Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cultures of the cyanobacterium Microcystis firma show rhythmic uptake and release of ammonia under conditions of carbon limitation. The massive removal of ammonia from the medium during the first light phase has little impact on the intracellular pH: a pH shift of less than 0.2 U towards the alkaline can be measured by in vivo ³¹P NMR. Furthermore, the energy status of the cells remains regulated. In vivo ¹⁵N NMR of M. firma, cultivated either with labelled nitrate or ammonia as the sole nitrogen source, reveals only gradual differences in the pool of free amino acids. Additionally both cultivation types show -aminobutyric acid, acid amides and yet unassigned secondary metabolites as nitrogen storing compounds. Investigating the incorporation of nitrogen under carbon limitation, however, only the amide nitrogen of glutamine is found permanently labelled in situ. While transamination reactions are blocked, nitrate reduction to ammonia can still proceed. Cation exchange processes in the cell wall are considered regarding the ammonia disappearance in the first phase, and the control of ammonia uptake is discussed with respect to the avoidance of intracellular toxification.Abbreviations GABA -aminobutyric acid - GOGAT glutamate synthase - GS glutamine synthetase - MDP methylene diphosphonate - MOPSO 3-(N-morpholino)-2-hydroxy-propanesulfonic acid - NDPS nucieoside diphosphosugars - NOE nuclear Overhauser effect - NMR nuclear magnetic resonance For convenience, the term ammonia is used throughout to denote ammonia or ammonium ion when there is no good evidence as to which chemical species is involved     

Mechanism of Cl- sensitivity in internal ion receptors of the leech; an inward current gated off by Cl- in the nephridial nerve cells

Summary The nephridial nerve cells of the leech, Hirudo medicinalis, 34 sensory cells, each associated with one nephridium, are sensitive to changes in extracellular Cl^- concentration, an important factor in ion homeostasis. Using single-electrode current- and voltage clamp and ion substitution techniques, the specificity and mechanism of Cl^- sensitivity of the nephridial nerve cell was studied in isolated preparations. Increase of the normally low external Cl^- concentration leads to immediate and sustained hyperpolarization, decrease of the frequency of bursts and decrease of membrane conductance. The response is halogen specific: Cl^- can be replaced by Br^–, but not by organic mono- or divalent anions or inorganic divalent anions.At physiological Cl^- concentrations (36mM extra-cellular Cl^-), the nephridial nerve cell has a high resting conductance for Cl^- and the membrane potential is governed by Cl^-. In high extracellular Cl^- concentrations (110–130 mM), membrane conductance is low, most likely due to the gating off of Cl^- channels. Under these conditions, membrane potential is dominated by the K⁺ distribution and the nephridial nerve cell hyperpolarizes towards E_K.Abbreviations NNC nephridial nerve cell - V _m membrane potential - E _Cl(k) equilibrium potential for Cl (K) - IV-curve current-voltage relationship     

Physiological roles of animal succinate thiokinases Specific association of the guanine nucleotide-linked enzyme with haem biosynthesis

The discovery of two distinct succinate thiokinases in mammalian tissues, one (G-STK) specific for GDP/GTP and the other (A-STK) for ADP/ATP, poses the question of their differential metabolic roles. Evidence has suggested that the A-STK functions in the citric acid cycle in the direction of succinyl-CoA breakdown (and ATP formation) whereas one role of the G-STK appears to be the re-cycling of succinate to succinyl-CoA (at the expense of GTP) for the purpose of ketone body activation. A third metabolic participation of succinyl-CoA is in haem biosynthesis. This communication shows that in chemically induced hepatic porphyria, when the demand for succinyl-CoA is increased, it is the level of G-STK only which is elevated, that of A-STK being unaffected. The results implicate G-STK in the provision of succinyl-CoA for haem biosynthesis, a conclusion which is further supported by the observation of a high G-STK/A-STK ratio in bone marrow.     

Comparison of the roles of Ostracods and Cladocerans in regulating community structure and metabolism in freshwater microcosms

Laboratory microcosms were used to compare the effects of the littoral ostracod Cypridopsis vidua and the planktonic cladoceran Daphnia magna on community structure and metabolism. Filter-feeding by cladocerans, both in the presence and absence of ostracods, greatly reduced the abundance of planktonic algae when D. magna reached peak density around day 50; rotifers and euglenids were then limited to flocculent matter on the container bottom. Both net production and community respiration rates decreased as community composition changed. Microcosms containing ostracods as the only microcrustacean showed little reduction in total algal numbers but the otherwise dominant alga, Scenedesmus spp., was replaced by Ankistrodesmus spp. when peak ostracod density was reached around day 100. Rotifers were completely eliminated but euglenids were able to coexist with ostracods. Ostracods impacted community metabolism less than cladocerans, but depressed respiration slightly more than net production.     

Degradation of 4-hydroxyphenylacetate by Xanthobacter 124X

Xanthobacter 124X when grom on 4-hydroxyphenylacetate was able to hydroxylate this compound yielding homogenisate. Ring fission of this latter compound gave maleylacetoacetate which was isomerized to fumarylacetoacetate. The isomerase involved resembled maleylacetoacetate isomerases in Gram-negative bacteria in that glutathione was required for activity. Fumarate and acetoacetate were both detected as products of the hydrolysis of fumarylacetoacetate.     

