Hematologic and Plasma Biochemical Values of Hyacinth Macaws (Anodorhynchus hyacinthinus)

The hyacinth macaw (Anodorhyncus hyacinthinus), considered the largest psittacine bird species in the world, is an endangered species, with a remaining population of approximately 6500 birds in the wild. To establish hematologic and plasma biochemical reference ranges and to verify differences related to sex, samples from 29 hyacinth macaws (14 males. 15 females) were obtained from birds apprehended from illegal wildlife trade and subsequently housed at the Sorocaba Zoo, Brazil. No significant differences in hematologic or plasma biochemical values were found between females and males. Compared with published reference values, differences were found in mean concentrations of total red blood cell count, corpuscular volume, corpuscular hemoglobin level, total white blood cell count, aspartate aminotransferase level, creatine kinase concentration, alkaline phosphatase concentration, and phosphorus level. Baseline hematologic and plasma biochemical ranges were established, which may be useful as reference values for clinicians working with this endangered species in captivity or rehabilitation centers.

Somaclonal variation: a morphogenetic and biochemical analysis of Mandevilla velutina cultured cells

Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A) than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation) and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60% shorter and a metabolic rate 33.6% higher than the control (F2 cell population). MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content.

Biochemical analysis of the methylic antigen of Paracoccidioides brasiliensis

Yeast forms of five strains of Paracoccidioides brasiliensis (SN, 2, 18, 192 and JT- 1) were cultured in a synthetic medium for obtaining methylic antigens. These antigens were lyophilized and studied for each strain, to determine their partial biochemical composition, through measurements of total lipid, protein and carbohydrate contents. Lipids of methylic antigens were purified and analysed for sterols, phospholipids, glycolipids, li-poproteins, and partial characterization of sterols. Significant differences were found among antigenic preparations derived from distinct P. brasiliensis strains, in relation to the quantitative determinations. On the other hand, sterol analysis revealed the presence of ergosterol, lanosterol and squalene in all samples. The diversity verified in the biochemical characteristics of antigens derived from different P. brasiliensis strains, confirm the need of using a pool of fungal samples in order to produce antigen preparations for serological procedures without hampering their sensitivity.

The influence of kaolin application on key metabolic pathways associated with grape berry quality: a molecular and biochemical analysis

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Biochemical analysis of female mice urine with reference to endocrine function: a key tool for estrus detection.

Species-specific chemical signals released through urine, sweat, saliva and feces are involved in communication between animals. Urinary biochemical constituents along with pheromones may contribute to variation across reproductive cycles and facilitate to estrus detection. Hence, the present study was designed to analyze such biochemical profiles, such as proteins, carbohydrates, lipids, fatty acids, in response with steroid hormones such as estradiol and progesterone. The experimental groups were normal, prepubertal, ovariectomized, and ovariectomized with estrogentreated female mice. In normal mice, the protein and lipid concentrations in urine were significantly higher in proestrus and estrus phases and the quantity of fatty acids was also comparatively higher in estrus. Furthermore, certain fatty acids, namely tridecanoic, palmitic and oleic acids, were present during proestrus and estrus phases, but were exclusively absent in ovariectomized mice. However, the carbohydrate level was equally maintained throughout the four phases of estrous cycle. For successful communication, higher concentrations of protein and specific fatty acids in estrus are directly involved. The significant increase in estradiol at estrus and progesterone at metestrus seems to be of greater importance in the expression pattern of biochemical constituents and may play a notable role in estrous cycle regulation. Thus, we conclude that the variations observed in the concentration of the biochemical constituents depend on the phase of the reproductive cycle as well as hormonal status of animals. The appearance of protein and specific fatty acids during estrus phase raises the possibility to use these as a urinary indicators for estrus detection.

