Thesis (Ph. D.)--University of Wisconsin--Madison, 1975. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 149-165).

Thesis (Ph. D.)--University of Wisconsin, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Protein kinases play important roles in regulating cellular functions in many organisms. This work deals specifically with the protein kinase CK2 (casein kinase II) and its role in regulating the activity of proteins involved in oocyte development in Xenopus laevis. Protein kinase CK2 is a tetrameric enzyme containing two catalytic subunits (alpha and alpha') and two identical regulatory subunits (beta) which forms the holoenzyme. CK2 phosphorylates many different proteins involved in many aspects of cellular functions. It phosphorylates serine and threonine sites and is considered to be a ubiquitous enzyme, expressed at different levels in different cell types. In this study CK2 activity was characterized in material from two sources: from isolated nuclei and messenger ribonucleoprotein (mRNP) particles from Xenopus oocytes. cDNAs expressing both the alpha and the beta subunits were cloned and antibodies were raised against the fusion protein containing the beta-subunit. The main objective of this study was to determine the effects that CK2 had on proteins involved in oocyte development. The interaction of CK2 with a protein known as histone deacetylase was studied in depth to determine how phosphorylation might influence its function and cellular compartmentalisation. Specifically, phosphorylation by CK2 is shown to improve the kinetics of nuclear unport, and the interaction of histone deacetylase with alpha-importin, a well-established nuclear transport protein, is revealed to be dependent on the phosphorylation state of histone deacetylase. Another aspect of this work is related to the association of CK2 with mRNP particles in the cytoplasm. mRNP particles function as long term storage units for mRNA to be used during oocyte maturation and early embryogenesis. It has been postulated that a protein kinase associated with these particles plays a role in controlling the binding of mRNA to proteins involved in translation repression (mRNA masking proteins). This study lends support to that theory, and the possible effects of CK2 phosphorylation on the masking "Y-box" proteins are discussed.

Ostrich organs/tissue/fluids were screened for plasma kallikrein-like, tissue kallikrein-like and tonin-like activity in a continuous-fluorogenic-assay system using Pro-Phe-Arg-7-amino-4-methylcoumarine, Phe- Arg-7-amino-4-methylcoumarine and Val-Leu-Arg--7-amino-4-trifluoro-methylcoumarine as substrates. Ostrich liver and kidney showed the highest specific plasma kallikrein-like activity. Ostrich adrenal glands and kidney showed the highest specific tissue kallikrein-like and tonin-like activity. Ostrich high molecular weight kininogen was purified from plasma and low molecular weight kininogen was partially purified. The N-terminal amino acid sequences of both high- and low molecular weight kininogens from ostrich plasma were determined. Ostrich plasma high molecular weight kininogen was purified as a 118 kD protein. The purified high molecular weight kininogen inhibits the cysteine proteinase papain at a ratio of one molecule HKG to two molecules of papain. Ornitho kinin-like molecules were detected in ostrich urine using reverse phase HPLC.

The little studied plasma kallikrein-kinin system of poikilotherm vertebrates was investigated in several species offish, amphibians, and reptiles, and compared to the well-known mammalian enzyme system. It was found that the plasmas of all fish and amphibians tested differed from reptilian and mammalian plasma in their inability to release a kinin-like factor when reacted with trypsin or glass, and no evidence was obtained to suggest that these plasmas contain enzymic machinery which can produce a kinin. However, it was shown that heat-denatured plasma from these animals did develop biological activity when treated with hog pancreas kallikrein, an enzyme specific for releasing kinins. Thus, the equivalent of a kininogen might exist in these plasmas. Since turtle plasma produced a kinin by endogenous enzymes, detailed studies of this system were conducted. By a variety of criteria, enzymic mechanisms for kinin production in this plasma were closely similar to those of mammalian plasma. However, purification of the turtle kinin released by endogenous enzymes, followed by pharmacological and chemical tests showed that this kinin was chemically different from bradykinin, its mammalian counterpart. Data obtained from amino acid analysis of the peptide, and from certain pharmacological tests, strongly suggested that the structure of turtle kininis 6-thr-bradykinin; i.e., that a threonine residue has been substituted for a serine . The possible significance of this finding is discussed. Preliminary studies of the pharmacological effects of bradykinin on aspects of blood pressure and flow in the turtle itself are described. Intra-arterial injections of bradykinin over a wide range of doses always produced a press or response which could be greatly reduced by adrenergic blockade. This is in contrast with the effect of, the peptide in mammals, where there is typically a hypotensive response which cannot be reduced by adrenergic blockade. The significance of this difference is discussed, and approaches to future investigations are suggested. / Science, Faculty of / Zoology, Department of / Graduate

