The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata

Four different bombesins (bombesin, His(6)-bombesin, Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin) have been identified by a systematic sequencing study of peptides in reverse phase HPLC fractions of the skin secretion of the European yellow-bellied toad, Bombina variegata, that had been solvated in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) and stored frozen at -20°C for 12 years. By using a 3'- and 5'-RACE PCR strategy, the corresponding biosynthetic precursor-encoding cDNAs of all four peptides were cloned from a cDNA library made from the same long-term frozen, acid-solvated skin secretion sample following thawing and lyophilization. Canonical bombesin and His(6)-bombesin are classical bombesin sub-family members, whereas Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin, belong to the litorin/ranatensin sub-family of bombesin-like peptides (BLPs). Assignment of these peptides to respective sub-families, was based upon both their primary structural similarities and their comparative pharmacological activities. An interesting observation in this study, was that the nucleotide sequences of the open-reading frames of cloned cDNAs encoding bombesin and its His(6)-substituted analog, were identical except for a single base that was responsible for the change observed at the position 6 residue in the mature peptide from Asn to His. In contrast, the precursor cDNA nucleotide sequences encoding the Phe(13)-bombesins, exhibited 53 base differences. The pharmacological activities of synthetic replicates of each bombesin were compared using two different mammalian smooth muscle preparations and all four peptides were found to be active. However, there were significant differences in their relative potencies.

Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.

Development of a Broad-Specificity Monoclonal Antibody-Based Immunoaffinity Chromatography Cleanup for Organophosphorus Pesticide Determination in Environmental Samples

An immunoaffinity chromatographic (IAC) method for the selective extraction and concentration of 13 organophosphorus pesticides (OPs, including coumaphos, parathion, phoxim, quinalphos, dichlofenthion, triazophos, azinphos-ethyl, phosalone, isochlorthion, parathion-methyl, cyanophos, disulfoton, and phorate) prior to analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The IAC column was prepared by covalently immobilizing a monoclonal antibody with broad specificity for OPs on CNBr-activated Sephrose 4B. The column capacity ranged from 884 to 2641 ng/mL of gel. The optimum elution solvent was 0.01 M phosphate-buffered saline containing 80% methanol. The breakthrough volume of the IAC column was found to be 400 mL. Recoveries of OPs from spiked environmental samples by IAC cleanup and HPLC-MS/MS analysis ranged from 60.2 to 107.1%, with a relative standard deviation below 11.1%. The limit of quantitation for 13 OPs ranged from 0.01 to 0.13 ng/mL (ng/g). The application of IAC cleanup coupled to HPLC-MS/MS in real environmental samples demonstrated the potential of this method for the determination of OP residues in environmental samples at trace levels.

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

Inorganic arsenic contents in rice-based infant foods from Spain, UK, China and USA

Spanish gluten-free rice, cereals with gluten, and pureed baby foods were analysed for total (t-As) and inorganic As (i-As) using ICP-MS and HPLC-ICP-MS, respectively. Besides, pure infant rice from China, USA, UK and Spain were also analysed. The i-As contents were significantly higher in gluten-free rice than in cereals mixtures with gluten, placing infants with celiac disease at high risk. All rice-based products displayed a high i-As content, with values being above 60% of the t-As content and the remainder being dimethylarsinic acid (DMA). Approximately 77% of the pure infant rice samples showed contents below 150 µg kg(-1) (Chinese limit). When daily intake of i-As by infants (4-12 months) was estimated and expressed on a bodyweight basis (µg d(-1) kg(-1)), it was higher in all infants aged 8-12 months than drinking water maximum exposures predicted for adults (assuming 1 L consumption per day for a 10 µg L(-1) standard).

