Temperature Dependence of Catecholamine Secretion from Cultured Bovine Chromaffin Cells

Abstract: Secretion of both epinephrine and norepinephrine by cultured chromaffin cells was studied at temperatures ranging from 0°C to 37°C. The percentage of epinephrine secreted was always lower than that of norepinephrine when the cells were stimulated with either acetylcholine or high K⁺ at any temperature. When the cells were stimulated with acetylcholine or carbachol the percentage of catecholamine secreted at 10 min increased with temperature from 4°C to 24°C and then decreased from 24°C to 37°C. Potassium-stimulated cells secreted increasing amounts of catecholamine as the temperature was increased to 37°C. We found, however, that the initial rates of secretion increased continuously as temperature increased throughout the range for both carbachol-and K⁺-stimulated cells. The temperature maximum of acetylcholine-stimulated secretion is caused by a faster shut-off of secretion at higher temperature. The Arrhenius plots of initial rates show an inflection point at approximately 17°C for carbachol-stimulated cells. The plot for K⁺-stimulated cells is a straight line over the entire temperature range. The transition could be caused by a conformational change in the cholinergic receptor/ion channel molecule.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

GO‐At : in silico prediction of gene function in Arabidopsis thaliana by combining heterogeneous data

Despite recent advances, accurate gene function prediction remains an elusive goal, with very few methods directly applicable to the plant Arabidopsis thaliana. In this study, we present GO‐At (gene ontology prediction in A. thaliana), a method that combines five data types (co‐expression, sequence, phylogenetic profile, interaction and gene neighbourhood) to predict gene function in Arabidopsis. Using a simple, yet powerful two‐step approach, GO‐At first generates a list of genes ranked in descending order of probability of functional association with the query gene. Next, a prediction score is automatically assigned to each function in this list based on the assumption that functions appearing most frequently at the top of the list are most likely to represent the function of the query gene. In this way, the second step provides an effective alternative to simply taking the ‘best hit’ from the first list, and achieves success rates of up to 79%. GO‐At is applicable across all three GO categories: molecular function, biological process and cellular component, and can assign functions at multiple levels of annotation detail. Furthermore, we demonstrate GO‐At’s ability to predict functions of uncharacterized genes by identifying ten putative golgins/Golgi‐associated proteins amongst 8219 genes of previously unknown cellular component and present independent evidence to support our predictions. A web‐based implementation of GO‐At ( http://www.bioinformatics.leeds.ac.uk/goat ) is available, providing a unique resource for plant researchers to make predictions for uncharacterized genes and predict novel functions in Arabidopsis.     

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

Protein structure topological comparison, discovery and matching service

We describe a fold level fast protein comparison and motif matching facility based on the TOPS representation of structure. This provides an update to a previous service at the EBI, with a better graph matching with faster results and visualization of both the structures being compared against and the common pattern of each with the target domain. AVAILABILITY: Web service at http://balabio.dcs.gla.ac.uk/tops or via the main TOPS site at http://www.tops.leeds.ac.uk. Software is also available for download from these sites.     

Recycling of manure nutrients: use of algal biomass from dairy manure treatment as a slow release fertilizer

An alternative to land spreading of manure is to grow crops of algae on the N and P present in the manure and convert manure N and P into algal biomass. The objective of this study was to evaluate the fertilizer value of dried algal biomass that had been grown using anaerobically digested dairy manure. Results from a flask study using two soils amended with algal biomass showed that 3% of total algal nitrogen (N) was present as plant available N at day 0. Approximately 33% of algal N was converted to plant available N within 21 days at 25 degrees C in both soils. Levels of Mehlich-3 extractable phosphorus (P) in the two soils rose with increasing levels of algal amendment but were also influenced by existing soil P levels. Results from plant growth experiments showed that 20-day old cucumber and corn seedlings grown in algae-amended potting mix contained 15-20% of applied N, 46-60% of available N, and 38-60% of the applied P. Seedlings grown in algae-amended potting mixes were equivalent to those grown with comparable levels of fertilizer amended potting mixes with respect to seedling dry weight and nutrient content. These results suggest that dried algal biomass produced from treatment of anaerobically digested dairy manure can substitute for commercial fertilizers used for potting systems.