Viability of an ectomycorrhizal fungus during cross-flow filtration

Factors affecting the viability and infectivity of an ectomycorrhizal fungus during moderate concentration by cross-flow filtration were determined. Mycelial suspensions were concentrated with three commercial membrane filters (Prostak Millipore Co., M14 Tech-Sep Co. and Ceraflo Norton Co.) under aseptic conditions. Medium components may reduce the filtration rate due to their low solubility. An antifoam agent did not reduce the average flux rates as much as did the malt extract. Clear unobstructed channels (I.D. 6mm) of the tubular modules (Tech-Sep) gave the best results both in terms of performance (filtration rate) and cell viability. Shear stresses caused by pumping and flow through narrow retentate channels were probably responsible for lowering viability and infectivity. There was no linear relationship between permeate fluxes and cell concentration. There is an optimum pore size both in terms of performance (filtration rate) and cell viability. Physical blockage of large pores by hyphae could explain lower permeate flux rates than those obtained with lower pore sizes membranes.     

Presence of a Na+/H+ exchanger in brush border membranes isolated from the kidney of the spiny dogfish,Squalus acanthias

Summary A membrane fraction, rich in brush border membranes, was prepared from renal proximal tubules of the spiny dogfish,Squalus acanthias, and the sodium-proton exchange mechanism in these membrane vesicles was investigated by both a rapid filtration technique and the fluorescence quenching of acridine organe.²²Na⁺ uptake was stimulated by an outwardly directed H⁺ gradient, and was inhibited by amiloride at a single inhibitory site with an apparentK _i of approximately 1.7×10^–5 M. In the presence of an H _i ⁺ >H _o ⁺ gradient, the of the Na⁺/H⁺ exchanger were 9.7±0.8 mM and 48.0±12.0 nmol·mg protein^–1·min^–1, respectively. The uptake of Na⁺ was electroneutral in the presence of a H⁺ gradient, indicating a stoichiometry of 1. In the fluorescence studies, quenching of acridine orange occurred in the presence of an outwardly directed Na⁺ gradient which was inhibited by amiloride. Thus, an electroneutral Na⁺/H⁺ exchanger with properties similar to those found in the mammalian kidney is also present in the spiny dogfish and may contribute to the urinary acidification of this marine animal.     

Periodic electric field as a biopolymer conformation switch: a possible mechanism

A theoretical model is proposed to describe the influence of a periodic electric field (PEF) upon a biopolymer. The biopolymer is treated as a classical mechanical system consisting of subsystems (molecular groups) which interact with each other through potential forces. The PEF is treated as a periodic driving force applied to a molecular group. The energy dissipation is considered using the model of fluid (viscous) friction. Arguments for the non-linear character of the friction-velocity dependence caused by the non-Newtonian rheology of a viscous medium are formulated.A forced molecular-group motion is investigated for the situation of a small driving-force period, with oscillations overdamped and a driving force consisting of more than one harmonic. As a result, it is established that the motion always gets to a terminal stage when only a small-scale vibration about some point, X ^*, takes place. The terminal motion is preceded by a transient characterized by the presence of a directional velocity component and so by a drift along a potential profile. the drift goes on until a barrier is met which has a sufficiently large steepness (the barrier height is not important). As a result, the point X ^* may happen to be remote from the conformation potential local minimum (conformational state). The physical reasons for the drift are described.If we consider the small-scale vibration about X ^* in the framework of the hierarchy of scales for intramolecular mobility, it can be regarded as an equilibrium mobility, whereas the drift can be regarded as a functionally important motion, and X ^* as a new conformational state which is realizable only in the presence of the PEF. It may be concluded, as the result of a consistent treatment and neglecting the small-scale vibration, that the conformational potential is modified by adding a linear term (in the one-dimensional case). In this connection, the set of conformational states can both deform (deviation of the positions of minima and their relative depth) and rearrange qualitatively (some minima can vanish and/or new minima can appear). In particular, the transition from one conformation to another one may happen due to rearrangement.     

A marker of acid-secreting membrane movement in rat gastric parietal cells

A monoclonal antibody (mab 146.14) marker of the movement of acid-secreting membranes in rat gastric parital cells has been produced and characterized. Mab 146.14 recognized a 95-kD major component of a purified membrane fraction of rat gastric mucosa, the protein composition of which was similar to that of well characterized porcine H+ -K+ ATPase-enriched membranes, and that presented the characteristic shift of density depending on whether it was purified from resting or stimulated tissues. Further biochemical analysis characterized the antigen as a membranous protein that might be in its native form, part of a higher multimolecular complex. Immunocytochemical localization of the antigen demonstrated that only membranes related to acid secretion in parietal cells expressed the 95-kD antigen. In resting conditions, the 95-kD antigen was diffusely distributed in the cell cytoplasm associated with inactive tubulovesicles. In stimulated cells, by contrast, all the antigen was recovered associated with secretory active microvilli formed by the apical insertion of the previously resting internal tubulovesicles. In conclusion, the 95-kD antigen, presumably a part of the rat gastric proton pump, is a marker of acid-secreting membranes in rat parietal cells. The translocation of antigen and membranes, observed by both light and electron microscopy supports the fusion model of membrane insertion from a cytoplasmic storage pool to the apical surface upon stimulation of acid secretion.     

The electrochemical proton potential generated by the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes grown on formate and elemental sulphur was found to use the polysulphide derivatives 2,2-tetrathiobispropionate (R₂S₄) or pentathionate (S₅O ₆ ⁼ ) as acceptors for formate oxidation. The specific activities of formate oxidation with these acceptors were similar to those with elemental sulphur. The main reaction products of R₂S₄ reduction were 2,2-dithiobispropionate (R₂S₂) and sulphide. Pentathionate was converted to thiosulphate and some elemental sulphur. The electrochemical proton potential across the cytoplasmic membrane of the bacterium was measured in the steady state of electron transport from formate to R₂S₄. The electrical proportion () of the determined through the distribution of labeled tetraphenylphosphonium cation was obtained as 0.17 Volt. The was zero, when a protonophore was present. The pH-difference across the membrane was negligible. Thus the generated by sulphur respiration is close to that measured earlier with fumarate as the terminal acceptor of electron transport.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - R₂S_n (n=2–5) 2,2-polythiobispropionate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TPP tetraphenylphosphonium cation     

A stochastic model for the membrane potential of a stimulated neuron

We present a simple model describing the transition between the prefiring, firing and postfiring phases of a single neuron in a large neural net. Using typical values for the physiological parameters that enter the model, we find average interspike times that are close to those reported in experimental measurements.     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase, located in mitochondiral outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.