in female rats. [source] Stringent testing identifies highly potent and escape-proof anti-HIV short hairpin RNAsTHE JOURNAL OF GENE MEDICINE, Issue 6 2009Karin J. von Eije Abstract Background RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences. Methods In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone and also infected shRNA-expressing T cell lines with HIV-1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA-expressing T cells upon challenge with increasing dosages of HIV-1, and measured the dose required to result in massive virus-induced syncytia formation in this 2-week assay. Results Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co-transfection assays. The use of increased dosages of HIV-1 selected the same highly potent shRNAs as the laborious and extended escape study. Conclusions These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs. Copyright © 2009 John Wiley & Sons, Ltd. [source] Aerodigestive Tract Invasion by Well-Differentiated Thyroid Carcinoma: Diagnosis, Management, Prognosis, and BiologyTHE LARYNGOSCOPE, Issue 1 2006Judith Czaja McCaffrey MD Objectives/Hypothesis: 1) To describe the clinical entity invasive well-differentiated thyroid carcinoma (IWDTC), 2) to determine prognostic factors for survival in patients with IWDTC, 3) to describe and compare types of surgical resection to determine treatment efficacy, 4) to offer a staging system and surgical algorithm for management of patients with IWDTC, 5) to examine alterations in expression of E-cadherin and ,-catenin adhesion molecules in three groups of thyroid tissue and propose a cellular mechanism for invasion of the aerodigestive tract. Study Design: Basic science: quantification of expression of E-cadherin and ,-catenin in three groups of thyroid tissue. Clinical: retrospective review of patients with IWDTC surgically treated and followed over a 45-year time period. Methods: Basic science: immunohistochemical staining was used with antibodies against E-cadherin and ,-catenin in three groups of tissue: group 1, normal control thyroid tissue (n = 10); group 2, conventional papillary thyroid carcinoma (n = 20); group 3, IWDTC (n = 12). Intensity scores were given on the basis of protocol. One-way analysis of variance (ANOVA) was used to evaluate differences between groups. Post hoc ANOVA testing was completed. P < .05 was significant. Clinical: patients were divided into three surgical groups within the laryngotracheal subset: group 1, complete resection of gross disease (n = 34); group 2, shave excision (n = 75); group 3, incomplete excision (n = 15). Cox regression analysis was used to determine significance of prognostic factors. Kaplan-Meier plots were used to evaluate survival. P < .05 was significant. Results: Basic science: a significant difference between the three thyroid tissue groups for E-cadherin expression was demonstrated on one-way ANOVA testing. When controls were compared with either experimental group in post hoc ANOVA testing, differences between all groups were demonstrated (P < .001). For ,-catenin, the intensities of the three groups were not different by one-way ANOVA testing. Similar nonsignificant results were found on post hoc ANOVA testing. Clinical: there was a statistically significant difference in survival for patients with and without involvement of any portion of the endolarynx or trachea (P < .01). There was a significant difference among all three surgical groups when compared (P < .001). When complete and shave groups were compared with gross residual group there was a significant decrease in survival in incomplete resection group (P < .01). Cox regression analysis demonstrated invasion of larynx and trachea were significant prognostic factors for poor outcome. The type of initial resection was significant on multivariate analysis. Removal of all gross disease is a major factor for survival. Conclusions: Basic science: there is a decrease in membrane expression of E-cadherin in IWDTC, and loss of this tumor suppressor adhesion molecule may contribute to the invasive nature of well-differentiated thyroid carcinomas. Clinical: laryngotracheal invasion is a significant independent prognostic factor for survival. Patients undergoing shave excision had similar survival when compared with those undergoing radical tumor resection if gross tumor did not remain. Gross intraluminal tumor should be resected completely. Shave excision is adequate for minimal invasion not involving the intraluminal surfaces of the aerodigestive tract. [source] Developmental Expression of Aquaporin 2 in the Mouse Inner Ear ,THE LARYNGOSCOPE, Issue 11 2000Michele Merves Abstract Objectives The maintenance of endolymph homeostasis is critical for the inner ear to perform