Importance of permafrost as a source of water for plants in east Siberian taiga

Stable oxygen isotope ratios of plant water (sap water) were observed at Spasskaya Pad experimental forest near Yakutsk, Russia in 1997–1999. The ¹⁸O of sap water in larch trees (Larix gmelinii) decreased soon after leaf unfolding every year, indicating that snowmelt water was used in the beginning of summer. During mid to late summer, a clear difference in the water source used by plants was observed between wet summers and severe drought summers. The ¹⁸O values of water in larch trees were high (–17.8 to –16.1) in August 1999 (wet summer), but low (–20.4 to –19.7) in August 1998 (drought summer). These results indicated that plants used rainwater during a wet summer, but meltwater from permafrost was used by plants during a drought summer. One important role of permafrost is to provide a direct source of water for plants in a severe drought summer; another role is to keep surplus water in the soil until the next summer. If this permafrost system is disturbed by future global warming, unique monotypic stands of deciduous larch trees in east Siberia might be seriously damaged in a severe drought summer.     

Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells

Apoptosis is involved in the pathogenesis of Sjögren’s syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H₄R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H₄R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H₄R agonist HST-10. Expression and internalization of H₄R were studied by immunofluorescence staining ± clathrin inhibitor methyl-β-cyclodextrin (MβCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H₄R internalization was inhibited by MβCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H₄R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H₄R-stimulated samples. H₄R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H₄R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H₄R activation on apoptosis of human salivary gland cells. Diminished H₄R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.     

Cytotype diversity and genome size variation in eastern Asian polyploid Cardamine (Brassicaceae) species

Background and Aims

Intraspecific ploidy-level variation is an important aspect of a species'' genetic make-up, which may lend insight into its evolutionary history and future potential. The present study explores this phenomenon in a group of eastern Asian Cardamine species.

Methods

Plant material was sampled from 59 localities in Japan and Korea, which were used in karyological (chromosome counting) and flow cytometric analyses. The absolute nuclear DNA content (in pg) was measured using propidium iodide and the relative nuclear DNA content (in arbitrary units) was measured using 4,6-diamidino-2-phenylindole fluorochrome.

Key Results

Substantial cytotype diversity was found, with strikingly different distribution patterns between the species. Two cytotypes were found in C. torrentis sensu lato (4x and 8x, in C. valida and C. torrentis sensu stricto, respectively), which displays a north–south geographical pattern in Japan. Hypotheses regarding their origin and colonization history in the Japanese archipelago are discussed. In Korean C. amaraeiformis, only tetraploids were found, and these populations may in fact belong to C. valida. C. yezoensis was found to harbour as many as six cytotypes in Japan, ranging from hexa- to dodecaploids. Ploidy levels do not show any obvious geographical pattern; populations with mixed ploidy levels, containing two to four cytotypes, are frequently observed throughout the range. C. schinziana, an endemic of Hokkaido, has hexa- and octoploid populations. Previous chromosome records are also revised, showing that they are largely based on misidentified material or misinterpreted names.

Conclusions

Sampling of multiple populations and utilization of the efficient flow cytometric approach allowed the detection of large-scale variation in ploidy levels and genome size variation attributable to aneuploidy. These data will be essential in further phylogenetic and evolutionary studies.     

Formation and temperature stability of G-quadruplex structures studied by electronic and vibrational circular dichroism spectroscopy combined with ab initio calculations

Variations in the structure of d(GGGA)(5) oligonucleotide in the presence of Li(+), Na(+), and K(+) ions and its temperature stability were studied using electronic and vibrational circular dichroism, IR absorption, and ab initio calculations with the Becke 3-Lee-Yang-Parr functional at the 6-31G** level. The samples were characterized by nondenaturing gel electrophoresis. Oligonucleotide d(GGGA)(5) in the presence of Li(+) forms a nonplanar single tetramer, with angles of 102 degrees and 171 degrees between neighboring guanine bases. This tetramer changes its geometry at temperatures >50 degrees C, but does not form a quadruplex structure. In the presence of Na(+), the d(GGGA)(5) structure was optimized to almost planar tetramers with an angle of 177 degrees between neighboring guanines. The spectral results suggest that it stacks into a quadruplex helical structure. This quadruplex structure decayed to a single tetramer at temperatures >60 degrees C. The Hartree-Fock energies imply that d(GGGA)(5) prefers to form complexes with Na(+) rather than Li(+). The d(GGGA)(5) structure in the presence of monovalent ions is stabilized against thermal denaturation in the order Li(+) < Na(+) < K(+).     

