Destruction of the ventral noradrenergic pathway elicited by administration of 6-hydroxydopamine (6-OHDA, 5 micrograms into each side of the ventral pons) reduced the content of norepinephrine (NE) in the anterior hypothalamus (-80%) and... more
Destruction of the ventral noradrenergic pathway elicited by administration of 6-hydroxydopamine (6-OHDA, 5 micrograms into each side of the ventral pons) reduced the content of norepinephrine (NE) in the anterior hypothalamus (-80%) and induced an increase in arterial blood pressure (ABP) and in heart rate. These hypertensive rats, showed hypersensitivity to the hypotensive effect of NE (0.5-2 micrograms) and clonidine (0.75-1.5 micrograms) administered into the anterior hypothalamic preoptic (AH/PO) region. Methysergide (1-2 micrograms) and, to a lesser extent, ketanserin (1-2 micrograms) administered into the anterior hypothalamic preoptic region also reduced the arterial blood pressure in these rats treated with 6-OHDA. Bilateral administration of 5,7-dihydroxytryptamine (5,7-DHT, 8 micrograms) into the median forebrain bundle decreased the content of serotonin (5-HT) in the hypothalamus (-85%) without change in arterial blood pressure but largely prevented the development of hypertension after treatment with 6-OHDA in the ventral pons. These results suggest that neurogenic hypertension is produced after the removal of NE tonic depressor activity in the anterior hypothalamus and that serotonergic mechanisms play a major role in the development of the increased arterial blood pressure in this preparation.
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Epithelial tight junctions form a regulated barrier that seals the paracellular space and prevents mixing of luminal contents with the interstitium. This barrier is composed of a group of proteins including the putative... more
Epithelial tight junctions form a regulated barrier that seals the paracellular space and prevents mixing of luminal contents with the interstitium. This barrier is composed of a group of proteins including the putative "sealing" protein occludin that appears to bind directly to a cytoplasmic junction protein, ZO-1. To study the interaction and regulation of these two components when paracellular integrity is altered, we assessed protein expression and immunofluorescent (IF) localization of ZO-1 and occludin in a rat model of hepatocyte tight junction damage induced by common bile duct ligation (CBDL). Protein levels were detected in liver by immunoblotting and IF localization by 3-dimensional reconstruction of serial 0.5-micron confocal microscopic optical sections. As previously described, ZO-1 protein levels progressively increased to threefold control levels 9 days after CBDL. In contrast, occludin protein levels decreased by 50% within 2 days after CBDL and returned to control values by 9 days. In control IF sections, ZO-1 and occludin colocalized, forming thin continuous staining outlining canaliculi. After CBDL, ZO-1 staining appeared discontinuous, and a punctate pericanalicular accumulation of signal developed around junctional areas. Occludin staining was also discontinuous after CBDL, but, in contrast to ZO-1, was not punctate and remained localized either in a linear fashion along canalicular margins or in a homogeneous fashion in immediately surrounding areas. CBDL results in changes in the expression and localization of the putative tight junction sealing protein occludin in hepatocytes that are distinct from those observed for the peripheral membrane tight junction protein ZO-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Research Interests: Physiology, Biology, Membrane Proteins, Immunohistochemistry, Cell Biology, and 15 moreMedicine, Liver, Animals, Male, Medical Physiology, Rats, Occludin, Base Sequence, Tight Junction, Biochemistry and cell biology, Molecular Sequence Data, Molecular probes, Tissue distribution, Ligation, and Common bile duct
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Introduction and methods: Olfactory epithelium (OE) consists of multiple cell types such as olfactory neurons, supporting cells, gland cells and basal cells and is derived from nasal placodes. Six1, a member of Six homeobox gene family,... more
Introduction and methods: Olfactory epithelium (OE) consists of multiple cell types such as olfactory neurons, supporting cells, gland cells and basal cells and is derived from nasal placodes. Six1, a member of Six homeobox gene family, is first expressed in the nasal placodes and continues to be expressed throughout the OE development in mice. To understand how Six1 is involved in OE development, we analyzed Six1-deficient homozygous mice (Six1) by in situ hybridization with probes of various olfactory neuron marker genes as well as by immunohistochemistry. We also assessed the effects of Six1 on neural differentiation using P19EC cells. Results: Six1 showed defects in differentiation of olfactory neurons, supporting cells and gland cells. The expression of proneural marker Mash1 was unchanged, while those of Ngn1, NeuroD and Lhx2 were severely reduced at E10.5. Olfactory neuron specific marker protein OMP was missing in the Six1 at E16.5. The expression of Six1 was unaltered in the olfactory epithelium of Mash1 mice, indicating that Six1 and Mash1 regulate OE differentiation independently. The expressions of Hes1 and Hes5 in OE were augmented in the Six1 at E10.5. No supporting cells were observed in Six1 at E18.5. The expression of Ngn1, Mash1 and Hes1 upon neural differentiation induction of P19 cells was altered when Six1 was constitutively overexpressed. Discussion: Six1 is involved in differentiation of olfactory neuron and supporting cells in OE through regulation of Ngn1, NeuroD, Lhx2, Hes1 and Hes5.
