Matrix effects observed during pesticides residue analysis in fruits by GC

The influence of the sample matrix in the CC-electron-capture detection analysis of the pesticides dimethoate, diazinon, chlorothalonil.. parathion methyl and fenitrothion in fruits samples has been studied. Experiments have been carried out where the pesticide responses in standard solutions prepared in selected solvent were compared with their response when present in apple, mango, papaya, banana, pineapple and melon extracts. The presence of matrix effects (MEs) and their extent were shown to be simultaneously influenced by several factors (matrix concentration, matrix type, pesticide concentration, analytical range). Pronounced MEs were observed particularly for dimethoate and diazinon in all matrices tested; in lower concentrations, all pesticides presented significant ME. The other pesticides presented variable ME. Higher ME enhancement was detected at lower pesticide concentration levels of and/or at higher matrix concentration solutions. The ME detected for fenitrothion, in the analytical range evaluated, were dependent on matrix type. For each pesticide, solvent and matrix-matched calibrations were compared for all fruit samples, and it could be concluded that quantitation based on standard solutions prepared in blank matrix extract (matrix-matched calibration) should be used to compensate the MEs and to obtain more accurate results for the pesticides studied.

Fate of Some New Fungicides (Cyprodinil, Fludioxonil, Pyrimethanil, and Tebuconazole) from Vine to Wine

The fate of four new fungicides (cyprodinil, fludioxonil, pyrimethanil, and tebuconazole) from the treatment on vine to the production of wine was studied. The influence of clarifying agents (bentonite, charcoal, potassium caseinate, gelatin, and polyvinylpolypyrrolidone) on residue concentrations in wine was also studied. The fungicide residues on grapes showed different decay rates after treatment, with first-order kinetics and half-lives ranging from 8 to 57 days. Grape processing into wine caused considerable residue reduction with cyprodinil (ca. 80%), fludioxonil (ca. 70%), and tebuconazole (ca. 50%) and no reduction with pyrimethanil. The two wine-making techniques employed (with and without maceration) had the same influence on the residue concentrations in wine, except for fludioxonil which showed maximum residue reduction with vinification with maceration. Among the clarifying agents tested, only charcoal showed effective action on the elimination of residue content in wine, proving complete elimination, or almost, of fungicide residues.

Effect of heated solutions on decay control and residues of imazalil in lemons

Freshly harvested lemons [(Citrus limon (L.) Burm)] were dipped 3 min in water with and without imazalil (IMZ) at 50, 100, and 200 ppm at 50 degrees C and at 1000 ppm IMZ at 20 degrees C. Following treatments fruit were kept at 9 degrees C and 90%-95% relative humidity (RH) for 13 weeks and an additional week at 21 degrees C and ca. 75% RH, to simulate a marketing period (SMP). No decay control was observed with fruit dipped in water at 50 degrees C. In contrast, IMZ treatments provided 90%-96% control of Penicillium rots during cold storage and SMP. Fungi other than Penicillium spp. were also found in all samples as differences among treatments were negligible. IMZ treatment caused some external damage to the fruit (peel browning), and the percentage of damaged fruit was related to the amount of active ingredient (AI) present in it. Dipping in 200 or 1000 ppm IMZ promoted off-flavor development after 10 weeks of storage, and fruit were judged to be unacceptable for consumption after 13 weeks of cold storage. After 1000 ppm IMZ dipping at 20 degrees C, residue concentration in fruit was 8.20 ppm; this value doubled that found in a previous investigation on lemons treated with comparable IMZ levels. Residue concentrations in fruit after treatment at 50 degrees C was strictly related to the amount of fungicide employed. After 13 weeks Al residues in fruit decreased to average ca. 35% of the initial values. During the 1 week SMP, residue levels decreased by a further ca. 25%. It was concluded that it is possible to achieve significant control of decay in lemons during longterm storage by dipping fruit in 50 ppm IMZ mixtures at 50 degrees C. Such treatment should be advised to remarkably reduce potential pollution in the environment due to packinghouse wastewater disposal.

An alternative LC-UV procedure for the determination of prochloraz residues in fruits

An alternative method using liquid chromatography with UV detection for the determination of prochloraz as 2,4,6-trichlorophenol in mango, papaya and orange is described. Ethyl acetate, acetone and dichloromethane were tested for extraction of prochloraz from the fruits. After extraction the residue of prochloraz was derivatized with pyridine hydrochloride. The analysis was carried out using liquid chromatography with UV detection and gas chromatography with electron-capture detection. Average recoveries of prochloraz from spiked fruits (0.1 and 0.2 mg kg-1) ranged from 80% to 94% with relative standard deviations between 5.6% and 12.6% (n=8). Detection and quantification limits were 0.05 and 0.1 mg kg-1, respectively. The LC-UV method was applied to mango and papaya samples submitted to dip treatment with a prochloraz formulation under laboratory conditions. In addition, fruit samples obtained from local markets were analysed. ©2005 Sociedade Brasileira de Química.

The effect of high concentrations of calcium hydroxide in neutralised synthetic supernatant liquor : implications for alumina refinery residues

The presence of calcium hydroxide (Ca(OH)2) in Bayer residue slurry inhibits the effectiveness of the seawater neutralisation process to reduce the pH and aluminium concentration in the residue. An increase in the slurry pH (reversion), after seawater neutralisation, is caused by the dissolution of calcium hydroxide and hydrocalumite (solid components found in bauxite refinery residue). Reversion was not observed when the final solution pH was greater than 10.5, due to hydrocalumite being in a state of equilibrium at high pH. Hydrocalumite has been found to form during the neutralisation process when high concentrations of calcium hydroxide are present in the residue liquor. The dissolution of hydrocalumite releases hydroxyl (OH-) and aluminium ions back into solution after the seawater neutralisation (SWN) process, which causes pH and aluminium reversion to occur. This investigation looks at the effect of Ca(OH)2 and subsequently hydrocalumite on the pH and aluminium concentration in bauxite refinery residue liquors after the SWN process.

