Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Comparative bioenergetics of permanent and temporary pond populations of the freshwater clam,Musculium partumeium (Say)

The population energetics of a temporary and a permanent pond population of Musculium partumeium in Southwest Ohio were studied. In the permanent pond (surface area = 396 m², maximum depth = 0.7 m) the population was bivoltine and iteroparous whereas in the temporary pond (surface area = 1042 m², maximum depth = 0.9 m) the population was usually univoltine and semelparous.Growth and biomass were assessed as total organic carbon and total nitrogen to provide estimates of productivity and seasonal changes in C:N for each generation. Productivity (non-respired assimilation = growth + reproduction; N-R.A. = G + R) was 6939 mgC·m^–2·a^–1 (3858 and 3353 mgC·m^–2·a^–1 for each generation) and 1661 mgC·m^–2·a^–1 for the permanent and temporary pond populations respectively. The average standing crop biomass (B) was 606.8 mgC·m^–2 (357.5 and 249.3 mgC·m^–2 for each generation) and 231.9 mgC·m^–2 with overall productivity: biomass ratios of 11.4 and 7.2 for the permanent pond and temporary pond populations respectively.Respiration rates were converted to carbon equivalents (respired assimilation = R.A.) and used to evaluate the components of total assimilation (T.A. = R.A. + N-R.A.) and the efficiency of partitioning this energy to N-R.A. for G and R. When expressed as a percentage, the production efficiencies (100 × N-R.A.:T.A.) were 50.4 and 62%, and the reproductive efficiencies (100 × R:N-R.A.) were 26.4 and 18% for the permanent and temporary pond populations respectively. The reproductive efficiencies for populations of these viviparous clams are greater than those for most oviparous molluscs.The comparative information on the energetics of these populations does not completely fit any theoretical consideration of reproductive effort or life-history strategy. These data are discussed in relation to selection for population success in temporary ponds.Funded in part by grants to Albert J. Burky from the Ohio Biological Survey and the University of Dayton Research CouncilFunded in part by grants to Albert J. Burky from the Ohio Biological Survey and the University of Dayton Research Council     

Effects of particle concentration and season on the filtration rates of the freshwater clam,Sphaerium striatinum Lamarck (Bivalvia: Pisidiidae)

The effects of particle concentration and season on the filtration rates of the freshwater clamSphaerium striatinum Lamarck were assessed by measuring clearance rates of small (2.02 μm) latex beads from dilute suspensions. Filtration rates decreased as particle concentration increased over a range of 2–64 mg 1⁻¹, with rates decreasing in similar proportion for clams of all sizes. For a 1-mg clam, rates decreased from approximately 8.4 to 0.57 ml clam⁻¹ h⁻¹. Seasonal filtration rates for adult clams peaked during periods of greatest reproduction. The patterns for smaller clams are similar, though proportional changes in filtration rates differ for various sizes of clams. It is estimated that clams occupying 1 m² of stream substrate removed about 3.67 gCa⁻¹. This is equivalent to 0.0004% of the carbon that flows past them annually. Filter-feeding provided only 24% of the calculated energy needs of the population, suggesting that another mode of feeding (e.g. deposit-feeding) may provide an important energy source for these forms. Funded in part by a grant-in-aid to D. J. Hornbach from Sigma-Xi, The Research Society. Funded in part by a grant-in-aid to D. J. Hornbach from Sigma-Xi, The Research Society.     

Larval habitat preference of the endemic Hawaiian midge,Telmatogeton torrenticola Terry (Telmatogetoninae)

Telmatogeton torrenticola Terry is a large endemic chironomid (lastinstar >20 mm) commonly found in high gradient Hawaiian streams on smoothrock surfaces with torrential, shallow flow and in the splash zones ofwaterfalls. We have quantified benthic water flow in larval habitat in a 50m segment of Kinihapai Stream, Maui using a thermistor-based microcurrentmeter. Under base flow conditions at sites suitable for larval attachment,depth was measured and bottom water velocity measurements were made 2 mmabove populations. Larval densities ranged from 386.9–1178m^–2, habitat bottom water velocities from 13.4–64.2 cms^–1, and water depths from 1.5–50 cm. Bottom velocitiesof sites with zero larvae ranged from 20.8–21.8 cm s^–1with depths from 50 to >160 cm. Larval densities were greatest inareas with high bottom water velocities and shallow depths. Stepwisemultiple regression analyses showed that density could be confidentlypredicted best by Froude number (r=0.81; p=0.008). In the absence of Froudenumber as a regression term, the best variable to predict larval density wasbottom velocity ratio: relative depth ratio (r=0.75; p=0.019). In addition,the torrential habitat of the larvae was always characterized by aperiphyton community that appeared to be the primary food resource for thelarvae. These data suggest that torrential flows over appropriate substratesare important factors regulating habitat availability for T. torrenticolaand that reduced discharge (e.g. affected by water diversions) couldsignificantly reduce the amount of available habitat for this organism andother flow sensitive stream fauna.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Reproductive strategies in the freshwater sphaeriid clam,Musculium partumeium (say), from a permanent and a temporary pond

