The importance of nitrogen and carbohydrate storage for plant growth of the alpine herb Veratrum album

We examined whether nitrogen (N) and carbohydrates reserves allow Veratrum album, an alpine forb, to start spring growth earlier than the neighbouring vegetation and to survive unpredictable disturbances resulting in loss of above-ground biomass. * Seasonal dynamics of plant reserves, soil N availability and vegetation growth were monitored. Veratrum album shoots were experimentally removed when carbohydrate reserves were at a seasonal minimum and the subsequent changes in biomass and reserves were compared with those in control plants. Reserves did not give V. album a competitive advantage in spring; however, they did function as a buffer against the impact of calamities. Shoot removal resulted in significantly lower root dry weight, higher N concentration in rhizome and roots and lower starch concentrations in rhizome and roots but no plant mortality was observed. Veratrum album used stored N reserves to supplement N uptake and establish high leaf N concentrations, which facilitated a rapid refilling of depleted carbohydrate reserves. The primary function of N reserves appears to be to allow V. album to complete the growing cycle in as short a period as possible, thus minimizing exposure to above-ground risks.     

Irradiance and temperature affect the competitive interference of blackberry on the physiology of European beech seedlings

The potential negative influence of competition from early successional species like blackberry (Rubus fruticosus) may be decisive for the natural regeneration success of drought-sensitive beech (Fagus sylvatica), especially in the light of climate change. With a split plot glasshouse experiment, we investigated the influence of two air temperature and irradiance levels on the competitive interference of blackberry on the water, nitrogen (N) and carbon (C) balance of beech seedlings under moderate drought. When increased temperature was accompanied by low irradiance the biomass, root-to-shoot ratio, N uptake and assimilation rates of blackberry were lower compared with beech, either grown alone or with blackberry. By contrast, when elevated temperature and high irradiance were combined, the root-to-shoot ratio and specific N uptake rate of blackberry were substantially increased, while the N acquisition of beech was impaired. Under lower temperature, with either full light or shade, the presence of blackberry had no significant effects on beech, for almost all tested parameters. Under elevated air temperature beech was impaired by the presence of blackberry at high irradiance. These findings emphasize the interacting effects between environmental factors and competition on the establishment of beech regeneration, which should be considered for future forest management in the frame of climate change.     

The effect of wortmannin on radiation-induced chromosome aberration formation in the radioresistant tumor cell line WiDr

We analyzed the formation of radiation-induced chromosome aberrations in the cells of the radioresistant colon carcinoma cell line WiDr after treatment with wortmannin, an inhibitor of PI-3 kinases, including DNA-PK. Cells irradiated in G0/G1 phase with 200 kV X rays were treated with wortmannin before or after irradiation. Chromosome-type and chromatid-type aberrations were scored in metaphase cells by either Giemsa staining or FISH. Moreover, DNA-PK activity was measured in the absence and presence of wortmannin. In irradiated G0/G1-phase WiDr cells, only chromosome-type aberrations, including simple and complex exchanges and excess acentrics, were observed. After addition of 1 to 20 microM wortmannin, the formation of chromosome-type exchange aberrations was completely suppressed. The irradiated cells displayed exclusively chromatid-type aberrations including simple and complex chromatid exchanges and chromatid/isochromatid breaks. Whether the chromatid-type aberrations arise during G0/G1 as a result of homologous recombination processes coping with damaged DNA or whether DNA damage induced during G0/G1 phase persists until S and G2 phase and is then processed by homologous recombination pathways must be investigated further.     

