Fluoroquinolone antibiotics determination in piggeries environmental waters.

To trace the contamination and evaluate the environmental impact of piggeries, the occurrence of norfloxacin (NOR), ciprofloxacin (CIPRO), enrofloxacin (ENRO) and sarafloxacin (SARA) residues were evaluated in water for swine consumption, piggeries' wastewaters, drainage waters and a river receiving piggery effluents. Water samples were acidified before being percolated through Oasis HLB cartridges or through a tandem system consisting of strong anion-exchange and Oasis HLB cartridges. A liquid chromatography with fluorescence detection (LC-FD) method developed in previous studies, based on the use of a monolithic column, was successfully applied. NOR, CIPRO, ENRO and SARA had good linear responses, with mean regression coefficients higher than 0.9989. The limits of quantification (LOQ) for waters for swine consumption, and a river receiving piggery effluents, were 0.025 ug L(-1) for NOR, CIPRO and ENRO and 0.05 ug L(-1) for SARA. For wastewaters and drainage waters, the LOQs were 0.05 ug L(-1) for CIPRO and ENRO and 0.1 ug L(-1) for SARA. Recoveries obtained for spiked samples ranged from 70.8 to 89.1%, and precision data were under 12.7%. The method was successfully applied to 18 environmental water samples, collected from piggeries in Alentejo, Portugal, and the first Portuguese data are presented. ENRO was detected in a total of 8 samples, at concentrations ranging between 0.31 microg L(-1) and 63 microg L(-1). CIPRO, its degradation product, and SARA were present in 2 samples, in the ranges 0.48-0.83 microg L(-1) and 1.8-14 microg L(-1), respectively. No fluoroquinolones were detected in the waters for swine consumption or in the river water samples.

Determination of fluoroquinolone residues in poultry muscle in Portugal.

A total of 98 poultry samples, including chicken and turkey muscle, were analysed, using a sensitive and reliable analytical method based on liquid chromatography (LC) with spectrofluorimetric detection, for simultaneous determination of four fluoroquinolone (FQ) antibiotics, namely enrofloxacin (ENRO), ciprofloxacin (CIPRO), norfloxacin (NOR), and sarafloxacin (SARA). The method involved extraction with 0.15 mol L(-1) HCl and clean-up by solid-phase extraction using Oasis HLB cartridges. Chromatographic separation was carried out on a C(18) TSK gel column, in isocratic mode, with 0.025 mol L(-1) H(3)PO(4) solution, adjusted to pH 3.0 with tetrabutylammonium hydroxide-methanol (78:22) as mobile phase. Good linearity over the investigated concentration range was observed, with mean values of correlation coefficients higher than 0.9989 for all the analytes studied. The limits of quantification (LOQ), expressed as the lowest fortification level with acceptable precision were 15 microg kg(-1) for ENRO, CIPRO, and NOR, and 30 microg kg(-1) for SARA; these values are in compliance with requirements for monitoring of maximum residues levels (MRLs). Overall recoveries from spiked samples ranged from 80% to 92% with relative standard deviations (RSD) lower than 6.1%. Of the chicken and turkey samples analysed, 44.2% and 37.8%, respectively, were contaminated. The levels found in the analysed poultry samples, collected from markets of Oporto and Coimbra, located in the north and central zones of Portugal, respectively, were lower than 114.2 and 87.6 microg kg(-1) in chicken and turkey muscle samples, respectively. One positive chicken sample was contaminated with ENRO at levels higher than the MRL.

Isolation and comparative analysis of the wheat TaPT2 promoter: identification in silico of new putative regulatory motifs conserved between monocots and dicots.

Phosphorus deficiency is one of the major nutrient stresses affecting plant growth. Plants respond to phosphate (Pi) deficiency through multiple strategies, including the synthesis of high-affinity Pi transporters. In this study, the expression pattern of one putative wheat high-affinity phosphate transporter, TaPT2, was examined in roots and leaves under Pi-deficient conditions. TaPT2 transcript levels increased in roots of Pi-starved plants. A 579 bp fragment of the TaPT2 promoter is sufficient to drive the expression of the GUS reporter gene specifically in roots of Pi-deprived wheat. This TaPT2 promoter fragment was also able to drive expression of the GUS reporter gene in transgenic Arabidopsis thaliana, under similar growth conditions. Conserved regions and candidate regulatory motifs were detected by comparing this promoter with Pi transporter promoters from barley, rice, and Arabidopsis. Altogether, these results indicate that there are conserved cis-acting elements and trans-acting factors that enable the TaPT2 promoter to be regulated in a tissue-specific and Pi-dependent fashion in both monocots and dicots.

