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.

Organic and conventional fertilisation procedures on the nitrate, antioxidants and pesticide content in parts of vegetables

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

Improving the Fluorescence Detectability of Polycyclic Aromatic Hydrocarbons for Evaluation of Workplace Environments of Cement Industries Processing Organic Residues

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

Model approach for estimating potato pesticide bioconcentration factor

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

Residues of Some Pesticides in Fresh and Dried Apricots

The persistence of three pesticides (fenitrothion, dimethoate, and ziram) in apricots in field conditions and their fate during the drying process were studied. After the treatments, the pesticides showed fast decay rates with pseudo-first-order kinetics and half-lives ranging from 6.9 to 9.9 days. The drying process showed a different effect on residue concentrations in dried apricots: omethoate (metabolite of dimethoate) and ziram residues had almost doubled, while fenitrothion disappeared and dimethoate remained constant.

Inhibitory effects of jackbean (Canavalia ensiformis L.) leaf residues on germination and vigour of crops and weeds

The effects of jackbean leaf residues incorporated in the soil on germination and seedlings growth of cucumber, radish and some weeds was examined. Trials were carried out under greenhouse conditions to (a) determine the amount of incorporated residue that is inhibitory to two test plants, (b) to determine if decomposition time changes the inhibitory levels of jackbean residues on test plants and (c) to determine the amount of residue that is inhibitory to the weed species. Jackbean leaf residues incorporated in soil at concentration of 2% or higher and allowed to decompose for a period of 0 to 2 weeks before sowing, reduced the initial growth of cucumber and radish and at different concentrations, reduced germination and growth of three weed species. These results suggest the presence of allelopathic components in Jackbean leaves that could affect seed germination and seedling development.

A SequenceSpace analysis of Lys49 phospholipases A2: Clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a subfamily of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Determination of 2,4-D and dicamba in food crops by MEKC

The determination of 2,4-D (2,4-dichlorophenoxyacetic acid) and Dicamba (2-methoxy-3,6-dichlorobenzoic acid) residues in sugar cane, rice and corn was performed by a supercritical fluid extraction (SFE) method using CO2/acetone as extraction mix and an SFE apparatus developed in our laboratory. The extracts were cleaned up after extraction by both liquid- liquid partition and a Florisil column. Micellar electrokinetic capillary chromatography (MEKC) coupled with ultraviolet on-column detection was used for the analysis of these pesticides. The detection limits were improved by the preparation of a special detection cell with an increased pathlength that gave detection limits of ca. 0.6 pg for 2,4-D and Dicamba. Our results demonstrated that capillary electrophoresis can be a powerful new analytical tool for pesticide residue analysis.

The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence

To find the regions having a major influence on the bioluminescence spectra of railroad worm luciferases, we constructed new chimeric luciferases switching the fragments from residues 1-219 and from 220-545 between Phrixotrix viviani (PxvGR; λmax = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; λmax = 623 nm) red light-emitting luciferases. The emission spectrum (λmax = 571 nm) and KM for luciferin in the chimera PxRE220GR (1-219, PxhRE; 220-545, PxvGR) suggested that the region above residue 220 of PxvGR had a major effect on the active site. However, switching the sequence between the residues 226-344 from PxvGR luciferase into PxhRE (PxREGRRE) luciferase resulted in red light emission (λmax = 603 nm), indicating that the region 220-344 by itself does not determine the emission spectrum. Furthermore, the sequence before residue 220 of the green-emitting luciferase is incompatible for light emission with the sequence above residue 220 of PxhRE. These results suggest that the fragments before and after residue 220, which correspond to distinct subdomains, may fold differently in the green- and red-emitting luciferases, affecting the active site conformation.

A simplified procedure for determination of clofentezine residues in fruits by liquid chromatography with UV detection

A rapid and sensitive method is described for the determination of clofentezine residues in apple, papaya, mango and orange. The procedure is based on the extraction of the sample with a hexane:ethyl acetate mixture (1:1, v/v) and liquid chromatographic analysis using UV detection. Mean recoveries from 4 replicates of fortified fruit samples ranged from 81% to 96%, with coefficients of variation from 8.9% to 12.5%. The detection and quantification limits of the method were of 0.05 and 0.1 mg kg-1, respectively.