Toxicological and biochemical analysis of the susceptibility of sylvatic Triatoma infestans from the Andean Valley of Bolivia to organophosphate insecticide

To increase our knowledge of the natural susceptibility of Triatoma infestans to an organophosphate insecticide, we performed toxicological and biochemical studies on three sylvatic populations from Bolivia and two populations from domestic dwellings from Bolivia and Argentina. Fifty-per-cent lethal doses (LD50) were determined based on the topical application of fenitrothion on first instar nymphs and mortality was assessed at 24 h. Both type of populations exhibited LD50ratios significantly higher than 1 with a range of the values (1.42-2.47); the maximum value were found in a sylvatic (-S) population, Veinte de Octubre-S. Samples were biochemically analysed using a glutathione S-transferase activity assay. The highest significant activity was obtained for Veinte de Octubre-S and the lowest activity was obtained for the reference population (102.69 and 54.23 pmol per minute per mg of protein respectively). Two out of the three sylvatic populations (Veinte de Octubre-S and Kirus Mayu-S) exhibited significantly higher glutathione S-transferase activity than that of the reference population. Based on this analysis of the natural susceptibility of this organism to organophosphate insecticides, continental and focal surveys of organophosphate susceptibility should be conducted to evaluate the evolution and distribution of this phenomenon.

GENETIC AND BIOCHEMICAL ANALYSIS OF THE TESTIS-SPECIFIC ZINC FINGER PROTEIN CTCFL/BORIS

Abstract en FrançaisCTCFL a d'abord été identifié comme un paralogue de la protéine ubiquitaire CTCF en raison de sa forte homologie entre leurs onze « zinc fingers », un domaine de liaison à l'ADN. Parmi ses nombreux rôles, la liaison des zinc fingers de CTCF à la région de contrôle de l'empreinte (ICR) maternelle non-méthylée Igf2/H19, contrôle l'expression empreinte (monoallélique) de H19 et IGF2 dans les cellules somatiques. La méthylation de l'ICR Igf2/H19 paternelle est nécessaire à l'expression empreinte de ces deux gènes. Bien que le mécanisme par lequel l'ICR est méthylé soit mal compris, il est connu que l'établissement de la méthylation se produit pendant le développement des cellules germinales mâles et que les ADN méthyltransférases de novo DNMT3A et DNMT3L sont essentiels. Par conséquent, CTCFL fournit un bon candidat pour un rôle dans la méthylation de l'ICR paternelle Igf2/H19 en raison de son expression restreinte à certains types de cellules où la méthylation de l'ICR a lieu (spermatogonies et spermatocytes) ainsi qu'en raison sa capacité à lier les ICR lgf2/HÎ9 dans ces cellules. Les premiers travaux expérimentaux de cette thèse portent sur le rôle possible des mutations de CTCFL chez les patients atteints du syndrome de Silver-Russell (SRS), où une diminution de la méthylation de l'ICR IGF2/H19 a été observée chez 60% d'entre eux. Admettant que CTCFL pourrait être muté chez ces patients, j'ai examiné les mutations possibles de CTCFL chez 35 d'entre eux par séquençage de l'ADN et analyse du nombre de copies d'exons. N'ayant trouvé aucune mutation chez ces patients, cela suggère que les mutations de CTCFL ne sont pas associées au SRS. Les travaux expérimentaux suivants ont porté sur les modifications post-traductionnelles de CTCFL par la protéine SU MO « small ubiquitin-like modifier » (SUMO). La modification de protéines par SU MO change les interactions avec d'autres molécules (ADN ou protéines). Comme CTCFL régule sans doute l'expression d'un certain nombre de gènes dans le cancer et que plusieurs facteurs de transcription sont régulés par SUMO, j'ai mené des expériences pour déterminer si CTCFL est sumoylé. En effet, j'ai observé que CTCFL est sumoylated in vitro et in vivo et j'ai déterminé les deux résidus d'attachement de SUMO aux lysines 181 et 645. Utilisant les mutants de CTCFL K181R et K645R ne pouvant pas être sumoylated, j'ai évalué les conséquences fonctionnelles de la modification par SUMO. Je n'ai trouvé aucun changement significatif dans la localisation subcellulaire, la demi-vie ou la liaison à l'ADN, mais ai constaté que la sumoylation module à la fois {'activation CTCFL-dépendante et la répression de l'expression génique. Il s'agit de la première modification post-traductionnelle décrite pour CTCFL et les conséquences possibles de cette modification sont discutées pour le cancer et les testicules normaux. Avec cette thèse, j'espère avoir ajouté des résultats importants à l'étude de CTCFL et donné quelques idées pour de futures recherches.AbstractJeremiah Bernier-Latmani, Institute of Pathology, University of Lausanne, CHUVCTCFL was first identified as a paralog of the ubiquitous protein CTCF because of high homology between their respective eleven zinc fingers, a DNA binding domain. Among its many roles, CTCF zinc finger-mediated binding to the unmethylated maternal Igf2/H19 imprinting control region (ICR), controls the imprinted (monoallelic) expression of Igf2 and H19 in somatic cells. Methylation of the paternal Igf2/H19 ICR is necessary for the imprinted expression of the two genes. Although the mechanism by which the ICR is methylated is incompletely understood, it is known that establishment of methylation occurs during male germ cell development and the de novo DNA methyltransferases DNMT3A and DNMT3L are essential. Therefore, CTCFL provided a good candidate to play a role in methylation of the paternal Igf2/H19 ICR because of its restricted expression to cell types where ICR methylation takes place (spermatogonia and spermatocytes) and its ability to bind the Igf2/H19 ICR in these cells. The first experimental work of this thesis investigated the possible role of CTCFL mutations in Silver-Russell syndrome (SRS) patients, where it has been observed that 60% of the patients have reduced methylation of the IGF2/HÎ9 ICR. Reasoning that CTCFL could be mutated in these patients, I screened 35 patients for mutations in CTCFL by DNA sequencing and exon copy number analysis, I did not find any mutations in these patients suggesting that mutations of CTCFL are not associated with SRS. The next experimental work of my thesis focused on posttranslational modification of CTCFL by small ubiquitin-like modifier (SUMO) protein. SUMO modification of proteins changes the interactions with other molecules (DNA or protein). As CTCFL arguably regulates the expression of a number of genes in cancer and many transcription factors are regulated by SUMO, I conducted experiments to assess whether CTCFL is sumoylated. I found that CTCFL is sumoylated in vitro and in vivo and determined the two residues of SUMO attachment to be lysines 181 and 645. Using K181R, K645R mutated CTCFL- which cannot be detected to be sumoylated-1 assessed the functional consequences of SUMO modification. I found no significant changes in subcellular localization, half-life or DNA binding, but found that sumoylation modulates both CTCFL-dependent activation and repression of gene expression. This is the first posttranslational modification described for CTCFL and possible consequences of this modification are discussed in both cancer and normal testis. With this thesis, I hope I have added important findings to the study of CTCFL and provide some ideas for future research.