Molecular evolution of glutamate receptors: a primitive signaling mechanism that existed before plants and animals diverged.

We performed a genealogical analysis of the ionotropic glutamate receptor (iGluR) gene family, which includes the animal iGluRs and the newly isolated glutamate receptor-like genes (GLR) of plants discovered in Arabidopsis. Distance measures firmly placed the plant GLR genes within the iGluR clade as opposed to other ion channel clades and indicated that iGluRs may be a primitive signaling mechanism that predated the divergence of animals and plants. Moreover, phylogenetic analyses using both parsimony and neighbor joining indicated that the divergence of animal iGluRs and plant GLR genes predated the divergence of iGluR subtypes (NMDA vs. AMPA/KA) in animals. By estimating the congruence of the various glutamate receptor gene regions, we showed that the different functional domains, including the two ligand-binding domains and the transmembrane regions, have coevolved, suggesting that they assembled together before plants and animals diverged. Based on residue conservation and divergence as well as positions of residues with respect to functional domains of iGluR proteins, we attempted to examine structure-function relationships. This analysis defined M3 as the most highly conserved transmembrane domain and identified potential functionally important conserved residues whose function can be examined in future studies.

Switching of gene expression: analysis of the factors that spatially and temporally regulate plant gene expression.

In this chapter, we have reviewed the present research and understanding of several families of transcription factors in plants. From this information, it appears there is good conservation between the types of transcription factors in plants and animals. However, there are several types of factors which have been isolated in plants that remain to be documented in animals (e.g., HD-Zip and GT). These as well as the presence of two types of TATA-binding proteins (TBPs) in plants suggest that although transcription in eukaryotes is highly conserved, fundamental differences may exist. Despite the differences, the modes of regulating transcription are well conserved. Figure 3 summarizes these modes of regulation. In recent years, the role of chromatin structure as well as subcellular localization have been the focus of a vast amount of research in mammals, Drosophila and yeast. However, very little research in these areas has been done in plants. Isolation of genes such as Curly leaf suggest a conservation of genes that influence the formation of heterochromatin-like structures. Whether or not this gene influences chromatin/heterochromatin structure in plants, however, remains to be tested. The study of nuclear localization of factors such as COP1 and KN1 is now leading to models for regulating nuclear transport as well as intercellular transport of transcription factors. Further study of the inter- and intracellular movement of these and other transcription factors may provide information on new modes of regulating transcription. In addition to understanding the role chromatin structure and subcellular localization of transcription factors may have on transcription initiation, the biological role of many plant transcription factors remains to be identified. Several approaches may be taken to understand the mechanisms by which transcription factors influence biochemical and physiological processes in the plant. These steps include 1) identification of the DNA-binding sites of the factors as well as the promoter regions which contain these sites. Presently, this approach is limiting in that not many non-coding regions have been sequenced and characterized in detail. Furthermore, the presence of a putative binding site within a promoter does not necessarily indicate that the factor will bind to the site in vivo. 2) Analysis of the binding affinity for a particular factor to a binding site in comparison to other related factors, via in vitro competition assays and quantitative titrations. This will provide information on how strongly these factors are binding to the sites, but without knowledge of all the factors present in a single cell it is difficult to recreate the in vivo conditions. 3) Generation of transgenic plants or microinjection of DNA/RNA to express a particular factor ectopically, reduce expression of the factor via antisense expression, and creation of dominant negative mutants by overexpression of key dimerization domains may provide information concerning what biological pathways these factors influence. 4) Isolation of mutations in particular transcription factors has been extremely informative in floral development. However, this approach usually entails isolation of a mutant due to a phenotype and eventual mutated locus. The cloning of the locus may or may not involve a transcription factor. 5) Many plant transcription factors have been isolated via sequence similarity to other previously identified and/or characterized transcription factors. However, the biological role of may of these factors is not known. In addition to ectopic expression of these factors by creating transgenic plants, isolation of a loss-of-function mutation may provide valuable information concerning the role of this factor in vivo. Many loss-of-function mutations in MADS box genes have led to a better understanding of how the MADS domain proteins interact with one another as well as how they influence floral development. (ABSTRACT TRUNCATED)

Pathogen-induced programmed cell death in plants, a possible defense mechanism.

