School of Biomedical Sciences Publications - UQ eSpace

An automatic image based single dilution method for end point titre quantitation of antinuclear antibodies tests using HEp-2 cells

2012-01-31T00:00:00Z

Indirect Immunofluorescence (IIF) on Human epithelial (HEp-2) cells test has been the golden standard for identifying the presence of Anti-Nuclear Antibodies (ANA) due to its high sensitivity and the large range of antigens that can be detected. Furthermore, IIF ANA test allows the positive sample strength (sample end point titre) to be reported. Despite its advantages, the IIF ANA test needs to be performed manually, and therefore it is perceived as an expensive and laborious process. This also applies to determining the strength of positive samples (end point titre) which traditionally is done by serially diluting the specimen. In this paper, we present an image-based method which is able to automatically determine the end point titre of positive samples based only on a single screening dilution. This can be done by simulating the manual titration process using a mathematical model of the exposure-density curve. Technically, a new Image Titration Endpoint (ITE) unit based on the model is introduced. Each specimen image is then measured in terms of this unit. Finally, the end point titre for the specimen is determined through a standard curve which specifies the end point titre given an ITE unit. This process is fully automated which would give an advantage over the current digital titration methods. The overall endpoint titre agreement between the proposed approach and the manual serial dilution method in the evaluation of 134 positive samples was 100%. This high agreement demonstrates that the proposed approach is suitable for routine ANA IIF testing in the clinical settings.

Ancient colour vision: Multiple opsin genes in the ancestral vertebrates

2007-08-15T00:00:00Z

Molecular investigation of the origin of colour vision has discovered five visual pigment (opsin) genes, all of which are expressed in an agnathan (jawless) fish, the lamprey Geotria australis. Lampreys are extant representatives of an ancient group of vertebrates whose origins are thought to date back to at least the early Cambrian, approximately 540 million years ago [1.]. Phylogenetic analysis has identified the visual pigment opsin genes of G. australis as orthologues of the major classes of vertebrate opsin genes. Therefore, multiple opsin genes must have originated very early in vertebrate evolution, prior to the separation of the jawed and jawless vertebrate lineages, and thereby provided the genetic basis for colour vision in all vertebrate species. The southern hemisphere lamprey Geotria australis (Figure 1A,B) possesses a predominantly cone-based visual system designed for photopic (bright light) vision [2. S.P. Collin, I.C. Potter and C.R. Braekevelt, The ocular morphology of the southern hemisphere lamprey Geotria australis Gray, with special reference to optical specializations and the characterisation and phylogeny of photoreceptor types. Brain Behav. Evol. 54 (1999), pp. 96–111.2. and 3.]. Previous work identified multiple cone types suggesting that the potential for colour vision may have been present in the earliest members of this group. In order to trace the molecular evolution and origins of vertebrate colour vision, we have examined the genetic complement of visual pigment opsins in G. australis.

An easy, cheap reliable method of monitoring coral bleaching

2007-08-23T00:00:00Z

An evaluation of potential mechanism-based inactivation of human drug metabolizing cytochromes P450 by monoamine oxidase inhibitors, including isoniazid

2007-08-15T00:00:00Z

To characterize potential mechanism-based inactivation (MBI) of major human drug-metabolizing cytochromes P450 (CYP) by monoamine oxidase (MAO) inhibitors, including the antitubercular drug isoniazid. Human liver microsomal CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities were investigated following co- and preincubation with MAO inhibitors. Inactivation kinetic constants (K-I and k(inact)) were determined where a significant preincubation effect was observed. Spectral studies were conducted to elucidate the mechanisms of inactivation. Hydrazine MAO inhibitors generally exhibited greater inhibition of CYP following preincubation, whereas this was less frequent for the propargylamines, and tranylcypromine and moclobemide. Phenelzine and isoniazid inactivated all CYP but were most potent toward CYP3A and CYP2C19. Respective inactivation kinetic constants (K-I and k(inact)) for isoniazid were 48.6 mu M and 0.042 min(-1) and 79.3 mu M and 0.039 min(-1). Clorgyline was a selective inactivator of CYP1A2 (6.8 mu M and 0.15 min(-1)). Inactivation of CYP was irreversible, consistent with metabolite-intermediate complexation for isoniazid and clorgyline, and haeme destruction for phenelzine. With the exception of phenelzine-mediated CYP3A inactivation, glutathione and superoxide dismutase failed to protect CYP from inactivation by isoniazid and phenelzine. Glutathione partially slowed (17%) the inactivation of CYP1A2 by clorgyline. Alternate substrates or inhibitors generally protected against CYP inactivation. These data are consistent with mechanism-based inactivation of human drug-metabolizing CYP enzymes and suggest that impaired metabolic clearance may contribute to clinical drug-drug interactions with some MAO inhibitors.

