Dietary glutamine supplementation increases the activity of peritoneal macrophages and hemopoiesis in early-weaned mice inoculated with Mycobacterium bovis bacillus Calmette-Guerin

Infants who are breast-fed have been shown to have a lower incidence of certain infectious diseases compared with formula-fed infants. Glutamine is one of the most abundant amino acids found in maternal milk and it is essential for the function of immune system cells such as macrophages. The purpose of this study was to investigate the effect of glutamine supplementation on the function of peritoneal macrophages and on hemopoiesis in early-weaned mice inoculated with Mycobacterium bovis bacillus Calmette-Guerin (BCG). Mice were wearied at 14 d of age and distributed to 2 groups and fed either a glutamine-free diet (n = 16) or a glutamine-supplemented diet (+Gln (n = 16). Both diets were isonitrogenous (with addition of a mixture of nonessential amino acids) and isocaloric. At d 21, 2 subgroups of mice (n = 16) were intraperitoneally injected with BCG and all mice were killed at d 28. Plasma, muscle and liver glutamine concentrations and muscle glutamine synthetase activity were not affected by diet or inoculation with BCG. The +GIn diet led to increased leukocyte and lymphocyte counts in the peripheral blood (P < 0.05) and granulocyte and lymphocyte counts in the bone marrow and spleen (P < 0.05). The +GIn diet increased spreading and adhesion capacities, hydrogen peroxide, nitric oxide, and tumor necrosis factor-alpha (TNF alpha) syntheses and the phagocytic and fungicidal activity of peritoneal macrophages (P < 0.05). The interaction between the +GIn diet and BCG inoculation increased the area under the curve of interleukin (IL)-1 beta and TNF alpha syntheses (P < 0.05). In conclusion, the intake of glutamine increases the function of peritoneal macrophages and hemopoiesis in early-weaned and BCG-inoculated mice. These data have important implications for the design of breast milk substitutes for human infants.

Amino acid synthesis in the symbiotic sea anemone Aiptasia pulchella

Symbiotic Aiptasia pulchella and freshly isolated zooxanthellae were incubated in (NaHCO3)-C-14 and NH4Cl for 1 to 240 min, and samples were analysed by reverse-phase high-performance liquid chromatography (HPLC) and an online radiochemical detector. NH4+ was first assimilated into C-14-glutamate and C-14-glutamine in the zooxanthellae residing in A. pulchella. The specific activities (dpm nmol(-1)) of C-14-glutamate and C-14-glutamine in vivo, were far greater in the zooxanthellae than in the host tissue, indicating that NH4+ was principally incorporated into the glutamate and glutamine pools of the zooxanthellae. C-14-alpha-ketoglutarate was taken up from the medium by intact A. pulchella and assimilated into a small amount of C-14-glutamate in the host tissue, but no C-14-glutamine was detected in the host fraction. The C-14-glutamate that was synthesized was most likely produced from transamination reactions as opposed to the direct assimilation of NH4+. The free aminoacid composition of the host tissue and zooxanthellae of A. pulchella was also measured. The results presented here demonstrate that NH4+ was initially assimilated by the zooxanthellae of A. pulchella.

The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages

Mycobacterium tuberculosis is an important pathogen of mammals that relies on 2-hydroxyphenyloxazoline-containing siderophore molecules called mycobactins for the acquisition of iron in the restrictive environment of the mammalian macrophage, These compounds have been proposed to be biosynthesized through the action of a cluster of genes that include both nonribosomal peptide synthase and polyketide synthase components. One of these genes encodes a protein, MbtB, that putatively couples activated salicylic acid with serine or threonine and then cyclizes this precursor to the phenyloxazoline ring system. We have used gene replacement through homologous recombination to delete the mbtB gene and replace this with a hygromycin-resistance cassette in the virulent strain of M. tuberculosis H37Rv, The resulting mutant is restricted for growth in iron-limited media but grows normally in iron-replete media. Analysis of siderophore production by this organism revealed that the biosynthesis of all salicylate-derived siderophores was interrupted. The mutant was found to be impaired for growth in macrophage-like THP-1 cells, suggesting that siderophore production is required for virulence of M. tuberculosis, These results provide conclusive evidence linking this genetic locus to siderophore production.

Th structure of the P-II-ATP complex

P-II is a signal transduction protein that is part of the cellular machinery used by many bacteria to regulate the activity of glutamine synthetase and the transcription of its gene. The structure of P-II was solved using a hexagonal crystal form (form I). The more physiologically relevant form of P-II is a complex with small molecule effecters. We describe the structure of P-II with ATP obtained by analysis of two different crystal forms (forms II and III) that were obtained by co-crystallization of P-II with ATP. Both structures have a disordered recognition (T) loop and show differences at their C termini. Comparison of these structures with the form I protein reveals changes that occur on binding ATP. Surprisingly, the structure of the P-II/ATP complex differs with that of GlnK, a functional homologue. The two proteins bind the base and sugar of ATP in a similar manner but show differences in the way that they interact with the phosphates. The differences in structure could account for the differences in their activities, and these have been attributed to a difference in sequence at position 82. It has been demonstrated recently that P-II and GlnK form functional heterotrimers in vivo. We construct models of the heterotrimers and examine the junction between the subunits.

Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYPO2OB were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii. (C) 2001 John Wiley & Sons, Inc.

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications. (C) 2010 Elsevier GmbH. All rights reserved.

Microcystin production by a freshwater spring cyanobacterium of the genus Fischerella

We investigated the production of a hepatotoxic, cyclic heptapeptide, microcystin, by a filamentous branched cyanobacterium belonging to the order Stigonematales, genus Fischerella. The freshwater Fischerella sp. strain CENA161 was isolated from spring water in a small concrete dam in Piracicaba, Sao Paulo State, Brazil, and identified by combining a morphological description with 16S rRNA gene sequencing and phylogenetic analysis. Microcystin (MCYST) analysis performed using an ELISA assay on cultured cells gave positive results. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis detected 33.6 mu g MCYST-LR per gram dry weight of cyanobacterial cells. Microcystin profile revealed by quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) analysis confirmed the production of MCYST-LR. Furthermore, genomic DNA was analyzed by PCR for sequences similar to the ketosynthase (KS) domain of the type I polyketide synthase gene, which is involved in microcystin biosynthesis. This revealed the presence of a KS nucleotide fragment similar to the mcyD and ndaD genes of the microcystin and nodularin synthetase complexes. Phylogenetic analysis grouped the Fischerella KS sequence together with mcyD sequences of the three known microcystin synthetase operon (Microcystis, Planktothrix and Anabaena) and ndaD of the nodularin synthetase operon, with 100% bootstrap support. Our findings demonstrate that Fischerella sp. CENA161 produces MYCST-LR and for the first time identify a nucleotide sequence putatively involved in microcystin synthesis in this genus. (C) 2009 Elsevier Ltd. All rights reserved.

Protein crystallography and examples of its applications in medicinal chemistry

The field of protein crystallography inspires and enthrals, whether it be for the beauty and symmetry of a perfectly formed protein crystal, the unlocked secrets of a novel protein fold, or the precise atomic-level detail yielded from a protein-ligand complex. Since 1958, when the first protein structure was solved, there have been tremendous advances in all aspects of protein crystallography, from protein preparation and crystallisation through to diffraction data measurement and structure refinement. These advances have significantly reduced the time required to solve protein crystal structures, while at the same time substantially improving the quality and resolution of the resulting structures. Moreover, the technological developments have induced researchers to tackle ever more complex systems, including ribosomes and intact membrane-bound proteins, with a reasonable expectation of success. In this review, the steps involved in determining a protein crystal structure are described and the impact of recent methodological advances identified. Protein crystal structures have proved to be extraordinarily useful in medicinal chemistry research, particularly with respect to inhibitor design. The precise interaction between a drug and its receptor can be visualised at the molecular level using protein crystal structures, and this information then used to improve the complementarity and thus increase the potency and selectivity of an inhibitor. The use of protein crystal structures in receptor-based drug design is highlighted by (i) HIV protease, (ii) influenza virus neuraminidase and (iii) prostaglandin H-2-synthetase. These represent, respectively, examples of protein crystal structures that (i) influenced the design of drugs currently approved for use in the treatment of HIV infection, (ii) led to the design of compounds currently in clinical trials for the treatment of influenza infection and (iii) could enable the design of highly specific non-steroidal anti-inflammatory drugs that lack the common side-effects of this drug class.

Resistance to antiparasitic drugs: The role of molecular diagnosis

Chemotherapy is central to the control of many parasite infections of both medical and veterinary importance. However, control has been compromised by the emergence of drug resistance in several important parasite species. Such parasites cover a broad phylogenetic range and include protozoa, helminths and arthropods. In order to achieve effective parasite control in the future, the recognition and diagnosis of resistance will be crucial. This demand for early, accurate diagnosis of resistance to specific drugs in different parasite species can potentially be met by modern molecular techniques. This paper summarises the resistance status of a range of important parasites and reviews the available molecular techniques for resistance diagnosis. Opportunities for applying successes in some species to other species where resistance is less well understood are explored. The practical application of molecular techniques and the impact of the technology on improving parasite control are discussed. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Macropodid herpesvirus 1 encodes genes for both thymidylate synthase and ICP34.5

Macropodid herpesvirus 1 (MaHV-1) is an unclassified alphaherpesvirus linked with the fatal infections of kangaroos and other marsupials. During the characterisation of the internal repeat region of MaHV-1, an open reading frame (ORF) encoding for thymidylate synthase (TS) gene was identified and completely sequenced. Southern blot analysis confirmed the presence of two copies of the TS gene in the MaHV-1 genome as expected. Computer analysis of the TS ORF showed it was 948 nucleotides in length. A putative polyadenylation signal was identified 17-22 bp inside the ORF implying a minimal or absent 3' untranslated region. The predicted polypeptide was 316 amino acid residues in length and contained the highly conserved motifs for folate binding and F-dUMP binding, typical of all TS enzymes. Interestingly, MaHV-1 TS polypeptide had highest similarity to the human TS polypeptide (81%) compared to the TS polypeptides of other herpesviruses (72-75%). Immediately upstream of the TS gene, a second ORF of 510 bp, encoding a polypeptide with 170 amino acid residues, was identified. The carboxyl domain of this MaHV-1 polypeptide shared 68% similarity to a 59 amino acid motif of human herpesvirus 1 ICP34.5, identifying it as the MaHV-1 ICP34.5 homologue. This is the first report of a herpesvirus that encodes for both TS and ICP34.5.