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Research Interests: Molecular Biology, Fluorescence Microscopy, Biology, Medicine, Biological Sciences, and 15 moreDogs, Cell line, Animals, Epithelium, Human Fibroblasts, Electric Impedance, Adherens junctions, Immunoblotting, Cell Membrane, fibroblasts, Adherens Junction, CHO cells, Cricetinae, coculture techniques, and Medical and Health Sciences
Research Interests: Biology, Membrane Proteins, Cell Biology, Biological Chemistry, Medicine, and 15 moreBiological Sciences, Dogs, Cell line, Animals, CHEMICAL SCIENCES, Neutrophils, Fluorescent Antibody Technique, Epithelial cells, Occludin, Electric Impedance, Tight Junction, Claudin, Tight Junctions, Cell Membrane, and Medical and Health Sciences
Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High concentrations of ethanol trigger mucosal hyperregeneration, disrupt cell adhesion, and increase the sensitivity to carcinogens. Most of these... more
Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High concentrations of ethanol trigger mucosal hyperregeneration, disrupt cell adhesion, and increase the sensitivity to carcinogens. Most of these effects are thought to be mediated by acetaldehyde, a genotoxic metabolite produced from ethanol by alcohol dehydrogenases. Here, we studied the role of low ethanol concentrations, more likely to mimic those found in the intestine in vivo, and used intestinal cells lacking alcohol dehydrogenase to identify the acetaldehyde-independent biological effects of ethanol. Under these conditions, ethanol did not stimulate the proliferation of nonconfluent cells, but significantly increased maximal cell density. Incorporation of phosphatidylethanol, produced from ethanol by phospholipase D, was instrumental to this effect. Phosphatidylethanol accumulation induced claudin-1 endocytosis and disrupted the claudin-1/ZO-1 association. The resulting nuclear translocation of ZONAB was shown to mediate the cell density increase in ethanol-treated cells. In vivo, incorporation of phosphatidylethanol and nuclear translocation of ZONAB correlated with increased proliferation in the colonic epithelium of ethanol-fed mice and in adenomas of chronic alcoholics. Our results show that phosphatidylethanol accumulation after chronic ethanol exposure disrupts signals that normally restrict proliferation in highly confluent intestinal cells, thus facilitating abnormal intestinal cell proliferation. (Mol Cancer Res 2007;5(11):1147–57)
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Research Interests: Endocrinology, Chemistry, Life Sciences, Medicine, Internal Medicine, and 15 moreFemale, Animals, Male, Acetylcholine, Rats, Stimulation, Gastrointestinal motility, Receptor, Hydrogen-Ion Concentration, Biochemistry and cell biology, Ileum, binding sites, Pharmacology and pharmaceutical sciences, In Vitro Techniques, and Electric stimulation
Cells from transporting epithelia have two basic properties: 1) a plasma membrane divided into an apical and a basolateral domain that confers vectoriality, and 2) tight junctions that transform epithelia into diffusion barriers. The... more
Cells from transporting epithelia have two basic properties: 1) a plasma membrane divided into an apical and a basolateral domain that confers vectoriality, and 2) tight junctions that transform epithelia into diffusion barriers. The expression of both properties depends on extracellular Ca2+.
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A low molecular weight substance which behaves like ouabain as inhibitor of brain membrane Na-K-ATPase and 3H-ouabain binding was found in plasma after saline expansion of extracellular fluid or angiotensin II infusion into the third... more
A low molecular weight substance which behaves like ouabain as inhibitor of brain membrane Na-K-ATPase and 3H-ouabain binding was found in plasma after saline expansion of extracellular fluid or angiotensin II infusion into the third brain ventricle in the rat. Intracerebroventricular infusion of angiotensin II antagonist, saralasin, blocks the increase of the Na-K-ATPase inhibitor produced by infusion of angiotensin II into the third ventricle or extracellular fluid saline expansion.