An evaluation of training procedures for casual demonstrators in anatomy laboratory classes

2011-10-24T00:00:00Z

A new category of eye movements in a small fish

Fritsches, KA og Marshall, J — 2007-08-13T00:00:00Z

A new GLT1 splice variant: cloning and immunolocalization of GLT1c in the mammalian retina and brain

2008-01-25T00:00:00Z

We have identified a novel carboxyl-terminal splice-variant of the glutamate transporter GLT1, which we denote as GLT1c. Within the rat brain only low levels of protein and message were detected, protein expression being restricted to end feet of astrocytes apposed to blood vessels or some astrocytes adjacent to the ventricles. Conversely, within the retina, this variant was selectively and heavily expressed in the synaptic terminals of both rod- and cone-photoreceptors in both humans and rats. Double-immunolabelling with antibodies to the carboxyl region of GLT1b/GLT1v, which is strongly expressed in apical dendrites of bipolar cells and in cone photoreceptors revealed that in the rat GLT1c was co-localised with GLT1b/GLT1v in cone photoreceptors but not with GLT1b/GLT1v in bipolar cells. GLT1c expression was developmentally regulated, only appearing at around postnatal day 7 in the rat retina, when photoreceptors first exhibit a dark current. Since the glutamate transporter EAAT5 is also expressed in terminals of rod photoreceptor terminals these data indicate that rod photoreceptors express two glutamate transporters with distinct properties. Similarly, cone photoreceptors express two glutamate transporters. We suggest that differential usage of these transporters by rod and cone photoreceptors may influence the kinetics of glutamate transmission by these neurons. (C) 2004 Elsevier Ltd. All rights reserved.

A new insight of mechanisms, diagnosis and treatment of diabetic cardiomyopathy

Zhang, Xinli og Chen, Chen — 2012-05-25T15:01:22Z

Diabetes mellitus is one of the most common chronic diseases across the world. Cardiovascular complication is the major morbidity and mortality among the diabetic patients. Diabetic cardiomyopathy, a new entity independent of coronary artery disease or hypertension, has been increasingly recognized by clinicians and epidemiologists. Cardiac dysfunction is the major characteristic of diabetic cardiomyopathy. For a better understanding of diabetic cardiomyopathy and necessary treatment strategy, several pathological mechanisms such as impaired calcium handling and increased oxidative stress, have been proposed through clinical and experimental observations. In this review, we will discuss the development of cardiac dysfunction, the mechanisms underlying diabetic cardiomyopathy, diagnostic methods, and treatment options.

A new marker for the meningeo-glial network: a two pore domain K channel TASK1

2009-05-18T00:00:00Z

A new model of development of the mammalian ovary and follicles

2013-05-10T19:03:54Z

Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.

A New Rig for Standardized Craniofacial Photography Put to the Test

2009-01-19T00:00:00Z

This article describes and tests a photography rig that has been built at the University of Melbourne, Australia, specifically for the purpose of taking rapid and highly standardized craniofacial photographs, in simultaneous views of front and profile. The rig uses a novel projected light range-finding system that has been designed for easy and accurate positioning of subjects, in the natural head position, at precise distances from the frontal camera. Results of experiments examining the intraobserver error of multiple photographs taken on the rig indicate that high-quality, repeatable photographs can be taken after a reasonably large amount of time has lapsed between photography sessions (e.g., 30 days). This study also indicates that some variability remains between photographs even when highly standardized protocols are followed. Consequently, it is expected that the variation between photographs with limited standardization is much larger and more likely to cause significant errors in any comparisons.

