Alterations in brainstem a2 adrenoreceptor activity in pyridoxine-deficient rat model of hypertension

Moderate pyridoxine deficiency in adult male Sprague-Dawley rats results in significant hypertension, associated with a general sympathetic stimulation , including an increase in the turnover of norepinephrine in the heart. Treatment of these rats with pyridoxine reversed blood pressure to normal within 24 h. Treatment of pyridoxine-deficient rats with clonidine or x-methyl dihydroxyphenylalanine (x-methyl DOPA) also reduced the blood pressure of these animals to normal . There was also a significant increase in the Bma, of high and low affinity [3H]p-amino-clonidine binding to crude synaptosomal membrane preparations of the brain stem of deficient rats indicating chronic underexposure of)(, adrenoreceptors to endogenous norepinephrin.

Modelling of Ultraviolet and Vissible Emissions in Comets

Comets are the spectacular objects in the night sky since the dawn of mankind. Due to their giant apparitions and enigmatic behavior, followed by coincidental calamities, they were termed as notorious and called as `bad omens'. With a systematic study of these objects modern scienti c community understood that these objects are part of our solar system. Comets are believed to be remnant bodies of at the end of evolution of solar system and possess the material of solar nebula. Hence, these are considered as most pristine objects which can provide the information about the conditions of solar nebula. These are small bodies of our solar system, with a typical size of about a kilometer to a few tens of kilometers orbiting the Sun in highly elliptical orbits. The solid body of a comet is nucleus which is a conglomerated mixture of water ice, dust and some other gases. When the cometary nucleus advances towards the Sun in its orbit the ices sublimates and produces the gaseous envelope around the nucleus which is called coma. The gravity of cometary nucleus is very small and hence can not in uence the motion of gases in the cometary coma. Though the cometary nucleus is a few kilometers in size they can produce a transient, extensive, and expanding atmosphere with size several orders of magnitude larger in space. By ejecting gas and dust into space comets became the most active members of the solar system. The solar radiation and the solar wind in uences the motion of dust and ions and produces dust and ion tails, respectively. Comets have been observed in di erent spectral regions from rocket, ground and space borne optical instruments. The observed emission intensities are used to quantify the chemical abundances of di erent species in the comets. The study of various physical and chemical processes that govern these emissions is essential before estimating chemical abundances in the coma. Cameron band emission of CO molecule has been used to derive CO2 abundance in the comets based on the assumption that photodissociation of CO2 mainly produces these emissions. Similarly, the atomic oxygen visible emissions have been used to probe H2O in the cometary coma. The observed green ([OI] 5577 A) to red-doublet emission ([OI] 6300 and 6364 A) ratio has been used to con rm H2O as the parent species of these emissions. In this thesis a model is developed to understand the photochemistry of these emissions and applied to several comets. The model calculated emission intensities are compared with the observations done by space borne instruments like International Ultraviolet Explorer (IUE) and Hubble Space Telescope (HST) and also by various ground based telescopes.

Physiological analysis of central and peripheral insect circadian pacemaker neurons

