Inoculation of cyprinid herpesvirus 3 (CyHV-3) on common carp brain cells-influence of process parameters on virus yield.

Research of cyprinid herpesvirus 3 (CyHV-3) is focused on the infection mechanism and disease development in animals using genetic and immunological approaches to improve treatments and diagnostics. In contrast, only few tried to investigate the CyHV-3 replication behaviour in available cell cultures. Whereas, obtaining high virus yields by in vitro replication enables achieving of the mentioned above goals easier and more reliable. The following work presents an attempt to illuminate the KHV replication in common carp brain (CCB) cell cultures from the engineering point of view. The isolate KHV-TP30 was used testing the influence on process parameters, such as multiplicity of infection (MOI), time of infection (TOI) and time of harvest (TOH). Virus concentrations and infectivity at different time points of infection were examined using hydrolyzed probe qPCR (Gilad et al. 2004) and 50% tissue culture infectivity dose (TCID50). The data obtained show that while the amount of the virus DNA remains constant after reaching its maximum, the infectivity of the virus decreases. Thus, especially, TOH can be crucial for generating a high-quality virus stock. Applying optimized parameters improved the infectivity of the harvested virus and reached a robust titre as high as 1.9 × 108 TCID50/mL. To our knowledge, so far, there is no information in the peer-reviewed literature showing comparably high virus titres. Such virus yields not only facilitate conduction of further studies, including stability tests of the virus stock under various supplementation or disinfection trails, but also provide enough virus material to perform more detailed examinations of the infection mechanism.

Tetracycline-encapsulated P(3HB) microsphere-coated 45S5 Bioglass(®)-based scaffolds for bone tissue engineering.

Bioglass(®)-based scaffolds for bone tissue engineering have been developed, which can also serve as carriers for drug delivery. For this, P(3HB) microspheres (PMSs) loaded with tetracycline were fabricated and immobilised on the scaffold surfaces by a modified slurry dipping technique. The sustained drug delivery ability in simulated body fluid was confirmed by using UV-Vis absorption spectroscopy measurements. The MTT assay using mouse fibroblast cells provided evidence that the tetracycline loaded microspheres produced in this study show limited cytotoxicity. The scaffolds developed in this work provide mechanical support, adequate 3D surface roughness, bioactivity and controlled drug delivery function, and are thus interesting candidates for bone tissue engineering applications.

Phylogenetic relationships within cation transporter families of Arabidopsis.

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.

The wheat cDNA LCT1 generates hypersensitivity to sodium in a salt-sensitive yeast strain.

Salinity affects large areas of agricultural land, and all major crop species are intolerant to high levels of sodium ions. The principal route for Na(+) uptake into plant cells remains to be identified. Non-selective ion channels and high-affinity potassium transporters have emerged as potential pathways for Na(+) entry. A third candidate for Na(+) transport into plant cells is a low-affinity cation transporter represented by the wheat protein LCT1, which is known to be permeable for a wide range of cations when expressed in yeast (Saccharomyces cerevisiae). To investigate the role of LCT1 in salt tolerance we have used the yeast strain G19, which is disrupted in the genes encoding Na(+) export pumps and as a result displays salt sensitivity comparable with wheat. After transformation with LCT1, G19 cells became hypersensitive to NaCl. We show that LCT1 expression results in a strong decrease of intracellular K(+)/Na(+) ratio in G19 cells due to the combined effect of enhanced Na(+) accumulation and loss of intracellular K(+). Na(+) uptake through LCT1 was inhibited by K(+) and Ca(2+) at high concentrations and the addition of these ions rescued growth of LCT1-transformed G19 on saline medium. LCT1 was also shown to mediate the uptake of Li(+) and Cs(+). Expression of two mutant LCT1 cDNAs with N-terminal truncations resulted in decreased Ca(2+) uptake and increased Na(+) tolerance compared with expression of the full-length LCT1. Our findings strongly suggest that LCT1 represents a molecular link between Ca(2+) and Na(+) uptake into plant cells.

K+-Selective inward-rectifying channels and apoplastic pH in barley roots

Recent structure-function analysis of heterologously expressed K+-selective inward-rectifying channels (KIRCs) from plants has revealed that external protons can have opposite effects on different members of the same gene family. An important question is how the diverse response of KIRCs to apoplastic pH is reflected at the tissue level. Activation of KIRCs by acid external pH is well documented for guard cells, but no other tissue has yet been studied. In this paper we present, for the first time to our knowledge, in planta characterization of the effects of apoplastic pH on KIRCs in roots. Patch-clamp experiments on protoplasts derived from barley (Hordeum vulgare) roots showed that a decrease in external pH shifted the half-activation potential to more positive voltages and increased the limit conductance. The resulting enhancement of the KIRC current, together with the characteristic voltage dependence, strongly relates the KIRC of barley root cells to AKT1-type as opposed to AKT3-type channels. Measurements of cell wall pH in barley roots with fluorescent dye revealed a bulk apoplastic pH close to the pK values of KIRC activation and significant acidification of the apoplast after the addition of fusicoccin. These results indicate that channel-mediated K+ uptake may be linked to development, growth, and stress responses of root cells via the activity of H+-translocating systems.

