Mutational analyses of human eIF5A-1-identification of amino acid residues critical for eIF5A activity and hypusine modification

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.

Lightning activity in Brazilian thunderstorms during TROCCINOX: implications for NOx production

During the TROCCINOX field experiment in January and February 2005, the contribution of lightning-induced nitrogen oxides (LNOx) from tropical and subtropical thunderstorms in Southern Brazil was investigated. Airborne trace gas measurements (NO, NOy, CO and O-3) were performed up to 12.5 km with the German research aircraft Falcon. During anvil penetrations in selected tropical and subtropical thunderstorms of 4 and 18 February, NOx mixing ratios were on average enhanced by 0.7-1.2 and 0.2-0.8 nmol mol(-1) totally, respectively. The relative contributions of boundary layer NOx (BL-NOx) and LNOx to anvil-NOx were derived from the NOx-CO correlations. on average similar to 80-90% of the anvil-NOx was attributed to LNOx. A Lightning Location Network (LINET) was set up to monitor the local distribution of cloud-to-ground (CG) and intra-cloud (IC) radiation sources (here called 'strokes') and compared with lightning data from the operational Brazilian network RINDAT (Rede Integrada Nacional de Deteccao de Descargas Atmosfericas). The horizontal LNOx mass flux out of the anvil was determined from the mean LNOx mixing ratio, the horizontal outflow velocity and the size of the vertical cross-section of the anvil, and related to the number of strokes contributing to LNOx. The values of these parameters were derived from the airborne measurements, from lightning and radar observations, and from a trajectory analysis. The amount of LNOx produced per LINET stroke depending on measured peak current was determined. The results were scaled up with the Lightning Imaging Sensor (LIS) flash rate (44 flashes s(-1)) to obtain an estimate of the global LNOx production rate. The final results gave similar to 1 and similar to 2-3 kg(N) per LIS flash based on measurements in three tropical and one subtropical Brazilian thunderstorms, respectively, suggesting that tropical flashes may be less productive than subtropical ones. The equivalent mean annual global LNOx nitrogen mass production rate was estimated to be 1.6 and 3.1 Tg a(-1), respectively. By use of LINET observations in Germany in July 2005, a comparison with the lightning activity in mid-latitude thunderstorms was also performed. In general, the same frequency distribution of stroke peak currents as for tropical thunderstorms over Brazil was found. The different LNOx production rates per stroke in tropical thunderstorms compared with subtropical and mid-latitude thunderstorms seem to be related to the different stroke lengths (inferred from comparison with laboratory data and observed lengths). In comparison, the impact of other lightning parameters as stroke peak current and stroke release height was assessed to be minor. The results from TROCCINOX suggest that the different vertical wind shear may be responsible for the different stroke lengths.

A2 Noradrenergic Lesions Prevent Renal Sympathoinhibition Induced by Hypernatremia in Rats

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influence of Tumour Condition on the Macrophage Activity in Candida albicans Infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Isolation and evaluation of the antioxidant activity of phenolic constituents of the Garcinia brasiliensis epicarp

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The effect of ions and adenosine nucleotides on the activity of lactate dehydrogenase from the epaxial muscle of fish

Lactate dehydrogenase was partially purified from the epaxial muscle of Piaractus mesopotamicus (pacu) and its hybrid Piaractus mesopotamicus x Colossoma macropomus (tambacu). This preparation was used for kinetic studies carried out at pH 6.0 and 7.5. It was also used for the study of the inhibition properties of adenosine nucleotides = ATP, ADP, AMP =, divalent ions Ni2+, Cu2+, Co2+ and the anions oxamate and oxalate.

nor-Lignans from the leaves of Styrax ferrugineus (Styracaceae) with antibacterial and antifungal activity

Chemical examination of the leaves of Styrax ferrugineus yielded 5-[3'-(β-D-glucopyranosyloxy)propyl]-7-methoxy-2-(3',4'-dime-thoxyphenyl) benzofuran, along with the known nor-lignans 5-(3'-hydroxypropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl) benzofuran, 5-(3'-hydroxypropyl)-7-methoxy-2-(3',4'-dimethoxyphenyl)benzofuran, 5-[3'-(β-D-glucopyranosyloxy)propyl]-7-methoxy-2-(3', 4'-methylenedioxyphenyl)benzofuran and the lignan, dihydrodehydrodiconiferyl alcohol. All arylpropanoids isolated showed antibacterial and antifungal activities. The structures of the isolates were established by spectroscopic analysis. (C) 2000 Elsevier Science Ltd.

