Estudos recentes têm demonstrado que a suplementação de beta-alanina (BA) pode melhorar o desempenho físico. O mecanismo proposto para tal resultado envolve o aumento das concentrações intramusculares de carnosina, um dipeptídeo cuja função mais bem atribuída é a de manutenção do equilíbrio ácido-básico. Apesar do emergente corpo literário acerca dos efeitos ergogênicos da suplementação de BA, a maior parte das evidências provém de estudos conduzidos com indivíduos não treinados ou fisicamente ativos, enquanto os estudos com indivíduos treinados são escassos, e seus resultados, controversos. Tem sido especulado que a diferença na capacidade tamponante muscular entre indivíduos treinados e não treinados é um possível fator mascarando o efeito ergogênico da suplementação de BA em indivíduos treinados, já que têm sido demonstrado que este perfil de indivíduos possui maior capacidade tamponante e conteúdo muscular de carnosina. Assim, o objetivo do presente estudo foi investigar a influência do estado de treinamento sobre o desempenho físico intermitente de membros inferiores em resposta à suplementação de BA. Para tanto, 40 homens jovens e saudáveis foram recrutados para participar do estudo, e divididos em dois grupos de acordo com o seu estado de treinamento [ciclistas treinados (T) ou indivíduos não treinados (NT)]. Os participantes foram aleatoriamente designados a um grupo suplementado com BA ou placebo (dextrose - PL), provendo quatro condições experimentais: NTPL, NTBA, TPL e TBA. A suplementação foi realizada com a ingestão de 6.4 gramas de BA ou PL por dia, durante 4 semanas. Antes e após o período de suplementação, os participantes completaram 4 séries do teste de Wingate para membro inferior, com 30 segundos de duração cada uma e 3 minutos de descanso entre elas. O trabalho total realizado foi significantemente aumentado após o período de suplementação em ambos os grupos NTBA (+1349 ± 1411 kJ; P = 0.03) e TBA (+1978 ± 1508 kJ; P = 0.002), foi significantemente reduzido no grupo NTPL (-1385 ± 2815 kJ; P = 0.03), e não se alterou no grupo TPL (-219 ± 1507 kJ; P = 0.73). Comparada ao período pré-suplementação, a potência média no período pós-suplementação foi significantemente maior na série 4 para o grupo NTBA (P = 0.0004), enquanto a mesma foi maior nas séries 1, 2 e 4 (P <= 0.05) para o grupo TBA. Não foram observadas diferenças na potência média entre o período pré- e pós-suplementação para os grupos NTPL e TPL. Em conclusão, quatro semanas de suplementação de BA foram efetivas em melhorar o desempenho físico intermitente de membros inferiores em ambos os participantes treinados e não treinados. Estes dados ressaltam a eficácia ergogênica da suplementação de BA para exercícios de alta-intensidade, independentemente do estado de treinamento do indivíduo / Recent studies have demonstrated that beta-alanine (BA) supplementation can improve performance. The proposed mechanisms for this result involve an increased muscle carnosine content, a dipeptide whose function is attributed to the maintenance of acid-base balance. Even though the body of evidence surrounding the ergogenic effects of BA supplementation is increasing, most of the evidences come from studies conducted with physically active or untrained individuals, while studies with trained participants are scarce, and their results, controversial. It has been speculated that the difference in muscle buffering capacity between trained and untrained individuals is a possible factor masking the ergogenic effect of BA supplementation in trained individuals, who have already been demonstrated to have greater buffering capacity and muscle carnosine content. Therefore, the aim of this study was to investigate the influence of training status on intermittent lower-body performance in response to BA supplementation. For this purpose, forty young males were divided into two groups according to their training status (trained - T, and untrained - NT cyclists). Participants were further randomly allocated to BA or placebo (dextrose - PL) groups, providing four experimental conditions: NTPL, NTBA, TPL, TBA. BA or PL was ingested by 6.4 g·d-1, during for 4 weeks. Before and after the supplementation period, participants completed four 30-seconds lower-body Wingate bouts, separated by 3 minutes. Total work done was significantly increased following supplementation in both NTBA (+1349 ± 1411 kJ; P = 0.03) and TBA (+1978 ± 1508 kJ; P = 0.002), and it was significantly reduced in NTPL (-1385 ± 2815 kJ; P = 0.03) with no difference for TPL (-219 ± 1507 kJ; P = 0.73). Compared to pre-supplementation, post-supplementation mean power output was significantly higher in bout 4 for NTBA (P = 0.0004), and higher in bouts 1, 2 and 4 (P <= 0.05) for TBA. No differences were observed in mean power output for NTPL and TPL from pre- to post-supplementation period. In conclusion, four weeks of BA supplementation was effective at improving intermittent lower-body performance in both untrained and trained individuals. These data highlight the efficacy of BA as an ergogenic aid for high-intensity exercise regardless of the training status of the individual

No description available.