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The paracellular pathway is an aqueous, extracellular route across endothelia and epithelia that is followed by substances according to their size and charge. Although under certain circumstances it may be traversed by objects as large as... more
The paracellular pathway is an aqueous, extracellular route across endothelia and epithelia that is followed by substances according to their size and charge. Although under certain circumstances it may be traversed by objects as large as leukocytes, it is generally used by water and small solutes. Under normal circumstances, proteins can only permeate through endothelia and a few types of epithelia, such as the glomerular and choroidal ones (Renkin and Gilmore, 1973; Kluge et al., 1986). It is limited by a tight junction (TJ) placed at the outermost end of the intercellular space (Fig. 1). Since this structure was first—and for many years—studied in epithelia like the frog skin and the urinary bladder, where it almost completely obstructs the passage of all substances, it is usually called a “tight” or “occluding” junction. In epithelia that separate two compartments with different compositions (e.g., the frog skin, the colon mucosa, the kidney collecting tubule), the TJ is in fact very restrictive and the amount of substances flowing through the paracellular pathway is almost negligible. On the contrary, in epithelia separating two compartments with similar ionic composition, and that have to translocate a relatively large volume of fluid in a short time (e.g., the kidney proximal tubule, the gallbladder mucosa), the TJ is quite leaky and the paracellular pathway may account for up to 90% of the transepithelial flux. When the TJ is so leaky, the rate of permeation through the paracellular pathway may also be restricted by the narrowness and tortuosity of the intercellular space, as well as by the composition of the extracellular matrix (Cereijido, 1991).
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Supplementary Figures S1-S6 from Phosphatidylethanol Accumulation Promotes Intestinal Hyperplasia by Inducing ZONAB-Mediated Cell Density Increase in Response to Chronic Ethanol Exposure
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<p>In LNT.shGFP treated eyes (<b>A</b>), the RPE monolayer lies on Bruch's membrane, has apical microvilli towards the photoreceptor outer segments the cells are interconnected by tight junctions... more
<p>In LNT.shGFP treated eyes (<b>A</b>), the RPE monolayer lies on Bruch's membrane, has apical microvilli towards the photoreceptor outer segments the cells are interconnected by tight junctions (<b>A,</b> black arrow. <b>B</b>, white arrows). In eyes either with depleted levels of ZO-1 (<b>C</b>) or overexpressing ZONAB (<b>D</b>), the RPE monolayer was highly disorganised with a marked loss of the epithelial monolayer characteristics, areas of RPE cells located on top of each other (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015730#pone-0015730-g006" target="_blank">Figure 6</a>: RPE pyknosis) and accumulation of extracellular debris was also seen (<b>C,</b> asterisk). RPE cells appeared flattened and elongated with absent microvilli (black arrows indicate the flat apical side of the cell), reduced basal infoldings, and mesenchymal-like morphology. In addition, numerous vacuoles were present within the cells (<b>D,</b> asterisk). In areas adjacent to RPE breaks (<b>E</b>), the RPE retained some of its epithelial characteristics, such as, microvilli present on the apical membrane and intracellular basal infoldings. However, melanin vesicle maturation was defective (asterisks). R, RPE cell nuclei. OS, photoreceptor outer segments. Mv, microvilli. BM, Bruch's membrane. BI, basal infoldings. Ph, phagosome. Size bar, 1 µm (except in <b>B</b>, 200 nm). n = 4 per treatment group.</p
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ur na l o f C el l B io lo gy
ZO-1 is a peripheral membrane protein of approximately 225 kDa located on the cytoplasmic side of all tight junctions. ZO-1 cDNA sequencing disclosed the presence of a 240-bp sequence in only some of the ZO-1 cDNAs studied. This 240-bp... more
ZO-1 is a peripheral membrane protein of approximately 225 kDa located on the cytoplasmic side of all tight junctions. ZO-1 cDNA sequencing disclosed the presence of a 240-bp sequence in only some of the ZO-1 cDNAs studied. This 240-bp region encoded an inframe insertion of 80 amino acids, named motif-alpha. Expression of the predicted transcripts in normal rat and human tissues and in human epithelial cell lines (Caco-2, T84, Hep G2) was shown by reverse transcription of RNA and then DNA amplification. Immunoblot analysis showed both protein isoforms were present; however, in different cell lines, their amounts differed markedly relative to each other. Immunolocalization at light and ultrastructural levels, using antibodies generated against motif-alpha or shared sequences flanking it, indicated both forms localized indistinguishably to tight junctions. These observations demonstrate the existence and variable expression of ZO-1 isoforms and raise the question whether these isoform...
