Root Morphology and Zn2+ Uptake Kinetics of the Zn Hyperaccumulator of Sedum alfredii Hance

Root morphology and Zn2 uptake kinetics of the hyperaccumulating ecotype (HE) and nonhyperaccumulating ecotype (NHE) of Sedum alfredii Hance were investigated using hydroponic methods and the radiotracer flux technique. The results indicate that root length, root surface area, and root volume of NHE decreased significantly with increasing Zn2 concentration in growth media, whereas the root growth of HE was not adversely affected, and was even promoted, by 500 μmol/L Zn2 . The concentrations of Zn2 in both ecotypes of S. alfredii were positively correlated with root length, root surface area and root volumes, but no such correlation was found for root diameter. The uptake kinetics for 65Zn2 in roots of both ecotypes of S. alfredii were characterized by a rapid linear phase during the first 6 h and a slower linear phase during the subsequent period of investigation. The concentration-dependent uptake kinetics of the two ecotypes of S. alfredii could be characterized by the Michaelis-Menten equation, with the Vmax for 65Zn2 influx being threefold greater in HE compared with NHE, indicating that enhanced absorption into the root was one of the mechanisms involved in Zn hyperaccumulation. A significantly larger Vmax value suggested that there was a higher density of Zn transporters per unit membrane area in HE roots.     

Gating in iodate-modified single cardiac Na+ channels

Summary Elementary Na⁺ currents were recorded at 19°C during 220-msec lasting step depolarizations in cell-attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the modifying influence of iodate, bromate and glutaraldehyde on single cardiac Na⁺ channels.Iodate (10 mmol/liter) removed Na⁺ inactivation and caused repetitive, burst-like channel activity after treating the cytoplasmic channel surface. In contrast to normal Na⁺ channels under control conditions, iodate-modified Na⁺ channels attain two conducting states, a short-lasting one with a voltage-independent lifetime close to 1 msec and, likewise tested between –50 and +10 mV, a long-lasting one being apparently exponentially dependent on voltage. Channel modification by bromate (10 mmol/liter) and glutaraldehyde (0.5 mmol/liter) also included the occurrence of two open states. Also, burst duration depended apparently exponentially on voltage and increased when shifting the membrane in the positive direction, but there was no evidence for two bursting states. Chemically modified Na⁺ channels retain an apparently normal unitary conductance (12.8±0.5 pS). Of the two substates observed, one of them is remarkable in that it is mostly attained from full-state openings and is very short living in nature; the voltage-independent lifetime was close to 2 msec. Despite removal of inactivation, open probability progressively declined during membrane depolarization. The underlying deactivation process is strongly voltage sensitive but, in contrast to slow Na⁺ inactivation, responds to a voltage shift in the positive direction with a retardation in kinetics. Chemically modified Na⁺ channels exhibit a characteristic bursting state much shorter than in DPI-modified Na⁺ channels, a difference not consistent with the hypothesis of common kinetic properties in noninactivating Na⁺ channels.     

Developing a cyclin blueprint as a tool for mapping the cell cycle in GS-NS0

The cell cycle is at the center of growth, productivity, and death of mammalian cell cultures. There exists a need to identify and quantify major landmarks in the cell cycle of industrially relevant mammalian cell lines and its association with productivity; central for designing productivity optimization strategies. Herein, we studied the expression of three cyclins, under both perturbed and unperturbed growth, by flow cytometry in batch cultures of GS-NS0. The perturbed systems involved two different DNA synthesis inhibitors, thymidine and dimethyl sulfoxide (DMSO). This approach enables the establishment of characteristic cyclin profiles, timings, and thresholds. In particular, two G₁ class cyclins (D1 and E1), and one G₂ cyclin (B1) were investigated. Cyclin B1 showed a clear cell cycle phase-specific expression increasing during G₂ phase where it was approximately 40% higher when compared to G₁ phase. Similarly, cyclin E1 showed a clear pattern being expressed approximately 10% higher in G₁ compared to G₂ phase and decreased through S phase. Cyclin D1 expression was fairly invariable throughout the cell cycle phases. The observed patterns provide a blueprint of the cell line's cell cycle, which can be used for the development of biologically accurate and experimentally validated distributed cell cycle models.     

