Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri

The cellular compartmentation of elements was analysed in the Zn hyperaccumulator Arabidopsis halleri (L.) O'Kane & Al-Shehbaz (=Cardaminopsis halleri) using energy-dispersive X-ray microanalysis of frozen-hydrated tissues. Quantitative data were obtained using oxygen as an internal standard in the analyses of vacuoles, whereas a peak/background ratio method was used for quantification of elements in pollen and dehydrated trichomes. Arabidopsis halleri was found to hyperaccumulate not only Zn but also Cd in the shoot biomass. While large concentrations of Zn and Cd were found in the leaves and roots, flowers contained very little. In roots grown hydroponically, Zn and Cd accumulated in the cell wall of the rhizodermis (root epidermis), mainly due to precipitation of Zn/Cd phosphates. In leaves, the trichomes had by far the largest concentrations of Zn and Cd. Inside the trichomes there was a striking sub-cellular compartmentation, with almost all the Zn and Cd being accumulated in a narrow ring in the trichome base. This distribution pattern was very different from that for Ca and P. The epidermal cells other than trichomes were very small and contained lower concentrations of Zn and Cd than mesophyll cells. In particular, the concentrations of Cd and Zn in the mesophyll cells increased markedly in response to increasing Zn and Cd concentrations in the nutrient solution. This indicates that the mesophyll cells in the leaves of A. halleri are the major storage site for Zn and Cd, and play an important role in their hyperaccumulation. Received: 4 April 2000 / Accepted: 16 May 2000     

Role of Calcineurin in Ca2+-Induced Release of Catecholamines and Neuropeptides

Abstract: Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca²⁺-regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munc18, MARCKS, dynamin I, and B-50/GAP-43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin-neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca²⁺-induced release of [³H]noradrenaline and neuropeptide cholecystokinin-8 in a specific and concentration-dependent manner. Our data show that the Ca²⁺/calmodulin-dependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin-8, transmitters stored in large dense-cored vesicles.     

Skeletal development in the Chinese soft‐shelled turtle Pelodiscus sinensis (Testudines: Trionychidae)

We investigated the development of the whole skeleton of the soft‐shelled turtle Pelodiscus sinensis, with particular emphasis on the pattern and sequence of ossification. Ossification starts at late Tokita‐Kuratani stage (TK) 18 with the maxilla, followed by the dentary and prefrontal. The quadrate is the first endoskeletal ossification and appears at TK stage 22. All adult skull elements have started ossification by TK stage 25. Plastral bones are the first postcranial bones to ossify, whereas the nuchal is the first carapacial bone to ossify, appearing as two unstained anlagen. Extensive examination of ossification sequences among autopodial elements reveals much intraspecific variation. Patterns of ossification of cranial dermal elements are more variable than those of endochondral elements, and dermal elements ossify before endochondral ones. Differences in ossification sequences with Apalone spinifera include: in Pelodiscus sinensis the jugal develops relatively early and before the frontal, whereas it appears later in A. spinifera; the frontal appears shortly before the parietal in A. spinifera whereas in P. sinensis the parietal appears several stages before the frontal. Chelydrids exhibit an early development of the postorbital bone and the palatal elements as compared to trionychids. Integration of the onset of ossification data into an analysis of the sequence of skeletal ossification in cryptodirans using the event‐pairing and Parsimov methods reveals heterochronies, some of which reflect the hypothesized phylogeny considered taxa. A functional interpretation of heterochronies is speculative. In the chondrocranium there is no contact between the nasal capsules and planum supraseptale via the sphenethmoid commissurae. The pattern of chondrification of forelimb and hind limb elements is consistent with a primary axis and digital arch. There is no evidence of anterior condensations distal to the radius and tibia. A pattern of quasi‐ simultaneity is seen in the chondrogenesis of the forelimb and the hind limb. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Hygienisation and Nutrient Conservation of Sewage Sludge or Cattle Manure by Lactic Acid Fermentation

Manure from animal farms and sewage sludge contain pathogens and opportunistic organisms in various concentrations depending on the health of the herds and human sources. Other than for the presence of pathogens, these waste substances are excellent nutrient sources and constitute a preferred organic fertilizer. However, because of the pathogens, the risks of infection of animals or humans increase with the indiscriminate use of manure, especially liquid manure or sludge, for agriculture. This potential problem can increase with the global connectedness of animal herds fed imported feed grown on fields fertilized with local manures. This paper describes a simple, easy-to-use, low-tech hygienization method which conserves nutrients and does not require large investments in infrastructure. The proposed method uses the microbiotic shift during mesophilic fermentation of cow manure or sewage sludge during which gram-negative bacteria, enterococci and yeasts were inactivated below the detection limit of 3 log₁₀ cfu/g while lactobacilli increased up to a thousand fold. Pathogens like Salmonella, Listeria monocytogenes, Staphylococcus aureus, E. coli EHEC O:157 and vegetative Clostridium perfringens were inactivated within 3 days of fermentation. In addition, ECBO-viruses and eggs of Ascaris suum were inactivated within 7 and 56 days, respectively. Compared to the mass lost through composting (15–57%), the loss of mass during fermentation (< 2.45%) is very low and provides strong economic and ecological benefits for this process. This method might be an acceptable hygienization method for developed as well as undeveloped countries, and could play a key role in public and animal health while safely closing the nutrient cycle by reducing the necessity of using energy-inefficient inorganic fertilizer for crop production.     