Biochemical analysis of the major vaccinia virus envelope antigen.

The major envelope antigen of vaccinia virus is an acylated protein of M(r) 37,000 (p37K) which is required for the formation of extracellular enveloped virions (EEV). Despite its important role in the wrapping process, p37K has not been studied in much detail. In order to better characterize this protein we have undertaken a detailed biochemical analysis. Sodium carbonate treatment showed that p37K is tightly bound to the viral envelope. Its resistance to proteinase K digestion indicates that it is not exposed on the surface of EEV but lines the inner side of the envelope. Since p37K does not contain a signal peptide characteristic of most membrane proteins, we examined the possibility that the protein acquires its membrane affinity through the addition of fatty acids. Indeed, Triton X-114 phase partitioning experiments demonstrated that p37K is hydrophobic when acylated, but hydrophilic in the absence of fatty acids. Three other viral proteins have been shown to be required for virus envelopment and release from the host cell and we therefore tested whether p37K interacts with viral proteins. In EEV and in absence of reducing agents, an 80-kDa complex reacting with an anti-37K antiserum was found. Analysis of this complex showed that it most likely consists of a p37K homodimer. Interestingly, only a small amount of p37K occurs as a complex, most of it is present in the viral envelope as monomers.

Blood pressure reductions following catheter-based renal denervation are not related to improvements in adherence to antihypertensive drugs measured by urine/plasma toxicological analysis.

Renal denervation can reduce blood pressure in patients with uncontrolled hypertension. The adherence to prescribed antihypertensive medication following renal denervation is unknown. This study investigated adherence to prescribed antihypertensive treatment by liquid chromatography-high resolution tandem mass spectrometry in plasma and urine at baseline and 6 months after renal denervation in 100 patients with resistant hypertension, defined as baseline office systolic blood pressure ≥140 mmHg despite treatment with ≥3 antihypertensive agents. At baseline, complete adherence to all prescribed antihypertensive agents was observed in 52 patients, 46 patients were partially adherent, and two patients were completely non-adherent. Baseline office blood pressure was 167/88 ± 19/16 mmHg with a corresponding 24-h blood pressure of 154/86 ± 15/13 mmHg. Renal denervation significantly reduced office and ambulatory blood pressure at 6-month follow-up by 15/5 mmHg (p < 0.001/p < 0.001) and 8/4 mmHg (p < 0.001/p = 0.001), respectively. Mean adherence to prescribed treatment was significantly reduced from 85.0 % at baseline to 80.7 %, 6 months after renal denervation (p = 0.005). The blood pressure decrease was not explained by improvements in adherence following the procedure. Patients not responding to treatment significantly reduced their drug intake following the procedure. Adherence was highest for angiotensin-converting enzyme inhibitors/angiotensin receptor blockers and beta blockers (>90 %) and lowest for vasodilators (21 %). In conclusion, renal denervation can reduce office and ambulatory blood pressure in patients with resistant hypertension despite a significant reduction in adherence to antihypertensive treatment after 6 months.

Vasectomy effect on canine seminal plasma biochemical components and their correlation with seminal parameters

Three semen samples were collected at 48 It intervals from 20 mature research dogs previously conditioned to manual semen collection. Vasectomy was performed in all dogs, and 15 days after surgery, another three ejaculates were similarly collected. The semen was evaluated, and centrifuged to obtain seminal plasma for measurement of pH, and concentrations of total proteins (TP), total chlorides (Cl), calcium (Ca), potassium (K), and sodium (Na). The seminal plasma protein profile was evaluated by SDS-PAGE; molecular weights and the integrated optical density (IOD) of each band were estimated. There was a negative correlation between K concentration and progressive motility (r = -0.49, P = 0.027), sperm vigor (r = -0.60, P = 0.0053), and plasma integrity, evaluated by both the hypo-osmotic swelling test (r = -0.50, P = 0.026) and a fluorescent stain (r = -0.45, P = 0.046). Positive correlations between Na and K pre- and post-vasectomy (r = 0.88, P < 0.001; r = 0.56, P < 0.01, respectively) were verified. There were a total of 37 bands pre-vasectomy and 35 post-vasectomy (range, 100.6-3.6 kDa). Bands B9 and B13 (42.6 and 29.2 kDa) were not present post-vasectomy. The IOD of band B3 (73.5 kDa) was higher (P 0.03) pre-vasectomy, compared to post-vasectomy; conversely, the IODs of bands B29 and B37 (7.8 and 3.6 kDa) increased (P 0.026 and 0.047). Pre-vasectomy, there was a positive correlation (r = 0.49, P = 0.029) between band B37 band (3.6 kDa) and the Na:K ratio. In conclusion, K appeared to be involved in sperm motility in dogs and could be a tool to evaluate sperm function. The prostate contributed several elements to canine seminal plasma. Vasectomy changed Ca concentrations and the protein profile of the seminal plasma. Further studies must be performed to clarify the function of these elements on the in vivo fertility of dogs. (c) 2006 Elsevier B.V. All rights reserved.