A new small molecule C5a receptor antagonist inhibits the reverse-passive arthus reaction and endotoxic shock in rats

2007-08-13T00:00:00Z

C5a is implicated as a pathogenic factor in a wide range of immunoinflammatory diseases, including sepsis and immune complex disease, Agents that antagonize the effects of C5a could be useful in these diseases. We have developed some novel C5a antagonists and have determined the acute anti-inflammatory properties of a new small molecule C5a receptor antagonist against C5a- and LPS-induced neutrophil adhesion and cytokine expression, as well as against some hallmarks of the reverse Arthus reaction in rats. We found that a single i.v. dose (1 mg/kg) of this antagonist inhibited both C5a- and LPS-induced neutropenia and elevated levels of circulating TNF-alpha, as well as polymorphonuclear leukocyte migration, increased TNF-alpha levels and vascular leakage at the site of immune complex deposition. These results indicate potent anti-inflammatory activities of a new C5a receptor antagonist and provide more evidence for a key early role for C5a in sepsis and the reverse Arthus reaction. The results support a role for antagonists of C5a receptors in the therapeutic intervention of immunoinflammatory disease states such as sepsis and immune complex disease.

A new species of the genus Pupinella (Mollusca: Pupinidae) from Taiwan

Ueng, Y. T. og Chiou, Tsyr-.Huei — 2009-12-09T00:00:00Z

Shellfish Family Name: Gastropoda (Gastropoda) of the bean snail Branch (Pupinidae). The kinds of type specimens in Kaohsiung County's Taoyuan Township, Mei Village (elevation about 1,700 meters) were taken, at present only in Kaohsiung County's Taoyuan Township, Mei Village to adopt to this new species, found in damp moss at the wet while. Pupinella （ Pupinopsis ） masuhoearuensis n. sp. Pupinella （ Pupinopsis ） swinhoei H. Adams, 1866. Pupinella morrisonia H. Adams. Pupinella merdionalis Schmacker et Boettger, 1891. Pupinella adamsi Sowerby, 1878. In this paper, shell characteristics, identified as new species - Meishan bean snail Pupinella (Pupinopsis) masuhoearuensis n. sp., Produced in Taiwan with Taiwan bean snail Pupinella (Pupinopsis) swinhoei H. Adams, 1866 compared to appearance, but also referred to as Yu-Shan bean snail Pupinella morrisonia H. Adams, golden bean snail Pupinella merdionalis Schmacker et Boettger, 1891, Adams bean snail Pupinella adamsi Sowerby, 1878, and other types of names. (10.5x5.1 mm) NMMNS004284001. NMMNS004284002 。 Gold-plated type specimen (10.5x5.1 mm) stored at the National Museum of Natural Science in Taichung NMMNS004284001, dry specimens are deposited deputy National Museum of Natural Science in Taichung, Taiwan NMMNS004284002. ( Gastropoda .（ sp. nov. ）、（ Taiwan）.

A new tetraphyllidean genus and species, Caulopatera pagei n. g., n. sp (Tetraphyllidea: Phyllobothriidae), from the grey carpetshark Chiloscyllium punctatum Muller & Henle (Orectolobiformes: Hemiscylliidae)

2010-08-29T00:00:28Z

A new genus and species of tetraphyllidean cestode, Caulopatera pagei n. g., n. sp., is described from the grey carpetshark Chiloscyllium punctatum Müller & Henle in Moreton Bay, Australia. The new genus is placed in the Phyllobothriidae, subfamily Phyllobothriinae. Caulopatera n. g. is distinct from all other phyllobothriine genera in having stalked, circular, non-loculate bothridia that lack an apical sucker, testes that are restricted to the region anterior to the cirrus-sac and circum-medullary vitelline follicles. The new genus most closely resembles Carpobothrium Shipley & Hornell, 1906, with which it shares non-loculate, stalked, unhooked bothridia without an accessory sucker and testes that are entirely anterior to the cirrus-sac, but differs from it in that it lacks a slit-like opening in each bothridium and flaps surrounding the opening. The possession of bothridial stalks is consistent with two cestode orders, the Tetraphyllidea and the Rhinebothriidea. The morphology of the bothridial stalks is consistent with other tetraphyllidean genera, in that Caulopatera possesses triangular bothridial stalks surrounding the back of the bothridia, indicating that it belongs in the Tetraphyllidea senso stricto, rather than in the recently recognised Rhinebothriidea. © 2010 Springer Science+Business Media B.V.

