Recovery of brain noradrenaline after 5,7-dihydroxytryptamine-induced axonal lesions in the rat

Time-dependent changes in regional CNS noradrenaline (NA) concentration, 3H-NA uptake and fluorescence morphology of CNS NA neurons were analysed in the adult rat up to 6 months after intraventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), and compared with the time-course of changes in brain and spinal cord indolamine neurons. Following a substantial depletion of both amines in all CNS regions (telodiencephalon, brainstem and spinal cord) at 10 days after 150 mug 5,7-DHT, brain NA--but not 5-HT--levels recovered to near-normal values in brainstem and forebrain (35% below the age-matched controls) within 4 months. This was accompanied by a total restoration of the initially decreased capacity of the brain tissue to accumulate 3H-NA in vitro. Within 10 days after 5,7-DHT, there was a disappearance of NA terminals from many telencephalic, diencephalic and lower brain stem nuclei, from the cerebral and cerebellar cortices, and the grey matter of the spinal cord, concomitant with the appearance of numerous distorted, highly fluorescent swellings along the non-terminal axons of the major noradrenergic projection pathways. The recovery of the NA levels was paralleled by a re-appearance of fluorescent fibres, signifying an intense sprouting and regrowth of the drug-lesioned axons, which eventually re-innervated some of the previously denervated telodiencephalic regions. Except for a permanent loss of some surface-near perikarya in group A1 (the main source of the bulbospinal projections) there was no evidence of a retrograde degeneration of noradrenergic cell bodies in the rat CNS. The results are compatible with the idea that 5,7-DHT mainly causes a lesion of NA axons at a distance from the cell bodies, and this is followed by sprouting and regrowth of axons from the lisioned neurites, and formation of new terminal-like fibres in some previously denervated telodiencephalic regions. These findings indicate that chemical axotomy of central NA neurons induced by 5,7-DHT is--in contrast to that induced by 6-hydroxydopamine--followed by extensive axonal regeneration.     

Uncertainty in predicting range dynamics of endemic alpine plants under climate warming

Correlative species distribution models have long been the predominant approach to predict species’ range responses to climate change. Recently, the use of dynamic models is increasingly advocated for because these models better represent the main processes involved in range shifts and also simulate transient dynamics. A well‐known problem with the application of these models is the lack of data for estimating necessary parameters of demographic and dispersal processes. However, what has been hardly considered so far is the fact that simulating transient dynamics potentially implies additional uncertainty arising from our ignorance of short‐term climate variability in future climatic trends. Here, we use endemic mountain plants of Austria as a case study to assess how the integration of decadal variability in future climate affects outcomes of dynamic range models as compared to projected long‐term trends and uncertainty in demographic and dispersal parameters. We do so by contrasting simulations of a so‐called hybrid model run under fluctuating climatic conditions with those based on a linear interpolation of climatic conditions between current values and those predicted for the end of the 21st century. We find that accounting for short‐term climate variability modifies model results nearly as differences in projected long‐term trends and much more than uncertainty in demographic/dispersal parameters. In particular, range loss and extinction rates are much higher when simulations are run under fluctuating conditions. These results highlight the importance of considering the appropriate temporal resolution when parameterizing and applying range‐dynamic models, and hybrid models in particular. In case of our endemic mountain plants, we hypothesize that smoothed linear time series deliver more reliable results because these long‐lived species are primarily responsive to long‐term climate averages.     

Shb promotes blood vessel formation in embryoid bodies by augmenting vascular endothelial growth factor receptor-2 and platelet-derived growth factor receptor-beta signaling

The mechanisms controlling blood vessel formation during early embryonal development have only partly been elucidated. Shb is an adaptor protein previously implicated in the angiogenic response to vascular endothelial growth factor (VEGF). To elucidate a possible role of Shb in embryonic vascular development, wild-type and SH2 domain mutated (R522K) Shb were overexpressed in murine embryonic stem (ES) cells. Embryoid bodies (EBs) differentiating from Shb-overexpressing ES cells in vitro were stained for CD31 or VEGFR-2 to visualize the formation of vascular structures. We found that Shb promotes the outgrowth of blood vessels in EBs both in the absence and presence of growth factors. This response may be the consequence of an increased number of VEGFR-2 positive cells at an early stage of EB development, a finding corroborated by both immunostaining and real-time RT-PCR. In addition, Shb overexpression upregulated the expression of PDGFR-beta, CD31, CD41 and Tal1. Cells co-expressing VEGFR-2 and PDGFR-beta were commonly observed when Shb was overexpressed and inhibition of PDGF-BB signaling reduced the amount of VEGFR-2 mRNA under these conditions. EBs expressing the Shb R522K-mutant did not form vascular structures. Microarray analysis of VEGFR-2/CD31 positive cells after 6 days of differentiation revealed numerous changes of expression of genes relating to an endothelial/hematopoietic phenotype in response to Shb overexpression. The findings suggest that Shb may play a crucial role during early ES cell differentiation to vascular structures by transducing VEGFR-2 and PDGFR-beta signals.     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation

Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG-binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG-binding domain (MBD) only. Similar to MeCP2, another methyl CpG-binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.     

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA⁻ cell lines are shown.     

Studies on the root associations of the truffle Terfezia terfezioides

The paper contains an overview of the results of the studies made on the truffle Terfezia terfezioides, particularly the investigations related to the associations of this fungus with plants. Twelve plant species originated from a natural habitat of the fungus were supposed to be connected with T. terfezioides based on the anatomy of the endogenous fungal structures in their roots. Aseptic experiments were carried out on modified MMN substrates with different phosphate concentrations to study the interaction of T. terfezioides with Robinia pseudoacacia and Helianthemum ovatum. The colonization of the roots of black locust was always weaker than that of Helianthemum. The main characteristics were the intracellular coiled, branched, frequently septated hyphae in dead root cells. The intercellular hyphae formed Hartig-net with finger like structures only in Helianthemum, the interactions could not be considered unambiguously as mycorrhizae. There was no difference between the RFLP profiles of the nr DNA ITS of nineteen fruit bodies collected at the same time from the habitat and the ITS of three randomly chosen specimens were identical on sequence level, too. These invariability makes to design species specific PCR primers possible to check unambiguously the host plants.     

Asn229 in the third helix of VPAC1 receptor is essential for receptor activation but not for receptor phosphorylation and internalization: comparison with Asn216 in VPAC2 receptor

After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation.

We evaluated in CHO cells expressing the VPAC₁ receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y²²⁴, N²²⁹, F²³⁰, W²³², E²³⁶, G²³⁷, Y²³⁹, L²⁴⁰. N²²⁹A VPAC₁ mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca²⁺]_i increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N²²⁹D mutant was not expressed at the membrane, and the N²²⁹Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N²²⁹A and N²²⁹Q VPAC₁ were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking.

Mutation of that conserved amino acid in VPAC₂ could be investigated only by a conservative mutation (N²¹⁶Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.     

Hydroxyproline-Rich Protein Material in Wood and Lignin of Fagus sylvatica

The nitrogen content, distribution, and amino acid composition of protein material were determined in wood and lignin of Fagus sylvatica. The data indicated that the nitrogen originated from hydroxyproline-rich cell wall glycoprotein, about half of which may be bound to the lignin polymer. The implications for lignocellulose biodegradation are discussed.