In insects, the cuticle provides protection against physical injury and water loss, rigidness for muscle attachment and mechanical support, and flexibility in inter-segmental and joint areas for mobility. As most insects undergo metamorphosis, they need to shred off old cuticle and synthesize new cuticle to fit the body shape and size throughout their life cycles. The newly formed cuticle, mainly composed of cuticular proteins, chitin, and sclerotizing reagents, needs to be hardened through the crosslinks between cuticular proteins and sclerotizing reagents. This dissertation concerns the biochemical activities of several pyridoxal 5-phosphate (PLP)-dependent decarboxylases with most of them involved in insect cuticle hardening. Herein, we first present a detailed overview of topics in reactions and enzymes involved in insect cuticle hardening. Aspartate 1-decarboxylase (ADC) is at the center of this dissertation. beta-alanine, the product of ADC-catalyzed reaction from aspartate, is the component of an important sclerotizing reagent, N-beta-alanyldopamine; the levels of beta-alanine in insects regulate the concentrations of dopamine, therefore affecting insect sclerotization and tanning (collectively referred as cuticle hardening in this dissertation). Biochemical characterization of insect ADC has revealed that this enzyme has typical mammalian cysteine sulfinic acid decarboxylase (CSADC) activity, able to generate hypotaurine and taurine. The result throws lights on research in the physiological roles of insect ADC and the pathway of insect taurine biosynthesis. Cysteine was found to be  an inactivator of several PLP-dependent decarboxylases, such as ADC, glutamate decarboxylase (GAD) and CSADC. This study helps to understand symptoms associated with the abnormal cysteine concentrations in several neurodegenerative diseases. A mammalian enzyme, glutamate decarboxylase like-1 (GADL1), has been shown to have the same substrate usage as insect ADC does, potentially contributing to the biosynthesis of taurine and/or beta-alanine in mammalian species. Finally, the metabolic engineering work of L-3, 4-dihydroxyphenylalanine decarboxylase (DDC) and 3, 4-dihydroxylphenylacetaldehyde (DHPAA) synthase has revealed that the reactions of these enzymes could be determined by a few conserved residues at their active site. As both enzymes have been implicated in the biosynthesis of sclerotizing reagents, it is of great scientific and practical importance to understand the similarity and difference in their reaction mechanisms. The results of this dissertation provide valuable biochemical information of ADC, DDC, DHPAA synthase, and GADL1, all of which are PLP-dependent decarboxylases. ADC, DDC, DHPAA synthase are important enzymes in insect cuticle hardening by contributing to the biosynthesis of sclerotizing reagents. Knowledge towards understanding of these enzymes will promote the comprehension of insect cuticle hardening and help scientists to search for ideal insecticide targets. The characterization of GADL1 lays groundwork for future research of its potential role in taurine and beta-alanine metabolism. / Ph. D.

No description available.

No description available.