Natural rock roughness as a key parameter for optimizing seedling adhesion and restoration of the seagrass Posidonia oceanica in its native environment

Seagrasses provide various ecosystem functions in coastal areas of the world. In the Mediterranean Sea, Posidonia oceanica is an endemic species threatened by several activities despite being protected by national and international laws. Currently, several transplanting initiatives have been carried out using different methods, among which those including seeds and seedlings are considered the most ecological and low-cost ones. Beach-cast fruits and seeds can be found in spring and their appearance can easily be reported, through a citizen science approach, by the community. One of the obstacles in using these methods is identifying the best substrate in which to place P. oceanica seeds to facilitate root adhesion of the seedlings prior to their transplantation into the sea. In the present study, we analyzed, using a 3D surface optical microscope, the roughness of natural rocks to identify the availability of specific roughness ranges suitable for adhesion and root anchoring of P. oceanica seedlings. Conventional roughness parameters and roughness power spectral density were calculated for the inner and outer surfaces of 9 different rock samples. Among the rock samples examined, the calcarenitic ones and in particular marsala calcarenite, due to the presence of the “ideal roughness for seedlings” can be considered one of the best consolidated substrates to be used for the construction of ad hoc devices on which plantlet of P. oceanica can grow for the purpose of restoration.     

Utilization of Citrate, Acetylcarnitine, Acetate, Pyruvate and Glucose for the Synthesis of Acetylcholine in Rat Brain Slices

Slices of rat caudate nuclei were incubated in saline media containing choline, paraoxon, unlabelled glucose, and [1,5-14C] citrate, [1-14C-acetyl]carnitine, [1-14C]acetate, [2-14C]pyruvate, or [U-14C]glucose. The synthesis of acetyl-labelled acetylcholine (ACh) was compared with the total synthesis of ACh. When related to the utilization of unlabelled glucose (responsible for the formation of unlabelled ACh), the utilization of labelled substrates for the synthesis of the acetyl moiety of ACh was found to decrease in the following order: [2-14C]pyruvate greater than [U-14C]glucose greater than [1-14C-acetyl]carnitine greater than [1,5-14C]citrate greater than [1-14C]acetate. The utilization of [1,5-14C]citrate and [1-14C]acetate for the synthesis of [14C]ACh was low, although it was apparent from the formation of 14CO2 and 14C-labelled lipid that the substrates entered the cells and were metabolized. The utilization of [1,5-14C]citrate for the synthesis of [14C]ACh was higher when the incubation was performed in a medium without calcium (with EGTA); that of glucose did not change, whereas the utilization of other substrates for the synthesis of ACh decreased. The results indicate that earlier (indirect) evidence led to an underestimation of acetylcarnitine as a potential source of acetyl groups for the synthesis of ACh in mammalian brian; they do not support (but do not disprove) the view that citrate is the main carrier of acetyl groups from the intramitochondrial acetyl-CoA to the extramitochondrial space in cerebral cholinergic neurons.     

Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis

Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.     

New inhibitors of fungal 17β-hydroxysteroid dehydrogenase based on the [1,5]-benzodiazepine scaffold

The synthesis and activity of a new series of non-steroidal inhibitors of 17β-hydroxysteroid dehydrogenase that are based on a 1,5-benzodiazepine scaffold are presented. Their inhibitory potential was screened against 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl), a model enzyme of the short-chain dehydrogenase/reductase superfamily. Some of these compounds are potent inhibitors of 17β-HSDcl activity, with IC₅₀ values in the low micromolar range and represent promising lead compounds that should be further developed and investigated as inhibitors of human 17β-HSD isoforms, which are the enzymes associated with the development of many hormone-dependent and neuronal diseases.