Accumulation of heavy metals in epidermal glands of the waterlily (Nymphaeaceae)

This study investigates the anatomical aspects of heavy-metal accumulation in the waterlily (Nymphaea `Aurora', Nymphaeaceae). Epidermal glands were identified by light microscopy on the abaxial side of the leaf laminae and on the epidermis of the rhizome; glandular trichomes were observed in the petiole epidermis. Glands were not observed in the roots. Accumulation of heavy metals in these glands was monitored using a scanning electron microscope equipped for energy-dispersive spectroscopy. Further experiments showed maximal cadmium and calcium accumulation in the mature leaf lamina in daylight, and this accumulation was inhibited by the herbicide 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. These results suggest that, in Nymphaea, heavy metals are accumulated primarily in association with glands found in plant organs that have direct contact with water or mud. Deposition and storage of heavy metals by these glands may represent a stage in the sequestration and detoxification of the metals. Our results raise the possibility of utilizing waterlilies for the removal of heavy metals from polluted environments. Received: 29 April 2000 / Accepted: 8 June 2000     

Microbial recognition via Toll-like receptor-dependent and -independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.     

High-performance thin-layer chromatography-bioautography for multiple antibiotic residues in cow's milk

An analytical method to identify and quantify multiple antibiotic residues (chloramphenicol, ampicillin, benzylpenicillin, dicloxacillin and erythromycin) in cow's milk by high-performance thin-layer chromatography (HPTLC) combined with bioautography was developed. The test microorganism used for bioautography was Bacillus subtilis ATCC 6633. Antibiotic residues were extracted with acetonitrile, fat eliminated with petroleum ether and residues isolated with dichloromethane The sensitivity of the method guarantees the detection of the above-mentioned antibiotics at levels below maximum residue limits (MRL) allowed for milk. Percentage recoveries ranged between 90 and 100%, with coefficients of variation between 7.2 and 21.3%. Some advantages of this methodology over thin-layer chromatography (TLC)/bioautography are also discussed.     

Cutting edge: IL-12 is required for the maintenance of IFN-gamma production in T cells mediating chronic resistance to the intracellular pathogen, Toxoplasma gondii

IL-12 is required for the development of IFN-gamma-dependent resistance to intracellular pathogens but is not thought to play a major role in its maintenance. To directly assess the requirement for continuous IL-12 signaling in long-term cell-mediated immunity, recombinant cytokine was transiently administered to IL-12 p40-deficient mice during the first 2 wk of infection with the intracellular pathogen Toxoplasma gondii. As expected, these animals survived the acute phase and established chronic infections. However, 4-6 wk after IL-12 withdrawal, the mice exhibited increased brain cyst burdens and succumbed to toxoplasmic encephalitis. Reactivation was associated with a loss of T-dependent IFN-gamma production without a concomitant increase in Th2 cytokine expression. Importantly, parasite Ag-induced IFN-gamma synthesis by purified T cells from these animals could be restored by in vitro exposure to IL-12. These results argue that endogenous IL-12 is required for the long-term maintenance of IFN-gamma-dependent resistance against intracellular pathogens.     

Peptide neurotoxins, small-cell lung carcinoma andneurological paraneoplastic syndromes

Peptide neurotoxins isolated from the venom of snakes, spiders and snails have represented invaluable tools for the identification and characterisation of membrane ion channels and receptors in vertebrate cells, including human neurons. We here report on the use of these toxins for the characterisation of membrane ion channels and receptors expressed by one of the most aggressive human cancers, small-cell lung carcinoma. This tumour shares many properties with other neuro-endocrine cell types, including the ability of firing action potentials and release hormones in a calcium-dependent manner. Toxins such as alpha-bungarotoxin and omega-conotoxins, among others, have been successfully used to characterise neuronal nicotinic receptors and voltage-dependent calcium channels, respectively, in human small-cell lung carcinoma cells. These receptors and ion channels are not only crucial for the growth of this specific tumour, but also represent autoantigens against which cancer patients build an autoimmune response. Although the aim of this autoimmune response is eventually the destruction of the cancer cells, the circulating antibodies cross-react with similar ion channels and receptors present in normal neurons or other cells, causing a number of different paraneoplastic diseases, the best characterised of which is the Lambert-Eaton myasthenic syndrome. Conotoxin-based radioimmunoassays have become an invaluable tool for the diagnosis and follow up of these paraneoplastic disorders and could represent a step forward in the early diagnosis of small-cell lung carcinoma itself.     