Στην παρούσα εργασία συνθέσαμε ένα άμινο-άκυλο- και ένα πεπτίδυλο- ανάλογο της χλωραμφαινικόλης. Τα ανάλογα αυτά ήταν η β-αλανίνη-χλωραμφαινικόλη (β-alaCAM) και η φαινυλαλανίνη-φαινυλαλανίνη-χλωραμφαινικόλη (PhePheCAM). Στην συνέχεια μελετήσαμε την βιολογική συμπεριφορά των αναλόγων αυτών μέσα από την μελέτη της κινητικής της αναστολής του σχηματισμού πεπτιδικού δεσμού που επιφέρουν τα εν λόγω ανάλογα. Σε πρωτεϊνοσυνθετικό σύστημα ριβοσωμάτων εκπορευόμενων από Escherichia coli η σύνθεση ακέτυλο-φαινυλαλάνυλο-πουρομυκίνης πραγματοποιείται μέσω μιας αντίδρασης ψευδοπρώτης τάξεως μεταξύ συμπλέγματος C, δηλαδή ακέτυλο-φαινυλαλάνυλο-poly(U)-ριβοσωμάτων, και περίσσειας πουρομυκίνης. Τόσο η β-alaCAM, όσο και η PhePheCAM μελετήθηκαν ως αναστολείς της αντίδρασης σύνθεσης ακέτυλο-φαινυλαλάνυλο-πουρομυκίνης και τα αποτελέσματα της κινητικής της αναστολής που επέφεραν συγκρίθηκαν με γνωστά από την βιβλιογραφία αντίστοιχα αποτελέσματα που αφορούν τόσο την μητρική ένωση, όσο και άλλα άμινο-άκυλο- και πεπτιδικά ανάλογα αυτής. Αρχικά παρατηρήσαμε ότι, απουσία αναστολέα, η αντίδραση ακολουθεί κινητική πρώτης τάξεως καθόλη την χρονική διάρκεια της χημικής αντίδρασης. Ωστόσο, στη συνέχεια παρατηρήσαμε ότι η παρουσία τόσο της β-alaCAM, όσο και της PhePheCAM είχε σαν αποτέλεσμα διφασικές λογαριθμικές συναρτήσεις συγκέντρωσης – χρόνου, όπου υφίστατο μία αρχική ή πρώτη χρονική φάση και μία τελική ή δεύτερη χρονική φάση της χημικής αντίδρασης πουρομυκίνης. Ακολούθησε λεπτομερής κινητική ανάλυση, αρχικά μέσω διαγραμμάτων διπλού αντιστρόφου για τις αρχικές και τις τελικές κλίσεις των λογαριθμικών χρονοκαμπυλών, καθώς και στη συνέχεια μέσω επαναδιαγραμμάτων αρχικών και τελικών κλίσεων έναντι της συγκέντρωσης του αναστολέα. Με τον τρόπο αυτό υπολογίστηκαν οι κινητικές σταθερές αναστολής Κi οι οποίες και συγκρίθηκαν με την κινητική σταθερά αναστολής της μητρικής ένωσης. Τέλος, μέσω υπολογιστικού προγράμματος προσομοίωσης, σχεδιάστηκαν οι συναρτήσεις της φαινομενικής σταθεράς εξισορρόπησης keq έναντι της συγκέντρωσης του αναστολέα και υπολογίστηκαν οι σταθερές k6 και k7. Tόσο η β-alaCAM όσο και η PhePheCAM εμφάνισαν συμπεριφορά βραδέως προσδενομένου συναγωνιστικού αναστολέα ανεξάρτητα από την συγκέντρωσή τους, σε αντίθεση με την μητρική ένωση η οποία εμφανίζει συμπεριφορά συναγωνιστικού αναστολέα σε μικρές συγκεντρώσεις αυτής και συμπεριφορά μικτού μη-συναγωνιστικού αναστολέα σε μεγαλύτερες συγκεντρώσεις αυτής. H β-alaCAM ευρέθηκε 4,6 φορές περισσότερο βιολογικά δραστική από την PhePheCAM και 14,3 βιολογικά ασθενέστερη από την μητρική ένωση. Σε αντίθεση με τη μητρική ένωση, η οποία δεν υφίσταται ισομερισμό, τόσο η β-alaCAM όσο και η PhePheCAM δίνουν, στην τελική ή δεύτερη χρονική φάση της αντίδρασης πουρομυκίνης, γένεση στο ισομερισμένο σύμπλοκο C*I. Αξιοσημείωτη είναι η παρατήρηση ότι ο σχηματισμός του ισομερισμένου συμπλόκου C*I λαμβάνει χώρα μέσω δύο κινητικών βημάτων στην περίπτωση της β-alaCAM, αλλά μέσω ενός μόνο κινητικού βήματος στην περίπτωση της PhePheCAM. Προτείνουμε, ως μοντέλο επεξηγηματικό του μηχανισμού βιολογικής δράσης και χημικής κινητικής των μελετηθέντων συνθετικών αναλόγων, ότι τόσο η β-alaCAM όσο και η PhePheCAM παρουσιάζουν αυξημένη στερεοχημική ομοιότητα με το 3΄-άκρο του άμινο-άκυλο-tRNA ή με το 3΄-άκρο του πεπτίδυλο-tRNA συγκριτικά με τη μητρική ένωση. Η αυξημένη αυτή στερεοχημική ομοιότητα πιθανότατα εξηγεί τον εκσεσημασμένο συναγωνιστικό χαρακτήρα της αναστολής που εμφανίζουν τα μελετηθέντα ανάλογα συγκριτικά με τη μητρική ένωση, ασχέτως του γεγονότος ότι η συνολική αναστολή που επιφέρουν δεν αποδεικνύεται