its functions of hearing and maintaining balance. The identification and cloning of aquaporins (a family of water channel proteins) has allowed the study of a novel cellular mechanism potentially involved in endolymph homeostasis. The objective of the present study was to define the developmental temporal and spatial e-pression pattern of aquaporin 2 (Aqp2) in the developing mouse inner ear. Study Design A systematic immunohistochemical study of Aqp2 protein e-pression was performed on embryonic mouse inner ears ranging from embryonic day 10 (otocyst stage) to embryonic day 18 (just before birth). Methods Serial cryosections of embryonic mouse inner ears were used for immunohistochemical e-periments. A rabbit polyclonal antisera raised against a synthetic Aqp2 peptide was used with a standard nickel intensified 3,3-diaminobenzidine reaction protocol for immunolocalization of Aqp2 in tissue sections. Results Aquaporin 2 is e-pressed diffusely in the early otocyst, then becomes progressively restricted as the inner ear matures. During early cochlear duct formation (embryonic days 12 and 13), e-pression of Aqp2 is homogeneous; later, it becomes restricted to specific regions of the endolymphatic compartment (embryonic days 15 and 18). Similar restriction of e-pression patterns could be noted for the vestibular structures. Endolymphatic duct and sac and stria vascularis e-pression of Aqp2 was noted to occur fairly late during development but demonstrated a distinct pattern of immunolabeling. Conclusions Aquaporin 2 shows an early and specific pattern of e-pression in the developing mouse inner ear, suggesting a significant role for this water channel protein in the development of endolymph homeostasis and meriting further functional studies of Aqp2 in the inner ear. [source] Insights into the cell of origin in breast cancer and breast cancer stem cellsASIA-PACIFIC JOURNAL OF CLINICAL ONCOLOGY, Issue 2 2010Geoffrey J LINDEMAN Abstract The precise cell types that give rise to tumors and mechanisms that underpin tumor heterogeneity are poorly understood. There is increasing evidence to suggest that diverse solid tumors are hierarchically organized and may be sustained by a distinct subpopulation of cancer stem cells (CSCs). The CSC hypothesis provides an attractive cellular mechanism that can account for the therapeutic refractoriness and dormant behavior exhibited by many tumor types. Breast cancer was the first solid malignancy from which CSCs were identified and isolated. Direct evidence for the CSC hypothesis has also recently emerged from mouse models of mammary tumorigenesis, although alternative models to explain heterogeneity also seem to apply. Our group has found that the luminal epithelial progenitor marker CD61/,3 integrin identified a CSC population in mammary tumors from MMTV- wnt-1 mice. However, no CSCs could be identified in the more homogeneous MMTV- neu/erbB2 model, suggesting an alternate (clonal evolution or stochastic) model of tumorigenesis. It seems likely that both paradigms of tumor propagation exist in human cancer. From a clinical perspective, the CSC concept has significant implications. Quiescent CSCs are thought to be more resistant to chemotherapy and targeted therapy. Enrichment of putative CSCs has been noted in studies of chemotherapy-treated patients, lending support to the CSC hypothesis and their potential role in chemoresistance. Although many unresolved questions on CSCs remain, ongoing efforts to identify and characterize CSCs continue to be an important area of investigation, with the potential to identify novel tumor targeting strategies. [source] 900 MHz modulated electromagnetic fields accelerate the clathrin-mediated endocytosis pathwayBIOELECTROMAGNETICS, Issue 3 2009Mihaela G. Moisescu Abstract We report new data regarding the molecular mechanisms of GSM-induced increase of cell endocytosis rate. Even though endocytosis represents an important physical and biological event for cell physiology, studies on modulated electromagnetic fields (EMF) effects on this process are scarce. In a previous article, we showed that fluid phase endocytosis rate increases when cultured cells are exposed to 900 MHz EMF similar to mobile phones' modulated GSM signals (217 Hz repetition frequency, 576 µs pulse width) and to electric pulses similar to the GSM electrical component. Trying to distinguish the mechanisms sustaining this endocytosis stimulation, we exposed murine melanoma cells to Lucifer Yellow (LY) or to GSM,EMF/electric pulses in the presence of drugs inhibiting the clathrin- or the caveolin-dependent endocytosis. Experiments were performed at a specific absorption rate (SAR) of 3.2 W/kg in a wire patch cell under homogeneously distributed