Resource pulses,species interactions,and diversity maintenance in arid and semi-arid environments

Arid environments are characterized by limited and variable rainfall that supplies resources in pulses. Resource pulsing is a special form of environmental variation, and the general theory of coexistence in variable environments suggests specific mechanisms by which rainfall variability might contribute to the maintenance of high species diversity in arid ecosystems. In this review, we discuss physiological, morphological, and life-history traits that facilitate plant survival and growth in strongly water-limited variable environments, outlining how species differences in these traits may promote diversity. Our analysis emphasizes that the variability of pulsed environments does not reduce the importance of species interactions in structuring communities, but instead provides axes of ecological differentiation between species that facilitate their coexistence. Pulses of rainfall also influence higher trophic levels and entire food webs. Better understanding of how rainfall affects the diversity, species composition, and dynamics of arid environments can contribute to solving environmental problems stemming from land use and global climate change.     

Transcriptional status of known and novel genes tagged with consensus of 33.15 repeat loci employing minisatellite-associated sequence amplification (MASA) and real-time PCR in water buffalo, Bubalus bubalis

We conducted minisatellite-associated sequence amplification (MASA) with an oligo (5' CACCTCTCCACCTGCC 3') based on consensus of 33.15 repeat loci using cDNA from the testis, ovary, spleen, kidney, heart, liver, and lung of water buffalo Bubalus bubalis and uncovered 25 amplicons of six different sizes (1,263, 846/847, 602, 576, 487, and 324 base pairs). These fragments, cloned and sequenced, were found to represent several functional, regulatory, and structural genes. Blast search of all the 25 amplicons showed homologies with 43 transcribing genes across the species. Of these, the 846/847-bp fragment, having homology with the adenylate kinase gene, showed nucleotide changes at six identical places in the ovary and testis. The 1,263; 324; and 487-bp fragments showed homology with the secreted modular calcium binding protein (SMOC-1), leucine-rich repeat neuronal 6A (LRRN6A) mRNA, and human TTTY5 mRNA, respectively. Real-time PCR showed maximum expression of AKL, LRRN6A, and T-cell receptor gamma (TCR-gamma)-like genes in the testis, SMOC-1 in the liver, and the T-cell receptor-like (TCRL) gene in the spleen compared to those used as endogenous control. We construe that these genes have evolved from a common progenitor and conformed to various biological functions during the course of evolution. MASA approach coupled with real-time PCR has potentials to uncover accurate expression of a large number of genes within and across the species circumventing the screening of cDNA library.     

A Conserved Methionine Residue Controls the Substrate Selectivity of a Neuronal Glutamate Transporter

Glutamate transporters located in the brain maintain low synaptic concentrations of the neurotransmitter by coupling its flux to that of sodium and other cations. In the binding pocket of the archeal homologue Glt_Ph, a conserved methionine residue has been implicated in the binding of the benzyl moiety of the nontransportable substrate analogue threo-β-benzyloxyaspartate. To determine whether the corresponding methionine residue of the neuronal glutamate transporter EAAC1, Met-367, fulfills a similar role, M367L, M367C, and M367S mutants were expressed in HeLa cells and Xenopus laevis oocytes to monitor radioactive transport and transport currents, respectively. The apparent affinity of the Met-367 mutants for d-aspartate and l-glutamate, but not for l-aspartate, was 10–20-fold reduced as compared with wild type. Unlike wild type, the magnitude of I_max was different for each of the three substrates. d-Glutamate, which is also a transportable substrate of EAAC1, did not elicit any detectable response with M367C and M367S but acted as a nontransportable substrate analogue in M367L. In the mutants, substrates inhibited the anion conductance as opposed to the stimulation observed with wild type. Remarkably, the apparent affinity of the blocker d,l-threo-β-benzyloxyaspartate in the mutants was similar to that of wild type EAAC1. Our results are consistent with the idea that the side chain of Met-367 fulfills a steric role in the positioning of the substrate in the binding pocket in a step subsequent to its initial binding.     

Engineered vascular beds provide key signals to pancreatic hormone-producing cells

The mechanisms underlying early islet graft failure are not entirely clear, but are thought to involve ischemic injury due to delayed vascularization. We hypothesize that blood vessels play an active role in cell-cell communications supporting islet survival and engraftment. To test this hypothesis and to uncouple endothelial cell (EC)-generated signaling stimuli from their nutritional and gas exchange functions, we developed three dimensional (3D) endothelial vessel networks in engineered pancreatic tissues prepared from islets, fibroblasts and ECs. The tri-culture setup, seeded on highly porous biocompatible polymeric scaffolds closely mimics the natural anatomical context of pancreatic vasculature. Enhanced islet survival correlating with formation of functional tube-like endothelial vessels was demonstrated. Addition of foreskin fibroblasts to islet-endothelial cultures promoted tube-like structure formation, which further supported islet survival as well as insulin secretion. Gene expression profiles of EC growth factors, extracellular matrix (ECM), morphogenes and differentiation markers were significantly different in 2D versus 3D culture systems and were further modified upon addition of fibroblasts. Implantation of prevascularized islets into diabetic mice promoted survival, integration and function of the engrafted engineered tissue, supporting the suggested role of ECs in islet survival. These findings present potential strategies for preparation of transplantable islets with increased survival prospects.