Radioactive residues associated with water treatment, use and disposal in Australia

Water resources are known to contain radioactive materials, either from natural or anthropogenic sources. Treatment, including wastewater treatment, of water for drinking, domestic, agricultural and industrial purposes has the potential to concentrate radioactive materials. Inevitably concentrated radioactive material is discharged to the environment as a waste product, reused for soil conditioning, or perhaps recycled as a new potable water supply. This thesis, presented as a collection of peer reviewed scientific papers, explores a number of water / wastewater treatment applications, and the subsequent nature and potential impact of radioactive residues associated with water exploitation processes. The thesis draws together research outcomes for sites predominantly throughout Queensland, Australia, where it is recognised that there is a paucity of published data on the subject. This thesis contributes to current knowledge on the monitoring, assessment and potential for radiation exposure from radioactive residues associated with the water industry.

Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.

Neutralization of acid sulfate solutions using bauxite refinery residues and its derivatives

This investigation has shown that by transforming free caustic in red mud (RM) to Bayer hydrotalcite (during the seawater neutralization (SWN) process) enables a more controlled release mechanism for the neutralization of acid sulfate soils. The formation of hydrotalcite has been confirmed by X-ray diffraction (XRD) and differential thermalgravimetric analysis (DTG), while the dissolution of hydrotalcite and sodalite has been observed through XRD, DTG, pH plots, and ICP-OES. Coupling of all techniques enabled three neutralization mechanisms to be determined: (1) free alkali, (2) hydrotalcite dissolution, and (3) sodalite dissolution. The mechanisms are determined on the basis of ICP-OES and kinetic information. When the mass of RM or SWN-RM is greater than 0.08 g/50 mL, the pH of solution increases to a suitable value for plant life with aluminum leaching kept at a minimum. To obtain a neutralization pH greater than 6 in 10 min, the following ratio of bauxite residue (g) in 50 mL with a known iron sulfate (Fe2(SO4)3) concentration can be determined as follows: 0.04 g:50 mL:0.1 g/L of Fe2(SO4)3.

Investigations Into the Significance of Lysine Residues in IGF-I

This PhD thesis presents novel and original research in the field of Insulin-like Growth Factor-I (or IGF-I) biology. IGF-I plays an essential role in promoting normal human growth and development; it also represents both a target and treatment for various diseases. This thesis provides interesting insights into previously uncharacterised mechanisms of action that underlie IGF-I biology. Such findings may lead to improved and novel treatments across a broad range of medical conditions.

Lysine residues of IGF-I are substrates for transglutaminases and modulate downstream IGF-I signalling

Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.

PEGylation of lysine residues reduces the pro-migratory activity of IGF-I

Background: The insulin-like growth factor (IGF) system is composed of ligands and receptors which regulate cell proliferation, survival, differentiation and migration. Some functions are regulated via intracellular signaling cascades, others by involvement of the extracellular milieu, including binding proteins and other extracellular matrix proteins. However, understanding of their functions and the exact nature of these interactions remains incomplete. Methods: IGF-I was PEGylated at its lysine sites - K27, K65 and K68. Binding of PEG-IGF-I to the IGFBPs was analyzed using BIAcore and its ability to activate the IGF-IR was assessed using IGF-IR phosphorylation assay. Furthermore, functional consequences of PEGylating the lysine residues of IGF-I was investigated using cell viability and cell migration assays. In addition, particular downstream signaling pathways regularly implicated in these mechanisms were also dissected using phospho-AKT and phospho-ERK1/2 assays. Results: In this study, IGF-I specifically PEGylated at lysine 27 (PEG-K27), 65 (PEG-K65) or 68 (PEG-K68) were employed. Receptor phosphorylation was only reduced by 2-fold with PEG-K65 and PEG-K68 over all the time points tested, and as observed in two cell types, 3T3 fibroblasts and MCF-7 breast cancer cells. PEGylation at K27 resulted in a much larger effect, with more than 10-fold lower activation for 3T3 fibroblasts and a ~3 fold reduced IGF-IR activation for MCF-7 breast cancer cells over 15 minutes. In addition, all PEG-IGF-I variants demonstrated a ten-fold reduction in the association rate to IGF binding proteins (IGFBPs). Functionally, all PEG variants completely lost their ability to induce cell migration in the presence of IGFBP-3/vitronectin (VN) complexes as compared to IGF-I; in contrast, cell viability was fully preserved. Further investigations into the downstream signaling pathways revealed that the PI3-K/AKT pathway was preferentially affected upon treatment with the PEG-IGF-I variants compared to the MAPK/ERK pathway. Conclusion: PEGylation of IGF-I has an impact on cell migration but not cell viability. General significance: PEG-IGF-I may differentially modulate IGF-I mediated functions that are dependent on its interaction with its receptor as well as key extracellular proteins such as VN and IGFBPs.

Preventing industrial scale using bauxite refinery residues

An Australian green power (AGP) company produces energy from burning biomass from the sugar industry and recycled wood waste, however alkali in biomass is released into a recirculating stream that forms a scale as it becomes more concentrated. This investigation has shown that the addition of Bayer liquor (alumina waste residue) successfully removes scale-forming species from the recirculating stream and thus has the potential to reduce the rate of scaling. Characterisation of the scale and Bayer precipitates has been performed using X-ray diffraction (XRD), infrared spectroscopy (IR) and inductively coupled plasma optical emission spectroscopy (ICP-OES).

Mechanisms of mono- and poly-ubiquitination : ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/ E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.