Introduction: Alpha-2-macroglobulin (α2m) is a plasma glycoprotein serine protease inhibitor. Previous studies have shown that coagulation factor concentrations are highly variable with age and α2m levels have been found to be twice as high in newborns compared to adults. This may contribute to a resistance towards thrombotic events observed in young populations. Protein glycosylation is known to affect protein activity and the glycosylation profile of adult α2m has previously been analyzed. Information regarding glycosylation of α2m in other age groups has yet to be elucidated. Therefore, the purpose of this study is to examine the differences in the glycosylation profiles between newborn and adult α2m. Methods: To evaluate glycan macroheterogeneity, plasma samples were enzymatically deglycosylated by PNGaseF, followed by SDS PAGE and western blotting (WB) to detect α2m. To evaluate microheterogeneity, plasma samples were incubated with Neuraminidase (Clostridium perfringens) followed by native PAGE and WB to determine sialic acid content. To detect non-sialylated terminal galactose residues, plasma samples were incubated with immobilized RCA120, and lectin-bound molecules were separated from unbound molecules. Additionally, the affinity of α2m for ricin was evaluated by eluting bound proteins with increasing concentrations of galactose. All fractions were subjected to SDS-PAGE and WB to detect α2m. 2D gel electrophoresis was completed to examine differences in pI and molecular weight of α2m in both age groups. Purification by immunoprecipitation was also performed and eluted α2m was analyzed by fluorescence-assisted carbohydrate electrophoresis (FACE) to determine the glycan fingerprint in the two populations. Results: Deglycosylation of both newborn and adult α2m with PNGaseF resulted in a change in migration and apparent molecular weight, however no statistically significant difference was found between newborn and adult. On native PAGE following treatment with neuraminidase, newborn α2m exhibited a statistically significant change in migration compared to adult. Additionally, newborn α2m exhibited a higher percentage of molecules bound to RCA120 than adult (no statistical difference) and elution of α2m from RCA120 with a galactose step gradient produced similar profiles for newborn and adult molecules. 2D electrophoresis and WB revealed a difference in pI of α2m in newborns as compared to adults. Finally, purified newborn and adult α2m were analyzed by FACE and quantification of prominent fluorescent bands revealed a higher secondary:primary band ratio in newborns when compared to adults. Conclusions: To our knowledge, this is the first study investigating glycosylation differences between newborn and adult α2m molecules. The results from PNGaseF analyses indicate no significant difference in total N-glycan content. Neuraminidase results suggest significantly greater sialic acid presence on newborn α2m, however there was no significant difference in galactose content. 2D electrophoresis revealed a difference in pI as well as the way in which newborn and adult α2m degrade when exposed to experimental conditions. A2m was successfully purified from both newborn and adult plasma, and FACE results indicate that the proportion of more branched glycans present in the two major fluorescent bands are of higher quantity in newborns than adults. / Thesis / Master of Science (MSc)

Zugl.: Giessen, Universiẗat, Diss., 2007.

Universiẗat, Diss., 2007--Giessen.

Thesis (Ph. D.)--University of Virginia, 1998. / Neurobiology of alpha-2 receptors. Includes bibliographical references. Also available online through Digital Dissertations.

The glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a structurally related family of neurotrophic factors. NTN exerts its effect through a multi-component receptor system consisting of the GDNF family receptor alpha 2 (GFRα2), proto-oncogene RET and/or NCAM. GFRα2 is spliced into at least three isoforms, GFRα2a, GFRα2b and GFRα2c. The present study investigated the expression and functional differences of GFRα2 isoforms. These receptor isoforms are differentially expressed in specific human brain regions. Using Neuro2A model, GDNF and NTN promote neurite outgrowth via GFRα2a and GFRα2c, but not GFRα2b. These GFRα2 isoforms regulate different early response genes when stimulated with GDNF and NTN. Interestingly, using co-expression models, GFRα2b inhibits ligand induced neurites outgrowth of GFRα2a and GFRα2c, and also the related receptor, GFRα1a. More intriguingly, ligands activated GFRα2b was also able to attenuate neurite extension induced by an unrelated stimulation using retinoic acid. MAPK activation induced by GDNF was not attenuated by GFRα2b in a co-expression model, while the early response genes expression profile (up-regulation of FosB) was similar to that induced by GFRα2b alone. This study suggest that GFRα2b is not merely a dominant negative isoform, but signals through a yet to be determined mechanism to antagonize and inhibit neuritogenesis. Together, these data suggest a new paradigm for the regulation of growth factor signaling and neurite outgrowth via an inhibitory splice variant of the receptor. / Singapore-MIT Alliance (SMA)