Arsenic accumulation and phosphorus status in two rice (Oryza sativa L.) cultivars surveyed from fields in South China

The consumption of paddy rice (Oryza sativa L.) is a major inorganic arsenic exposure pathway in S.E. Asia. A multi-location survey was undertaken in Guangdong Province, South China to assess arsenic accumulation and speciation in 2 rice cultivars, one an Indica and the other a hybrid Indica. The results showed that arsenic concentrations in rice tissue increased in the order grain <husk <straw <root. Rice grain arsenic content of 2 rice cultivars was significant different and correlated with phosphorus concentration and molar ratio of P/As in shoot, being higher for the Indica cultivar than for the hybrid Indica, which suggests altering shoot phosphorus status as a promising route for breeding rice cultivars with reduced grain arsenic. Speciation of grain arsenic, performed using HPLC-ICP-MS, identified inorganic arsenic as the dominant arsenic species present in the rice grain.

Quantitative and qualitative trapping of volatile methylated selenium species entrained through nitric acid

Quantification and speciation of volatile selenium (Se) fluxes in remote areas has not been feasible previously, due to the absence of a simple and easily transportable trapping technique that preserves speciation. This paper presents a chemo-trapping method with nitric acid (HNO3) for volatile Se species, which preserves speciation of trapped compounds. The recovery and speciation of dimethylselenide (DMSe) and dimethyl diselenide (DMDSe) entrained through both concentrated nitric acid and hydrogen peroxide (H2O2) were compared by HPLC-ICP-MS and HPLC-HG-AFS analyses. It was demonstrated that trap reproducibility was better for nitric acid and a recovery of 65.2 +/- 1.9% for DMSe and 81.3 +/- 3.9% for DMDSe was found in nitric acid traps. HPLC-ES-MS identified dimethyl selenoxide (DMSeO) as the trapped product of DMSe. Methylseleninic acid (MSA) was identified to be the single product of DMDSe trapping. These oxidized derivatives have a high stability and low volatility, which makes nitric acid a highly attractive trapping liquid for volatile Se species and enables reconstruction of the speciation of those species. The presented trapping method is simple, quantifiable, reproducible, and robust and can potentially be applied to qualitatively and quantitatively study Se volatilization in a wide range of natural environments.

Quantitative and qualitative trapping of arsines deployed to assess loss of volatile arsenic from paddy soil

Arsenic volatilization in the environment is thought to be an important pathway for transfer from terrestrial pools to the atmosphere. However, this phenomenon is not well characterized due to inherent sampling issues in trapping, quantifying and qualifying these arsine gases; including arsine (AsH(3)), monomethyl arsine (MeAsH(2)), dimethyl arsine (Me(2)AsH) and trimethyl arsine (TMAs). To quantify and qualify arsines in air we developed a novel technique based on silver nitrate impregnated silica gel filled tubes. The method was characterized by measuring the recovery of trapped arsines after elution of this chemo-trap with hot boiling diluted nitric acid. Results from three separate experiments, measured by ICP-MS, showed that the method is reproducible and quantitative. Arsine species recovery ranged from 80.1 to 95.6%, with limit of detection as low as 3.8 ng per chemo-trap tube. Moreover, HPLC-ICP-MS analysis of hot boiling water eluted traps showed that the corresponding oxy ions of the arsines were formed with the As-C bonds of the molecule intact, hence, allowing qualification of trapped arsine species. A microcosm study examining volatile arsenic evolution from field contaminated Bangladeshi paddy soils (24.2 mg/kg arsenic) was used to show the application of silver nitrate chemo-trapping approach. Traps were placed on the inlet and the outlet of microcosms containing the soils that were either (cattle derived) manured or not, or flooded or not, in a factorial design. The headspace was purged with air at a flow rate of 12 mL/min. Results showed that as much as 320 ng of arsenic (0.014% of total soil content) could be emitted in a 3 week period for manured and flooded soils and that TMAs was the dominant species evolved, with lesser quantities of Me(2)AsH. No volatile arsenic evolution was observed for nonmanured treatments, and arsine release from the nonflooded, manured treatment was much less than the flooded treatment.