Morphological and biochemical analysis of the stored and larval food of an obligate necrophagous bee, Trigona hypogea

Trigona hypogea, T. crassipes, and T. necrophaga are obligate necrophagous bees that differ from the majority of bees by using animal material instead of pollen as a protein resource. Since T. hypogea does not store protein in cerumen pots, it was thought that glandular secretions were its only larval protein source. This is in contrast to T. necrophaga which stores a yellowish proteinaceous jelly in the pots. Our results show that the larval food of T. hypogea has a higher protein content than the food stored in the pots and that it presents an electrophoretical protein pattern similar to that of the hypopharyngeal gland, indicating that workers add glandular secretions to the larval food while provisioning the brood cells. Thus, it can be suggested that T. hypogea has a provisioning behavior similar to other Meliponinae. The presence of several bands of proteins in the food stored in the pots shows that this species stores carrion mixed with honey in storage pots. Morphological data suggest that both larvae and adults make use of the same foodstuffs. These results also suggest that T. hypogea is more similar to other necrophagous species than it is to T. necrophaga (a more specialized bee).

Biochemical Analysis of a Castor Bean Leaf Extract and its Insecticidal Effects Against Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae)

Plant extracts represent a great source of molecules, with insecticidal activity, which are used for pest control in several crop production systems. This work aimed to evaluate the toxicity of an aqueous extract of leaves of castor bean against larvae of Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) in search for different classes of molecules with insecticidal activities by using in vitro assays. The effects of the castor bean leaf extract on the food utilization, development, and survival of S. frugiperda larvae was evaluated by feeding the larvae an artificial diet supplemented with different concentrations of the extract (0%, 1%, 2.5%, 5%, and 10% w/v). The effects observed were dose-dependent, and the highest concentration evaluated (10% w/v) was the one the most affected food utilization by altering the nutritional indices, as well as larval weight gain, development time, and survivorship. In vitro assays to detect saponins, lectins, and trypsin inhibitors in the castor bean leaf extract were performed, but only trypsin inhibitors were detected. No preference for the diet source was detected in S. frugiperda by feeding the larvae in choice experiments with diets containing different concentrations of the castor bean extract tested. The data obtained indicate the existence of a potential molecule in the tested extract of castor bean to be used as an alternative insecticide to be integrated in the management of S. frugiperda.

Biochemical analysis of the phosphatase domain of the human soluble epoxide hydrolase (sEH)