Alle bisher untersuchten Lebewesen besitzen (circadiane) innere Uhren, die eine endogene Perioden-länge von ungefähr 24 Stunden generieren. Eine innere Uhr kann über Zeitgeber mit der Umwelt synchronisiert werden und ermöglicht dem Organismus, rhythmische Umweltveränderungen vorweg zu nehmen. Neben einem zentralen Schrittmacher, der Physiologie und Verhalten des Organismus steuert, gibt es in unterschiedlichen Organen auch periphere Uhren, die die zeitlichen Abläufe in der spezifischen Funktion dieser Organe steuern. In dieser Arbeit sollten zentrale und periphere Schrittmacherneurone von Insekten physiologisch untersucht und verglichen werden. Die Neurone der akzessorischen Medulla (AME) von Rhyparobia maderae dienten als Modellsystem für zentrale Schrittmacher, während olfaktorische Rezeptorneurone (ORNs) von Manduca sexta als Modellsystem für periphere Schrittmacher dienten. Die zentralen Schrittmacherneurone wurden in extrazellulären Ableitungen an der isolierten AME (Netzwerkebene) und in Patch-Clamp Experimenten an primären AME Zellkulturen (Einzelzellebene) untersucht. Auf Netzwerkebene zeigten sich zwei charakteristische Aktivitätsmuster: regelmäßige Aktivität und Wechsel zwischen hoher und niedriger Aktivität (Oszillationen). Es wurde gezeigt, dass Glutamat ein Neurotransmitter der weitverbreiteten inhibitorischen Synapsen der AME ist, und dass in geringem Maße auch exzitatorische Synapsen vorkommen. Das Neuropeptid pigment-dispersing factor (PDF), das von nur wenigen AME Neuronen exprimiert wird und ein wichtiger Kopplungsfaktor im circadianen System ist, führte zu Hemmungen, Aktivierungen oder Oszillationen. Die Effekte waren transient oder langanhaltend und wurden wahrscheinlich durch den sekundären Botenstoff cAMP vermittelt. Ein Zielmolekül von cAMP war vermutlich exchange protein directly activated by cAMP (EPAC). Auf Einzelzellebene wurde gezeigt, dass die meisten AME Neurone depolarisiert waren und deshalb nicht feuerten. Die Analyse von Strom-Spannungs-Kennlinien und pharmakologische Experimente ergaben, dass unterschiedliche Ionenkanäle vorhanden waren (Ca2+, Cl-, K+, Na+ Kanäle sowie nicht-spezifische Kationenkanäle). Starke, bei hohen Spannungen aktivierende Ca2+ Ströme (ICa) könnten eine wichtige Rolle bei Ca2+-abhängiger Neurotransmitter-Ausschüttung, Oszillationen, und Aktionspotentialen spielen. PDF hemmte unterschiedliche Ströme (ICa, IK und INa) und aktivierte nicht-spezifische Kationenströme (Ih). Es wurde angenommen, dass simultane PDF-abhängige Hyper- und Depolarisationen rhythmische Membranpotential-Oszillationen verursachen. Dieser Mechanismus könnte eine Rolle bei PDF-abhängigen Synchronisationen spielen. Die Analyse peripherer Schrittmacherneurone konzentrierte sich auf die Charakterisierung des olfaktorischen Corezeptors von M. sexta (MsexORCO). In anderen Insekten ist ORCO für die Membran-Insertion von olfaktorischen Rezeptoren (ORs) erforderlich. ORCO bildet Komplexe mit den ORs, die in heterologen Expressionssystemen als Ionenkanäle fungieren und Duft-Antworten vermitteln. Es wurde die Hypothese aufgestellt, dass MsexORCO in pheromonsensitiven ORNs in vivo nicht als Teil eines ionotropen Rezeptors sondern als Schrittmacherkanal fungiert, der unterschwellige Membranpotential-Oszillationen generiert. MsexORCO wurde mit vermeintlichen Pheromonrezeptoren in human embryonic kidney (HEK 293) Zellen coexprimiert. Immuncytochemie und Ca2+ Imaging Experimente zeigten sehr schwache Expressionsraten. Trotzdem war es möglich zu zeigen, dass MsexORCO wahrscheinlich ein spontan-aktiver, Ca2+-permeabler Ionenkanal ist, der durch den ORCO-Agonisten VUAA1 und cyclische Nucleotide aktiviert wird. Außerdem wiesen die Experimente darauf hin, dass MsexOR-1 offensichtlich der Bombykal-Rezeptor ist. Eine weitere Charakterisierung von MsexORCO in primären M. sexta ORN Zellkulturen konnte nicht vollendet werden, weil die ORNs nicht signifikant auf ORCO-Agonisten oder -Antagonisten reagierten.

Caracterización de la exposición ocupacional a los gases, dióxido de azufre (so2), sulfuro de hidrógeno (h2s), y monóxido de carbono (co), y la percepción de salud de los trabajadores, en una empresa del sector hidrocarburo en Colombia