In vitro evaluation of antimicrobial activity of sealers and pastes used in endodontics

The antimicrobial activity of four root canal sealers (AH Plus, Sealapex, Ketac Endo, and Fill Canal), two calcium hydroxide pastes (Calen and Calasept), and a zinc oxide paste was evaluated. Seven bacterial strains were used, six of them standard; Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922, and Enterococcus faecalis ATCC 10541. There was a wild strain of Streptococcus mutans isolated from saliva obtained in an adult dental clinic. Activity was evaluated using the agar diffusion method with Brain Heart Infusion agar and Müller Hinton medium seeded by pour plate. Calcium hydroxide-based sealers and pastes were either placed directly into 4.0 × 4.0 mm wells or by using absorbent paper points. The plates were kept at room temperature for 2 hr for diffusion. After incubation at 37°C for 24 hr, the medium was optimized with 0.05 g% TTC gel and inhibition haloes were measured. All bacterial strains were inhibited by all materials using the well method. However, when the materials were applied with absorbent paper points, Enterococcus faecalis was not inhibited by zinc oxide, and Pseudomonas aeruginosa was not inhibited by AH Plus, Fill Canal, and the zinc oxide-based paste. We conclude that sealers and pastes presented antimicrobial activity in vitro and culture medium optimization with 0.05 g% TTC gel facilitated observation of the inhibition haloes. Copyright © 2000 by The American Association of Endodontists.

Two short peptides including segments of subunit A of Escherichia coli DNA gyrase as potential probes to evaluate the antibacterial activity of quinolones

Quinolones constitute a family of compounds with a potent antibiotic activity. The enzyme DNA gyrase, responsible for the replication and transcription processes in DNA of bacteria, is involved in the mechanism of action of these drugs. In this sense, it is believed that quinolones stabilize the so-called 'cleavable complex' formed by DNA and gyrase, but the whole process is still far from being understood at the molecular level. This information is crucial in order to design new biological active products. As an approach to the problem, we have designed and synthesized low molecular weight peptide mimics of DNA gyrase. These peptides correspond to sequences of the subunit A of the enzyme from Escherichia coli, that include the quinolone resistance-determining region (positions 75-92) and a segment containing the catalytic Tyr-122 (positions 116-130). The peptide mimic of the non-mutated enzyme binds to ciprofloxin (CFX) only when DNA and Mg2+ were present (Kd = 1.6 × 10 -6 m), a result previously found with DNA gyrase. On the other hand, binding was reduced when mutations of Ser-83 to Leu-83 and Asp-87 to Asn-87 were introduced, a double change previously found in the subunit A of DNA gyrase from several CFX-resistant clinical isolates of E. coli. These results suggest that synthetic peptides designed in a similar way to that described here can be used as mimics of gyrases (topoisomerases) in order to study the binding of the quinolone to the enzyme-DNA complex as well as the mechanism of action of these antibiotics. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.

Quick preparative separation of natural naphthopyranones with antioxidant activity by high-speed counter-current chromatography

The natural naphthopyranones paepalantine (1), paepalantine-9O-β-D-glucopyranoside (2) and paepalantine-9-O-β-D-allopyranosyl-(1→6)-O-β-D-glucopyranoside (3) were separated in a preparative scale from the ethanolic extract of the capitula of Paepalanthus bromelioides by high-speed counter-current chromatography (HSCCC). The solvent system used was composed of water-ethanol-ethyl acetate-hexane (10:4:10:4, v/v/v/v). This technique led to the separation of the three different naphthopyranone glycosides in pure form in approximately 7 hours. Paepalantine showed a good antioxidant activity when assayed by the DPPH radical spectrophotometric assay.