Die lösliche Epoxidhydrolase (sEH) gehört zur Familie der Epoxidhydrolase-Enzyme. Die Rolle der sEH besteht klassischerweise in der Detoxifikation, durch Umwandlung potenziell schädlicher Epoxide in deren unschädliche Diol-Form. Hauptsächlich setzt die sEH endogene, der Arachidonsäure verwandte Signalmoleküle, wie beispielsweise die Epoxyeicosatrienoic acid, zu den entsprechenden Diolen um. Daher könnte die sEH als ein Zielenzym in der Therapie von Bluthochdruck und Entzündungen sowie diverser anderer Erkrankungen eingesetzt werden. rnDie sEH ist ein Homodimer, in dem jede Untereinheit aus zwei Domänen aufgebaut ist. Das katalytische Zentrum der Epoxidhydrolaseaktivität befindet sich in der 35 kD großen C-terminalen Domäne. Dieser Bereich der sEH s wurde bereits im Detail untersucht und nahezu alle katalytischen Eigenschaften des Enzyms sowie deren dazugehörige Funktionen sind in Zusammenhang mit dieser Domäne bekannt. Im Gegensatz dazu ist über die 25 kD große N-terminale Domäne wenig bekannt. Die N-terminale Domäne der sEH wird zur Haloacid Dehalogenase (HAD) Superfamilie von Hydrolasen gezählt, jedoch war die Funktion dieses N-terminal Domäne lange ungeklärt. Wir haben in unserer Arbeitsgruppe zum ersten Mal zeigen können, dass die sEH in Säugern ein bifunktionelles Enzym ist, welches zusätzlich zur allgemein bekannten Enzymaktivität im C-terminalen Bereich eine weitere enzymatische Funktion mit Mg2+-abhängiger Phosphataseaktivität in der N-terminalen Domäne aufweist. Aufgrund der Homologie der N-terminalen Domäne mit anderen Enzymen der HAD Familie wird für die Ausübung der Phosphatasefunktion (Dephosphorylierung) eine Reaktion in zwei Schritten angenommen.rnUm den katalytischen Mechanismus der Dephosphorylierung weiter aufzuklären, wurden biochemische Analysen der humanen sEH Phosphatase durch Generierung von Mutationen im aktiven Zentrum mittels ortsspezifischer Mutagenese durchgeführt. Hiermit sollten die an der katalytischen Aktivität beteiligten Aminosäurereste im aktiven Zentrum identifiziert und deren Rolle bei der Dephosphorylierung spezifiziert werden. rnrnAuf Basis der strukturellen und möglichen funktionellen Ähnlichkeiten der sEH und anderen Mitgliedern der HAD Superfamilie wurden Aminosäuren (konservierte und teilweise konservierte Aminosäuren) im aktiven Zentrum der sEH Phosphatase-Domäne als Kandidaten ausgewählt.rnVon den Phosphatase-Domäne bildenden Aminosäuren wurden acht ausgewählt (Asp9 (D9), Asp11 (D11), Thr123 (T123), Asn124 (N124), Lys160 (K160), Asp184 (D184), Asp185 (D185), Asn189 (N189)), die mittels ortsspezifischer Mutagenese durch nicht funktionelle Aminosäuren ausgetauscht werden sollten. Dazu wurde jede der ausgewählten Aminosäuren durch mindestens zwei alternative Aminosäuren ersetzt: entweder durch Alanin oder durch eine Aminosäure ähnlich der im Wildtyp-Enzym. Insgesamt wurden 18 verschiedene rekombinante Klone generiert, die für eine mutante sEH Phosphatase Domäne kodieren, in dem lediglich eine Aminosäure gegenüber dem Wildtyp-Enzym ersetzt wurde. Die 18 Mutanten sowie das Wildtyp (Sequenz der N-terminalen Domäne ohne Mutation) wurden in einem Expressionsvektor in E.coli kloniert und die Nukleotidsequenz durch Restriktionsverdau sowie Sequenzierung bestätigt. Die so generierte N-terminale Domäne der sEH (25kD Untereinheit) wurde dann mittels Metallaffinitätschromatographie erfolgreich aufgereinigt und auf Phosphataseaktivität gegenüber des allgemeinen Substrats 