A unified procedure for the correction of liquid junction potentials in patch clamp experiments on endo- and plasma membranes.

Correct determination of absolute values and polarities of liquid junction potentials (LJPs) is essential in patch clamp experiments for the estimation of ionic selectivities and activation voltages of ion channels. A simple approach for the correction of membrane voltages for LJPs has been developed. The method covers all combinations of LJPs between the solutions of bath, patch pipette and reference salt bridge and is applicable to all patch configurations on plasma membranes and endomembranes.

Multiple inward channels provide flexibility in Na+/K+ discrimination at the plasma membrane of barley suspension culture cells.

Ion transport across the plasma membrane of suspension-culture cells derived from immature barley embryos has been studied in low (15 mM KCl) and high (additional 150 mM NaCl) salt conditions to understand how plants discriminate between K(+) and Na(+) during ion uptake. In both media about 50% of the cells exhibited resting potentials more negative than any of the passive diffusion potentials. In whole-cell patch clamp experiments membrane hyperpolarization activated large inward currents. Whilst the instantaneous current components did not discriminate between K(+) and Na(+), the time-dependent current, I(in), was selective for K(+) over Na(+). Further analysis of I(in) revealed the following properties: double exponential current activation (time-constants 0.03 s and 0.3 s, half activation potential - 171 mV); no inactivation; complete block by Ba(2+) (30 mM in 100 mM KCl) and part block by TEA(+) (maximum 50% with 20 mM); dependence on millimolar concentrations of cytoplasmic ATP; no block by external or cytoplasmic Na(+). The selectivity sequences K(+) ≫ Rb(+) > NH(+)(4) > Na(+) ≫ Cl(-) and K(+) ≫ NH(+)(4) > Na(+) > Rb(+) were determined from measurements of reversal potentials and relative steady-state currents respectively. P(Na):P(K) was 0.07 ± 0.02 (from reversal potentials) and I(Na):I(K) was 0.17 + 0.05 (from relative currents). A high variance among the observed permeability ratios suggested that several channels with different ion-selectivities contributed to the time-dependent whole-cell currents. In single channel experiments, several inward channels with distinct properties were found. The major channels were (i) a voltage-gated, K(+)-selective channel (12 pS), (ii) an ATP-activated non-selective cation channel (7 pS) and (iii) an inward-rectifying anion-channel (150 pS, all unitary conductances given for 100 mM KCI). No significant differences were found in whole-cell currents or single-channel characteristics between cells that had been adapted to a high-salt growth-medium (150 mM NaCl) and non-adapted cells. The idea that differential regulation of plasma membrane ion channels gives rise to a physiological flexibility, allowing the cells to control Na(+) uptake under varying external conditions, is discussed.

Na+ transport in Acetabularia bypasses conductance of plasmalemma.

Na(+)-selective microelectrodes with the sensor ETH 227 have been used to measure the cytoplasmic Na+ concentration, [Na+]c, in Acetabularia. In the steady-state, [Na+]c is about 60 mM (external 460 mM). Steps in external Na+ concentration, [Na+]o, cause biexponential relaxations of [Na+]c which have formally been described by a serial three-compartment model (outside<==>compartment 1<==>compartment 2). From the initial slopes (some mMsec-1) net uptake and release of about 3 mumolm-2sec-1 Na+ are determined. Surprisingly, but consistent with previous tracer flux measurements (Mummert, H., Gradmann, D. 1991. J. Membrane Biol, 124:255-263), these Na+ fluxes are not accompanied by corresponding changes of the transplasmalemma voltage. [Na+]c is neither affected by the membrane voltage, nor by electrochemical gradients of H+ or Cl- across the plasmalemma, nor by cytoplasmic ATP. The results suggest a powerful vesicular transport system for ions which bypasses the conductance of the plasmalemma. In addition, transient increases of [Na+]c have been observed to take place facultatively during action potentials. The exponential distribution of the amplitudes of these transients (many small and few large peaks) points to local events in the more ore less close vicinity of the Na+ recording electrode. These events are suggested to consist of disruption of endoplasmic vesicles due to a loss of pressure in the cytoplasm.