Contamination and sub-lethal toxicological effects of persistent organic pollutants in the European eel (<Emphasis Type="Italic">Anguilla anguilla</Emphasis>) in the Orbetello lagoon (Tuscany,Italy)

This paper reports on contamination levels and their sub-lethal toxicological effects in specimens of the European eel (Anguilla anguilla) in the Orbetello Lagoon, (Tuscany, Italy). Organochlorine pesticides (OC) and polychlorinated biphenyls (PCBs) were investigated as priority pollutants in muscle tissue. Phase I P450 enzymes, i.e., EROD, B(a)PMO and the two reductases (NADH ferryred and cyt c.), and cholinesterase (ChE) were assayed in liver and muscle as sensitive biological indicators of fish health. PCBs, lindane and p,p′DDE in muscles showed a wide concentration range (0.001–0.025 μg g⁻¹ wet weight) and attained the lowest levels in the eastern basin. High homogeneity and relatively low values were observed for phase I P450 enzymes, suggesting that no significant detoxification process of OC pesticides and PCBs occurred. The threat posed by organophosphate insecticides (OP) and CB compounds was also evidenced by ChE activity. The integrated response of phase I P450 enzymes and ChE activity being an indicator of potential effects of toxic contaminant levels on reproductive success and population decline of eels, can be used to assess the overall lagoon quality.     

Abeta(31-35) peptide induce apoptosis in PC 12 cells: contrast with Abeta(25-35) peptide and examination of underlying mechanisms

The toxic behaviour of the two shorter sequences of the native Abeta amyloid peptide required for cytotoxicity i.e., Abeta(31-35) and Abeta(25-35) peptides, was studied. We have shown that Abeta(31-35) peptide induces neurotoxicity in undifferentiated PC 12 cell via an apoptotic cell death pathway, including caspase activation and DNA fragmentation. Abeta(25-35) peptide, like the shorter amyloid peptide has the ability to induce neurotoxicity, as evaluated by the MTS reduction assay and by adherent cell count, but the Abeta(25-35) peptide-induced neurotoxicity is not associated with any biochemical features of apoptosis. The differences observed between the neurotoxic properties of Abeta(31-35) and Abeta(25-35) peptides might result on their different ability to be internalised within the neuronal cells. Furthermore, this study reveals that the redox state of methionine residue, C-terminal in Abeta(31-35) and Abeta(25-35) peptides affect in a different way the toxic behaviour of these two short amyloid fragments. Taken together our results suggest that Abeta(31-35) peptide induces cell death by apoptosis, unlike the Abeta(25-35) peptide and that role played by methionine-35 in Abeta induced neurotoxicity might be related to the Abeta aggregation state.     

In vitro setting of dose-effect relationships of 32 metal compounds in the Balb/3T3 cell line, as a basis for predicting their carcinogenic potential

The results are reported of the second stage in a programme for a systematic in vitro study on the carcinogenic potential of metal compounds with Balb/3T3 clone A31-1-1 mouse fibroblasts. Nineteen metal compounds that exhibited a strong cytotoxic effect during a previous screening run with a 100 microM fixed dose were tested with a 72-hour exposure over a wide range of concentrations (from 0.1 microM to 1000 microM), to produce dose-effect curves to permit extrapolation of the 50% inhibition concentration (IC50) values for each metal compound. This allows the establishment of a suitable range of doses for individual metal species, for use in the subsequent Balb/3T3 assay based on a two-stage concurrent cytotoxicity and morphological transformation protocol. Another 13 metal compounds were also tested, to determine whether the Balb/3T3 cell transformation assay is really a valuable in vitro model in relation to the problem of metal speciation. Of the metal compounds assayed, 26 showed a dose related cytotoxic response with calculated IC50 values ranging from 0.25 microM (CH3HgCl) to 140 microM [(C5H5)2TiCl2], whereas six metal compounds, namely (NH4)6Mo7O24*4H2O, CH3AsO(OH)2, C2H6AsNaO2(3H2O, KBr, CrCl3*6H2O and (NH4)2[TiO(C2O4)2]*H2O, displayed no observable cytotoxicity or low cytotoxicity at all the doses tested. The determination of IC50 values permits a ranking of the cytotoxicity responses of metal compounds with the highest cytotoxicities. Dose-effect curves and IC50 values of different chemical forms of individual metal compounds of As, Br, Cr, Hg, Ir, Pt, Te, Ti and V (cationic/anionic inorganic or organometallic species) showed clearly how the chemical nature of the metal strongly influences the toxic response. This confirms that the Balb/3T3 cell line is a valuable in vitro model with respect to the problem of metal speciation. This is a fundamental aspect to be considered when incorporating the results from in vitro cell transformation assays of the carcinogenic potential of metal compounds into regulatory testing schemes. In this context, the choice of test metal species for the development and validation of such assays cannot disregard the possibility that humans will be exposed to specific chemical forms of individual metal compounds (different oxidation states, and inorganic or organometallic natures) that can profoundly affect their toxicity.     

