Grass cover on forest clear-cut areas ameliorates some soil chemical properties

Clear-cut areas formed after forest decline due to acid deposition, pest attacks, or wind-breaks in temperate mountainous regions are often populated by grass (mainly Calamagrostis villosa). This study focused on the changes of soil chemical characteristics under the grass cover replacing the forest, focusing mainly on aluminium (Al) speciation. Clear-cut area due to strong acid deposition in the Jizera Mountains (Northern Bohemia) was studied. The soils under grass cover exhibit higher pH values and lower exchangeable Al content compared to adjacent surviving forest. Mobile Al species under the grass have larger proportion of non-toxic organic complexes. The content of exchangeable base cations is slightly higher under the grass. The positive effect of grass on soil chemistry was enhanced by liming. The temporary grass cover can therefore improve soil chemical quality for following reforestation. However, the differences are generally limited to surface organic horizons. Similar results were found also on a bark-beetle clear-cut area in the Bohemian Forest (Southern Bohemia) with smaller acid deposition; nevertheless, most differences were not significant there.     

Synthesis and evaluation of phenylcarbamate derivatives as ligands for nicotinic acetylcholine receptors

Phenylcarbamate derivatives were synthesized and evaluated in radioligand binding assays for different nicotinic acetylcholine receptor (nAChR) subtypes. Carbamate derivatives bearing a pyrrolidine or piperidine moiety 8-20 exhibited much lower affinity for alpha7* nAChR than the analogues in the quinuclidine series 21-25, although the same structural elements are present. Furthermore, in contrast to the quinuclidine analogues 21-25, all (S)-pyrrolidine derivatives 8-12 and the piperidine analogues 15 and 16 exhibited higher affinities for alpha4beta2* nAChR.     

Rab9 GTPase regulates late endosome size and requires effector interaction for its stability

Rab9 GTPase resides in a late endosome microdomain together with mannose 6-phosphate receptors (MPRs) and the tail-interacting protein of 47 kDa (TIP47). To explore the importance of Rab9 for microdomain establishment, we depleted the protein from cultured cells. Rab9 depletion decreased late endosome size and reduced the numbers of multilamellar and dense-tubule-containing late endosomes/lysosomes, but not multivesicular endosomes. The remaining late endosomes and lysosomes were more tightly clustered near the nucleus, implicating Rab9 in endosome localization. Cells displayed increased surface MPRs and lysosome-associated membrane protein 1. In addition, cells showed increased MPR synthesis in conjunction with MPR missorting to the lysosome. Surprisingly, Rab9 stability on late endosomes required interaction with TIP47. Rabs are thought of as independent, prenylated entities that reside either on membranes or in cytosol, bound to GDP dissociation inhibitor. These data show that Rab9 stability is strongly influenced by a specific effector interaction. Moreover, Rab9 and the proteins with which it interacts seem critical for the maintenance of specific late endocytic compartments and endosome/lysosome localization.     

Endocytosis of epidermal growth factor receptor regulated by Grb2-mediated recruitment of the Rab5 GTPase-activating protein RN-tre

The Grb2 adaptor protein is best known for its role in signaling to the small GTPase p21(ras), mediated through its interaction with the SOS guanine nucleotide exchange factor. Here, we demonstrate that Grb2 also signals to Rab5, a small GTPase that plays a key role in early endocytic trafficking. Grb2 functions through association with RN-tre, a GTPase-activating protein for Rab5. Grb2 and RN-tre associate both in vitro and in vivo, with interaction mediated by both SH3 domains of Grb2 and extended proline-rich sequences in RN-tre. Association between Grb2 and RN-tre is constitutive and occurs independently of Eps8, a previously identified binding partner of RN-tre. Epidermal growth factor (EGF) stimulates recruitment of RN-tre to the EGF receptor (EGFR) in a Grb2-dependent manner. Grb2 and the EGFR are internalized and co-localized in endocytic vesicles in response to EGF. Overexpression of RN-tre blocks the internalization of both proteins, consistent with its function as a negative regulator of Rab5 and endocytosis. Strikingly, RN-tre does not block EGF-induced internalization of a Grb2 mutant deficient in RN-tre binding. These results 1) suggest that the ability of RN-tre to inhibit internalization of the EGFR requires Grb2-mediated binding to the receptor and 2) identify Grb2 as a critical regulator of Rab5 and EGFR endocytosis.     

Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor

The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket.     

Neural stem cells transplanted into intact brains as neurospheres form solid grafts composed of neurons, astrocytes and oligodendrocyte precursors

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential that can give rise to neurons and glia in vivo and in vitro. The aim of this study was to transplant NSCs as whole neurospheres into intact brain and assess the fate and phenotype of their progeny generated in vivo. We isolated NSCs from E14 foetal rat forebrains and cultured them in basic fibroblast and epidermal growth factor-supplemented serum-free medium in the form of neurospheres in vitro. Neurospheres were transplanted into the intact brains of 2 Wistar rats and after a period of 3 weeks, grafted brains were examined immunohistochemically. Neurospheres formed solid grafts that were found in the lateral ventricle and in the velum interpositum under the hippocampus. The majority of cells in the transplanted tissue were identified as beta-III-tubulin(+), NeuN(+), PanNF(+) and synaptophysin(+) neurons and were accumulated throughout the graft centre. GFAP(+) astrocytes were scattered throughout the entire graft and astrocyte processes delimited the outer and perivascular surfaces. A great number of NG2(+) oligodendrocyte precursors was detected. Nestin(+) endothelial cells were found to line capillaries growing in the transplant. These data indicate that nestin(+) NSCs prevailing in neurospheres differentiate following transplantation into nestin(-) neuronal and glial cells which confirms the multipotency of NSCs. Three weeks posttransplantation neuronal and astrocyte cells reached terminal differentiation (formation of synaptic vesicles and superficial and perivascular limiting membranes) while elements of oligodendroglial cell lineage remained immature. Grafting stem cells as non-dissociated neurospheres provide cells with favourable conditions which facilitate cell survival, proliferation and differentiation. However, in the intact brain, grafted neurosphere cells were not found to integrate with the brain parenchyma and formed a compact structure demarcated from its surroundings.     

Measuring Biomass and Carbon Stock in Resprouting Woody Plants

Resprouting multi-stemmed woody plants form an important component of the woody vegetation in many ecosystems, but a clear methodology for reliable measurement of their size and quick, non-destructive estimation of their woody biomass and carbon stock is lacking. Our goal was to find a minimum number of sprouts, i.e., the most easily obtainable, and sprout parameters that should be measured for accurate sprout biomass and carbon stock estimates. Using data for 5 common temperate woody species, we modelled carbon stock and sprout biomass as a function of an increasing number of sprouts in an interaction with different sprout parameters. The mean basal diameter of only two to five of the thickest sprouts and the basal diameter and DBH of the thickest sprouts per stump proved to be accurate estimators for the total sprout biomass of the individual resprouters and the populations of resprouters, respectively. Carbon stock estimates were strongly correlated with biomass estimates, but relative carbon content varied among species. Our study demonstrated that the size of the resprouters can be easily measured, and their biomass and carbon stock estimated; therefore, resprouters can be simply incorporated into studies of woody vegetation.