Effects of co-cropping Bidens pilosa (L.) and Tagetes minuta (L.) on bioaccumulation of Pb in Lactuca sativa (L.) growing in polluted agricultural soils

Polluted agricultural soils are a serious problem for food safety, with phytoremediation being the most favorable alternative from the environmental perspective. However, this methodology is generally time-consuming and requires the cessation of agriculture. Therefore, the purpose of this study was to evaluate two potential phytoextractor plants (the native species Bidens pilosa and Tagetes minuta) co-cropped with lettuce growing on agricultural lead-polluted soils. The concentrations of Pb, as well as of other metals, were investigated in the phytoextractors, crop species, and in soils, with the potential risk to the health of consumers being estimated. The soil parameters pH, EC, organic matter percentage and bioavailable lead showed a direct relationship with the accumulation of Pb in roots. In addition, the concentration of Pb in roots of native species was closely related to Fe (B. pilosa, r = 0.81; T. minuta r = 0.75), Cu (T. minuta, r = 0.93), Mn (B. pilosa, r = 0.89) and Zn (B. pilosa, r = 0.91; T. minuta, r = 0.91). Our results indicate that the interaction between rhizospheres increased the phytoextraction of lead, which was accompanied by an increase in the biomass of the phytoextractor species. However, the consumption of lettuce still revealed a toxicological risk from Pb in all treatments.     

Comparison of several ELISA tests for detecting the presence of IgG and IgM against herpes simplex viruses

Four enzyme-linked immunosorbent assays designated test 1 (ETI-HSVK-G 1/2); test 2 (ETI-HSVK-M 1/2); test 3 (ETI-HSVK-G 2), and test 4 (BioElisa HSV2 IgG) were studied to evaluate different stages of herpes simplex virus (HSV) infection. Samples (50 sera and 14 cerebrospinal fluid) were included in four groups. Group 1 consisted of samples from patients with primary HSV infections; group 2 comprised samples from patients with recurrent HSV infections; group 3 were samples nonreactive to HSV; and group 4 were samples from patients with infections by other herpes viruses (4a, chickenpox; 4b, herpes zoster; and 4c, infectious mononucleosis by Epstein-Barr virus). The percentages of agreement between tests 1 and 2 were 100 and 72.1%, respectively. The total diagnostic values of tests 1 and 2 were: 100 and 50% sensitivity, respectively; and 100 and 89% specificity, respectively. Few positive results for HSV-2 infection were found, and so, tests 3 and 4 were not evaluated. The results of tests 3 and 4 for a chickenpox patient, and a herpes zoster patient were not in agreement.     

Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques

The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).     

The role of flow velocity combined with habitat complexity as a top–down regulator in seagrass meadows

Large‐scale losses of seagrass areas have been associated with eutrophication events, which have led to an overproduction of photosynthetic organisms including epiphytes. Grazers that feed on epiphytes can exert a significant top–down control in the system, but the effects of physical factors on grazing activity and feeding behaviour have been rarely examined. We addressed the combination of hydrodynamic regime and seagrass shoot density can alter the feeding and foraging behaviours of mesograzers. A full factorial experiment, with flow velocity (high, medium and low) and shoot density (high versus low) as main factors, was conducted in a racetrack flume using artificial seagrass plots. The results showed that when high flow velocity conditions were combined with low shoot density, consumption of epiphytes by mesograzers was strongly reduced. In contrast, when flow velocity was low or shoot density was high, mesograzers exhibited high feeding rates and vigorous swimming behaviour. These results clearly indicate that hydrodynamic stress reduces the time that mesograzers can spend feeding, since it inhibits their swimming behaviour, and thus indirectly affecting to the density of epiphytes. Therefore, the triggering of trophic cascade effects in seagrass communities under these experimental conditions depended on the interrelationship and feedbacks among shoot density, abiotic (flow velocity) and biotic (epiphytes and mesograzers) compartments, with flow velocity exerting a top–down control on seagrass ecosystems.     

