Conductance Ratios and Cellular Identity

Recent experimental evidence suggests that coordinated expression of ion channels plays a role in constraining neuronal electrical activity. In particular, each neuronal cell type of the crustacean stomatogastric ganglion exhibits a unique set of positive linear correlations between ionic membrane conductances. These data suggest a causal relationship between expressed conductance correlations and features of cellular identity, namely electrical activity type. To test this idea, we used an existing database of conductance-based model neurons. We partitioned this database based on various measures of intrinsic activity, to approximate distinctions between biological cell types. We then tested individual conductance pairs for linear dependence to identify correlations. Contrary to experimental evidence, in which all conductance correlations are positive, 32% of correlations seen in this database were negative relationships. In addition, 80% of correlations seen here involved at least one calcium conductance, which have been difficult to measure experimentally. Similar to experimental results, each activity type investigated had a unique combination of correlated conductances. Finally, we found that populations of models that conform to a specific conductance correlation have a higher likelihood of exhibiting a particular feature of electrical activity. We conclude that regulating conductance ratios can support proper electrical activity of a wide range of cell types, particularly when the identity of the cell is well-defined by one or two features of its activity. Furthermore, we predict that previously unseen negative correlations and correlations involving calcium conductances are biologically plausible.     

Proton-metal distance determination in cobalt(II) stellacyanin by 1H nuclear magnetic resonance relaxation measurements including Curie-spin effects: a proposed structure of the metal-binding region

1H nuclear magnetic resonance (1H NMR) experiments on Co(II)-substituted stellacyanin have been performed. Large paramagnetic hyperfine shifts are observed, the whole spectrum covering a range of 190 ppm. Experiments were mainly performed at 270 MHz from which temperature and pH* dependencies of the out-shifted resonances were reported, as well as determinations of the longitudinal (T1) and transverse (T2) relaxation times. These relaxation times are among other things, dependent on the individual proton-metal distance, and the aim of this work has been to determine these distances, by use of the Solomon-Bloembergen equations modified to include the so-called "Curie spin". The application of this method to a protein has not been reported earlier. Experiments were also performed at 100, 400, and 500 MHz in order to estimate the size of the Curie spin from the field dependence of the line widths. Furthermore, determination of the values for the rotational correlation time, tau r, and the effective magnetic moment, mu eff, was necessary for the present approach. With apostellacyanin, tau r was found to be (6.0 +/- 0.4) X 10-8 s. From the paramagnetic susceptibility of Co(II) stellacyanin, the value (4.53 +/- 0.03)beta was determined for mu eff. The proposed assignments of several paramagnetically out-shifted resonances. the proton-metal distances obtained, and the known peptide sequence of stellacyanin have allowed us to build a three-dimensional model of the metal site and its surrounding structure consistent with all the experimental data. It is revealed that both histidine ligands bind the metal with their 3-nitrogens. Also we find strong indications that a second sulfur atom is actually binding the metal, this being the long-sought-after fourth ligand. The model suggests that this sulfur belongs to Cys-59, which together with Cys-93 constitutes the disulfide bridge known to be present in the structure. A potential fifth ligand, an amide oxygen from Asn-47, is also found.     

Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C

The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.     

User requirements on CT-based computed dose planning systems in radiation therapy. Presentation of 'check lists'

The expanding use of computers in radiation therapy procedures, especially the rapidly increasing use of digital CT-information, necessitates the coordination of the different systems in order to facilitate their developments. In order to define necessary demands for tomorrow a Nordic cooperation was initiated 1981 by NORDFORSK (Nordic co-operative organization for applied research), and a group of physicians and physicists having their daily work in this field of medicine and physics was invited to produce a report on 'User requirements on CT-based computed dose planning systems on radiation therapy'. The work has been done within the frame of NORDFORSK's activities and has been independent of the existing commissions and associations in the radiology field, but it has taken into consideration recommendations that have been given by or are being produced by other organizations. This report is a short summary of the complete paper which will be published in Acta Radiologica. The aim of this short version is to get an early presentation of the 'requirement lists' (see Appendix) which we think are of immediate importance.     

Change in climatically suitable breeding distributions reduces hybridization potential between Vermivora warblers

Aim

Climate change is affecting the distribution of species and subsequent biotic interactions, including hybridization potential. The imperiled Golden-winged Warbler (GWWA) competes and hybridizes with the Blue-winged Warbler (BWWA), which may threaten the persistence of GWWA due to introgression. We examined how climate change is likely to alter the breeding distributions and potential for hybridization between GWWA and BWWA.

Location

North America.

