Characterization of electron transfer reactions of microperoxidase assembled at short-chain thiol-monolayers on gold

In this study thiol-monolayers were used in order to modify gold and provide suitable chemical functionalities for the immobilization of the small redox-active haem-containing peptide, microperoxidase (MP-11). Initially, we assembled a variety of thiol-containing species on the gold electrodes and measured a series of electron transfer reactions, each characteristic of the surface-modifier. Using suitable immobilization strategies, we subsequently covalently bound MP-11 to appropriate monolayers and found two characteristic electrochemical responses (i.e. using MP-11 bound to mercaptopropionic acid, redox peaks were seen at E^0′ = −315 mV and at +179 mV versus Ag|AgCl, with the former being attributed to the haem and the latter with the thiol monolayer). Exposure of the peptide-thiol surface to UV irradiation resulted in cleavage of the Au---S bond, leading to a decrease in the magnitude of both responses. Our work is supported by corroborative evidence showing the immobilization of the peptide, obtained using both X-ray photoelectron and reflectance Fourier transform infra-red spectroscopies. We hypothesize that differences in the ionic charges on the protein backbone account for the shift in E^0′ for MP-11, as observed in our system, when compared to that found for MP-11 immobilized by different strategies.     

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

Structural alterations in synaptosomal membrane-associated proteins and lipids by transient middle cerebral artery occlusion in the cat

We have previously reported that ischemia reperfusion injury results from free radical generation following transient global ischemia, and that this radical induced damage is evident in the synaptosomal membrane of the gerbil. [Hall et al, (1995) Neuroscience 64: 81–89] In the present study we have extended these observations to transient focal ischemia in the cat. We prepared synaptosomal membranes from frontal, parietal-temporal, and occipital regions of the cat cerebral cortex with reperfusion times of 1 and 3 hours following 1 hour right middle cerebral artery occlusion. The membranes were selectively labeled with protein and lipid specific paramagnetic spin labels and analyzed using electron paramagnetic resonance spectrometry. There were significant motional changes of both the protein and lipid specific spin labels in the parietal-temporal and occipital regions with 1 hour reperfusion; but, both parameters returned to control values by 3 hours reperfusion. No significant changes were observed in the normally perfused frontal pole at either reperfusion time. These results support the argument that free radicals play a critical role in cell damage at early reperfusion times following ischemia.     

Calcium fluxes at plasmalemma and tonoplast

Abstract. An account is given of the characterization of calcium fluxes across plasmalemma and tonoplast membranes of root cortical cells, using compartmental analysis. Some of the assumptions associated with the method are discussed. Recent evidence regarding the concentration of free Ca²⁺ in plant cells, and the mechanisms driving active calcium transport across cell membranes, is reviewed. It is proposed that the evidence from whole cell studies and work at the molecular level is mutually supportive, and some speculation is ventured about the general pattern of calcium transport in higher plant cells.     

The transport of morphologically abnormal sperm in the female reproductive tract of mice

Mice of the PL/J strain exhibit a high percentage of morphologically abnormal sperm and provide a model for studying the function of abnormal sperm. The ability of such sperm to reach the site of fertilization within the female reproductive tract has been investigated. We have found a decrease in the percentage of structurally abnormal sperm within the population that reaches the oviduct. This observation suggests either that there is an active selection against abnormal sperm or that they are physiologically disadvantaged in reaching the site of fertilization.     

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-β-hydroxylase

Presynaptic muscarinic receptors labeled with [³H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [³H]quinuclidinyl benzilate or [³H]dexetimide binding revealed a distribution profile similar to that of dopamine-β-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-β-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-β-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.     

Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap,leaves and fruits of Lupinus albus L.

Quinolizidine alkaloids formed in the leaves of Lupinus albus L. are translocated via the phloem to the other plant organs, especially the maturing fruits. Compared with amino-acid transport in the phloem, the alkaloids contribute about 8% to the overall nitrogen being exported from the leaf. Since it is likely that the alkaloids are subsequently degraded in the target tissues a minor role of quinolizidine alkaloids might be nitrogen transport. A marked diurnal fluctuation of alkaloids was observed in the leaves, the phloem sap, the roots and the fruits with an increase during the day and an amplitude of several hundred percent thus providing evidence for a rapid turnover of endogenous alkaloids.Abbreviations QA quinolizidine alkaloids - GLC gas-liquid chromatography     

Amino acid transport in the rat exocrine pancreas

Summary Amino acid transport and incorporation have been studied in vitro in rat pancreatic lobules after maximal and supramaximal hormonal stimulation with caerulein. Incorporation into proteins was increased already after 30 and 120 min of maximal stimulation, but was decreased after the infusion of a supramaximal dose. Uptake of neutral amino acids was monitored using labeled leucine and -aminoisobutyric acid (AIB). In the case of leucine the free pool was consistently reduced after maximal stimulation, while supramaximal doses led to an increase which could be potentiated by the addition of 2mM tetracaine. Using AIB, a significant increase in the intracellular pool was observed after maximal stimulation, conversely a decrease after supramaximal stimulation. Release of labeled leucine and AIB from preloaded lobules during incubation in the cold was significantly reduced after maximal secretory stimulation, but was found enhanced by 200 to 300 percent after supramaximal stimulation. No fine structural alterations at junctional complexes or at both the lateral and luminal plasma membranes were observed after maximal stimulation except an increased number of exocytotic figures at the luminal face. However, supramaximal stimulation led to progressive rarefaction of the tight junctional network and disintegration of the gap junctions. Concomitantly, an equal distribution of membrane particles on both faces of the plasma membrane together with a random occurrence of exocytotic figures were observed.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (SFB 122, project C 5). Dedicated to Professor Dr. Gerhard Petry, Marburg, on the occasion of his 65th birthday     

The goblet cavity matrix in the larval midgut of Hyalophora cecropia

The larval midgut of the silkmoth Hyalophora cecropia was examined using scanning electron microscopy. Goblet cells were observed to contain within their cavities a matrix plug. This matrix material was extruded onto the lumen side of the epithelium when the tissue was stretched. The rôle of this matrix material in maintenance of the capacity of the midgut to transport ions in vivo and in vitro is discussed.     

Some characteristics of a fast movement of auxin in intact tomato seedlings (Lycopersicon esculentum)

Using exogenous ¹⁴C-IAA, a fast movement of auxin was demonstrated in intact tomato plants ( Lycopersicon esculentum Mill, cv. Craigella) cultured in vitro. This movement is insensitive to or weakly enhanced by cold temperature (0°C) and not influenced by gravity. In short time experiments (10 min) the main part of the radioactivity which travels through the plant is IAA; in long time ones (6 h) the metabolism is much higher and leads mainly to the formation of IAAsp. The integrity of the plant is necessary for the possibility to observe such a fast movement. Isolated hypocotyl segments show only the classical polar and slow movement of auxin. Microautoradiographic techniques show that the fast movement takes place inside the plant. In the epicotyl, the vascular bundles, mainly phloem cells, are labelled; in contrast, the radioactivity detected in the hypocotyl is low, and the tracers can be visualized only inside the epidermal cells.