MALDI Imaging Mass Spectrometry: STATE OF THE ART TECHNOLOGY IN CLINICAL PROTEOMICS*

A decade after its inception, MALDI imaging mass spectrometry has become a unique technique in the proteomics arsenal for biomarker hunting in a variety of diseases. At this stage of development, it is important to ask whether we can consider this technique to be sufficiently developed for routine use in a clinical setting or an indispensable technology used in translational research. In this report, we consider the contributions of MALDI imaging mass spectrometry and profiling technologies to clinical studies. In addition, we outline new directions that are required to align these technologies with the objectives of clinical proteomics, including: 1) diagnosis based on profile signatures that complement histopathology, 2) early detection of disease, 3) selection of therapeutic combinations based on the individual patient''s entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single “node” in the pathway.MS has become a versatile tool that we are familiar with in large part due to important electronic and informatics advancements. The ability to obtain the molecular weight is one of the first steps in the identification of a molecule. With the addition of primary structural information mass spectrometry has become a useful technique to identify molecules within complex mixtures.Biological specimens, such as tissues, urine, or plasma, are complex and highly heterogeneous, which makes them inherently difficult to analyze. Further research and developments are necessary to achieve reliable biological models for understanding and studying pathologies. Therefore, it is of primary importance to identify the constituents of these systems and subsequently understand how they function within the framework of the tissue. With regard to clinical proteomics, there is the added dimension of disease, and therefore, the main goal is to characterize the cellular circuitry with a focus on the impact of the disease and/or therapy on these cellular networks.Mass spectrometry has become a centerpiece technology predominantly in the field of proteomics. Nonetheless a more comprehensive understanding of the constituents of biological systems will be aided by determining the constituent distribution. This anatomical dimension has been added through mass spectrometry imaging (MSI)¹ especially using MALDI-MSI.MALDI is an ion source that is well compatible with the introduction of raw materials and surfaces. Shortly after its introduction, MALDI was used for direct tissue profiling. The first applications were neurobiological studies on dissected organs from the mollusk Lymnaea stagnalis (1–8), crustaceans (9), and other mollusks (10, 11). More recently, MALDI was used to generate profiles from tissue sections and ion images using a scanning method to analyze the surface (12) (Fig. 1). This led to the first MALDI MS tissue section imaging micrographs in 1997 (13–15). These studies were followed by 10 years of intense efforts to improve the sensitivity, reproducibility, data processing, tissue preservation, and preparation treatments to fully characterize the proteome leading to a clear improvement of molecular images (16–39) (Fig. 2).Open in a separate windowFig. 1.Schematic representation of the MALDI-MSI work flow. After tissue sectioning and transfer onto a conductive and transparent sample plate, the MALDI matrix is deposited, and data are acquired by recording mass spectra according to a raster of points covering the surface to be analyzed. Mass spectra recorded with their coordinates on the tissue are processed, and molecular images of the localization of molecules can be reconstructed. a.u., arbitrary units; ITO, idium tin oxide.Open in a separate windowFig. 2.Ten years'' evolution from one of the first MALDI images presented in 1999 at the 47th ASMS Conference on Mass Spectrometry and Allied Topics (left) (reprinted with permission of Caprioli and co-workers (84)) and molecular images obtained by our group for mouse stem cells injected in brain tissue sections (right) (M. Wisztorski, C. Meriaux, M. Salzet, and I. Fournier, unpublished results).These developments led to clinical studies using MALDI-MSI technology. Clinical proteomics has many objectives including 1) diagnosis based on signatures as a complement to histopathology, 2) early disease detection, 3) individualized selection of therapeutic combinations that best target the patient''s entire disease-specific protein network, 4) real time assessment of therapeutic efficacy and toxicity, 5) rational redirection of therapy based on changes in the diseased protein network that are associated with drug resistance, and 6) combinatorial therapy in which the signaling pathway itself is viewed as the target rather than any single “node” in the pathway.Based on these key objectives, can we consider MALDI-MSI a mature technology for use in clinical studies? What is the potential impact of this technology in anatomy/pathology and disease? By reviewing each objective, do we have sufficient evidence that MALDI-MSI satisfies the criteria imposed by clinical proteomics? We will now specifically address each of these key points.     

A study of histocompatibility-2 antigens in wild mice from Santiago,Chile

LyM-1 is the provisional designation given to a system of murine cell-surface alloantigens which are controlled by genes closely linked to those of theMls system. Formal genetic analysis has failed to disclose separation of genes determiningMls and LyM-1 antigens, but studies of the distribution of these antigens among inbred strains shows that the LyM-1 polymorphism is not primarily responsible for the MLR activity associated with Mls differences, and suggests that LyM-1 and Mls substances are products of genes at closely linked, but probably separate loci. Absorption analysis shows that strains whose cells react with anti-LyM-1.2 can be divided into at least two classes on the basis of the efficiency with which their cells remove anti-LyM-1.2 antibodies. This provides evidence for the existence of two LyM-1 alleles in addition to the one(s) possessed by nonreactive mouse strains.     

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 μg CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.     

Binding kinetics of calbindin-D(28k) determined by flash photolysis of caged Ca(2+)

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.