Methods

We used GWWA and BWWA occurrence data to model climatically suitable conditions under historical and future climate scenarios. Models were parameterized with 13 bioclimatic variables and 3 topographic variables. Using ensemble modeling, we estimated historical and modern distributions, as well as a projected distribution under six future climate scenarios. We quantified breeding distribution area, the position of and amount of overlap between GWWA and BWWA distributions under each climate scenario. We summarized the top explanatory variables in our model to predict environmental parameters of the distributions under future climate scenarios relative to historical climate.

Results

GWWA and BWWA distributions are projected to substantially change under future climate scenarios. GWWA are projected to undergo the greatest change; the area of climatically suitable breeding season conditions is expected to shift north to northwest; and range contraction is predicted in five out of six future climate scenarios. Climatically suitable conditions for BWWA decreased in four of the six future climate scenarios, while the distribution is projected to shift east. A reduction in overlapping distributions for GWWA and BWWA is projected under all six future climate scenarios.

Main Conclusions

Climate change is expected to substantially alter the area of climatically suitable conditions for GWWA and BWWA, with the southern portion of the current breeding ranges likely to become climatically unsuitable. However, interactions between BWWA and GWWA are expected to decline with the decrease in overlapping habitat, which may reduce the risk of genetic introgression.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25°C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.     

The primary structure of NG2, a novel membrane-spanning proteoglycan

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.     

Neuropeptide Y and catecholamine release in the piglet during hypoxia: enhancement by theophylline.

Sympathoadrenal activity was studied in 13 young piglets during hypoxia. The piglets were anaesthetized with chloralose/urethane, tracheostomized, paralyzed with gallamine and artificially ventilated. A femoral artery catheter was inserted and used for blood sampling. The piglets were challenged with 6 min of 6% CO2, 10 min of 12% O2 and 6 min of 6% O2 before and after theophylline (an adenosine receptor antagonist) treatment 20 mg/kg (n = 9) or saline (n = 4). Plasma samples were obtained before, during and after each hypercapnic or hypoxic period and analysed for their content of noradrenaline, adrenaline and neuropeptide Y. Hypercapnia with 6% CO2 and moderate hypoxia with 12% O2 did not lead to any significant increase of either noradrenaline (NA), adrenaline (A) or neuropeptide Y (NPY). However, severe hypoxia with 6% O2 increased the NA level from 30 to 66 nmol/l; the A level from 1 to 28 nmol/l and NPY from 140 to 213 pmol/l. After treatment with theophylline the baseline NA increased from 27 to 40 nmol/l, A rom 1.5 to 4.0 and NPY concentration from 65 to 171 pmol/l. Theophylline moderately enhanced the release of NPY, NA and A during the 12% O2 challenge. However, during the severe hypoxia (6%), the increase of NA (from 49 to 333 nmol/l), A (from 8 to 214 nmol/l) and NPY (from 184 to 385 pmol/l) showed considerably enhancement after the theophylline treatment. The results obtained before and after saline were similar showing that the duration of the experiments per se did not change the baseline levels or the effect of the challenges on NA, A or NPY levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Choice of hydrogen uptake (Hup) status in legume‐rhizobia symbioses

The H₂ is an obligate by-product of N-fixation. Recycling of H₂ through uptake hydrogenase (Hup) inside the root nodules of leguminous plants is often considered an advantage for plants. However, many of the rhizobium-legume symbioses found in nature, especially those used in agriculture are shown to be Hup⁻, with the plants releasing H₂ produced by nitrogenase activity from root nodules into the surrounding rhizosphere. Recent studies have suggested that, H₂ induces plant-growth-promoting rhizobacteria, which may explain the widespread of Hup⁻ symbioses in spite of the low energy efficiency of such associations. Wild legumes grown in Nova Scotia, Canada, were surveyed to determine if any plant-growth characteristics could give an indication of Hup choice in leguminous plants. Out of the plants sampled, two legumes, Securigera varia and Vicia cracca, showed Hup⁺ associations. Securigera varia exhibited robust root structure as compared with the other plants surveyed. Data from the literature and the results from this study suggested that plants with established root systems are more likely to form the energy-efficient Hup⁺ symbiotic relationships with rhizobia. Conversely, Hup⁻ associations could be beneficial to leguminous plants due to H₂-oxidizing plant-growth-promoting rhizobacteria that allow plants to compete successfully, early in the growing season. However, some nodules from V. cracca tested Hup⁺, while others were Hup⁻. This was similar to that observed in Glycine max and Pisum sativum, giving reason to believe that Hup choice might be affected by various internal and environmental factors.