σε καμία περίπτωση ισχυρότερη της αναστολής που επιφέρει η μητρική ένωση. Για τον λόγο αυτό τα εν λόγω συνθετικά ανάλογα της χλωραμφαινικόλης θα πρέπει να θεωρηθούν παλίνδρομα-ανάστροφα ανάλογα (retro-inverso analogs). / One aminoacyl and one peptidyl analog of chloramphenicol (Cl2CHCO-CAM) were prepared. These are L-β-alaCAM and L-PhePheCAM. The kinetics of inhibition of peptide bond formation by these analogs were examined in a cell-free system which had been used previously for the study of Cl2CHCO-CAM [Drainas et al, Eur. J. Biochem. 1987, 164, 53-58]. In a model cell-free system, derived from Escherichia coli, acetylphenylalanyl-puromycin is produced in a pseudo-first-order reaction between the preformed acetylphenylalanyl/tRNA/poly(U)/ribosome complex (complex C) and excess puromycin. Both L-β-alaCAM and L-PhePheCAM were tested as inhibitors in this reaction. In the absence of inhibitor, the reaction follows first-order kinetics for the entire course of the reaction. In the presence of the analog the reaction gives biphasic log-time plots. The kinetic informations pertaining to the initial and the terminal slopes of the plot are analyzed (initial-slope and terminal-slope analysis). Μοreover, through a computer simulation non-linear regression fitting program, the plots between the keq values and the concentration of the inhibitor [I] were constructed, and consequently the values of k6 and k7 were estimated. Detailed kinetic analysis suggests that both these analogs (I) behave as slow-binding inhibitors and react competitively with complex C to form the complex C*I which is inactive towards puromycin. In the presence of L-β-alaCAM, C*I is formed via a two-step mechanism in which C*I is the product of a slow conformational change of the initial encounter complex CI according to the equation C + I CI C*I. Our results, concerning the two-step mechanism of L-β-alaCAM are in agreement with the results of previous investigations evaluating the potency and kinetic mechanisms of other aminoacyl and peptidyl analogs of chloramphenicol [Michelinaki et al, Mol. Pharmacol. 1997, 51, 139-146]. However, in the presence of L-PhePheCAM, our results are unique because we found evidences that C*I is formed via a one-step mechanism as a product of a slow conformational change according to the equation C + I C*I. The parent compound gives complex inhibition kinetics; increasing the concentration of the parent compound changes the inhibition from competitive to mixed noncompetitive [Drainas et al, Eur. J. Biochem. 1987, 164, 53-58]. In contrast, the analogs give competitive kinetics even at high concentrations of the inhibitor. The following Ki and Ki* values have been determined: Ki = 45 μΜ for L-β-alaCAM, Ki* = 10 μΜ for L-β-alaCAM and Ki* = 46 μM for L-PhePheCAM. If we were to assume that both L-β-alaCAM and L-PhePheCAM behave as classical competitive inhibitors, we could say that L-β-alaCAM is 4.6 times more potent than L-PhePheCAM. On this assumption we could also compare chloramphenicol with L-β-alaCAM and see that L-β-alaCAM is 14.3 times weaker than chloramphenicol (Ki = 0.7 μΜ). It is suggested that as compared with chloramphenicol, both L-β-alaCAM and L-PhePheCAM have increased structural similarity to the 3΄-terminus of aminoacyl-tRNA or of peptidyl-tRNA and this similarity results in a more pronounced competitive inhibition. The results are compared with previous data and discussed on the basis of a possible retro-inverso relationship between chloramphenicol analogs and puromycin.

No description available.