Dopamine Metabolites in Rat Cisternal Cerebrospinal Fluid: Major Contribution from Extrastriatal Dopamine Neurones

The interpretation of central 3,4-dihydroxyphenylethylamine (dopamine, DA) metabolism, as indicated by determinations in rat cisternal CSF, was investigated using intrastriatal injection of the DA neurotoxin 6-hydroxydopamine (6-OHDA) and intraperitoneal injection of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). DA turnover was subsequently determined by measurement of the rate of accumulation of total 3,4-dihydroxyphenylacetic acid and homovanillic acid (DOPAC + HVA) in the CSF after probenecid was given. Two days later the rats were killed, and metabolism of DA and 5-hydroxytryptamine (5-HT) was investigated by determining levels of the amines and their metabolites in brain regions. Although 6-OHDA greatly decreased striatal DA metabolism, this was not paralleled by DA turnover as indicated by CSF, as this fell only moderately and approximately in parallel with results for the brain as a whole. 5-HT metabolism was essentially unaltered. DSP4 considerably depleted noradrenaline and caused smaller decreases of 5-HT metabolism in some regions. However, DA metabolism was not significantly affected, either in brain or CSF, which suggests that noradrenaline neurones make only a small contribution to central DA metabolism. Results as a whole suggest that DOPAC and HVA concentrations in rat cisternal CSF reflect whole brain DA metabolism and derive predominantly from DA neurones in extrastriatal regions of the brain.     

Investigation of Dopamine Content, Synthesis, and Release in the Rabbit Retina In Vitro: I. Effects of Dopamine Precursors, Reserpine, Amphetamine, and l-DOPA Decarboxylase and Monoamine Oxidase Inhibitors

The basal catecholamine content of rabbit retina was determined by liquid chromatography with electrochemical detection (LC-EC) and 3,4-dihydroxyphenylethylamine (dopamine, DA) found to be the major catecholamine. The immediate DA precursor, 3,4-dihydroxyphenylalanine (L-DOPA), and the metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were also detected at about 2.8% and 17% of DA levels, respectively. When added exogenously, L-tyrosine did not increase the rate of DA synthesis over the basal level. In contrast, exogenous L-DOPA led to a 3.5-fold increase in DA, and to a 20-fold increase in DOPAC content. The monoamine oxidase inhibitors pargyline and (-)-deprenyl differentially affected the degradation of DA, since 100 microM pargyline was apparently more effective than 100 microM (-)-deprenyl. Reserpine and (+/-)-amphetamine each induced a Ca2+-independent decrease of DA stores. The separate actions of reserpine and (+/-)-amphetamine in lowering tissue DA levels were additive, suggesting two separate pools of DA available for release from presynaptic stores. The present study demonstrates that the LC-EC technique may be used to investigate the modulation of the synthesis and release of retinal DA in vitro, without the prior uptake of radiolabelled transmitter.     

Differentiation among populations of Sedum wrightii (Crassulaceae) in response to limited water availability: water relations,CO2 assimilation,growth and survivorship

Summary Sedum wrightii is one of only a few species in the Crassulaceae for which there is evidence for a high degree of variability in the ratio of daytime to nighttime CO₂ assimilation. There are both environmental and genetic components to this variability. S. wrightii grows over a wide altitudinal gradient. The purpose of this study was to compare low, intermediate, and high altitude populations with respect to the degree of CAM expression and the capability to tolerate limited water availability. We utilized clonallyreplicated genotypes of plants from each population in common environment greenhouse experiments. Genetic differences among the populations were found in long-term water use efficiency, in 24 hour CO₂ exchange patterns, in biomass ¹³C values, in carbon allocation, and in water status and ultimately survival during prolonged drought. The differences among the populations appear to be closely related to differences in the native habitats. The low altitude, desert plants had the greatest ability to grow and survive under conditions of limited water availability and appear to have the greatest shift to nighttime CO₂ uptake during periods without water, while the high altitude plants had the poorest performance under these conditions and appear to shut down net carbon uptake when severely water limited.     

Mannitol metabolism in cultured plant cells

Non-structural storage carbohydrates were measured in 9-day-old barley ( Hordeum vulgare L. cv. Brant) primary leaves. Accumulation rates of starch, sucrose and total non-structural carbohydrates (TNC) were approximately linear when measured between 2- and 12-h of light. Progressively higher TNC accumulation rates were observed at higher irradiance levels (i.e., comparing 250, 550 and 1050 ·mol m⁻² s⁻¹). Synthesis of a low-molecular-weight fructan also was enhanced by high irradiances. Low irradiance treatments decreased leaf sucrose levels and there was a corresponding increase in the lag period preceding starch synthesis in the light. Increased starch accumulation rates were usually observed when sucrose concentrations were high. These and other results suggested that cytosolic sucrose concentrations affected starch metabolism in the chloroplast. However, sucrose accumulation rates increased and starch storage decreased when barley seedlings were transferred from 20 to 10°C during the light period. Lowering the night temperature from 20 to 10°C for a single dark period 8-days after planting increased the TNC content of barley primary leaves at the beginning of day nine. In this experiment, TNC accumulation rates of treated and untreated leaves were similar. Changes in the accumulation rate of TNC were usually observed within 2- to 4-h after barley seedlings were exposed to altered environmental conditions. Monitoring rapid changes in leaf carbohydrate levels is a sensitive method for assessing the effects of environmental treatments on photosynthetic metabolism.