A cadmium resistance plasmid, pXU5, in Staphylococcus aureus, strain ATCC25923

A 25.9-kb plasmid, pXU5, encoding high level cadmium resistance was isolated from Staphylococcus aureus strain ATCC25923. A labelled cadA probe from plasmid pI258 hybridised to a 2.3-kb EcoRI fragment of pXU5. pXU5 was incompatible with an S. aureus incompatibility group 1 plasmid.     

Bassoon,a Novel Zinc-finger CAG/Glutamine-repeat Protein Selectively Localized at the Active Zone of Presynaptic Nerve Terminals

The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 M _r, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.     

Aggregation as a cost-reducing strategy for lycaenid larvae

If a mutualistic relationship entails providing services ata cost, selection will favor individuals that maximize the netbenefits of the interaction and minimize the costs. Larvae ofmany species of lycaenid butterflies secrete nutritious foodrewards to attending ants and, in return, receive protectionagainst predators and parasitoids. Because ants typically recruitmore workers to larger resources, by forming groups the larvaemay ensure more reliable access to ants and thereby gain betterprotection. A further consequence of aggregating should be achange of the cost-benefit relationship for individual larvae.The larger the group, the smaller a single larva's influencewill be on total ant density, which could lead to a smallerinvestment in secretion, thus reducing the per capita cost ofcooperation. In this study, die influence of ant attendance,group size, and companion quality on larval investment was investigated.The interaction between the obligately ant-dependent lycaenid,Jalmanus evagoras, and its attendant Iridomyrmax ants was manipulatedand the effect on larval secretion measured. As the level ofant attendance increased, the delivery of food rewards increased,bodi for solitary and for aggregated larvae. When aggregated,larvae provided less food rewards to ants dun when solitary,and secretion rate decreased with increasing group size. Furthermore,larvae had lower secretion rates when paired with a bigger,more attractive larva than when paired with a smaller one. Theconsiderable reduction in secretion rates for larvae in groupssuggests that gaining protection at a lower secretion cost couldbe one factor that promotes aggregation in myrmecophilous lycaenids.     

Cytokine inducible matrix metalloproteinase expression in immortalized rat chondrocytes is independent of nitric oxide stimulation

Summary The objective of this study was to determine if an immortalized mammalian chondrocyte cell line had a profile of matrix metalloproteinase (MMP) expression that was consistent with what has been reported for primary chondrocytes in vitro and in vivo. A combination of zymography, Western, and Northern analysis was used to examine the expression of MMPs that are relevant to cartilage degradation. Both interleukin-1β and tumor necrosis factor α induced a 4- to 9-fold increase in the level of MMP-9 expression in conditioned media, and a 17- to 24-fold increase in MMP-3 mRNA. Other compounds such as basic fibroblast growth factor and staurosporine each increased MMP-9 expression individually and potentiated the effects of the two cytokines. Transforming growth factor β had no positive or inhibitory effects. N-methyl arginine blocked the increase in nitric oxide observed following treatment with the cytokines but did not prevent the increased expression of MMPs. The pattern of metalloproteinase expression observed in IRC cells and the response to cytokines is very similar to what has been reported during the pathogenesis of osteoarthritis. The IRC cells should be useful as a model system to study basic mechanisms controlling chondrocyte MMP expression and to identify pharmacological modulators of this process.     

Estrogen Receptor and HER2 Status on Disseminated Tumor Cells and Primary Tumor in Patients with Early Breast Cancer

BACKGROUND: We evaluated both estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status on disseminated tumor cells (DTCs) in the bone marrow of 54 patients with early breast cancer and compared these with the corresponding primary tumor (PT). MATERIALS AND METHODS: Bone marrow aspirates were obtained at the time of first surgery, and ER and HER2 status on DTCs was assessed simultaneously by immunocytochemistry using a triple fluorescence staining method. RESULTS: The median number of DTCs was 13 (range 1-95). The concordance rate between ER status on DTC and PT was 74%. Patients with an ER-positive PT were significantly more likely to have at least one ER-positive DTC (34 out of 42) than patients with an ER-negative PT (6 out of 12; P = .031). Thirty-nine (93%) of the 42 patients with ER-positive PT had at least one ER-negative DTC. The concordance rate between HER2 status on DTC and PT was 52%. The probability of having at least one HER2-positive DTC was not related to the HER2 status of the PT (P = 0.56). Twenty-two (46%) of the 48 patients with a HER2-negative PT had at least one HER2-positive DTC. All the six patients with a HER2-positive PT had at least one HER2-negative DTC. CONCLUSION: Taken together, our study confirms that ER and/or HER2 status may differ between DTC and PT. This discordance could be important for patients lacking ER or HER2 expression on the PT but showing ER-positive or HER2-positive DTC because they might benefit from an endocrine and/or HER2-targeted therapy.     

Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.     

Development of homogeneous luminescence assays for histone demethylase catalysis and binding

Covalent modifications to histones play important roles in chromatin dynamics and the regulation of gene expression. The JumonjiC (JmjC)-containing histone demethylases (HDMs) catalyze the demethylation of methylated lysine residues on histone tails. Here we report the development of homogeneous luminescence-based assay methods for measuring the catalytic activity and the binding affinities of peptides to HDMs. The assays use amplified luminescent proximity homogeneous assay (ALPHA) technology, are sensitive and robust, and can be used for small molecule inhibitor screening of HDMs. We have profiled known inhibitors of JMJD2E and demonstrate a correlation between the inhibitor potencies determined by the ALPHA and other types of assays. Although this study focuses on the JMJD2E isoform, the catalytic turnover and binding assays described here can be used in studies on other HDMs. The assays should be useful for the development of small molecule inhibitors selective for HDM isoforms.