O controle neural da pressão arterial é essencial para a manutenção da homeostase do organismo. Este controle é realizado principalmente por núcleos bulbares e hipotalâmicos. Um dos principais sistemas de neurotransmissão envolvidos no controle da pressão arterial é o catecolaminérgico. Células noradrenérgicas e adrenérgicas estão presentes em todos os centros bulbares reguladores da pressão arterial, enquanto seus receptores, principalmente o receptor α2 adrenérgico (α2r), estão presentes nas mesmas regiões além de estarem presentes também nos núcleos hipotalâmicos. Estes receptores, quando ativados nestes núcleos, geram respostas cardiovasculares e atuam em conjunto com outros sistemas de neurotransmissão neste controle. Dentre estes sistemas de neurotransmissão, merecem destaque os sistemas angiotensinérgico e glutamatérgico, não apenas por estarem presentes nestes núcleos, mas também por atuarem no controle da pressão arterial. Contudo, pouco se sabe sobre como o sistema catecolaminérgico interage com estes sistemas. Desta forma, estudamos neste projeto a influência do sistema glutamatérgico e angiotensinérgico sobre o sistema catecolaminérgico no bulbo de ratos. Através de culturas de células bulbares, demonstramos que a ativação de receptores glutamatérgicos do tipo NMDA é capaz de elevar os níveis proteicos do α2R e que os receptores ionotrópicos do tipo não-NMDA precisam estar desbloqueados para tal. Em ratos adultos, microinjeções repetidas inibem a resposta bradicárdica induzida pela ativação dos α2rR no NTS. Contudo, o knockdown dos receptores AT1 de angiotensina restaura a resposta bradicárdica. A partir destes resultados, foi demonstrado que o sistema glutamatérgico é capaz de modular o sistema catecolaminérgico, enquanto o knockdown do receptor AT1 de angiotensina no NTS acentua a resposta bradicárdica dos α2R do NTS. Estes resultados sugerem que os sistemas de neurotransmissão bulbares interagem de diferentes formas e a compreensão deste controle pode vir a ser de grande valia para a compreensão de como se dá o controle da pressão arterial pelo sistema nervoso / The neural control of blood pressure is essential for the maintenance of the homeostasis of the organism. This control is performed mainly by nuclei in the medulla oblongata and in the hypothalamus. One of the main neurotransmitter system involved in this control is the catecholaminergic. Noradrenergic and adrenergic cells are present in all medullary nuclei involved in the arterial pressure regulation, while its receptors, especially the α2 adrenoceptor, are present in the same region plus hypothalamic nuclei. These receptors, upon activation in these nuclei, generate cardiovascular response, and act with other neurotransmission systems in this control. Among these systems, the glutamatergic and angiotensinergic deserve attention not only for also being present in the same nuclei, but for also acting in the control of the arterial pressure. Both angiotensinergic and glutamatergic systems interact with the catecholaminergic system throughout the nervous system. However, little is known about how the catecholaminergic system interacts with these systems in the modulation of blood pressure. Therefore, we studied in this project the influence of the glutamatergic and angiotensinergic systems over the catecholaminergic system in the medulla oblongata of rats. Through cell cultures of the medulla oblongata, we demonstrated that the activation of glutamatergic NMDA receptors is capable of elevating the proteic levels of α2-adrenoceptors, and that non-NMDA receptors need to be unblocked for such. In adult rats, repeated microinjections inhibit the bradycardic response elicited by the α2 adrenoceptors in the NTS. However, the angiotensin AT1 receptors knockdown restored the bradycardic response. Through the chronic knockout of angiotensinergic AT1 receptors in the NTS, we observed bradycardic response elicited by the activation of α2 adrenoceptors in the NTS of the knock-down rats. These results suggest that the medullary neurotransmission systems interact in different ways, and the comprehension of this control may be of great value for the comprehension of how the neural control of the blood pressure works