Cultivation of Chlorella vulgaris in tubular photobioreactors: A lipid source for biodiesel production

Chlorella vulgaris was cultivated in two different 2.0 L-helicoidal and horizontal photobioreactors at 5 klux using the bicarbonate contained in the medium and ambient air as the main CO₂ sources. The influence of bicarbonate concentration on biomass growth as well as lipid content and profile was first investigated in shake flasks, where the stationary phase was achieved in about one half the time required by the control. The best NaHCO₃ concentration (0.2 g L⁻¹) was then used in both photobioreactors. While the fed-batch run performed in the helicoidal photobioreactor provided the best result in terms of biomass productivity, which was (84.8 mg L⁻¹ d⁻¹) about 2.5-fold that of the batch run, the horizontal configuration ensured the highest lipid productivity (10.3 mg L⁻¹ d⁻¹) because of a higher lipid content of biomass (22.8%). These preliminary results suggest that the photobioreactor configuration is a key factor either for the growth or the composition of this microalga. The lipid quality of C. vulgaris biomass grown in both photobioreactors is expected to meet the standards for biodiesel, especially in the case of the helicoidal configuration, provided that further efforts will be made to optimize the conditions for its production as a biodiesel source.     

Kinetics of cellulose hydrolysis by halostable cellulase from a marine Aspergillus niger at different salinities

Kinetics of cellulose hydrolysis with halostable cellulase from a marine Aspergillus niger was analyzed at different salinities. Cellulase activity in 8% NaCl solution was 1.43 folds higher than that in NaCl free solution. Half saturation constant, K_m (15.6260 g/L) and the rate constant of deactivation, K_de (0.3369 g/L h) in 8% NaCl solution was lower than that (18.6364 g/L), 0.3754 (g/L h) in NaCl free solution. The maximum initial hydrolysis velocity, V_max (25.5295 g/L h), in 8% NaCl solution was higher than that in NaCl free solution (25.0153 g/L h). High salinity increased affinity to the cellulase to the substrate and thermostability. Halostable cellulase from a marine Aspergillus niger was valuable for cellulose hydrolysis under high salinity conditions.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Adsorption Optimization of Lead (II) Using Saccharum Bengalense as a Non-Conventional Low Cost Biosorbent: Isotherm and Thermodynamics Modeling

In the present study a novel biomass, derived from the pulp of Saccharum bengalense, was used as an adsorbent material for the removal of Pb (II) ions from aqueous solution. After 50 minutes contact time, almost 92% lead removal was possible at pH 6.0 under batch test conditions. The experimental data was analyzed using Langmuir, Freundlich, Timken and Dubinin-Radushkevich two parameters isotherm model, three parameters Redlich—Peterson, Sip and Toth models and four parameters Fritz Schlunder isotherm models. Langmuir, Redlich—Peterson and Fritz-Schlunder models were found to be the best fit models. Kinetic studies revealed that the sorption process was well explained with pseudo second-order kinetic model. Thermodynamic parameters including free energy change (ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) have been calculated and reveal the spontaneous, endothermic and feasible nature of the adsorption process. The thermodynamic parameters of activation (ΔG ^#, ΔH ^#and ΔS ^#) were calculated from the pseudo-second order rate constant by using the Eyring equation. Results showed that Pb (II) adsorption onto SB is an associated mechanism and the reorientation step is entropy controlled.     

Controlling the folding efficiency of an integral membrane protein

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.     

KINETICS OF NITRATE,AMMONIUM, AND UREA UPTAKE BY FOUR INTERTIDAL SEAWEEDS FROM NEW ZEALAND1

The competitive ability for N uptake by four intertidal seaweeds, Stictosiphonia arbuscula (Harvey) King et Puttock, Apophlaea lyallii Hook. f. et Harvey, Scytothamnus australis Hook. f. et Harvey, and Xiphophora gladiata (Labillardière) Montagne ex Harvey, from New Zealand is described by the uptake kinetics for NO₃^?, NH₄⁺, and urea. This is the first study to report uptake kinetics for N uptake by a range of southern hemisphere intertidal seaweeds in relation to season and zonation. Species growing at the highest shore positions had higher NO₃^? and urea uptake at both high and low concentrations and had unsaturable NH₄⁺ uptake in both summer and winter. Although there was evidence of some feedback inhibition of V_max for NO₃^? uptake by Stictosiphonia arbuscula growing at the lower vertical limits of its range, rates were high compared with species growing lower on the shore. Our results highlight the superior competitive ability for N uptake of certain high intertidal seaweeds, and consistent with our previous findings we can conclude that intertidal seaweeds in southeast New Zealand are adapted to maximizing N acquisition in a potentially N‐limiting environment.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.