Summary Reproduction was studied in two populations ofMusculium partumeium from temporary and permanent ponds. Adults of the single annual generation from the ephemeral pond have an annual selection ratio of 25:1 with 37.03 gC per newborn, and an intrinsic rate of increase (r) of 0.0084 day^-1. Fall-born adults from the permanent pond have an annual selection ratio of 38:1, 21.82 gC per newborn and anr of 0.0115 day^-1; springborn adults have an annual selection ratio of 136:1 (107:1 for their contribution to fall birth and 29:1 for the spring birth period) with newborns of 24.21 gC and anr of 0.0304. The trade off between quantity and quality of young is discussed in terms of adaptive strategies.     

Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.     

Modeling <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> metabolism: from genome to metabolic fluxes

Background  

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years.     

Simulation of human atherosclerotic femoral plaque tissue: the influence of plaque material model on numerical results

Background

Due to the limited number of experimental studies that mechanically characterise human atherosclerotic plaque tissue from the femoral arteries, a recent trend has emerged in current literature whereby one set of material data based on aortic plaque tissue is employed to numerically represent diseased femoral artery tissue. This study aims to generate novel vessel-appropriate material models for femoral plaque tissue and assess the influence of using material models based on experimental data generated from aortic plaque testing to represent diseased femoral arterial tissue.

Methods

Novel material models based on experimental data generated from testing of atherosclerotic femoral artery tissue are developed and a computational analysis of the revascularisation of a quarter model idealised diseased femoral artery from a 90% diameter stenosis to a 10% diameter stenosis is performed using these novel material models. The simulation is also performed using material models based on experimental data obtained from aortic plaque testing in order to examine the effect of employing vessel appropriate material models versus those currently employed in literature to represent femoral plaque tissue.

Results

Simulations that employ material models based on atherosclerotic aortic tissue exhibit much higher maximum principal stresses within the plaque than simulations that employ material models based on atherosclerotic femoral tissue. Specifically, employing a material model based on calcified aortic tissue, instead of one based on heavily calcified femoral tissue, to represent diseased femoral arterial vessels results in a 487 fold increase in maximum principal stress within the plaque at a depth of 0.8 mm from the lumen.

Conclusions

Large differences are induced on numerical results as a consequence of employing material models based on aortic plaque, in place of material models based on femoral plaque, to represent a diseased femoral vessel. Due to these large discrepancies, future studies should seek to employ vessel-appropriate material models to simulate the response of diseased femoral tissue in order to obtain the most accurate numerical results.

     

Review and classification of variability analysis techniques with clinical applications

Background

Representation of independent biophysical sources using Fourier analysis can be inefficient because the basis is sinusoidal and general. When complex fractionated atrial electrograms (CFAE) are acquired during atrial fibrillation (AF), the electrogram morphology depends on the mix of distinct nonsinusoidal generators. Identification of these generators using efficient methods of representation and comparison would be useful for targeting catheter ablation sites to prevent arrhythmia reinduction.

Method

A data-driven basis and transform is described which utilizes the ensemble average of signal segments to identify and distinguish CFAE morphologic components and frequencies. Calculation of the dominant frequency (DF) of actual CFAE, and identification of simulated independent generator frequencies and morphologies embedded in CFAE, is done using a total of 216 recordings from 10 paroxysmal and 10 persistent AF patients. The transform is tested versus Fourier analysis to detect spectral components in the presence of phase noise and interference. Correspondence is shown between ensemble basis vectors of highest power and corresponding synthetic drivers embedded in CFAE.

Results

The ensemble basis is orthogonal, and efficient for representation of CFAE components as compared with Fourier analysis (p ≤ 0.002). When three synthetic drivers with additive phase noise and interference were decomposed, the top three peaks in the ensemble power spectrum corresponded to the driver frequencies more closely as compared with top Fourier power spectrum peaks (p ≤ 0.005). The synthesized drivers with phase noise and interference were extractable from their corresponding ensemble basis with a mean error of less than 10%.

Conclusions

The new transform is able to efficiently identify CFAE features using DF calculation and by discerning morphologic differences. Unlike the Fourier transform method, it does not distort CFAE signals prior to analysis, and is relatively robust to jitter in periodic events. Thus the ensemble method can provide a useful alternative for quantitative characterization of CFAE during clinical study.