O controle neural da pressão arterial é essencial para a manutenção da homeostase do organismo. Este controle é realizado principalmente por núcleos bulbares e hipotalâmicos. Um dos principais sistemas de neurotransmissão envolvidos no controle da pressão arterial é o catecolaminérgico. Células noradrenérgicas e adrenérgicas estão presentes em todos os centros bulbares reguladores da pressão arterial, enquanto seus receptores, principalmente o receptor α2 adrenérgico (α2r), estão presentes nas mesmas regiões além de estarem presentes também nos núcleos hipotalâmicos. Estes receptores, quando ativados nestes núcleos, geram respostas cardiovasculares e atuam em conjunto com outros sistemas de neurotransmissão neste controle. Dentre estes sistemas de neurotransmissão, merecem destaque os sistemas angiotensinérgico e glutamatérgico, não apenas por estarem presentes nestes núcleos, mas também por atuarem no controle da pressão arterial. Contudo, pouco se sabe sobre como o sistema catecolaminérgico interage com estes sistemas. Desta forma, estudamos neste projeto a influência do sistema glutamatérgico e angiotensinérgico sobre o sistema catecolaminérgico no bulbo de ratos. Através de culturas de células bulbares, demonstramos que a ativação de receptores glutamatérgicos do tipo NMDA é capaz de elevar os níveis proteicos do α2R e que os receptores ionotrópicos do tipo não-NMDA precisam estar desbloqueados para tal. Em ratos adultos, microinjeções repetidas inibem a resposta bradicárdica induzida pela ativação dos α2rR no NTS. Contudo, o knockdown dos receptores AT1 de angiotensina restaura a resposta bradicárdica. A partir destes resultados, foi demonstrado que o sistema glutamatérgico é capaz de modular o sistema catecolaminérgico, enquanto o knockdown do receptor AT1 de angiotensina no NTS acentua a resposta bradicárdica dos α2R do NTS. Estes resultados sugerem que os sistemas de neurotransmissão bulbares interagem de diferentes formas e a compreensão deste controle pode vir a ser de grande valia para a compreensão de como se dá o controle da pressão arterial pelo sistema nervoso / The neural control of blood pressure is essential for the maintenance of the homeostasis of the organism. This control is performed mainly by nuclei in the medulla oblongata and in the hypothalamus. One of the main neurotransmitter system involved in this control is the catecholaminergic. Noradrenergic and adrenergic cells are present in all medullary nuclei involved in the arterial pressure regulation, while its receptors, especially the α2 adrenoceptor, are present in the same region plus hypothalamic nuclei. These receptors, upon activation in these nuclei, generate cardiovascular response, and act with other neurotransmission systems in this control. Among these systems, the glutamatergic and angiotensinergic deserve attention not only for also being present in the same nuclei, but for also acting in the control of the arterial pressure. Both angiotensinergic and glutamatergic systems interact with the catecholaminergic system throughout the nervous system. However, little is known about how the catecholaminergic system interacts with these systems in the modulation of blood pressure. Therefore, we studied in this project the influence of the glutamatergic and angiotensinergic systems over the catecholaminergic system in the medulla oblongata of rats. Through cell cultures of the medulla oblongata, we demonstrated that the activation of glutamatergic NMDA receptors is capable of elevating the proteic levels of α2-adrenoceptors, and that non-NMDA receptors need to be unblocked for such. In adult rats, repeated microinjections inhibit the bradycardic response elicited by the α2 adrenoceptors in the NTS. However, the angiotensin AT1 receptors knockdown restored the bradycardic response. Through the chronic knockout of angiotensinergic AT1 receptors in the NTS, we observed bradycardic response elicited by the activation of α2 adrenoceptors in the NTS of the knock-down rats. These results suggest that the medullary neurotransmission systems interact in different ways, and the comprehension of this control may be of great value for the comprehension of how the neural control of the blood pressure works

