In Saccharomyces cerevisiae a short amino acid sequence facilitates excretion in the growth medium of periplasmic proteins

In Saccharomyces cerevisiae the cell wall is a barrier to excretion of proteins in the growth medium. Although small proteins are more easily released than bigger ones, other factors besides molecular sieving may play a role in partitioning of periplasmic proteins. By using several complementary approaches including enzyme-activity assays, quantitative immunoblotting on subcellular fractions and growth media, as well as a novel approach involving the use of flow cytometry and specific antibodies, we show that residues 1–8 of mature glucoamylase greatly enhance excretion of both glucoamylase and β-galactosidase in vivo and facilitate extraction of periplasmic proteins in vitro . Immunological data obtained by flow cytometry on whole cells indicate that this amino acid sequence increases the fraction of enzyme reaching the outer cell-wall layers. This amino acid sequence may define a novel type of topogenic sequence, facilitating the crossing of the yeast cell wall in vivo and facilitating extraction of periplasmic proteins by non-disruptive means in vitro .     

Structure-Activity Analyses of β-Amyloid Peptides: Contributions of the β25–35 Region to Aggregation and Neurotoxicity

Abstract: The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of β-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of β-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of β-amyloid, β25–35, and the full-length protein, β1–42. We examine the effects of amino acid residue deletions and substitutions on the ability of β-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant β-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of β25–35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the β33–35 region. In β1–42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of β-sheet secondary structure in aggregating, toxic β-amyloid peptides but not in nonaggregating, nontoxic β-amyloid peptides. Together, these data further define the primary and secondary structures of β-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.     

Molecular Cloning and Differential Expression Patterns of Avian Glutamate Receptor mRNAs

Abstract: We have identified and studied potential ionotropic glutamate receptor genes in pigeon brain. Three cDNA clones exhibit significant amino acid sequence identity to members of a rodent ligand-gated ion channel family. One of them, GluP-II, encodes a full-length AMPA-sensitive glutamate receptor GluR2 (GluR-B) homologue, whereas the other two partial clones, designated as GluP-III and -IV, are nearly identical to rodent GluR3 (GluR-C) and GluR4 (GluR-D) receptor subunits. Northern analysis demonstrated that the avian genes are widely expressed in the brain. Within the brain regions analyzed by in situ hybridization histochemistry, the three avian GluR subunits showed distinct and regionally specific mRNA expression patterns in the adult. Most of the differences in their expression were observed in cell types of the telencephalon, certain thalamic nuclei, the optic tectum, and the cerebellar cortex. A particularly striking finding was the expression of GluP-II in Golgi epithelial/Bergmann glial cells. In contrast, Bergmann glial cells in rat cerebellum do not express GluR2 (GluR-B) subunit genes. Immunoreactivity for a monoclonal sequence-specific antipeptide antibody was widespread and most prominent in Purkinje cell perikarya and their dendrites, neuronal cell bodies of the ectostriatum, and the deep optic tectum. These results demonstrate the existence of multiple subunits of the ionotropic glutamate receptor channel family in avians. Excitatory amino acid receptor genes appear to be highly conserved during evolution.     

Applications of ecological stoichiometry for sustainable acquisition of ecosystem services

Human activities have differentially altered biogeochemical cycling at local, regional and global scales. We propose that a stoichiometric approach, examining the fluxes of multiple elements and the ratio between them, may be a useful tool for better understanding human effects on ecosystem processes and services. The different scale of impacts of the elements carbon, nitrogen and phosphorus and the different nature of their biogeochemical cycles, imply a large variation of their stoichiometric ratios in space and time and thus divergent impacts on biota.
In this paper, we examine the effects of anthropogenic perturbations on nutrient ratios in ecosystems in two examples and one case study. Altered stoichiometry in agricultural systems (example 1) can affect not only crop yield and quality but also the interactions between plants and their pollinators, pests and pathogens. Human activities have also altered stoichiometry in coastal ecosystems (example 2). Increased N loading has especially lead to increased N:P and reduced Si:N ratios, with detrimental effects on ecosystem services derived from coastal pelagic food webs, such as fish yield and water quality. The terrestrial–aquatic linkage in stoichiometric alterations is illustrated with a case study, the Mississippi River watershed, where anthropogenic activities have caused stoichiometric changes that have propagated through the watershed into the northern Gulf of Mexico.
Coupled with altered stoichiometric nutrient inputs are the inherent differences in variation and sensitivity of different ecosystems to anthropogenic disturbance. Furthermore, the connections among the components of a watershed may result in downstream cascades of disrupted functioning. Applying a multiple element perspective to understanding and addressing societal needs is a new direction for both ecological stoichiometry and sustainability.     