As much as the definition of life may be controversial, the definition of death also may prove problematic. In recent years it became apparent that the death of a living cell may follow more than one possible scenario: it may result from an externally applied physical injury (an accidental death), or it may be the outcome of activating an internal pathway for cell suicide (a programmed death). That cells can participate in their own execution may indicate that certain types of cell deaths that were previously considered to be caused by foreign agents such as pathogens or drugs may actually result from the activation of a programmed cell death pathway that is normally latent in cells. Here, we describe the activation of such a cell suicide pathway in plant cells upon the recognition of an invading pathogen. We discuss the possible use of this pathway as a defense mechanism against infection and the possibility that in many ways the use of this type of cell death in plants is functionally analogous to that used by mammalian cells in response to infection by pathogens.

lem7, a novel temperature-sensitive Arabidopsis mutation that reversibly inhibits vegetative development.

An important question in developmental biology concerns the mechanisms by which a few cells coordinate division and differentiation to yield the complex structures and organs found in multicellular organisms. During vegetative growth in plants, cells in the apical meristem must coordinate division and differentiation to yield the fully mature leaf organ. Alterations in these processes may result in an abnormal leaf. In this paper we present the isolation and characterization of an EMS-generated, cold-temperature-sensitive mutation in Arabidopsis thaliana, designated lem7 (leaf morphogenesis). lem7 is a semidominant mutation that maps to a novel locus on chromosome 2. When grown at 16 degrees C, lem7 reversibly arrests leaf development at the shoot apex. In contrast, lem7 grown at 30 degrees C appears phenotypically normal. Our data also suggest that the Lem7 locus may not be involved solely in leaf organogenesis, but may also play a role in floral development and the maintenance of patterns and structures after cellular differentiation. At an intermediate temperature of 23 degrees C, leaves on the lem7 plant emerged phenotypically normal but began to show drastic changes at about 13 days postgermination. These changes include a reduced bilateral symmetry, a rough leaf lamina, a reduced number of trichomes, and an altered vascular network. Leaves that developed at the permissive temperature (30 degrees C) and shifted to the nonpermissive temperature (16 degrees C) form tumor-like outgrowths. Histological analysis of these tumor-like outgrowths and leaves grown at the intermediate temperature reveal abnormally large mesophyll cells, a disorganized mesophyll layer, and collapsed epidermal cells. We propose that the reversible inhibition of leaf development in lem7 under nonpermissive temperatures may serve as a useful tool for identifying genes involved in Arabidopsis leaf organogenesis.

The conserved ELK-homeodomain of KNOTTED-1 contains two regions that signal nuclear localization.

Nuclear localization serves as a regulatory mechanism in the activity of several transcription factors. KNOTTED-1 (Kn1) is a homeodomain protein likely to regulate vegetative development in maize. At least twelve genes related to Kn1 are known in maize and six in Arabidopsis. Ectopic expression of the maize, rice and Arabidopsis Kn1-related genes have been shown to alter cell fate determination. In this paper, we study the nuclear localization capabilities of the Kn1 homeodomain and the proximal amino acid residues (the ELK region) which is highly conserved among Kn1-related homeodomain proteins. The ELK homeodomain (ELK-HD) of Kn1 was fused to the reporter gene uidA encoding the bacterial enzyme beta-glucuronidase (GUS) and transformed into tobacco and onion cells. Quantitation of GUS activity in nuclear and total protein extracts from transgenic tobacco revealed a highly localized GUS activity in the nucleus for the ELK-HD/GUS fusion protein, as compared to the basal level of GUS activity in the nucleus for the GUS only protein. The ELK-HD/GUS transformants showed no unusual characteristics, thus indicating that expression of the putative Kn1 DNA-binding domain fused to GUS may be insufficient to create a dominant negative phenotype. Histochemical analysis of the onion epidermal cells transfected by particle bombardment demonstrated that greater than 50 % of the transformed onion epidermal cells showed higher levels of GUS staining in the nucleus relative to the cytoplasm. Deletion analysis of the ELK-HD revealed that the Kn1 homeodomain comprising the three predicted alpha-helices and the conserved ELK domain can each function independently as nuclear localization signals.

Assessment of cognitive functioning in poly-substance abusers.

Neuropsychological deficits of memory and spatial perception have been associated with substance abuse. The present study assessed the degree to which verbal abstraction is impaired in substance abusers because this function typically is reduced in diffuse cerebral disorders. Test scores of 100 consecutively admitted substance-abusing patients indicated that performance on the Similarities test of the WAIS was actually higher than Vocabulary scores. This finding was replicated with the WAIS-R on a second sample of 100 patients who demonstrated impaired memory and psychomotor speed. These findings indicated that verbal abstraction was unimpaired in two large samples of poly-substance abusers. Further, the superiority of Similarities over Vocabulary scores suggests that in poly-substance abusers, Similarities scores may be a better index of premorbid intellectual competence than the more typically used Vocabulary scores.