O presente estudo objetivou avaliar o efeito da dexmedetomidina, associada ou não à bupivacaína, na raquianestesia de fêmeas felinas submetidas à ovariohisterectomia. Foram utilizadas 34 gatas, jovens e adultas, sem raça definida, saudáveis submetidas a anestesia inalatória com isofluorano e aos seguintes tratamentos, após distribuição aleatória: grupo bupivacaína (GB) - raquianestesia com a bupivacaína isolada (0,5 mg/kg), grupo dexmedetomidina (GD) - raquianestesia com dexmedetomidina (1 mcg/kg) e grupo de dexmedetomidina bupivacaína (GDB) - raquianestesia com dexmedetomidina (1 mcg/kg) e bupivacaína (0,5 mg/kg). Após a indução da anestesia e manutenção com isofluorano, os animais foram posicionados em decúbito lateral direito para punção subaracnoide realizada no espaço lombossacro com agulha espinhal 25G. Os animais foram mantidos em decúbito dorsal até o final do procedimento cirúrgico e os atributos fisiológicos foram avaliados no período pré, trans e por 3 horas no período pós-operatório. Nenhum animal apresentou arritmia ou hipotensão arterial. O GDB apresentou redução significativa da frequência cardíaca e incremento pressórico quando comparado ao GB (p<0,01). Não houve diferença significativa no consumo de fentanil e no requerimento de isofluorano entre os grupos durante o procedimento cirúrgico. Na dose e diluição empregadas, a bupivacaína não determinou bloqueio motor significativo. As associações utilizadas promoveram analgesia adequada no período pós-operatório. O GDB apresentou maior grau de sedação durante parte da recuperação da anestesia (90min) (p<0,05), sem aumento no tempo de extubação. A adição da dexmedetomidina à bupivacaína na raquianestesia não aumentou o bloqueio motor e sensitivo; entretanto aumentou o grau de sedação dos animais promovendo melhor qualidade na recuperação anestésica sem deflagrar complicações cardiorrespiratória ou neurológica. / The present study aimed to evaluate the effect of dexmedetomidine, associated or not to bupivacaine, on spinal anesthesia in female felines submitted to ovariohysterectomy. Thirty four mixed breed healthy cats, young and adult, underwent inhalation anesthesia with isoflurane and the following treatments, after random distribution: bupivacaine group (BG) - spinal anesthesia with bupivacaine alone (0.5 mg/kg), dexmedetomidine group (DG) - spinal anesthesia with dexmedetomidine (1 mcg/kg). and dexmedetomidine/bupivacaine group (DBG) - spinal anesthesia with dexmedetomidine (1 mcg/kg) and bupivacaine (0.5 mg/kg). After anesthetic induction and maintenance with isoflurane, the animals were positioned in the right lateral recumbency for subarachnoid puncture performed in the lumbosacral space with a 25G spinal needle. The animals were kept in dorsal recumbency until the end of the surgical procedure and the physiological parameters were assessed in the pre, trans and 3 hours postoperative period. No animal presented arrhythmia or arterial hypotension. DBG presented a significant reduction in heart rate and pressure increase when compared to BG (p <0.01). There was no significant difference in fentanyl consumption and in the isoflurane requirement between groups during the surgical procedure. At the dose and dilution used, bupivacaine did not determine significant motor blockage. The associations used promoted adequate analgesia in the postoperative period. DBG had a higher degree of sedation during part of the anesthetic recovery (90min) (p <0.05), without an increase in extubation time. The addition of dexmedetomidine to bupivacaine in spinal anesthesia did not increase motor and sensory blockage; however, it increased the sedation level of the animals, promoting better quality of anesthetic recovery without triggering cardiorespiratory or neurological complications.