Selenium in higher plants: understanding mechanisms for biofortification and phytoremediation:understanding mechanisms for biofortification and phytoremediation

Selenium (Se) is an essential micronutrient for many organisms, including plants, animals and humans. As plants are the main source of dietary Se, plant Se metabolism is therefore important for Se nutrition of humans and other animals. However, the concentration of Se in plant foods varies between areas, and too much Se can lead to toxicity. As we discuss here, plant Se uptake and metabolism can be exploited for the purposes of developing high-Se crop cultivars and for plant-mediated removal of excess Se from soil or water. Here, we review key developments in the current understanding of Se in higher plants. We also discuss recent advances in the genetic engineering of Se metabolism, particularly for biofortification and phytoremediation of Se-contaminated environments.

The molecular form of mercury in biota:identification of novel mercury peptide complexes in plants

The molecular structure of a variety of novel mercury-phytochelatin complexes was evidenced in rice plants exposed to inorganic mercury (Hg2+) using RP-HPLC with simultaneous detection via ICP-MS and ES-MS.

Speciation and distribution of arsenic and localization of nutrients in rice grains

Arsenic (As) contamination of rice grains and the generally low concentration of micronutrients in rice have been recognized as a major concern for human health. Here, we investigated the speciation and localization of As and the distribution of (micro)nutrients in rice grains because these are key factors controlling bioavailability of nutrients and contaminants. Bulk total and speciation analyses using high-pressure liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICP-MS) and X-ray absorption near-edge spectroscopy (XANES) was complemented by spatially resolved microspectroscopic techniques (micro-XANES, micro-X-ray fluorescence (micro-XRF) and particle induced X-ray emission (PIXE)) to investigate both speciation and distribution of As and localization of nutrients in situ. The distribution of As and micronutrients varied between the various parts of the grains (husk, bran and endosperm) and was characterized by element-specific distribution patterns. The speciation of As in bran and endosperm was dominated by As(III)-thiol complexes. The results indicate that the translocation from the maternal to filial tissues may be a bottleneck for As accumulation in the grain. Strong similarities between the distribution of iron (Fe), manganese (Mn) and phosphorus (P) and between zinc (Zn) and sulphur (S) may be indicative of complexation mechanisms in rice grains.

Survey of arsenic and its speciation in rice products such as breakfast cereals, rice crackers and Japanese rice condiments

Rice has been demonstrated to be one of the major contributors to arsenic (As) in human diets in addition to drinking water, but little is known about rice products as an additional source of As exposure. Rice products were analyzed for total As and a subset of samples were measured for arsenic speciation using high performance liquid chromatography interfaced with inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). A wide range of rice products had total and inorganic arsenic levels that typified those found in rice grain including, crisped rice, puffed rice, rice crackers, rice noodles and a range of Japanese rice condiments as well as rice products targeted at the macrobiotic, vegan, lactose intolerant and gluten intolerance food market. Most As in rice products are inorganic As (75.2-90.1%). This study provides a wider appreciation of how inorganic arsenic derived from rice products enters the human diet. (C) 2008 Elsevier Ltd. All rights reserved.

High Percentage Inorganic Arsenic Content of Mining Impacted and Nonimpacted Chinese Rice