EMF field and controlled temperature (in the range of 28.5,29.5 °C). Thus, the observed increase in LY uptake was not a thermal effect. Chlorpromazine and ethanol, but not Filipin, inhibited this increase. Therefore, the clathrin-dependent endocytosis is stimulated by the GSM,EMF, suggesting that the cellular mechanism affected by the modulated EMF involves vesicles that detach from the cell membrane, mainly clathrin-coated vesicles. Bioelectromagnetics 30:222,230, 2009. © 2008 Wiley-Liss, Inc. [source] Ecological correlates of body size in relation to cell size and cell number: patterns in flies, fish, fruits and foliageBIOLOGICAL REVIEWS, Issue 2 2007Jeff Arendt Abstract Body size is important to most aspects of biology and is also one of the most labile traits. Despite its importance we know remarkably little about the proximate (developmental) factors that determine body size under different circumstances. Here, I review what is known about how cell size and number contribute to phenetic and genetic variation in body size in Drosophila melanogaster, several fish, and fruits and leaves of some angiosperms. Variation in resources influences size primarily through changes in cell number while temperature acts through cell size. The difference in cellular mechanism may also explain the differences in growth trajectories resulting from food and temperature manipulations. There is, however, a poorly recognized interaction between food and temperature effects that needs further study. In addition, flies show a sexual dimorphism in temperature effects with the larger sex responding by changes in cell size and the smaller sex showing changes in both cell size and number. Leaf size is more variable than other organs, but there appears to be a consistent difference between how shade-tolerant and shade-intolerant species respond to light level. The former have larger leaves via cell size under shade, the latter via cell number in light conditions. Genetic differences, primarily from comparisons of D. melanogaster, show similar variation. Direct selection on body size alters cell number only, while temperature selection results in increased cell size and decreased cell number. Population comparisons along latitudinal clines show that larger flies have both larger cells and more cells. Use of these proximate patterns can give clues as to how selection acts in the wild. For example, the latitudinal pattern in D. melanogaster is usually assumed to be due to temperature, but the cellular pattern does not match that seen in laboratory selection at different temperatures. [source] Spleen neoangiogenesis in patients with myelofibrosis with myeloid metaplasiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2004Giovanni Barosi Summary Neoangiogenesis is an integral component of bone marrow myeloproliferation in patients with myelofibrosis with myeloid metaplasia (MMM). As extramedullary haematopoiesis is a constitutive feature of MMM, we studied spleen neoangiogenesis by a computerized image analysis in MMM patients. Compared with five normal subjects, spleen CD34-staining capillary vascular density (CVD) was 2·1,3·03 times higher than the upper range of normal in six of the 15 (40%) MMM patients. CD8-staining sinusoidal vascular density (SVD) was constantly normal or lesser than normal and was inversely correlated with CVD (R = ,0·53; P = 0·04). In MMM patients who did not receive cytoreductive or radiation therapy in the month before splenectomy (n = 9), the CVD was a significant determinant of spleen size (R = 0·88; P = 0·04). In MMM patients, the number of spleen CD34+ haematopoietic stem cells was increased from 1·2 to 98 times the upper limit of normal, and predicted the expansion of CVD (R = 0·57; P = 0·03). A population of cells expressing the CD34+/CD133+/VEGFR-2+ angiopoietic phenotype was present in the blood and spleen of five of seven patients. These results document that neoangiogenesis is an integral component of spleen re-localization of haematopoietic stem cells and suggest a cellular mechanism for spleen neoangiogenesis. [source] Control of muscle blood flow during exercise: local factors and integrative mechanismsACTA PHYSIOLOGICA, Issue 4 2010I. Sarelius Abstract Understanding the control mechanisms of blood flow within the vasculature of skeletal muscle is clearly fascinating from a theoretical point of view due to the extremely tight coupling of tissue oxygen demands and blood flow. It also has practical implications as impairment of muscle blood flow and its prevention/reversal by exercise training has a major impact on widespread diseases such as hypertension and diabetes. Here we analyse the role of mediators generated by skeletal muscle activity on smooth muscle