Binding of elements of protein kinase C-alpha regulatory domain to lamin B1

Previous results from our laboratory have demonstrated that lamin B1 is a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are important for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of the regulatory domain of rat PKC-alpha, we have established that binding occurs through both the V1 region and a portion of the C2 region (i.e., the calcium-dependent lipid binding [CaLB] domain) of the kinase. In particular, we have found that amino acids 200-217 of the CaLB domain are essential for binding lamin B1, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain of PKC-alpha and lamin B1. In agreement with the results of other investigators, we have determined that binding of regulatory elements of PKC-alpha to lamin B1 does not require the presence of cofactors such as PS and Ca(2+). We have also found that the binding site of lamin B1 for PKC-alpha is localized in the carboxyl-terminus of the lamin. Our findings may prove to be important in shedding more light on the mechanisms that regulate PKC functions within the nuclear compartment and may also lead to the synthesis of isozyme-specific pharmacological tools to attenuate or reverse PKC-dependent nuclear signalling pathways important for the pathogenesis of cancer.     

The identification of a novel human homologue of the SH3 binding glutamic acid-rich (SH3BGR) gene establishes a new family of highly conserved small proteins related to Thioredoxin Superfamily

The SH3 binding glutamic acid-rich (SH3BGR) gene was cloned in an effort to identify genes located to human chromosome 21, within the congenital heart disease region, and expressed in the developing heart. After the identification of SH3BGR, two human homologous genes, SH3BGRL and SH3BGRL3, were identified and mapped to chromosome Xq13.3 and 1p34.3-35, respectively. SH3BGRL and SH3BGRL3 code for small proteins similar to the N-terminal region of the SH3BGR protein. SH3BGRL3 protein shows a significant similarity to Glutaredoxin 1 of Escherichia coli, and all the three proteins are predicted to belong to Thioredoxin-like protein Superfamily. Here we describe the identification and characterization of an additional human homologue of SH3BGR, named SH3BGRL2. The SH3BGRL2 gene maps to chromosome 6q13-15 and its messenger RNA has a large 3' untranslated region containing several AUUUA repeats. SH3BGRL2 codes for a protein of 107 amino acids, which, like SH3BGRL and SH3BGRL3 proteins, is highly homologous to the N-terminal region of the SH3BGR protein and appears to be related to Glutaredoxins and to PKC-interacting cousin of thioredoxin homology domain. We propose that the identification of SH3BGRL2 establishes a novel family of human genes, coding for highly conserved small proteins belonging to Thioredoxin-like protein Superfamily.     

Upper airway afferents are sufficient to evoke the early components of respiratory-related cortical potentials in humans.

Repeated inspiratory occlusions in humans elicit respiratory-related cortical potentials, the respiratory counterpart of somatosensory-evoked potentials. These potentials comprise early components (stimulus detection) and late components (cognitive processing). They are considered as the summation of several afferent activities from various part of the respiratory system. This study assesses the role of the upper airway as a determinant of the early and late components of the potentials, taking advantage of the presence of a tracheotomy in patients totally or partially deafferented. Eight patients who could breathe either through the mouth or through a tracheotomy orifice (whole upper airway bypassed) were studied (4 quadriplegic patients with phrenic pacing, 4 patients with various sources of inspiratory pump dysfunction). Respiratory-related evoked potentials were recorded in CZ-C3 and CZ-C4. They were consistently present after mouth occlusions, with a first positive P1 and a first negative N1 components of normal latencies (P1: 40.4 +/- 6.1 ms in CZ-C3 and 47.6 +/- 7.6 ms in CZ-C4; N1: 84.4 +/- 27.1 ms in CZ-C3 and 90.2 +/- 17.4 ms in CZ-C4) and amplitudes. Tracheal occlusions did not evoke any cortical activity. Therefore, in patients with inspiratory pump dysfunction, the activation of upper airway afferents is sufficient to produce the early components of the respiratory-related evoked cortical potentials. Per contra, in this setting, pulmonary afferents do not suffice to evoke these components.     

Molecular cloning, genomic organization and developmental expression of the Xenopus laevis hyaluronan synthase 3.

The content of hyaluronan (HA), a polymer of the extracellular matrix involved in a variety of physiological and pathological processes, depends on the activity of synthetic (HAS) and degrading enzymes. Since HA is also involved in embryogenesis, we have used Xenopus as a model organism because information is available for HAS1 and HAS2, but not for HAS3. We report the sequence of xlHAS3 mRNA, its genomic organization and its expression in adult tissues as well as during embryonic development. Interestingly, evidence from in situ hybridization indicates that xlHAS3 expression is restricted to the developing inner ear and cement gland. In addition, we have correlated the expression pattern of the enzymes involved in HA metabolism with the HA content during development.     

Live-cell 3D super-resolution imaging in thick biological samples

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.