En el proceso de extracción de petróleo (crudo) deben realizarse tratamientos físicos y químicos en estaciones de recolección del hidrocarburo con el fin de garantizar su calidad antes de su entrega para el transporte y comercialización. Para la realización de esta actividad el personal operativo requerido (operadores) debe realizar diferentes actividades, tales como ronda operacional, verificación de sistemas de almacenamiento del crudo, agua residual del proceso e insumos químicos utilizados en su tratamiento y manipulación de facilidades en las estaciones de recolección, entre otras. Como resultados de las actividades rutinarias los operadores están expuestos a factores de riesgo químico asociados a gases y vapores orgánicos generados en los procesos de tratamiento del crudo. En el presente trabajo se realizaron mediciones de calidad de aire e higiene industrial en diferentes estaciones tratamiento de crudo, con el propósito de evaluar los niveles de exposición de los operadores a gases y vapores de hidrocarburos durante el proceso de tratamiento de crudo y dar respuesta a la siguiente pregunta: ¿existe relación entre la exposición ocupacional, las emisiones atmosféricas de gases (SO2, CO, H2S) y la percepción de afectación de la salud de los trabajadores que se encuentran expuestos durante la actividad laboral, en una empresa del sector de hidrocarburos? Se realizó un estudio de corte transversal, mediante la aplicación de cuestionarios sobre las condiciones de trabajo y de salud a 30 trabajadores que laboran en una estación de tratamiento de crudo de una compañía del sector de hidrocarburos. Los operadores objeto de estudio laboran en turnos rotativos, han estado vinculados con la compañía por más de dos años y tienen contrato directo, adicionalmente, se identificaron los factores de riesgos ambientales y ocupacionales para el grupo de trabajadores y se realizó una revisión de los informes de medición de higiene industrial y de calidad de aire de las estaciones donde labora el personal seleccionado con el fin de establecer si los resultados se relacionan. Los resultados obtenidos indican que el 100% de los trabajadores son de género masculino y se desempeñan en cargos de operadores, recorredores de pozos de crudo y supervisores. El 97% de los operadores tiene más de cuarenta años de edad y el 80% de los mismos ha laborado por más de 6 años en la compañía. Acerca de la percepción de los trabajadores sobre su estado de salud el 90% afirma que su salud es buena, el 97% respondió que no presenta problemas respiratorios, el 23% manifiesta que presenta trastornos dermatológicos y el 27% indican que presenta dolor de cabeza constante. De la revisión de los informes de calidad de aire disponibles se encontró que las mediciones de Dióxido de Azufre SO2, Monóxido de Carbono CO se encuentran dentro del rango definido como el de menor impacto para la salud humana. De los datos del informe se puede concluir que la calidad del aire es buena en el 100% de las áreas de influencia de las estaciones de tratamiento de crudo. Según los informes de higiene industrial el 34% de las instalaciones presenta concentraciones de Sulfuro de Hidrógeno (H2S) en el límite permisible para exposiciones crónicas en un promedio ponderado de tiempo (TLV-TWA) y el límite permisible para exposiciones agudas en un límite de exposición a corto plazo (TLV-STEL). Solo el 37% de los trabajadores objeto de este estudio percibe el riesgo por la exposición a factores de riesgo químicos y son claramente consientes que se encuentran expuestos a estos riesgos por la manipulación de productos químicos y exposición a sustancias químicas producto de sus actividades rutinarias, el 73% no percibe el riesgo de exposición por su actividad laboral. Se recomienda que la compañía fortalezca su esquema de vigilancia para generar alternativas que eleven los niveles de consciencia del riesgo del trabajador. Los factores de riesgo ambiental y ocupacional, de los gases y vapores generados se deben al proceso de tratamiento de crudo, están mutuamente relacionados dado que al generarse una emisión y/o escape no controlado como consecuencia se tiene una afectación directa al medio ambiente y a los trabajadores.

Referencing of street-level flows measured during the DAPPLE 2004 campaign

Street-level mean flow and turbulence govern the dispersion of gases away from their sources in urban areas. A suitable reference measurement in the driving flow above the urban canopy is needed to both understand and model complex street-level flow for pollutant dispersion or emergency response purposes. In vegetation canopies, a reference at mean canopy height is often used, but it is unclear whether this is suitable for urban canopies. This paper presents an evaluation of the quality of reference measurements at both roof-top (height = H) and at height z = 9H = 190 m, and their ability to explain mean and turbulent variations of street-level flow. Fast response wind data were measured at street canyon and reference sites during the six-week long DAPPLE project field campaign in spring 2004, in central London, UK, and an averaging time of 10 min was used to distinguish recirculation-type mean flow patterns from turbulence. Flow distortion at each reference site was assessed by considering turbulence intensity and streamline deflection. Then each reference was used as the dependent variable in the model of Dobre et al. (2005) which decomposes street-level flow into channelling and recirculating components. The high reference explained more of the variability of the mean flow. Coupling of turbulent kinetic energy was also stronger between street-level and the high reference flow rather than the roof-top. This coupling was weaker when overnight flow was stratified, and turbulence was suppressed at the high reference site. However, such events were rare (<1% of data) over the six-week long period. The potential usefulness of a centralised, high reference site in London was thus demonstrated with application to emergency response and air quality modelling.