Research Interests: Physiology, Molecular Biology, Biology, Membrane Proteins, Transmission Electron Microscopy, and 15 moreMedicine, Cell line, Humans, Animals, American, Medical Physiology, Fluorescent Antibody Technique, Amino Acid Sequence, Base Sequence, Cytoplasm, Tight Junction, Biochemistry and cell biology, Isomerism, Molecular Sequence Data, and Tissue distribution
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The bilateral destruction of the ventral noradrenergic pathway induced by 6-hydroxydopamine (6-OHDA) administration into the ventral pons led to an increase in arterial blood pressure (ABP) and norepinephrine depletion in the amygdaloid... more
The bilateral destruction of the ventral noradrenergic pathway induced by 6-hydroxydopamine (6-OHDA) administration into the ventral pons led to an increase in arterial blood pressure (ABP) and norepinephrine depletion in the amygdaloid complex, nucleus accumbens, septal area and olfactory bulb. Specific angiotensin converting enzyme (ACE) activity was significantly increased only in the amygdaloid complex (Control: 4.56 +/- 0.95; Vehicle: 4.08 +/- 1.07; 6-OHDA: 11.76 +/- 1.84). A significant correlation between arterial blood pressure and specific ACE activity levels in the amygdaloid complex was observed (r: 0.775; p less than 0.002). These results suggest that an increase in specific ACE activity of the amygdaloid complex after norepinephrine depletion could play a role in the development of hypertension in this model.
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Formation and maintenance of tissue barriers require the coordination of cell mechanics and cell–cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to... more
Formation and maintenance of tissue barriers require the coordination of cell mechanics and cell–cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 disrupted junction assembly and morphogenesis in an ECM stiffness-dependent manner and led to a stiffness-dependant reorganisation of active myosin. Both junction formation and morphogenesis were rescued by inhibition of actomyosin contractility. ZO-1 depletion also impacted mechanical tension at cell-matrix and E-cadherin-based cell–cell adhesions. The effect on E-cadherin also depended on ECM stiffness and correlated with effects of ECM stiffness on actin cytoskeleton organisation. However, ZO-1 knockout also revealed tension-independent functions of ZO-1. ZO-1-deficient cells could assemble functional barriers ...
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Angiotensin converting enzyme was detectable in rat pineal gland and exhibited a circadian rhythm in activity with maximum at the end of the light phase of daily photoperiod. Superior cervical ganglionectomy (SCGx) or exposure to light... more
Angiotensin converting enzyme was detectable in rat pineal gland and exhibited a circadian rhythm in activity with maximum at the end of the light phase of daily photoperiod. Superior cervical ganglionectomy (SCGx) or exposure to light for 6 days increased enzyme activity and obliterated morning-evening differences, whereas injection of the beta-agonist isoproterenol depressed the high values observed in SCGx animals. These results indicate that angiotensin converting enzyme in the pineal gland is under negative control by the norepinephrine released from pineal sympathetic nerves.
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<p>MDA-MB-231 cells were plated on the indicated 2D matrices and migration was analyzed by time-lapse microscopy as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050188#pone-0050188-g002"... more
<p>MDA-MB-231 cells were plated on the indicated 2D matrices and migration was analyzed by time-lapse microscopy as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050188#pone-0050188-g002" target="_blank">figure 2</a> for 5 hours. In panels B and C, the cells had been transfected with the indicated siRNAs. All quantifications show means ± 1SD of four different fields (12 cells were tracked for each field). Note, only migration on collagen and Matrigel is p114RhoGEF-dependent. Bar, 30 µm.</p
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Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are... more
Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. Here we show that p114RhoGEF/ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating protein kinase A (PKA) signaling, and is required for PKA-induced actomyosin remodeling, cAMP-responsive element binding protein (CREB)-driven gene expression of proteins requ...
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<p>MDA-MB-231 cells were plated on the indicated 2D matrices and migration was analyzed by time-lapse microscopy as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050188#pone-0050188-g002"... more
<p>MDA-MB-231 cells were plated on the indicated 2D matrices and migration was analyzed by time-lapse microscopy as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050188#pone-0050188-g002" target="_blank">figure 2</a> for 5 hours. In panels B and C, the cells had been transfected with the indicated siRNAs. All quantifications show means ± 1SD of four different fields (12 cells were tracked for each field). Note, only migration on collagen and Matrigel is p114RhoGEF-dependent. Bar, 30 µm.</p
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Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are... more
Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. Here we show that p114RhoGEF/ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating protein kinase A (PKA) signaling, and is required for PKA-induced actomyosin remodeling, cAMP-responsive element binding protein (CREB)-driven gene expression of proteins requ...