Desenvolvimento de um biossensor para Ácido Glutâmico

A presente dissertação tem com objetivo o desenvolvimento de um biossensor com base nos polímeros de impressão molecular para a deteção de uma molécula alvo, o ácido glutâmico que é convertido em glutamina pela glutamina sintetase, recorrendo à potenciometria. Nas células neoplásicas a glutamina não é sintetizada podendo-se considerar que o ácido glutâmico é um potencial agente anti-cancro. A técnica de impressão molécular utilizada foi a polimerização em bulk, combinando a acrilamida e a bis acrilamida com o ácido glutâmico. Para se verificar se a resposta potenciométrica obtida era de facto da molécula alvo foram preparados em paralelo com os sensores, materiais de controlo, ou seja, moléculas sem impressão molécular (NIP). Para se controlar a constituíção química dos vários sensores nomeadamente, do NIP e do polímero de impressão molecular (MIP) antes e após a remoção bem como a molécula foram realizados estudos de Espetroscopia de Infravermelhos de Transformada de Fourier (FTIR), Scanning electron microscope (SEM) e Espetroscopia de Raios X por dispersão em energia (EDS). Os materiais desenvolvidos foram aplicados em várias membranas que diferiam umas das outras, sendo seletivas ao ião. A avaliação das características gerais das membranas baseou-se na análise das curvas de calibração, conseguidas em meios com pHs diferentes, comparando os vários elétrodos. O pH 5 foi o que apresentou melhor resultado, associado a uma membrana que continha um aditivo, o p-tetra-octilphenol, e com o sensor com percentagem de 3%. Posto isto, testou-se em material biológico, urina, com as melhores características quer em termos de sensibilidade (18,32mV/década) quer em termos de linearidade (1,6x10-6 a 1,48x10-3 mol/L). Verificou-se ainda que aplicando iões interferentes na solução, estes não interferem nesta, podendo ser aplicados na amostra sem que haja alteração na resposta potenciométrica. O elétrodo é capaz de distinguir o ácido glutâmico dos restantes iões presentes na solução.

The Role of Prostaglandins in the Lower Urinary Tract Dynamics of Prostatectomized Patients: a Trial with Indomethacin and Aspirin

Using 49 prostatectomized patients as experimental subjects, we studied the effects of Inclometnacin and acetylsalicylic acid — accredited prostaglandin synthetase inhibitors — from a urodynamic and clinical standpoint. Relevant urodynamic data was gathered 1 hr 30 mi after the patients had taken the drugs and placebo. Clinical results were further scrutinized after 8 days of treatment, at which time a new urodynamic workup was again performed on some patients. Results were again studied shortly after the end of treatment. The effect of the drugs on bladder and urethral structures was borne out by clear-ct!t clinical and urodynamic changes. After statistically analyzing such changes, we concluded that prostaglandin synthesis inhibition resulting in the inhibition of prostaglandin action had, at least in part, led to the changes noted. In the present report we shall discuss the role played by the highly complex mechanisms at work.

Clinical Outcomes and Genetic Expression Profile in Human Liver Graft Dysfunction During Ischemia/Reperfusion Injury

Introduction. This study aims to compare the molecular gene expression during ischemia reperfusion injury. Several surgical times were considered: in the beginning of the harvesting (T0), at the end of the cold ischemia period (T1), and after reperfusion (T2) and compared with graft dysfunction after liver transplant (OLT). Methods. We studied 54 patients undergoing OLT. Clinical, laboratory data, and histologic data (Suzuki classification) as well as the Survival Outcomes Following Liver Transplantation (SOFT) score were used and compared with the molecular gene expression of the following genes: Interleukin (IL)-1b, IL-6, tumor necrosis factor-a, perforin, E-selectin (SELE), Fas-ligand, granzyme B, heme oxygenase-1, and nitric oxide synthetase. Results. Fifteen patients presented with graft dysfunction according to SOFT criteria. No relevant data were obtained by comparing the variables graft dysfunction and histologic variables. We observed a statistically significant relation between SELE at T0 (P ¼ .013) and IL-1b at T0 (P ¼ .028) and early graft dysfunction. Conclusions. We conclude that several genetically determined proinflammatory expressions may play a critical role in the development of graft dysfunction after OLT.

Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Tese de Doutoramento em Ciências - Especialidade em Biologia