Internalins from the human pathogen Listeria monocytogenes combine three distinct folds into a contiguous internalin domain

The pathology of type II diabetes includes deposition of amyloid in the extra cellular space surrounding the beta-cells of the endocrine pancreas. The principle component of these deposits is an insoluble fibrillar form of a normally soluble 37 residue peptide hormone, islet amyloid polypeptide. Multiple sequence analysis and peptide synthesis have identified a core set of residues (20 to 29) as intrinsically amyloidogenic. As the fibrillogenesis of the 20-29 peptide often requires conditions that deviate considerably from physiological, residues 20 to 29 may be necessary, but not sufficient, for amyloidosis. We aim to determine the structural role of residues outside this core in the context of in vitro fibrillogenesis of the wild-type peptide at physiological pH and ionic strength. Specifically, we make use of an intrinsic fluorescent probe, tyrosine 37 (Y37), to explore the role of the C terminus in fibrillogenesis. Our protocol permits steady state measurement of the lag phase and fiber conformational states of the protein under identical conditions. These are compared to a non-amyloidogenic variant of islet amyloid polypeptide from rat and N-acetyl-tyrosinamide as models of the unfolded state under matched conditions. Spectral, quenching and anisotropic properties of Y37 in the fiber state indicate that the C terminus is packed in a well-defined environment with near frozen rigidity. The presence of a fluorescence resonance energy transfer pathway shows Y37 is near F15 and F23. The lag-phase conformation, while considerably less ordered than the fiber, is more ordered than unfolded models. Differences in anisotropy between the lag and fiber state were used to monitor fibrillogenesis in real time. Parallel assessment of fiber formation using the histological dye, ThT, indicate that ordering at the C terminus of islet amyloid polypeptide is coincident with, and thus indicative of, fiber formation.     

Mutational evidence for an internal fusion peptide in flavivirus envelope protein E

The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus promotes cell entry by inducing fusion of the viral membrane with an intracellular membrane after uptake by endocytosis. This protein differs from other well-studied viral and cellular fusion proteins because of its distinct molecular architecture and apparent lack of involvement of coiled coils in the low-pH-induced structural transitions that lead to fusion. A highly conserved loop (the cd loop), which resides at the distal tip of each subunit and is mostly buried in the subunit interface of the native E homodimer at neutral pH, has been hypothesized to function as an internal fusion peptide at low pH, but this has not yet been shown experimentally. It was predicted by examination of the X-ray crystal structure of the TBE virus E protein (F. A. Rey et al., Nature 375:291-298, 1995) that mutations at a specific residue within this loop (Leu 107) would not cause the native structure to be disrupted. We therefore introduced amino acid substitutions at this position and, using recombinant subviral particles, investigated the effects of these changes on fusion and related properties. Replacement of Leu with hydrophilic amino acids strongly impaired (Thr) or abolished (Asp) fusion activity, whereas a Phe mutant still retained a significant degree of fusion activity. Liposome coflotation experiments showed that the fusion-negative Asp mutant did not form a stable interaction with membranes at low pH, although it was still capable of undergoing the structural rearrangements required for fusion. These data support the hypothesis that the cd loop may be directly involved in interactions with target membranes during fusion.     

Expression of olfactory receptors in the cribriform mesenchyme during prenatal development

Olfactory receptors (ORs) are expressed in sensory neurons of the nasal epithelium, where they are supposed to be involved in the recognition of suitable odorous compounds and in the guidance of outgrowing axons towards the appropriate glomeruli in the olfactory bulb. During development, some olfactory receptor subtypes have also been found in non-sensory tissues, including the cribriform mesenchyme between the prospective olfactory epithelium and the developing telencephalon, but it is elusive if this is a typical phenomenon for ORs. Monitoring the onset and time course of expression for several receptor subtypes revealed that 'extraepithelial' expression of ORs occurs very early and transiently, in particular between embryonic stages E10.25 and E14.0. In later stages, a progressive loss of receptor expressing cells was observed. Molecular phenotyping demonstrated that the receptor expressing cells in the cribriform mesenchyme co-express key elements, including Galpha(olf), ACIII and OMP, characteristic for olfactory neurons in the nasal epithelium. Studies on transgenic OMP/GFP-mice showed that 'extraepithelial' OMP/GFP-positive cells are located in close vicinity to axon bundles projecting from the nasal epithelium to the presumptive olfactory bulb. Moreover, these cells are primarily located where axons fasciculate and change direction towards the anterior part of the forebrain.     