The Effect of Elevated [CO2] on Uptake and Allocation of 13C and 15N in Beech (Fagus sylvatica L.) during Leafing

Abstract: A continuous dual ¹³CO₂ and ¹⁵NH₄¹⁵NO₃ labelling experiment was undertaken to determine the effects of ambient (350μmol mol^-1) or elevated (700μmol mol^-1) atmospheric CO₂ concentrations on C and N uptake and allocation within 3-year-old beech ( Fagus sylvatica L.) during leafing. After six weeks of growth, total carbon uptake was increased by 63 % (calculated on total C content) under elevated CO₂ but the carbon partitioning was not altered. 56 % of the new carbon was found in the leaves. On a dry weight basis was the content of structural biomass in leaves 10 % lower and the lignin content remained unaffected under elevated as compared to ambient [CO₂]. Under ambient [CO₂] 37 %, and under elevated [CO₂] 51 %, of the lignin C of the leaves derived from new assimilates. For both treatments, internal N pools provided more than 90 % of the nitrogen used for leaf-growth and the partitioning of nitrogen was not altered under elevated [CO₂]. The C/N ratio was unaffected by elevated [CO₂] at the whole plant level, but the C/N ratio of the new C and N uptake was increased by 32 % under elevated [CO₂].     

A combined pathway of sulfur compound disproportionation in Desulfovibrio desulfuricans

The fates of the two different sulfur atoms of the thiosulfate molecule during anaerobic disproportionation by the sulfate-reducing bacterium Desulfovibrio desulfuricans were followed by isotope mass spectrometry. During disproportionation, ³²S-thiosulfate was preferentially metabolized, and the residual thiosulfate became enriched in ³⁴S. The sulfate formed was isotopically heavier than the inner sulfur of the consumed thiosulfate. Vice versa, the sulfide formed was isotopically lighter than the outer sulfur of the consumed thiosulfate. These results indicate that thiosulfate is cleaved to intermediates that undergo further disproportionation to sulfate and sulfide in a second step. These intermediates are probably elemental sulfur and sulfite. It is concluded that disproportionation of thiosulfate, sulfite and elemental sulfur includes a combined pathway.     

Long-term changes in population size,distribution and productivity of skuas (<Emphasis Type="Italic">Stercorarius</Emphasis> spp.) at Signy Island,South Orkney Islands

In this study, we investigate the numbers, productivity and territory distribution of the two species of skuas (brown Stercorarius lonnbergi and south polar Stercorarius maccormicki) breeding at Signy Island, South Orkneys, and compare the results with trends elsewhere. Comparison with previous counts indicates a biphasic increase in brown skuas at Signy Island; much faster from 1958/1959 to 1982/1983 (3.3 % per annum), than in subsequent years (0.4 % per annum from 1983/1984 to 2013/2014). Relative distribution of territories has changed little over time. The reduced rate of population growth in recent years was broadly coincident with a decrease in numbers of penguins (and therefore potential prey), which may also explain recent reductions in skua numbers at other Antarctic sites. As prey have become limiting, breeding success of brown skuas at Signy Island is now slightly lower than in the 1950s/early 1960s, but timing of breeding does not appear to have changed. Brown skuas at Signy Island may still have enough resources to start breeding, but as the season progresses and availability of resources declines, chick survival is reduced. South polar skuas have declined from ten pairs in 1982/1983 to one pair in 2013/2014, and mixed pairs have increased from one to three pairs. A review of the literature indicated that although population trend data are available for relatively few sites elsewhere in the subantarctic and Antarctic, numbers of brown skuas appear to be generally decreasing or stable, and of south polar skuas to be stable or increasing.     

Food intake rates of inactive fish are positively linked to boldness in three‐spined sticklebacks Gasterosteus aculeatus

To investigate the link between personality and maximum food intake of inactive individuals, food‐deprived three‐spined sticklebacks Gasterosteus aculeatus at rest in their home compartments were provided with ad libitum prey items. Bolder individuals ate considerably more than shyer individuals, even after accounting for body size, while sociability did not have an effect. These findings support pace‐of‐life theory predicting that life‐history strategies are linked to boldness.     

Seed dormancy and germination of the rare,high elevation Balkan endemic <Emphasis Type="Italic">Cerastium dinaricum</Emphasis> (Caryophyllaceae)

Short or long-term ex situ conservation is becoming increasingly important in conservation of plants in today’s changing environments. One of the important steps in ex situ conservation is the collection and storage of seeds and the consequent establishment of seed germination protocols. Cerastium dinaricum (Caryophyllaceae) is an endemic, high elevation and rare species of European conservation concern. Because of its severely fragmented distribution along the Dinaric Alps, the populations are likely to undergo further shrinkage in the future, which addresses the need of a long-term effective conservation management. From the potential ex situ population management perspective, we focused our study on germination ecology of C. dinaricum. The study revealed that temperature considerably affected the germination of seeds, which germinate better at 20 °C rather than 10 °C. A period of cold-wet stratification also significantly improved the final germination percentage with more pronounced increase at 20 °C, while addition of GA3 increased the final germination percentage by breaking the dormancy of non-stratified seeds. Mechanical scarification did not improve germination; on the contrary, it resulted in the lowest germination success. Seeds grown in complete darkness germinated significantly better compared to control when they were exposed to cold-wet stratification. Contrary to previous studies on some alpine species, which germinate better when exposed to light, dark treatment resulted in the highest germination percentages with 70 and 90% germination success after 4 and 8 weeks of stratification, respectively.     

In Adrenal Medulla Synaptophysin (Protein p38) Is Present in Chromaffin Granules and in a Special Vesicle Population

We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one-dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two-dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the "large granules" and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (less than 10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.