Angiotensin 1A receptors transfected into caudal ventrolateral medulla inhibit baroreflex gain and stress responses

2012-12-02T00:25:34Z

Angiotensin AT-1 receptor antagonism: complementary or alternative to ACE inhibition in cardiovascular and renal disease

Doggrell, Sheila A. — 2007-08-14T18:59:43Z

Both angiotensin-converting enzyme (ACE) inhibitors and AT-1 receptor antagonists reduce the effects of angiotensin II, however they may have different clinical effects. This is because the ACE inhibitors, but not the AT-1 receptor antagonists, increase the levels of substance P, bradykinin and tissue plasminogen activator. The AT-1 receptor antagonists, but not the ACE inhibitors, are capable of inhibiting the effects of angiotensin II produced by enzymes other than ACE. On the basis of the present clinical trial evidence, AT-1 receptor antagonists, rather than the ACE inhibitors, should be used to treat hypertension associated with left ventricular (LV) hypertrophy. Both groups of drugs are useful when hypertension is not complicated by LV hypertrophy, and in diabetes. In the treatment of diabetes with or without hypertension, there is good clinical support for the use of either an ACE inhibitor or an AT-1 receptor antagonist. ACE inhibitors are recommended in the treatment of renal disease that is not associated with diabetes, after myocardial infarction when left ventricular dysfunction is present, and in heart failure. As the incidence of cough is much lower with the AT-1 receptor antagonists, these can be substituted for ACE inhibitors in patients with hypertension or heart failure who have persistent cough. Preliminary studies suggest that combining an AT-1 receptor antagonist with an ACE inhibitor may be more effective than an ACE inhibitor alone in the treatment of hypertension, diabetes with hypertension, renal disease without diabetes and heart failure. However, further trials are required before combination therapy can be recommended in these conditions.

Angiotensin II exerts glucose-dependent effects on K-v currents in mouse pancreatic beta-cells via angiotensin II type 2 receptors

2010-02-07T00:09:38Z

Hyper-glycemia-associated glucotoxicity induces beta-cell apoptosis but the underlying mechanisms are unknown. Interestingly, prolonged exposure to high glucose upregulates the expression and function of the renin-angiotensin system (RAS). We hypothesize that the voltage-gated outward potassium (K-v) current, which governs beta-cell membrane potential and insulin secretion, has a role in glucotoxicity. In this study, we investigated the effects of prolonged exposure to high glucose on mouse pancreatic beta-cells and concurrent effects on the RAS by examining changes in expression of angiotensin II (ANG II) receptors and changes in the expression and activity of K-v channels. beta-Cells were incubated in high glucose medium for 1-7 days and then were examined with electrophysiological and molecular biology techniques. Prolonged exposure to high glucose produced a marked increase in beta-cell primary K-v channel subunit, K(v)2.1, expression and K-v current amplitude. Enhanced expression of ANG II type 1 receptor (AT(1)R) was also observed under high glucose conditions, whereas blockade of AT(1)R by losartan did not alter K-v channel expression. External application of ANG II reduced K-v current amplitude under normal, but not high, glucose conditions. The effect of ANG II on K-v channel gating was abolished by ANG II type 2 receptor (AT(2)R) antagonism. These data suggest that hyperglycemia alters beta-cell function through modification of the K-v channel which may be associated with the RAS.

ANGIOTENSIN II INCREASES OSTEOBLAST RANKL:OPG RATIO THROUGH TRANSACTIVATION OF ERBB RECEPTORS BY EGF-LIKE LIGANDS

MacRae, K og Thomas, W og Sernia C — 2011-11-18T00:00:00Z

Angiotensin II increases osteoblast RANKL:OPG ratio through transactivation of ErbB receptors by EGF-like ligands

2011-11-18T00:00:00Z

Angiotensin II mediates cardiomyocyte hypertrophic growth pathways via MMP-dependent HB-EGF liberation

2009-01-14T00:00:00Z

Abstract: Pathological cardiac stimulation by angiotensinII (AngII) can cause left ventricular hypertrophy, a major independent risk factor for heart attack and death. We have previously reported that AngII exerts its hypertrophic effects by usurping the epidermal growth factor (EGF) signalling pathway via metalloprotease-dependent transactivation. However, the EGF-like ligand responsible for AngII-mediated transactivation and cardiac hypertrophy remains to be identified. Using phosphorylated ERK1/2 as a read-out of growth pathway activation and an alkaline phosphatase-tagged Heparin-Binding EGF-like Growth Factor (HB-EGF) reporter construct to examine AngII-mediated liberation, we provide evidence that HB-EGF is the soluble growth factor involved in AngII-induced left ventricular hypertrophy.