Solution-phase synthesis of Aib-containing cyclic hexapeptides

The 18-membered Aib-containing cyclohexapeptides, cyclo(-Gly-Aib-Aib-Gly-Aib-Phe-) (22), cyclo(-Gly-Aib-Phe(2Me)-Gly-Aib-Aib-) (24a), cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Aib-) (24b), and cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Phe-) (25), have efficiently been synthesized by solution-phase techniques. The linear precursors 1a-1d were prepared by combining the 'azirine/oxazolone method' for incorporation of alpha,alpha-disubstituted alpha-amino acids (Aib, Phe(2Me)) into the peptide chains by classical peptide coupling methods for segment condensations. Deprotection of the amino and carboxy termini of 1a-1d, followed by cyclization with DEPC as the coupling reagent, gave the above-mentioned cyclic hexapeptides 22, 24a, 24b, and 25 in good yields (26-57%). The solid-state conformations of the linear hexapeptides 1d, 16 and 27, and of the cyclohexapeptides 22 and 25 have been established by X-ray crystallography.     

Evolution of class B floral homeotic proteins: obligate heterodimerization originated from homodimerization

The class B floral homeotic genes from the higher eudicot model systems Arabidopsis and Antirrhinum are involved in specifying the identity of petals and stamens during flower development. These genes exist in two different types termed DEF- and GLO-like genes. The proteins encoded by the class B genes are stable and functional in the cell only as heterodimeric complexes of a DEF- and a GLO-like protein. In line with this, heterodimerization is obligate for DNA binding in vitro. The genes whose products have to heterodimerize to be stable and functional are each other's closest relatives within their genomes. This suggests that the respective genes originated by gene duplication, and that heterodimerization is of relative recent origin and evolved from homodimerization. To test this hypothesis we have investigated the dimerization behavior of putative B proteins from phylogenetic informative taxa, employing electrophoretic mobility shift assays and the yeast two-hybrid system. We find that an ancestral B protein from the gymnosperm Gnetum gnemon binds DNA in a sequence-specific manner as a homodimer. Of the two types of B proteins from the monocot Lilium regale, the GLO-like protein is still able to homodimerize, whereas the DEF-like protein binds to DNA only as a heterodimeric complex with the GLO-like protein. These data suggest that heterodimerization evolved in two steps after a gene duplication that gave rise to DEF- and GLO-like genes. Heterodimerization may have originated after the gymnosperm-angiosperm split about 300 MYA but before the monocot-eudicot split 140-200 MYA. Heterodimerization may have become obligate for both types of flowering plant B proteins in the eudicot lineage after the monocot-eudicot split.     

The murine alpha(1)-proteinase inhibitor gene family: polymorphism,chromosomal location,and structure

alpha(1)-Proteinase inhibitor (alpha(1)-PI) is a member of the serpin superfamily of serine proteinase inhibitors, which function in maintaining homeostasis through regulation of numerous proteolytic processes. In laboratory mice (Mus musculus domesticus), alpha(1)-PI occurs in multiple isoforms encoded by a family of three to five genes that are polymorphic among inbred strains and that are located at the Serpina1 locus on chromosome 12. In the present study, we have characterized the alpha(1)-PI gene family of inbred mice in more detail. We show that mice express seven isoforms, all of which are encoded by genes that map to the Serpina1 locus. In addition, polymorphism at the locus is defined by three haplotypes (Serpina1(b), Serpina1(c), and Serpina1(l)) that differ with regard to both the number and identity of alpha(1)-PI genes. Finally, we present the complete sequence of an 84-kb region of Serpina1 containing a tandem repeat of two alpha(1)-PI genes.