The four major N- and C-terminal splice variants of the excitatory amino acid transporter GLT-1 form cell surface homomeric and heteromeric assemblies

The L-glutamate transporter GLT-1 is an abundant CNS membrane protein of the excitatory amino acid transporter (EAAT) family which controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using RT-PCR, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N- and C-termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected HEK-293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (V5, HA or FLAG) into the second extracellular domain of each isoform allowed co-immunoprecipitation and tr-FRET studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms are able to combine to form homomeric and heteromeric assemblies, each of which are expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

Effect of acyl chain length on transfection efficiency and toxicity of polyethylenimine

Polyethylenimine (PEI) is an efficient nonviral gene delivery vector because of its high buffering capacity and DNA condensation ability. In our study, the amino groups on the polymeric backbone were acylated using acetic or propionic anhydride to alter the protonation behaviour and the hydrophilic/hydrophobic balance of the polymer. The concentration of acylated primary amines was determined using trinitrobenzene sulphonic acid assay. Results showed that our modified polymers had lower buffering capacities in solutions compared to PEI. The polymers were complexed with plasmid encoding enhanced green fluorescent protein at three different ratios (1:1, 1:2 and 1:10 w/w DNA to polymer) to form polyplexes and their toxicities and transfection efficiencies were evaluated in HEK 293 cells. Acylation reduced the number of primary amines on the polymer and the surface charge, improving haemocompatibility and reducing cytotoxicity. The reduction in the concentration of amino groups helped to optimise DNA compaction and facilitated polyplex dissociation in the cell, which increased transfection efficiency of the modified polymers compared to the parent polymer. Polymers with buffering capacities greater than 50% and less than 80% relative to PEI, showed higher transfection efficiencies than PEI. The propionic anhydride modified polymers had appropriate interactions with DNA which provided both DNA compaction and polyplex dissociation. These systems interacted better with the cell membrane because of their slightly higher lipophilicity and formed polyplexes which were less cytotoxic than polyplexes of acetic anhydride modified polymers. Among the vectors tested, 1:0.3 mol/mol PEI:propionic anhydride in a 1:2 w/w DNA:polymer composition provided the best transfection system with improved transfection efficiency and reduced cytotoxicity.

Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains

The stable signal peptide (SSP) of the lymphocytic choriomeningitis virus surface glycoprotein precursor has several unique characteristics. The SSP is unusually long, at 58 amino acids, and contains two hydrophobic domains, and its sequence is highly conserved among both Old and New World arenaviruses. To better understand the functions of the SSP, a panel of point and deletion mutants was created by in vitro mutagenesis to target the highly conserved elements within the SSP. We were also able to confirm critical residues required for separate SSP functions by trans-complementation. Using these approaches, it was possible to resolve functional domains of the SSP. In characterizing our SSP mutants, we discovered that the SSP is involved in several distinct functions within the viral life cycle, beyond translocation of the viral surface glycoprotein precursor into the endoplasmic reticulum lumen. The SSP is required for efficient glycoprotein expression, posttranslational maturation cleavage of GP1 and GP2 by SKI-1/S1P protease, glycoprotein transport to the cell surface plasma membrane, formation of infectious virus particles, and acid pH-dependent glycoprotein-mediated cell fusion.

Comparison of the bacterial Enhancin-like proteins from Yersinia and Bacillus spp. with a baculovirus Enhancin

Enhancins are a class of metalloproteases found in some baculoviruses that enhance viral infection by degrading the peritrophic, membrane (PM) of the insect midgut. However, sequencing has revealed enhancin-like genes with 24-25% homology to viral enhancins, in the genomes of Yersinia pestis and Bacillus anthracis. AcMNPV does not encode enhancin therefore recombinant AcMNPV budded viruses (BVs) and polyhedra inclusion bodies (PIBs) were generated expressing the bacterial Enhancins. Bacterial Enhancins were found to be cytotoxic when compared to viral enhancin, however, larval bioassays suggested that the bacterial Enhancins did not enhance infection in the same way as viral Enhancin. This suggests that the bacterial Enhancins may have evolved a distinct biochemical function. (c) 2005 Elsevier Inc. All rights reserved.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INFalpha and INFgamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Improved display of synthetic IgG-binding domains on the baculovirus surface

