Plants,Birds and Butterflies: Short-Term Responses of Species Communities to Climate Warming Vary by Taxon and with Altitude

As a consequence of climate warming, species usually shift their distribution towards higher latitudes or altitudes. Yet, it is unclear how different taxonomic groups may respond to climate warming over larger altitudinal ranges. Here, we used data from the national biodiversity monitoring program of Switzerland, collected over an altitudinal range of 2500 m. Within the short period of eight years (2003–2010), we found significant shifts in communities of vascular plants, butterflies and birds. At low altitudes, communities of all species groups changed towards warm-dwelling species, corresponding to an average uphill shift of 8 m, 38 m and 42 m in plant, butterfly and bird communities, respectively. However, rates of community changes decreased with altitude in plants and butterflies, while bird communities changed towards warm-dwelling species at all altitudes. We found no decrease in community variation with respect to temperature niches of species, suggesting that climate warming has not led to more homogenous communities. The different community changes depending on altitude could not be explained by different changes of air temperatures, since during the 16 years between 1995 and 2010, summer temperatures in Switzerland rose by about 0.07°C per year at all altitudes. We discuss that land-use changes or increased disturbances may have prevented alpine plant and butterfly communities from changing towards warm-dwelling species. However, the findings are also consistent with the hypothesis that unlike birds, many alpine plant species in a warming climate could find suitable habitats within just a few metres, due to the highly varied surface of alpine landscapes. Our results may thus support the idea that for plants and butterflies and on a short temporal scale, alpine landscapes are safer places than lowlands in a warming world.     

Cloning, sequencing and heterologous expression of a new chitinase gene, chi92, from Streptomyces olivaceoviridis ATCC 11238

A new chitinase gene, chi92, encoding the largest known chitinase from Streptomyces olivaceoviridisATCC 11238 was sequenced by means of different PCR-methods. The cloned gene was expressed in E. coliand the recombinant protein could be detected by Western-blot analysis. The multiplicity of chitinolytic enzymes of this strain is discussed.     

Lectin binding sites in Paramecium tetraurelia cells. I. Labeling analysis predominantly of secretory components

Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis. When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in approximately 6-10 vacuoles, which are several microns in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (otherwise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts). Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of "tips"), while ConA (fluorescence concentrated over "bodies") and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (approximately 1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable "matrix". Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function.     

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca²⁺] _o (extracellular Ca²⁺ concentration) is between about 10^–4 to 10^–3 m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca²⁺ _o or increasing [Ca²⁺] _o to 10^–2 m inhibit exocytotic membrane fusion. Veratridine does not release Ca²⁺ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na⁺ _o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca²⁺] _o ) in the somatic, i.e., nonciliary cell membrane and that a Ca²⁺ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.We thank Dr. C. Kung (Madison, WI) for providing the pawn mutant, Drs. G. Lehle and R. Waldschütz-Schüppel (Konstanz, Germany) for their help with light scattering experiments, and Ms. E. Dassler and D. Bliestle for continuous help during the extensive photographic documentation. This work has been supported by Deutsche Forschungsgemeinschaft, Schwerpunkt Neue mikroskopische Techniken für Biologie und Medizin (grant P178/11) and SFB156/B4.     

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (< or = 80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to approximately 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i = 65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e = 500 microM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.     

Comparison of in vivo and in vitro phosphorylation of the exocytosis-sensitive protein PP63/parafusin by differential MALDI mass spectrometric peptide mapping.

PP63 (parafusin) is a 63 kDa phosphoprotein, which exists in at least two different isoforms. It is very rapidly (80 ms) dephosphorylated during triggered trichocyst exocytosis. This occurs selectively in exocytosis-competent Paramecium tetraurelia strains. At least two protein kinases isolated from Paramecium, casein kinase type II kinase and cGMP-dependent kinase, are able to phosphorylate the two recombinant PP63/parafusin isoforms, both with phosphoglucomutase activity, in vitro. By performing mass spectrometric peptide mapping, we have investigated in vitro phosphorylation of recombinant PP63/parafusin by these kinases in comparison to in vivo phosphorylation of native PP63/parafusin isolated from Paramecium homogenates. Low picomolar quantities of proteolytic digests of recombinant and native PP63/parafusin, prior to and following alkaline phosphatase treatment, were directly analyzed by MALDI mass spectrometry. In native PP63-1/parafusin-1, six of 64 serine and threonine residues (S-196, T-205, T-280, T-371, T-373, and T-469) were found definitely, 27 were found possibly phosphorylated, 28 were identified as nonphosphorylated, and three were not covered by mapping. Three of the six certainly phosphorylated amino acids represent consensus phosphorylation sites for casein kinase II or cGMP-dependent protein kinase. In vitro phosphorylation studies of recombinant PP63/parafusin confirm that some of the sites found were used in vivo; however, also significant differences with respect to in vivo phosphorylation of native PP63/parafusin were observed. The two Paramecium protein kinases that were used do not preferably phosphorylate expected consensus sites in vitro. Homology structure modeling of PP63/parafusin with rabbit phosphoglucomutase revealed that the majority of residues found phosphorylated is located on the surface of the molecule.     

Dynamics of polymorphism of acidocalcisomes in<Emphasis Type="Italic"> Leishmania</Emphasis> parasites

Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 m diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warrens medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-κB and HSP27/p38/AKT Pathways and by Inhibiting ABCB1

Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis.     

Synthesis and antiviral activity of novel HCV NS3 protease inhibitors with P4 capping groups

We have synthesized and evaluated a series of novel HCV NS3 protease inhibitors with various P4 capping groups, which include urea, carbamate, methoxy-carboxamide, cyclic carbamate and amide, pyruvic amide, oxamate, oxalamide and cyanoguanidine. Most of these compounds are remarkably potent, exhibiting single-digit to sub-nanomolar activity in the enzyme assay and cell-based replicon assay. Selected compounds were also evaluated in the protease-inhibitor-resistant mutant transient replicon assay, and they were found to show quite different potency profiles against a panel of HCV protease-inhibitor-resistant mutants.