Windrowing and prismatic baling of sugar cane vegetal residues: Some operational parameters and energetic efficiency

It was evaluated the energetic efficiency and operational parameters of a windrowing and prismatic baling, both from CASE NEW HOLLAND® operations in sugarcane vegetal residues (green leaves, dry leaves and tops) picked mechanically in green cane. The area belongs to COSTA PINTO MILL (COSAN® Group) which was harvested mechanically by combines in the State of Sao Paulo, Brazil. The geographic location of the area is: Latitude 22°40'30S, Longitude 47°36'38W and Altitude of 605m. The variety was RB 82-5336, planted in 1.40m row spacing, with 78t.ha-1 yield. The vegetal residues analysis obtained 69.93% of leaves, 21.44% of stalks fractions, 2.27% of tops and 6.36% of total strange matter. The vegetal residues values were: gross heat of 18.43MJ.kg-1, low heat of 17.00MJ.kg'1 and useful heat of 12.94MJ.kg-1. The vegetal residues average energetic potential was 342.48GJ.ha-1. The treatments were simple, double and triple windrowing. The use of the rake and prismatic baler to pick up the residues was viable. The simple windrowing treatment presented the best results: effective capacity of 83.06t.ha-1, fuel consumption of 0.18L.t -1 and 99.95% of positive energetic efficiency. The bales obtained in the treatment of triple windrowing presented the largest specific mass average of 221.11kg.m-3. The soil amount in the bales increased with successive windrowing. The baling operation in the triple windrowing treatment obtained better results, presenting the effective capacities of 20.29t.h -1 and 1.45ha.h-1 and fuel consumption of for baled in 1.37L.t-1. The high total energetic efficiency of 99.53% indicates that is technically viable the withdrawal of the vegetal residues.

The sugar cane vegetal residues unloaded in the sugar mill: Operational costs and physical characteristics

Brazil produced in 2002/03 season 317.87×106tons of sugar cane stalks and 36.88×106tons of vegetal residues (green leaves, dry leaves and tops) in a planted area of 4.61×106 hectares (ha). These residues have a useful heat of 3,613.14Mcal.t-1. Currently most of this biomass is burned as a pre-harvest practice. The doubt persists in the system type that it must be adopted to pick up, load, transport and unload this biomass at the sugar mill boilers. This study analyzed 22 variables related to operational costs and physical characteristics of these residues in a field situation using a JOHN DEERE® 6850 forage harvester with two different treatments: T1 and T2 (two types of rakes) with 6 repetitions each one. The geographic location of the studied area that belongs to COSTA PINTO MILL (COSAN® Group) is: Latitude 22°40'30S and Longitude 47°36'38W. The adopted methodology was proposed by Ripoli et al. (2002). The obtained results at a 5% level of significance showed that both treatments did not differed significantly between them. Some of the results were, where EBP stands for Oil Equivalent Barrel: Windrowing (T1=US$0.17.EBP-1 and US$9.59.ha-1, T2=US$0.08.EBP-1 and US$4.27.ha-1); Pick up (T1=US$1.31.EBP-1 and US$44.29.ha-1, T2 =US$1.37.EBP-1 and US$48.36.ha-1); Transportation (T1=US$1.27.EBP-1 and US$14,30.ha -1, T2=US$1.33.EBP-1 and US$14,80.ha -1), Unloading at the sugar mill (T1=US$0.30.EBP-1 and US$3.39.ha-1, T2=US$0.32.EBP-1 and US$3.51.ha-1); Total (T1=US$3.05.EBP-1 and US$71.57.ha-1, T2=US$3.10.EBP-1 and US$70.94.ha-1). Confronting the obtained data with the ones in the bibliography, this system revealed itself more expensive than the baling system or the integral harvest system using combines.

Windrowing and cylindrical baling of sugar cane vegetal residues: Some operational parameters and energetic efficiency

The objective of this study was to analyze the sugar cane vegetal residues collection, as well as determining its energetic potential, using a rake and cylindrical baler, both from NEW HOLLAND® under two different windrowing process (simple and double). The field tests were carried out in an area that belongs to COSTA PINTO MILL (COSAN® Group) in the city of Piracicaba, Sao Paulo State, Brazil. The geographic location of the area is: Latitude 22°4030'S, Longitude 47°3633'W and altitude of 605m. From the trash analysis, before the baling, the following average results were obtained: 69.93% of leaves; 2.27% of stalks fractions; 21.44% of tops and 6.36% of total strange matter. The estimated residues yield was 27.01 tons.ha -1 with a gross heat of 18.43 MJ.kg-1, low heat of 17.01 MJ.kg-1, useful heat of 13.32 MJ.kg-1, average moisture of 20.76% and an energetic potential of 494,875.09 MJ.ha-1. In the windrowing operations (simple and double) the averages of the 5 out of 13 analyzed variable presented differences between them in a 1% level of significance in the Tukey Test. The averages comparison of the results for bale's specific mass and the effective capacities (ton.h-1) e (ha.h-1) had been significant at a 5% level in the Tukey Test. The comparisons of the averages for the results had been significant to 1% level. The strange matter averages of the bales did not differed between them.