Improved display of foreign protein moieties in combination with beneficial alteration of the viral surface properties should be of value for targeted and enhanced gene delivery. Here, we describe a vector based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV) displaying synthetic IgG-bincling domains (ZZ) of protein A fused to the transmembrane anchor of vesicular stomatitis virus (VSV) G protein. This display vector was equipped with a GFP/EGFP expression cassette enabling fluorescent detection in both insect and mammalian cells. The virus construct displayed the biologically active fusion protein efficiently and showed increased binding capacity to IgG. As the display is carried out using a membrane anchor of foreign origin, gp64 is left intact for virus entry, which may increase gene expression in the transduced mammalian cells. In addition, the viral vector can be targeted to any desired cell type via binding of ZZ domains when an appropriate IgG antibody is available.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INF alpha and INF gamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Inhibitory effects of olive oil phenolics on invasion in human colon adenocarcinoma cells in vitro

Studies in human, animal and cellular systems suggest that phenols from virgin olive oil are capable of inhibiting several stages in carcinogenesis, including metastasis. The invasion cascade comprises cell attachment to extracellular matrix components or basement membrane, degradation of basement membrane by proteolytic enzymes and migration of cells through the modified matrix. In the present study, we investigated the effect of phenolics extracted from virgin olive oil (OVP) and its main constituents: hydroxytyrosol (3,4-dihydroxyphenylethanol), tyrosol (p-hydroxyphenylethanol), pinoresinol and caffeic acid. The effects of these phenolics were tested on the invasion of HT115 human colon carcinoma cells in a Matrigel invasion assay. OVP and its compounds showed different dose-related anti-invasive effects. At 25 mu g/ml OVP and equivalent doses of individual compounds, significant anti-invasive effects were seen in the range of 45-55% of control. Importantly, OVP, but not the isolated phenolics, significantly reduced total cell number in the Matrigel invasion assay. There were no significant effects shown on cell viability, indicating the reduction of cell number in the Matrigel invasion assay was not due to cytotoxicity. There were also no significant effects on cell attachment to plastic substrate, indicating the importance of extracellular matrix in modulating the anti-invasive effects of OVP. In conclusion, the results from this study indicate that phenols from virgin olive oil have the ability to inhibit invasion of colon cancer cells and the effects may be mediated at different levels of the invasion cascade. (c) 2007 Wiley-Liss, Inc.

Selective separation of the major whey proteins using ion exchange membranes

Synthetic microporous membranes with functional groups covalently attached were used to selectively separate beta-lactoglobulin, BSA, and alpha-lactalbumin from rennet whey. The selectivity and membrane performance of strong (quaternary ammonium) and weak (diethylamine) ion-exchange membranes were studied using breakthrough curves, measurement of binding capacity, and protein composition of the elution fraction to determine the binding behavior of each membrane. When the weak and strong anion exchange membranes were saturated with whey, they were both selective primarily for beta-lactoglobulin with less than 1% of the eluate consisting of alpha-lactalbumin or BSA. The binding capacity of a pure alpha-lactoglobulin solution was in excess of 1.5 mg/cm(2) of membrane. This binding capacity was reduced to approximately 1.2 mg/cm(2) when using a rennet whey solution (pH 6.4). This reduction in protein binding capacity can be explained by both the competitive effects of other whey proteins and the effect of ions present in whey. Using binary solution breakthrough curves and rennet whey breakthrough curves, it was shown that alpha-lactalbumin and BSA were displaced from the strong and weak anion exchange membranes by beta-lactoglobulin. Finally, the effect of ionic strength on the binding capacity of individual proteins for each membrane was determined by comparing model protein solutions in milk permeate (pH 6.4) and a 10 mM sodium phosphate buffer (pH 6.4). Binding capacities of beta-lactoglobulin, alpha-lactalbumin, and BSA in milk permeate were reduced by as much as 50%. This reduction in capacity coupled with the low binding capacity of current ion exchange membranes are 2 serious considerations for selectively separating complex and concentrated protein solutions.