In vitro activity of zinc oxide-eugenol and glass ionomer cements on Candida albicans

The aim of this study was to evaluate in vitro the antimicrobial activity of glass ionomer (GIC) and zinc oxide-eugenol (ZOE) cements against Candida albicans. Standardized GIC and ZOE specimens were maintained in contact with C. albicans suspension (1 x 10(6) cells/ml) at 37 degrees C for 24 h, 48 h or 7 days. A control group without any testing cement was included. After the incubation period, aliquots of 0.1 ml were plated on Sabouraud's agar, and then the number of colonies was counted. The results were expressed as values of logarithms of colony-forming units per milliliter (log CFU/mL) and were analyzed statistically by Kruskal-Wallis ANOVA. After 48 h of incubation, the ZOE group presented no growth of C. albicans. GIC and control groups presented similar mean values at all tested periods. According to the results obtained, it could be concluded that, under the experimental conditions, ZOE cement was more effective in vitro against C. albicans than GIC.

Biological control of sheep parasite nematodes by nematode-trapping fungi: In vitro activity and after passage through the gastrointestinal tract

The main method used for the control of gastrointestinal nematodes in sheep production is the application of chemotherapeutic agents, which often lead to the selection of parasites resistant to given active principles. Biological control can be considered a promising alternative, contributing to an increase in the efficacy of verminous control. We determined the in vitro activity and in situ survival of the predatory fungi Arthrobotrys musiformis and Arthrobotrys conoides during passage through the gastrointestinal tract of sheep after oral administration of conidia in microencapsulated form and as a liquid in natura. Initial in vitro tests showed that both fungi were efficient in the predation of trichostrongylid L3 larvae present in the faeces of sheep naturally infected with gastrointestinal nematodes. The fungi presented high nematophagous activity, which was 99.3% for A. conoides and 73.7% for A. musiformis. A. conoides did not survive passage through the gastrointestinal tract under the conditions of the present experiment. On the other hand, A. musiformis was reisolated after administration in either microencapsulated or liquid form, suggesting that this species is a promising alternative for the control of nematodes in sheep since it survives without any protection (in natura). © Springer 2005.

A new imidazole alkaloid and other constituents from Pilocarpus grandiflorus and their antifungal activity

The stems of Pilocarpus grandiflorus have afforded the new imidazole alkaloid 4,6-dehydro-1,2,4,5-tetrahydro-2,5-dioxopilocarpine in addition to the 17 known compounds germanicol, β-amiryn, ocotillone, stigmast-4-en-3-one, 3β-hydroxy-stigmast-5-en-7-one, 6β-hydroxy-stigmast-4-en-3-one, β-sitosterol, scopoletin, 3-(1′,1′-dimethylallyl)-scopoletin, elisin, dictamine, 4-methoxy-2-quinolone, platydesmine, syringaresinol, syringaldehyde, syringic acid and vanillic acid. Their structures were elucidated on the basis of chemical and spectroscopic evidence. The phenolic compounds vanillic acid and syringaldehyde and the furoquinoline alkaloid platydesmine exhibited antifungal activity against Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants (Atta sexdens rubropilosa). © 2005 Verlag der Zeitschrift für Naturforschung.

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors. © 2005 Elsevier Inc. All rights reserved.

Daily activity resources collection by the swarm-founding wasp Angiopolybia pallens (Hymenoptera: Vespidae)

Angiopolybia pallens (Lepeletier, 1836) exhibited an intense activity at the beginning (6:00-7:00 a.m.) and end of the day (5:30-6:00 p.m.) (bimodal daily activity) and a low efficiency index in resources collection. The species also exhibited an equivalence relation in the items collected (nectar, prey and pulp) and a negative reaction to temperature and luminosity increasing, and reacted positively to an increase in the relative air humidity.