In this thesis I investigated the possibility that the kinin system could be involved in the process of ovulation. This study was divided into four parts, these are outlined be 1ow. (1) To determine whether and when the kinin system is activated in relation to ovulation, plasma kininogen levels were estimated in female rats, guinea pigs, and humans at different stages of their estrus or menstrual cycles. Non-ovulating females (women using oral contraceptives,, or post-menopausal women) and male guinea pigs served as controls. The ovulating females of all three species showed a marked decline in kininogen levels shortly before ovulation, suggesting that the kinin system was activated at this time. The fall was absent in the non-ovulating controls, with the exception of women using oral contraceptives. In the latter subjects the fall occurred at a similar time in the 'cycle', and was of a similar magnitude as the fall in normal women. These results showed that the fall is a preovulatory change and raised the possibility that a mechanism more fundamental than the events obstructed by the oral contraceptives could be at least partially responsible for the decline. (2) After establishing the timing of the fall in plasma kininogen levels, an attempt was made to locate the enzymes responsible for the change. The kinin-forming enzymes of the two locations most likely to be involved in kinin release during ovulation, that is, the plasma and the ovary were examined. The evidence indicated that kinin-forming enzymes were present in both locations and suggested that their concentrations increased as ovulation neared. (3) In order to examine the possibility that an ovulatory stimulus can activate the kinin system, female rats were treated with an ovulatory dose of luteinizing hormone (LH) or estradiol -17β one day before the anticipated time of ovulation and kininogen level declines. Estimation of plasma kininogen levels revealed marked declines in the LH-treated animals, estradiol-17β had no observable effect. This evidence suggested that LH, but not estradiol-17β could be responsible, at least in part, for the decreased kininogen values just before ovulation. (4) Lastly, to establish the ability of a kinin to initiate some of the more important events of the ovulatory process, the effects of bradykinin on ovarian smooth muscle contractility and ovarian follicular blood vessel permeability in the rat were examined. Bradykinin stimulated ovarian contractility in in vitro preparations to a significantly greater degree in ovaries isolated during the ovulatory period than at any other stage of the cycle. Also, the degree of movement of the dye Trypan Blue from the general circulation throughout ovarian follicular tissue over a ten minute exposure period was significantly greater in tissue from animals treated with bradykinin than those that were not. This suggests that bradykinin can increase ovarian follicular blood vessel permeability in the rat. Both of these bradykinin-induced effects were reduced, but not eliminated by indomethacin, suggesting that prostaglandins may be involved. Results from this study indicate that the kinin system is activated during the preovulatory period, possibly at the level of the ovary, that LH may be partially responsible for this activation, and that kinins may play a role in triggering increases in ovarian contractility and blood vessel permeability both directly and possibly via the release of prostaglandins. More definite proof awaits the development of a satisfactory kinin antagonist. / Science, Faculty of / Zoology, Department of / Graduate

Pre-eclampsia is a pregnancy-related disorder characterised by high blood pressure, proteinuria and oedema. The aetiology of the disease is unclear but evidence suggests that endothelial dysfunction is central to the development of the maternal syndrome. Kinins are endogenous peptides released by the endothelium that contribute to the regulation of cardiovascular homeostasis by inducing vasodilation, fibrinolysis and angiogenesis. Given that pre-eclampsia is associated with reduced endotheliumdependent relaxation, coagulation abnormalities and an angiogenic imbalance, it was hypothesised that alterations of kinin receptor-mediated responses may be involved in the pathogenesis of the condition. To investigate whether changes in kinin receptor activity are involved in the impairment of endothelium-dependent relaxation observed in pre-eclampsia, the effects of specific B2 and B1 receptor agonists and antagonists on myometrial vascular tone were tested on arteries from healthy pregnancy and pre-eclampsia. The results demonstrated that in addition to classical bradykinin B2 receptor-mediated relaxation, a subset of healthy patients exhibited nitric oxide-dependent relaxation to the B1 receptor agonist Lys-des- Arg9-BK (LDABK) which could not be inhibited by either B1 or B2 receptor antagonists. Also, vessels that exhibited this novel response to LDABK were more sensitive to bradykinin. Furthermore, this study revealed that patients with pre-eclampsia had an attenuated response to both bradykinin and LDABK. Immunolocalisation and mRNA expression of the kinin receptors in the myometrium revealed no differences between healthy pregnancy and pre-eclampsia suggesting that disturbances of kinin receptor signalling rather than changes in receptor distribution or expression levels may be involved in the reduction of kinin-mediated responses in these patients. The role of kinins in mediating placental angiogenesis in healthy pregnancy and preeclampsia was determined using the endothelial tube formation assay in primary human umbilical vein endothelial cells (HUVECs) isolated from healthy women and women with pre-eclampsia. B2 and B1 receptor agonists induced endothelial tube formation via a VEGF-dependent, nitric oxide-independent mechanism in healthy HUVECs cultured in normoxic conditions. HUVECs isolated from women with pre-eclampsia cultured under normoxia and HUVECs from healthy pregnancies cultured under hypoxia exhibited greater levels of angiogenic branching compared with healthy normoxic cells, but were unresponsive to bradykinin and LDABK. Incubation of these cells with a VEGF receptor inhibitor reduced the elevated levels of tube formation indicating that this effect may be due to hypoxic upregulation of VEGF or an intrinsic difference in their angiogenic capacity. Further studies are required to determine the cause for the differences in angiogenic potential between healthy and pre-eclamptic cells and the impact this could have on placental vascular development and the pathogenesis of preeclampsia.