4-Nitophenylphosphat getestet. Diejenigen Mutanten, die Phosphataseaktivität zeigten, wurden anschließend kinetischen Tests unterzogen. Basiered auf den Ergebnissen dieser Untersuchungen wurden kinetische Parameter mittels vier gut etablierter Methoden berechnet und die Ergebnisse mit der „direct linear blot“ Methode interpretiert. rnDie Ergebnisse zeigten, dass die meisten der 18 generierten Mutanten inaktiv waren oder einen Großteil der Enzymaktivität (Vmax) gegenüber dem Wildtyp verloren (WT: Vmax=77.34 nmol-1 mg-1 min). Dieser Verlust an Enzymaktivität ließ sich nicht durch einen Verlust an struktureller Integrität erklären, da der Wildtyp und die mutanten Proteine in der Chromatographie das gleiche Verhalten zeigten. Alle Aminosäureaustausche Asp9 (D9), Lys160 (K160), Asp184 (D184) und Asn189 (N189) führten zum kompletten Verlust der Phosphataseaktivität, was auf deren katalytische Funktion im N-terminalen Bereich der sEH hindeutet. Bei einem Teil der Aminosäureaustausche die für Asp11 (D11), Thr123 (T123), Asn124 (N124) und Asn185 (D185) durchgeführt wurden, kam es, verglichen mit dem Wildtyp, zu einer starken Reduktion der Phosphataseaktivität, die aber dennoch für die einzelnen Proteinmutanten in unterschiedlichem Ausmaß zu messen war (2 -10% and 40% of the WT enzyme activity). Zudem zeigten die Mutanten dieser Gruppe veränderte kinetische Eigenschaften (Vmax allein oder Vmax und Km). Dabei war die kinetische Analyse des Mutanten Asp11  Asn aufgrund der nur bei dieser Mutanten detektierbaren starken Vmax Reduktion (8.1 nmol-1 mg-1 min) und einer signifikanten Reduktion der Km (Asp11: Km=0.54 mM, WT: Km=1.3 mM), von besonderem Interesse und impliziert eine Rolle von Asp11 (D11) im zweiten Schritt der Hydrolyse des katalytischen Zyklus.rnZusammenfassend zeigen die Ergebnisse, dass alle in dieser Arbeit untersuchten Aminosäuren für die Phosphataseaktivität der sEH nötig sind und das aktive Zentrum der sEH Phosphatase im N-terminalen Bereich des Enzyms bilden. Weiterhin tragen diese Ergebnisse zur Aufklärung der potenziellen Rolle der untersuchten Aminosäuren bei und unterstützen die Hypothese, dass die Dephosphorylierungsreaktion in zwei Schritten abläuft. Somit ist ein kombinierter Reaktionsmechanismus, ähnlich denen anderer Enzyme der HAD Familie, für die Ausübung der Dephosphorylierungsfunktion denkbar. Diese Annahme wird gestützt durch die 3D-Struktur der N-terminalen Domäne, den Ergebnissen dieser Arbeit sowie Resultaten weiterer biochemischer Analysen. Der zweistufige Mechanismus der Dephosphorylierung beinhaltet einen nukleophilen Angriff des Substratphosphors durch das Nukleophil Asp9 (D9) des aktiven Zentrums unter Bildung eines Acylphosphat-Enzym-Zwischenprodukts, gefolgt von der anschließenden Freisetzung des dephosphorylierten Substrats. Im zweiten Schritt erfolgt die Hydrolyse des Enzym-Phosphat-Zwischenprodukts unterstützt durch Asp11 (D11), und die Freisetzung der Phosphatgruppe findet statt. Die anderen untersuchten Aminosäuren sind an der Bindung von Mg 2+ und/oder Substrat beteiligt. rnMit Hilfe dieser Arbeit konnte der katalytischen Mechanismus der sEH Phosphatase weiter aufgeklärt werden und wichtige noch zu untersuchende Fragestellungen, wie die physiologische Rolle der sEH Phosphatase, deren endogene physiologische Substrate und der genaue Funktionsmechanismus als bifunktionelles Enzym (die Kommunikation der zwei katalytischen Einheiten des Enzyms) wurden aufgezeigt und diskutiert.rn