Angiotensin II regulates RANKL:OPG ratio potentially through transactivation of ErbB receptors by EGF-like ligands

MacRae, K. og Thomas, W. og Sernia C. — 2011-10-18T00:00:00Z

Angiotensin II Type 2 Receptor Antagonizes Angiotensin II Type 1 Receptor-Mediated Cardiomyocyte Autophagy

2009-07-07T00:00:00Z

Autophagy has emerged as an important process in the pathogenesis of cardiovascular diseases, but the proximal triggers for autophagy are unknown. Angiotensin II plays a central role in the pathogenesis of cardiac hypertrophy and heart failure. In this study, we used angiotensin II type 1 (AT1) and type 2 (AT2) receptor–expressing adenoviruses in cultured neonatal cardiomyocytes to provide the first demonstration that neonatal cardiomyocyte autophagic activity is differentially modulated by AT1 and AT2 receptor subtypes. Angiotensin II stimulation (48 hours) of neonatal cardiomyocytes expressing the AT1 receptor alone (Ad-AT1; 10 multiplicities of infection) induced a significant increase in the number of HcRed-LC3 autophagosomes per cell (17.3±1.6 versus 33.3±4.1 autophagosomes per cell; P<0.05). Coexpression of a high ratio of AT2:AT1 (Ad-AT2:Ad-AT1 multiplicity of infection ratio: 20:5) receptors completely abrogated the AT1-mediated increase in autophagy (9.3±1.4 versus 33.3±4.1 autophagosomes per cell; P<0.05). Treatment with the AT2 receptor antagonist PD123319 did not reverse the AT2-mediated antiautophagic effect. AT1- and AT2-mediated autophagic responses were also assessed in cardiomyocytes from a genetic model that exhibits neonatal myocardial growth suppression. In these neonate myocyte cultures, AT1 receptor activation induced a marked increase in the number of myocytes containing cytoplasmic vacuoles compared with the control (22.7±4.1% versus 1.1±0.6%; P<0.001) and was characterized by a nonapoptotic autophagic phenotype. The incidence of cardiomyocyte autophagic vacuolization in this myocyte population decreased dramatically to only 0.4±0.2% in myocytes infected with a high ratio of Ad-AT2:Ad-AT1. This study provides the first description of reciprocal regulation of cardiomyocyte autophagic induction by the AT1 and AT2 receptor subtypes.

Angiotensinogen expression by rat epididymis: evidence for an intrinsic, angiotensin-generating system

Leung, P. S. og Wong, T. P. og Sernia, C. — 2008-06-10T00:00:00Z

Previous studies have suggested the presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis with functions in epididymal activity and sperm maturation. In the present study, the localization and expression of angiotensinogen, the component of the RAS which is indispensable for intracellular angiotensin generation, were investigated by immunochemistry, hybridization histochemistry and by reverse-transcriptase polymerase chain reaction (RT-PCR). Western blot analysis of protein from the epididymis confirmed the presence of angiotensinogen with the expected molecular mass of about 60 kDa, in agreement with results from other tissues. Immunocytochemistry showed the regional localization of immunoreactivity for angiotensinogen in the rat epididymis. In situ hybridization histochemistry further demonstrated the expression of angiotensinogen mRNA by the epididymal epithelium in a region-specific manner along the length of the rat epididymis. RT-PCR confirmed that the rat epididymis expresses angiotensinogen mRNA. On the other hand, mRNA of renin was not detected in the rat epididymis using Northern blot and RT-PCR analyses. The present study strongly supports the existence of an intrinsic, angiotensin-generating system based on locally formed angiotensinogen as a precursor for angiotensin production. This epididymal RAS may have paracrine or autocrine roles in mediating the epididymal and sperm functions. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