relaxation in resistance vessels in vitro and in vivo. We summarize their cellular mechanisms of action and their relative roles in exercise hyperaemia with regard to early and late responses. We also discuss the consequences of interactions among mediators with regard to identifying their functional significance. We focus on (potential) mechanisms integrating the action of the mediators and their effects among the cells of the intact arteriolar wall. This integration occurs both locally, partly due to myoendothelial communication, and axially along the vascular tree, thus enabling the local responses to be manifest along an entire functional vessel path. Though the concept of signal integration is intriguing, its specific role on the control of exercise hyperaemia and the consequences of its modulation under physiological and pathophysiological conditions still await additional analysis. [source] Periostin promotes a fibroblastic lineage pathway in atrioventricular valve progenitor cellsDEVELOPMENTAL DYNAMICS, Issue 5 2009Russell A. Norris Abstract Differentiation of prevalvular mesenchyme into valve fibroblasts is an integral step towards the development of functionally mature cardiac valves. Although clinically relevant, little is known regarding the molecular and cellular mechanisms by which this process proceeds. Genes that are regulated in a spatio-temporal pattern during valve remodeling are candidates for affecting this differentiation process. Based on its expression pattern, we have focused our studies on the role of the matricellular gene, periostin, in regulating the differentiation of cushion mesenchymal cells into valve fibroblasts. Herein, we demonstrate that periostin expression is coincident with and regulates type I collagen protein production, a major component of mature valve tissue. Adenoviral-mediated knock-down of periostin in atrioventricular mesenchyme resulted in a decrease in collagen I protein expression and aberrant induction of myocyte markers indicating an alteration in AV mesenchyme differentiation. In vitro analyses using a novel "cardiotube" assay further demonstrated that expression of periostin regulates lineage commitment of valve precursor cells. In these cells, expression of periostin and collagen I are regulated, in part, by TGF,-3. We further demonstrate that TGF,-3, through a periostin/collagen pathway, enhances the viscoelastic properties of AV cushion tissue surface tension and plays a crucial role in regulating valve remodeling. Thus, data presented here demonstrate that periostin, a TGF,-3 responsive gene, functions as a crucial mediator of chick AV valve maturation via promoting mesenchymal-to-fibroblast differentiation while blocking differentiation of alternative cell types (myocytes). Developmental Dynamics 238:1052,1063, 2009. © 2009 Wiley-Liss, Inc. [source] Morphogenesis of the node and notochord: The cellular basis for the establishment and maintenance of left,right asymmetry in the mouseDEVELOPMENTAL DYNAMICS, Issue 12 2008Jeffrey D. Lee Abstract Establishment of left,right asymmetry in the mouse embryo depends on leftward laminar fluid flow in the node, which initiates a signaling cascade that is confined to the left side of the embryo. Leftward fluid flow depends on two cellular processes: motility of the cilia that generate the flow and morphogenesis of the node, the structure where the cilia reside. Here, we provide an overview of the current understanding and unresolved questions about the regulation of ciliary motility and node structure. Analysis of mouse mutants has shown that the motile cilia must have a specific structure and length, and that they must point posteriorly to generate the necessary leftward fluid flow. However, the precise structure of the motile cilia is not clear and the mechanisms that position cilia on node cells have not been defined. The mouse node is a teardrop-shaped pit at the distal tip of the early embryo, but the morphogenetic events that create the mature node from cells derived from the primitive streak are only beginning to be characterized. Recent live imaging experiments support earlier scanning electron microscopy (SEM) studies and show that node assembly is a multi-step process in which clusters of node precursors appear on the embryo surface as overlying endoderm cells are removed. We present additional SEM and confocal microscopy studies that help define the transition stages during node morphogenesis. After the initiation of left-sided signaling, the notochordal plate, which is contiguous with the node, generates a barrier at the embryonic midline that restricts the cascade of gene expression to the left side of the embryo. The field is now poised to dissect the genetic and