Biochemical analysis of mutations in P450 oxidoreductase

All microsomal P450s require POR (cytochrome P450 reductase) for catalytic activity. Most of the clinically used drugs are metabolized by a small number of P450s and polymorphisms in the cytochrome P450s are known to cause changes in drug metabolism. We have recently found a number of POR missense mutations in the patients with disordered steroidogenesis. Our initial report described five missense mutations (A284P, R454H, V489E, C566Y and V605F) identified in four patients. We built bacterial expression vectors for each POR variant, purified the membranes expressing normal or variant POR and characterized their activities with cytochrome c and P450c17 assays. We have recently completed an extensive study of the range of POR mutations and characterized the mutants/polymorphisms A112V, T139A, M260V, Y456H, A500V, G536R, L562P, R613X, V628I and F643del from sequencing of patient DNA. We also studied POR variants Y179D, P225L, R313W, G410S and G501R that were available in databases or the published literature. We analysed the mutations with a three-dimensional model of human POR that was based on an essentially similar rat POR with known crystal structure. The missense mutations found in patients with disordered steroidogenesis mapped to functionally important domains of POR and the apparent polymorphisms mapped to less crucial regions. Since a variation in POR can alter the activity of all microsomal P450s, it can also affect the drug metabolism even with a normal P450. Understanding the genetic and biochemical basis of POR-mediated drug metabolism will provide valuable information about possible differences in P450-mediated reactions among the individuals carrying a variant or polymorphic form of POR.