Angiotensinogen localization and secretion in the rat pancreas

2007-08-15T00:00:00Z

Renin and angiotensinogen have been previously found in the rat pancreas, and angiotensin receptors have been located in the apical domain of duct cells. To evaluate the possibility that angiotensin II could be generated within the duct system, we decided to determine whether angiotensinogen is present in rat pancreatic juice and the angiotensinogen-immunoreactive pancreatic cell types that could be responsible for its production. Angiotensinogen was detected in significant amounts by Western blotting in pancreatic juice collected from several individual rats. Different isoforms between plasma and pancreatic juice angiotensinogens were demonstrated by isoelectric focusing. Immunocytochemical experiments revealed angiotensinogen-immunoreactive cells at the periphery of the islets of Langerhans, and confocal microscopy demonstrated that most angiotensinogen-immunoreactive cells were glucagon-secreting cells. Secretion of angiotensinogen did not follow the regulated secretory pathway since it was absent from the glucagon-containing granules. This was confirmed by electron microscopy immunocytochemistry. Duct and acinar cells did not express angiotensinogen at an immunocytochemical detectable level. The present findings indicated an exocrine secretion of angiotensinogen by glucagon-secreting cells and suggest that one of the final targets of the local pancreatic renin-angiotensin system may be the duct epithelium.

Angiotensin receptors: form and function and distribution

Thomas, Walter G. og Mendelsohn, Frederick A. O. — 2009-01-14T00:00:00Z

The peptide hormone, angiotensin II, acts primarily via type I (AT1) and type II (AT2) angiotensin receptors. Proteolytic fragments of angiotensin II also have biological activity via these and other receptors, with actions that may mimic or antagonise angiotensin II. Most notably, a high affinity-binding site for angiotensin IV (the Val3-Phe8 fragment of angiotensin II) has recently been identified as the insulin-regulated aminopeptidase (IRAP). While AT1 and AT2 receptors are seven transmembrane-spanning, G protein-coupled receptors with some well-established features of relevance to health and disease, the existence of separate receptor systems for angiotensin fragments offers exciting possibilities for new therapeutics to target the diverse actions of the angiotensin peptides.

Angiotensin type 1A receptors in C1 neurons of the rostral ventrolateral medulla modulate the pressor response to aversive stress

2012-03-13T08:37:19Z

Animal Movement

Bennett, M. B. — 2007-08-14T00:00:00Z

Animal studies in stress research: the brain, accelerated ageing and impaired control of stress homones

Bradley, A. J. — 2008-06-06T00:00:00Z

An improved open-channel structure of MscL determined from FRET confocal microscopy and simulation

2010-12-19T00:10:09Z

Mechanosensitive channels act as molecular transducers of mechanical force exerted on the membrane of living cells by opening in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing, blood pressure regulation, and osmoregulation. Here, we determine the likely structure of the open state of the mechanosensitive channel of large conductance using a combination of patch clamp, fluorescence resonance energy transfer (FRET) spectroscopy, data from previous electron paramagnetic resonance experiments, and molecular and Brownian dynamics simulations. We show that structural rearrangements of the protein can be measured in similar conditions as patch clamp recordings while controlling the state of the pore in its natural lipid environment by modifying the lateral pressure distribution via the lipid bilayer. Transition to the open state is less dramatic than previously proposed, while the N terminus remains anchored at the surface of the membrane where it can either guide the tilt of or directly translate membrane tension to the conformation of the pore-lining helix. Combining FRET data obtained in physiological conditions with simulations is likely to be of great value for studying conformational changes in a range of multimeric membrane proteins. © 2010 Corry et al.

An inhibitor of phospholipase A2 group IIA modulates adipocyte signaling and protects against diet-induced metabolic syndrome in rats

2012-09-12T11:31:16Z

An inquiry-based practical for a large, foundation-level undergraduate laboratory that enhances student understanding of basic cellular concepts and scientific experimental design

2012-05-24T08:53:44Z

An integrated pharmacokinetic and imaging evaluation of vehicle effects on solute human epidermal flux and, retention characteristics

2008-07-29T16:23:44Z

An introduction to the physiology of hearing

Pickles, J. O. — 2009-04-14T11:46:39Z

An introduction to the physiology of hearing

Pickles, James O. — 2013-02-14T10:59:48Z

An investigation of organochlorine and polychlorobiphenyl concentrations in the blood and eggs of the carnivorous flatback turtle, Natator depressus, from Queensland, Australia

2013-03-17T00:16:55Z

An inwardly rectifying chloride current sensitive to extracellular osmolality is present in pig pancreatic acinar cells

Carew, M. A. og Thorn, P. — 2011-03-10T00:00:00Z

Anion Exchangers DTDST (SLC26A2), DRA (SLC26A3) and Pendrin (SLC26A4)