Made available in DSpace on 2015-07-22T20:49:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-29. Added 1 bitstream(s) on 2015-08-11T03:25:27Z : No. of bitstreams: 1 Publico-00272a.pdf: 1939032 bytes, checksum: 8909a1a58abf8a089d6c5e15f9ab0dc0 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:27Z : No. of bitstreams: 2 Publico-00272a.pdf: 1939032 bytes, checksum: 8909a1a58abf8a089d6c5e15f9ab0dc0 (MD5) Publico-00272b.pdf: 922324 bytes, checksum: 7763fc40b0d1d67d34b28da3e6cdf817 (MD5) / A síndrome metabólica é uma condição patológica de impacto global e recursos financeiros substanciais são gastos por governos no tratamento de doenças associadas como obesidade, diabetes mellitus e cardiopatias. O crescente índice de obesos no mundo não tem uma causa única e parece ter raízes evolutivas. É clara a participação do hipotálamo no controle da ingestão alimentar e do gasto energético, sendo ele um dos principais locais de integração de fatores centrais e periféricos que controlam a homeostase energética. Nosso grupo recentemente correlacionou o sistema calicreína-cinina como controle do gasto energético, mostrando que animais nocautes para o gene codificacador do receptor B1 de cininas (B1-/-) apresentam um fenótipo de resistência à obesidade induzida por dieta. Estes animais apresentam, por exemplo, níveis séricos basais reduzidos de leptina, porém são mais sensíveis a este hormônio em comparação a animais controles selvagens. Além disso, esses animais possuem também um aumento na atividade física quando submetidos a uma dieta rica em lipídeos. A descoberta de que a leptina tem uma ação trófica sobre estruturas hipotalâmicas estimulou-nos a investigar se anormalidades estruturais no hipotálamo têm papel importante na causação do fenótipo observado. As técnicas de imuno-histoquímica e hibridização in situ com sondas para peptídeos classicamente implicados no controle do balanço energético - Neuropeptídeo Y (NPY), transcrito relacionado à cocaína e anfetamina (CART), -melanocyte stimulating hormone (-MSH) e sonda para o receptor de leptina (Ob-Rb) - foram usadas. A região do núcleo paraventricular hipotalâmico e a região perifornicial da área hipotalâmica lateral (em que a ação do CART está corelacionada com o aumento do gasto energético e aumento da atividade locomotora, respectivamente) de animais B1-/- apresentam um aumento significativo na expressão de CART em comparação a animais WT corroborando nossas observações experimentais prévias sobre estes animais. / The metabolic syndrome is a global aiction and substantial financial resources are spent by governments in the treatment of related diseases such as obesity, diabetes mellitus and cardiopathies. The growing index of obese people in the world has not a single cause and seems to have evolutionary roots. There is a clear role of the hypothalamus in the control of food intake and energetic expenditure. It is one of the major integration sites of peripheral and central factors regulating energy homeostasis. Our group has recently correlated the kallikrein-kinin system with the control of energy expenditure showing that mice that had the kinin B1 receptor gene knockedout( B1-/-) display a phenotype of resistance to diet-induced obesity. These mice have reduced leptin serum levels and are more sensitive to this hormone as opposed to wild-type mice. In addition, they diplay an increased motor activity when submited to a high-lipid containing diet. The discovery that leptin has a trophic action on hypothalamic structures has prompted us to investigate whether structural abnormalities in the hipothalamic circuitry of B1-/- mice would have a role in the causation of the observed phenotype. Immunohistochemistry and in situ hybridization techniques using probes targeting peptides classically implicated in the control of energy balance - Neuropeptide Y (NPY), Cocaine-and-Amphetamine Related Transcript (CART), -melanocyte stimulating hormone (-MSH) and the leptin receptor (Ob-Rb) - were used. The paraventricular nucleus of the hypothalamus and the perifornical region of the lateral hipothalamic area are places at which the CART peptide activity is respectively correlated to increased energy expenditure and increased locomotor activity. An increased CART expression was demonstrated in these nuclei in B1-/- mice, satisfying our previous experimental observations about the B1 knock-out phenotype. / TEDE / BV UNIFESP: Teses e dissertações

No description available.

The present investigation was undertaken as a preliminary study to learn the response of Escherichia coli cells grown under identical experimental conditions to various concentrations of indoleacetic acid, gibberellic acid, and kinetin alone, and in combination with one another.