Intensified antineoplastic effect by combining an HDAC‐inhibitor,an mTOR‐inhibitor and low dosed interferon alpha in prostate cancer cells

A significant proportion of men diagnosed with prostate cancer (PCa) eventually develop metastatic disease, which progresses to castration resistance, despite initial response to androgen deprivation. As anticancer therapy has become increasingly effective, acquired drug resistance has emerged, limiting efficacy. Combination treatment, utilizing different drug classes, exemplifies a possible strategy to foil resistance development. The effects of the triple application of the histone deacetylase (HDAC) inhibitor valproic acid (VPA), the mammalian target of rapamycin inhibitor everolimus and low dosed interferon alpha (IFNα) on PCa cell growth and dissemination capacity were investigated. For that purpose, the human PCa cell lines, PC‐3, DU‐145 and LNCaP were treated with the combined regimen or separate single agents. Cell growth was investigated by the MTT dye reduction assay. Flow cytometry served to analyse cell cycle progression. Adhesion to vascular endothelium or immobilized collagen, fibronectin and laminin was quantified. Migration and invasion characteristics were determined by the modified Boyden chamber assay. Integrin α and β subtypes were investigated by flow cytometry, western blotting and RT‐PCR. Integrin related signalling, Epidermal Growth Factor Receptor (EGFr), Akt, p70S6kinase and extracellular signal‐regulated kinases (ERK)1/2 activation were also assessed. The triple application of VPA, everolimus and low dosed IFNα blocked tumour cell growth and dissemination significantly better than any agent alone. Antitumour effects were associated with pronounced alteration in the cell cycle machinery, intracellular signalling and integrin expression profile. Combining VPA, everolimus and low dosed IFNα might be a promising option to counteract resistance development and improve outcome in PCa patients.     

The Origin and Early Radiation of Archosauriforms: Integrating the Skeletal and Footprint Record

We present a holistic approach to the study of early archosauriform evolution by integrating body and track records. The ichnological record supports a Late Permian–Early Triassic radiation of archosauriforms not well documented by skeletal material, and new footprints from the Upper Permian of the southern Alps (Italy) provide evidence for a diversity not yet sampled by body fossils. The integrative study of body fossil and footprint data supports the hypothesis that archosauriforms had already undergone substantial taxonomic diversification by the Late Permian and that by the Early Triassic archosauromorphs attained a broad geographical distribution over most parts of Pangea. Analysis of body size, as deduced from track size, suggests that archosauriform average body size did not change significantly from the Late Permian to the Early Triassic. A survey of facies yielding both skeletal and track record indicate an ecological preference for inland fluvial (lacustrine) environments for early archosauromorphs. Finally, although more data is needed, Late Permian chirotheriid imprints suggest a shift from sprawling to erect posture in archosauriforms before the end-Permian mass extinction event. We highlight the importance of approaching palaeobiological questions by using all available sources of data, specifically through integrating the body and track fossil record.     

Dissection of the conformational cycle of the multidrug/lipidA ABC exporter MsbA

Recent crystal structures of the multidrug ATP‐binding cassette (ABC) exporters Sav1866 from Staphylococcus aureus, MsbA from Escherichia coli, Vibrio cholera, and Salmonella typhimurium, and mouse ABCB1a suggest a common alternating access mechanism for export. However, the molecular framework underlying this mechanism is critically dependent on assumed conformational relationships between nonidentical crystal structures and therefore requires biochemical verification. The structures of homodimeric MsbA reveal a pair of glutamate residues (E208 and E208′) in the intracellular domains of its two half‐transporters, close to the nucleotide‐binding domains (NBDs), which are in close proximity of each other in the outward‐facing state but not in the inward‐facing state. Using intermolecular cysteine crosslinking between E208C and E208C′ in E. coli MsbA, we demonstrate that the NBDs dissociate in nucleotide‐free conditions and come close on ATP binding and ADP·vanadate trapping. Interestingly, ADP alone separates the half‐transporters like a nucleotide‐free state, presumably for the following catalytic cycle. Our data fill persistent gaps in current studies on the conformational dynamics of a variety of ABC exporters. Based on a single biochemical method, the findings describe a conformational cycle for a single ABC exporter at major checkpoints of the ATPase reaction under experimental conditions, where the exporter is transport active. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.