Markovich, Daniel — 2010-04-12T00:00:00Z

Anion transporter links to urolithiasis and hepatotoxicity

2012-12-23T00:13:00Z

AnkyrinG is required to maintain axo-dendritic polarity in vivo

2010-11-17T16:12:11Z

Neurons are highly polarized cells that extend a single axon and several dendrites. Studies with cultured neurons indicate that the proximal portion of the axon, denoted as the axon initial segment (AIS), maintains neuronal polarity in vitro. The membrane-adaptor protein ankyrinG (ankG) is an essential component of the AIS. To determine the relevance of ankG for neuronal polarity in vivo, we studied mice with a cerebellum-specific ankG deficiency. Strikingly, ankG-depleted axons develop protrusions closely resembling dendritic spines. Such axonal spines are enriched with postsynaptic proteins, including ProSAP1/Shank2 and ionotropic and metabotropic glutamate receptors. In addition, immunofluorescence indicated that axonal spines are contacted by presynaptic glutamatergic boutons. For further analysis, double mutants were obtained by crossbreeding ankG−/− mice with L7/Purkinje cell-specific promoter 2 (PCP2) mice expressing enhanced green fluorescent protein (EGFP) in Purkinje cells (PCs). This approach allowed precise confocal microscopic mapping of EGFP-positive spiny axons and their subsequent identification at the electron microscopic level. Ultrastructurally, axonal spines contained a typical postsynaptic density and established asymmetric excitatory synapses with presynaptic boutons containing synaptic vesicles. In the shaft of spiny axons, typical ultrastructural features of the AIS, including the membrane-associated dense undercoating and cytoplasmic bundles of microtubules, were absent. Finally, using time-lapse imaging of organotypic cerebellar slice cultures, we demonstrate that nonspiny PC axons of EGFP-positive/ankG−/− mice acquire a spiny phenotype within a time range of only 3 days. Collectively, these findings demonstrate that axons of ankG-deficient mice acquire hallmark features of dendrites. AnkG thus is important for maintaining appropriate axo-dendritic polarity in vivo.

An MRI-based atlas and database of the developing mouse brain

2010-12-05T00:04:01Z

An outline of desensitization in pentameric ligand-gated ion channel receptors

Keramidas, Angelo og Lynch, Joseph W. — 2012-11-08T16:57:43Z

Pentameric ligand-gated ion channel (pLGIC) receptors exhibit desensitization, the progressive reduction in ionic flux in the prolonged presence of agonist. Despite its pathophysiological importance and the fact that it was first described over half a century ago, surprisingly little is known about the structural basis of desensitization in this receptor family. Here, we explain how desensitization is defined using functional criteria. We then review recent progress into reconciling the structural and functional basis of this phenomenon. The extracellular–transmembrane domain interface is a key locus. Activation is well known to involve conformational changes at this interface, and several lines of evidence suggest that desensitization involves a distinct conformational change here that is incompatible with activation. However, major questions remain unresolved, including the structural basis of the desensitization-induced agonist affinity increase and the mechanism of pore closure during desensitization.

A novel CYP2A6 allele, CYP2A6*23, impairs enzyme function in vitro and in vivo and decreases smoking in a population of Black-African descent

2008-07-29T00:00:00Z

A novel in vitro human microglia model: Characterization of human monocyte-derived microglia

2012-09-02T00:20:54Z

A novel large-conductance Ca(2+)-activated potassium channel and current in nerve terminals of the rat neurohypophysis