Alpha2-Rezeptoren, die weiter in alpha2A, alpha2B und alpha2C unterteilt werden, gehören zur Gruppe der adrenergen Rezeptoren innerhalb der Klasse der G-Protein-gekoppelten Rezeptoren. Sie sind maßgeblich an der Regulation vieler physiologischer Prozesse beteiligt. Vieles, was heute über alpha2-Rezeptoren bekannt ist, wurde mithilfe von alpha2-defizienten Mäusen, sogenannten „Knock-Out“-Mäusen (KO) herausgefunden, von denen bislang drei Einzel-KOs und der Doppel-KO der Subtypen A und C existieren. Im Rahmen dieser Arbeit wurden durch Kreuzung der vorhandenen KO-Linien Mauslinien generiert, die defizient für alpha2A und alpha2B, für alpha2B und alpha2C oder alle drei alpha2-Rezeptoren sind. Während alpha2AB-KO-Mäuse ungefähr entsprechend der Mendelschen Verteilung geboren wurden, zeigte sich, dass alpha2BC-KO-Mäuse teilweise und alpha2ABC-KO-Mäuse sogar komplett embryonal letal waren. Die morphologischen Unter-suchungen legten den Zeitpunkt der embryonalen Letalität der alpha2ABC-KO-Mäuse auf den Tag E10,5 der Embryonalentwicklung fest und konnten zeigen, dass diese Letalität in einem Vaskularisierungsdefekt innerhalb der extraembryonalen Organe Plazenta und Dottersack begründet lag. Diese Organe stellen die Versorgung des Embryos mit Nährstoffen und Sauerstoff sicher und sorgen somit für dessen Entwicklung. Durch RT-PCR-Experimente konnte die mRNS für alle drei alpha2-Rezeptorsubtypen an Tag E10,5 sowohl im Embryo als auch in Plazenta und Dottersack nachgewiesen werden. Autoradiographische Experimente und Radioligandenbindungsstudien an Plazenten machten deutlich, dass der Großteil an alpha2-Rezeptoren im embryonalen Teil der Plazenta exprimiert wird, nämlich in den Riesenzellen und in der sich daran anschließenden Spongiotrophoblastschicht, und dass hierbei alpha2-Rezeptoren vom B-Subtyp vorherrschen. In den genannten Zellen konnte mittels Immunhistochemie eine alpha2-Rezeptor-vermittelte Phosphorylierung der MAP-Kinasen ERK1/2 gezeigt werden, die auch in kultivierten WT-Dottersäcken beobachtet werden konnte. Unter basalen Bedingungen zeigte sich, dass die ERK1/2-Phosphorylierung in Gewebe von alpha2ABC-KO-Embryonen drastisch vermindert war, während andere Signalwege, die von alpha2-Rezeptoren angestoßen werden können, nicht beeinträchtigt waren. Versuche in einem Zellkulturmodell und mit kultivierten WT-Dottersäcken ergaben eine physiologisch relevante Wechselwirkung zwischen dem alpha2B-Rezeptor und dem PDGFbeta-Rezeptor, einer Rezeptortyrosinkinase, als deren Mechanismus sich in Co-Kultur-Experimenten mit alpha2B-Rezeptor-transfizierten Zellen und alpha2ABC-defizienten Dottersäcken die Transaktivierung von Rezeptortyrosinkinasen herausstellte. In dieser Arbeit konnte demonstriert werden, dass a2-Rezeptoren bei der Maus über eine Transaktivierung von ERK1/2 die Vaskularisierung der Plazenta und des Dottersacks bedingen und damit eine normale Embryonalentwicklung sicherstellen. / alpha2-Receptors belong to the familiy of adrenergic receptors within the superfamily of G-protein coupled receptors. They are involved in the regulation of many physiological processes. Most of the known functions have been investigated using mice deficient in alpha2-receptors. To date, single knockout mouse lines exist for each subtype of alpha2-receptors and also the alpha2AC-knockout. In this study the remaining double knockouts and the triple-knockout were generated by crossing the existing knockout mice. While mice deficient for the alpha2A- and the alpha2B-receptor were born with the expected Mendelian ratio, embryonic lethality occurred in the alpha2BC-knockout mice, and this was complete in mice lacking all three alpha2-receptors. Morphological examinations revealed that alpha2ABC-mice die around midgestation because of a defect in vascularisation in the extra-embryonic organs placenta and yolk sac. These are the organs which support the embryo with nutrients and oxygen and are therefore essential for embryonic development. RT-PCR-experiments detected mRNA for all three subtypes of alpha2-receptors on day E10.5 of embryonic development in embryo, placenta and yolk sac. Autoradiography and radioligand binding studies showed that most of the alpha2-receptors are expressed in the embryonic part of the placenta, in particular in giant cells and the underlying spongiotrophoblast layer. The alpha2B-receptor is the main subtype in these tissues. Immunohistochemistry of stimulated placenta slices demonstrated alpha2-receptor mediated phosphorylation of the MAP-kinase ERK1/2, which was also observed in cultivated yolk sacs of WT-mice. In freshly prepared tissue of alpha2ABC-knockout embryos ERK1/2-phosphorylation was dramatically decreased, while other signaling pathways of alpha2-receptors were unaffected. Experiments using cell culture and cultivated yolk sacs of WT-mice revealed a physiologically relevant interaction between alpha2B-receptors and PDGFbeta-receptors, a receptor tyrosine kinase. The mechanism of this interaction was illucidated in co-culture experiments of alpha2B-receptor transfected cells and alpha2 ABC-knockout yolk sacs as a G-protein coupled receptor initiated transactivation of receptor tyrosine kinases. In this study it was demonstrated that in mice alpha2-receptors are responsible for the vascularisation of placenta and yolk sac by transactivation of ERK1/2, and that they are, therefore, necessary for proper embryonic development.

Surfactant protein D (SP-D) is an important innate immune collectin involved in uptake and clearance of microbes and allergens in the lungs. SP-D has been shown to ameliorate allergic asthma reactions in mice; however, the mechanisms for this are not fully understood. We investigated the role of SP-D in the uptake and clearance of the model allergen ovalbumin (OVA) by macrophages. We discovered that SP-D does not bind OVA but binds fractions with contaminating proteins; ovomucin and ovomacroglobulin. We extended these findings to show that SP-D binds human alpha-2-macroglobulin (A2M) in its cleaved or intact state, in a concentration-, calcium-, and carbohydrate-dependent manner. A2M increases the innate immune potential of SP-D by increasing its ability to agglutinate the bacteria Escherichia coli and Bacillus subtilis. We found that SP-D does not increase the uptake of OVA by murine macrophage cell lines, or by alveolar macrophages in vivo in BALB/cJ mice.