Two approaches were undertaken to characterize the arsenic (As) content of Chinese rice. First, a national market basket survey (n = 240) was conducted in provincial capitals, sourcing grain from China's premier rice production areas. Second, to reflect rural diets, paddy rice (n = 195) directly from farmers fields were collected from three regions in Hunan, a key rice producing province located in southern China. Two of the sites were within mining and smeltery districts, and the third was devoid of large-scale metal processing industries. Arsenic levels were determined in all the samples while a subset (n = 33) were characterized for As species, using a new simple and rapid extraction method suitable for use with Hamilton PRP-X100 anion exchange columns and HPLC-ICP-MS. The vast majority (85%) of the market rice grains possessed total As levels <150 ng g(-1). The rice collected from mine-impacted regions, however, were found to be highly enriched in As, reaching concentrations of up to 624 ng g(-1). Inorganic As (As(i)) was the predominant species detected in all of the speciated grain, with As(i) levels in some samples exceeding 300 ng g(-1). The As(i) concentration in polished and unpolished Chinese rice was successfully predicted from total As levels. The mean baseline concentrations for As(i) in Chinese market rice based on this survey were estimated to be 96 ng g(-1) while levels in mine-impacted areas were higher with ca. 50% of the rice in one region predicted to fail the national standard.

Speciation and localization of arsenic in white and brown rice grains

Synchrotron-based X-ray fluorescence (S-XRF) was utilized to locate arsenic (As) in polished (white) and unpolished (brown) rice grains from the United States, China, and Bangladesh. In white rice As was generally dispersed throughout the grain, the bulk of which constitutes the endosperm. In brown rice As was found to be preferentially localized at the surface, in the region corresponding to the pericarp and aleurone layer. Copper, iron, manganese, and zinc localization followed that of arsenic in brown rice, while the location for cadmium and nickel was distinctly different, showing relatively even distribution throughout the endosperm. The localization of As in the outer grain of brown rice was confirmed by laser ablation ICP-MS. Arsenic speciation of all grains using spatially resolved X-ray absorption near edge structure (micro-XANES) and bulk extraction followed by anion exchange HPLC-ICP-MS revealed the presence of mainly inorganic As and dimethylarsinic acid (DMA). However, the two techniques indicated different proportions of inorganic:organic As species. A wider survey of whole grain speciation of white (n=39) and brown (n=45) rice samples from numerous sources (field collected, supermarket survey, and pot trials) showed that brown rice had a higher proportion of inorganic arsenic present than white rice. Furthermore, the percentage of DMA present in the grain increased along with total grain arsenic.

Can we trust mass spectrometry for determination of arsenic peptides in plants:comparison of LC-ICP-MS and LC-ES-MS/ICP-MS with XANES/EXAFS in analysis of Thunbergia alata

The weakest step in the analytical procedure for speciation analysis is extraction from a biological material into an aqueous solution which undergoes HPLC separation and then simultaneous online detection by elemental and molecular mass spectrometry (ICP-MS/ES-MS). This paper describes a study to determine the speciation of arsenic and, in particular, the arsenite phytochelatin complexes in the root from an ornamental garden plant Thunbergia alata exposed to 1 mg As L(-1) as arsenate. The approach of formic acid extraction followed by HPLC-ES-MS/ICP-MS identified different As(III)-PC complexes in the extract of this plant and made their quantification via sulfur (m/z 32) and arsenic (m/z 75) possible. Although sulfur sensitivity could be significantly increased when xenon was used as collision gas in ICP-qMS, or when HR-ICP-MS was used in medium resolution, the As:S ratio gave misleading results in the identification of As(III)-PC complexes due to the relatively low resolution of the chromatography system in relation to the variety of As-peptides in plants. Hence only the parallel use of ES-MS/ICP-MS was able to prove the occurrence of such arsenite phytochelatin complexes. Between 55 and 64% of the arsenic was bound to the sulfur of peptides mainly as As(III)(PC(2))(2), As(III)(PC(3)) and As(III)(PC(4)). XANES (X-ray absorption near-edge spectroscopy) measurement, using the freshly exposed plant root directly, confirmed that most of the arsenic is trivalent and binds to S of peptides (53% As-S) while 38% occurred as arsenite and only 9% unchanged as arsenate. EXAFS data confirmed that As-S and As-O bonds occur in the plants. This study confirms, for the first time, that As-peptides can be extracted by formic acid and chromatographically separated on a reversed-phase column without significant decomposition or de-novo synthesis during the extraction step.