cellular mechanisms that create and organize the specialized cells of the node and midline that are essential for left,right asymmetry. Developmental Dynamics 237:3464,3476, 2008. © 2008 Wiley-Liss, Inc. [source] Anteroposterior patterning in the limb and digit specification: Contribution of mouse geneticsDEVELOPMENTAL DYNAMICS, Issue 9 2006Benoît Robert Abstract The limb has been a privileged object of investigation and reflection for scientists over the past two centuries and continues to provide a heuristic framework to analyze vertebrate development. Recently, accumulation of new data has significantly changed our view on the mechanisms of limb patterning, in particular along the anterior-posterior axis. These data have led us to revisit the mode of action of the zone of polarizing activity. They shed light on the molecular and cellular mechanisms of patterning linked to the Shh-Gli3 signaling pathway and give insights into the mechanism of activation of these cardinal factors, as well as the consequences of their activity. These new data are in good part the result of systematic Application of tools used in contemporary mouse molecular genetics. These have extended the power of mouse genetics by introducing mutational strategies that allow fine-tuned modulation of gene expression, interchromosomal deletions and duplication. They have even made the mouse embryo amenable to cell lineage analysis that used to be the realm of chick embryos. In this review, we focus on the data acquired over the last five years from the analysis of mouse limb development and discuss new perspectives opened by these results. Developmental Dynamics 235:2337,2352, 2006. © 2006 Wiley-Liss, Inc. [source] Dopamine modulation of the In vivo acetylcholine response in the Drosophila mushroom bodyDEVELOPMENTAL NEUROBIOLOGY, Issue 11 2009Vitold Tsydzik Abstract Olfactory sensory information in Drosophila is transmitted through antennal lobe projections to Mushroom Body neurons (Kenyon cells) by means of cholinergic synapses. Application of acetylcholine (ACh) and odors produce significant increases in intracellular calcium ([Ca2+]i) in these neurons. Behavioral studies show that Kenyon cell activity is modulated by dopaminergic inputs and this modulation is thought to be the basis for an olfactory conditioned response. However, quantitative assessment of the synaptic inputs to Kenyon cells is currently lacking. To assess neuronal activity under in vivo conditions, we have used the endogenously-expressed camgaroo reporter to measure [Ca2+]i in these neurons. We report here the dose-response relationship of Kenyon cells for ACh and dopamine (DA). Importantly, we also show that simultaneous application of ACh and DA results in a significant decrease in the response to ACh alone. In addition, we show inhibition of the ACh response by cyclic adenosine monophosphate. This is the first quantitative assessment of the effects of these two important transmitters in this system, and it provides an important basis for future analysis of the cellular mechanisms of this well established model for associative olfactory learning. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] Reactive species and early manifestation of insulin resistance in type 2 diabetesDIABETES OBESITY & METABOLISM, Issue 2 2006L. E. Fridlyand The early stages of type 2 diabetes mellitus are characterized by the development of insulin resistance (IRe) in muscle cells and adipocytes with the concomitant loss of ,-cell compensation. We have extensively reviewed the literature related to metabolic and signalling pathways of reactive oxygen species (ROS) in regard to the coordinated development of oxidative stress and IRe. We considered the hypothesis that oxidative stress leads to IRe in muscle cells and adipocytes, but found that the data are more consistent with the hypothesis that the cellular mechanisms that protect against oxidative stress per se are capable of creating an ROS-dependent insulin-resistant state. Furthermore, ROS-induced mitochondrial dysfunction can lead to disruptions of lipid metabolism, increasing the intracellular lipid content, and, in addition, contribute to lipid-dependent IRe in myocytes. Together, these two ROS-activated pathways to IRe can contribute to a global state of profound resistance to insulin action. Therapeutic strategies should, therefore, be directed towards reducing insulin resistance without an increase in ROS production or concentration. Pharmacological or other approaches to IRe that result in the activation of mitochondrial biogenesis in particular could be highly beneficial in the prevention or treatment of both insulin resistance and type 2 diabetes. [source] | |||||||||||||||||||||||||||||||||||||