2008-06-19T10:47:00Z

1. Nerve terminals of the rat posterior pituitary were acutely dissociated and identified using a combination of morphological and immunohistochemical techniques. Terminal membrane currents were studied using the 'whole-cell' patch clamp technique and channels were studied using inside-out and outside-out patches. 2. In physiological solutions, but with 7 mM 4-aminopyridine (4-AP), depolarizing voltage clamp steps from different holding potentials (-90 or -50 mV) elicited a fast, inward current followed by a slow, sustained, outward current. This outward current did not appear to show any steady-state inactivation. 3. The threshold for activation of the outward current was -30 mV and the current-voltage relation was 'bell-shaped'. The amplitude increased with increasingly depolarized potential steps. The outward current reversal potential was measured using tail current analysis and was consistent with that of a potassium current. 4. The sustained potassium current was determined to be dependent on the concentration of intracellular calcium. Extracellular Cd2+ (80 microM), a calcium channel blocker, also reversibly abolished the outward current. 5. The current was delayed in onset and was sustained over the length of a 150 ms-duration depolarizing pulse. The outward current reached a peak plateau and then decayed slowly. The decay was fitted by a single exponential with a time constant of 9.0 +/- 2.2 s. The decay constants did not show a dependence on voltage but rather on intracellular Ca2+. The time course of recovery from this decay was complex with full recovery taking > 190 s. 6. 4-AP (7 mM), dendrotoxin (100 nM), apamin (40-80 nM), and charybdotoxin (10-100 nM) had no effect on the sustained outward current. In contrast Ba2+ (200 microM) and tetraethylammonium inhibited the current, the latter in a dose-dependent manner (apparent concentration giving 50% of maximal inhibition (IC50) = 0.51 mM). 7. The neurohypophysial terminal outward current recorded here corresponds most closely to a Ca(2+)-activated K+ current (IK(Ca)) and not to a delayed rectifier or IA-like current. It also has properties different from that of the Ca(2+)-dependent outward current described in the magnocellular neuronal cell bodies of the hypothalamus. 8. A large conductance channel is often observed in isolated rat neurohypophysial nerve terminals. The channel had a unit conductance of 231 pS in symmetrical 150 mM K+.

A novel mouse model of veno-occlusive disease provides strategies to prevent thioguanine-induced hepatic toxicity

2013-03-31T00:31:31Z

A novel threaded ring structure for the antimicrobial peptide microcin J25

2011-09-09T18:37:11Z

A Novel Transgenic Zebrafish Model for Studying Secretion in the Exocrine Pancreas

2011-11-27T00:00:00Z

A novel use of TAT-EGFP to validate techniques to alter osteosarcoma cell surface glycosaminoglycan expression

2008-02-18T00:00:00Z

Several methods to alter cell surface glycosaminoglycan (GAG) expression have previously been described, including treatments with chlorate to reduce the addition of charged sulfate groups, xyloside compounds to displace GAGs from their core proteins, and GAG lyases, such as heparinase and chondroitinase, to release GAG fragments from the cell layer. While these methods are useful in identifying cellular mechanisms which are dependent on GAGs, they must be stringently validated to assess results in the appropriate context. To determine the most useful technique for the evaluation of GAG function in osteogenesis, MG-63 osteosarcoma cells were systematically treated with these agents and evaluated for changes in cell surface GAGs using a TAT-EGFP fusion protein. TAT, a protein transduction domain from the HIV-1 virus, requires cell surface GAGs to traverse cell membranes. The EGFP component provides a method to assess protein entry into cells in both qualitative and quantitative tests. Here, TAT-EGFP transduction analysis confirmed radiochemical and physiological data that chlorate effectively disrupts GAG expression. TAT-EGFP entry into cells was also inhibited by the exogenous application of commercial heparin and GAGs extracted from MG-63 cells as well as by the pre-treatment of cells with chondroitinase ABC. However, neither heparinase III treatment nor the addition of exogenous chondroitin-6-sulfate affected TAT-EGFP entry into cells. In addition, xyloside-beta-D-naphthol and xyloside-beta-D-cis/trans-decahydro-2-naphthol treatment could not induce significant phenotypic change in these cells, and the unaffected TAT-EGFP transduction confirmed that this was due to an inability to efficiently prime GAG synthesis. The use of TAT-EGFP is thus a useful technique to specifically evaluate cell surface GAG expression in a simple, quantifiable manner, and avoids the complications involved with conventional radiochemical assays or analytical chromatography.

An overview of the sensory receptors regulating cough

Mazzone, Stuart B. — 2009-12-18T00:00:00Z

The cough reflex represents a primary defensive mechanism for airway protection in a variety of mammalian species. However, excessive and inappropriate coughing can emerge as a primary presenting symptom of many airway diseases. Cough disorders are characterized by a reduction in the threshold for reflex initiation and, as a consequence, the occurrence of cough in response to stimuli that are normally innocuous in nature. The current therapeutic strategies for the treatment of cough disorders are only moderately effective. This undoubtedly relates in part to limitations in our understanding of the neural components comprising the cough reflex pathway. The aim of this review is to provide an overview of current concepts relating to the sensory innervation to the mammalian airways, focusing particularly on the sensory receptors that regulate cough. In addition, the review will highlight particular areas and issues relating to cough neurobiology that are creating controversy in the field.

Antagonism of microRNA-126 suppresses the effector function of TH2 cells and the development of allergic airways disease

2011-03-09T14:30:09Z