Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K⁺ and Ca²⁺ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 μm, AA reduces the amplitude and accelerates the inactivation of the K⁺ current activated by depolarization; at higher concentrations (≥5 μm), AA still blocks this K⁺ current, but also induces a very large noninactivating K⁺ current. At 2 or 3 μm, AA enhances the T-type Ca²⁺ current, close to its threshold of activation, whereas at 10 μm, it blocks that current. AA (1–10 μm) also blocks the dihydropyridine-sensitive L-type Ca²⁺ current. Thus, the effect of AA on Ca²⁺ entry through voltage-gated Ca²⁺ channels can change qualitatively with the AA concentration: at 2 or 3 μm, AA will favor Ca²⁺ entry through T channels, both by lowering the voltage-gated K⁺ conductance and by increasing the T current, whereas at 10 μm, AA will prevent Ca²⁺ entry through voltage-gated Ca²⁺ channels, both by inducing a K⁺ conductance and by blocking Ca²⁺ channels.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Mechanisms of cell volume regulation in the proximal segment of the Malpighian tubule of Rhodnius neglectus

The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using videooptic techniques. When the medium osmolality was increased with addition of 100 mm mannitol the cells shrank to a minimum of 16.84±2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K⁺ or Cl^? or HCO ₃ ^? by Na⁺, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na⁺ by tetramethylammonium (TMA⁺) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10^?4 m, anthracene-9-COOH 5×10^?4 m, furosemide 5×10^?4 m or ethacrynic acid 5×10^?5 m, also abolished RVI. On the other hand, addition of either Ba²⁺ 10^?3 m, SITS 5× 10^?4 m, ouabain 10^?3 m or vanadate 10^?3 m, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mm or verapamil 10^?6 m, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mm induced a cell swelling to a maximum of 33.11+1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na⁺, K⁺, Cl^?, Ca²⁺ and HCO ₃ ^? transport pathways and a ouabain-insensitive ATPase stimulated by Na⁺. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP; Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq e Financiadora de Projetos e Pesquisas-FINEP.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+ and Zn2+) and water coordination on the structure and properties of l-histidine and zwitterionic l-histidine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. The effect of metal ions and water on the structure of l-histidine is examined. The effect of metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺) and water on structures of His·M(H₂O)m, m = 0.1 complexes have been determined theoretically employing density functional theories using extended basis sets. Of the five stable complexes investigated the relative stability of the gas-phase complexes computed with DFT methods (with one exception of K⁺ systems) suggest metallic complexes of the neutral l-histidine to be the most stable species. The calculations of monohydrated systems show that even one water molecule has a profound effect on the relative stability of individual complexes. Proton dissociation enthalpies and Gibbs energies of l-histidine in the presence of the metal cations Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ were also computed. Its gas-phase acidity considerably increases upon chelation. Of the Lewis acids investigated, the strongest affinity to l-histidine is exhibited by the Cu²⁺ cation. The computed Gibbs energies ΔG are negative, span a rather broad energy interval (from ?130 to ?1,300 kJ/mol), and upon hydration are appreciably lowered.     

The activity of erythrocyte and brain Na+/K+ and Mg2+-ATPases in rats subjected to acute homocysteine and homocysteine thiolactone administration

Hyperhomocysteinemia is associated with various pathologies including cardiovascular disease, stroke, and cognitive dysfunctions. Systemic administration of homocysteine can trigger seizures in animals, and patients with homocystinuria suffer from epileptic seizures. Available data suggest that homocysteine can be harmful to human cells because of its metabolic conversion to homocysteine thiolactone, a reactive thioester. A number of reports have demonstrated a reduction of Na⁺/K⁺-ATPase activity in cerebral ischemia, epilepsy and neurodegeneration possibly associated with excitotoxic mechanisms. The aim of this study was to examine the in vivo effects of d,l-homocysteine and d,l-homocysteine thiolactone on Na⁺/K⁺- and Mg²⁺-ATPase activities in erythrocyte (RBC), brain cortex, hippocampus, and brain stem of adult male rats. Our results demonstrate a moderate inhibition of rat hippocampal Na⁺/K⁺-ATPase activity by d,l-homocysteine, which however expressed no effect on the activity of this enzyme in the cortex and brain stem. In contrast,d,l-homocysteine thiolactone strongly inhibited Na⁺/K⁺-ATPase activity in cortex, hippocampus and brain stem of rats. RBC Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities were not affected by d,l-homocysteine, while d,l-homocysteine thiolactone inhibited only Na⁺/K⁺-ATPase activity. This study results show that homocysteine thiolactone significantly inhibits Na⁺/K⁺-ATPase activity in the cortex, hippocampus, and brain stem, which may contribute at least in part to the understanding of excitotoxic and convulsive properties of this substance.     

Uptake of amino acids by actidione-treated yeast cells

The uptake of glycine,l-aspartic acid andl-lysine at pH 5 was not systematically affected by the presence of up to 0.1m NaCl or KCl, without or with 0.3mm actidione. The observed effects, either positively or negatively altering the rate of amino acid uptake, depended on the concentration of the ion but were always qualitatively the same for Na⁺ and K⁺. The inhibition prevailing with 0.1m ions was significantly more pronounced with Na⁺ than with K⁺. The presence of other anions (phosphate, nitrate, sulphate) had a slight but significant effect on uptake as compared with the variant using chloride. It is concluded that inSaccharomyces cerevisiae the transport of amino acids is not essentially coupled with the transport of different ions.     

Endogenous,Ca2+-dependent cysteine-protease cleaves specifically the ryanodine receptor/Ca2+ release channel in skeletal muscle

The association of an endogenous, Ca²⁺-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and β-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca²⁺ with half-maximal activity obtained at 0.1 μm and maximal activity at 10 μm. Mg²⁺ is also an activator of this enzyme (CI₅₀=22 μm). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type. This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356–1367 of the RyR and the cleavage site, the calmodulin binding site, at residues 1383–1400. The RyR cleavage by the Ca²⁺-dependent thiol-protease is prevented in the presence of ATP (1–5 mm) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca²⁺ efflux. A possible involvement of this specific cleavage of the RyR/Ca²⁺ release channel in the control of calpain activity is discussed.     

K+-dependent 3H-d-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp

The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-d-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-d-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-d-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J _max and lowered K_m in both salts. ³H-d-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis–Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-d-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (d-galactose, α-methyl-d-gluco-pyranoside, unlabeled d-glucose, d-fructose, and d-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-d-glucose influx. Only unlabeled d-glucose, d-fructose, and d-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of d-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-d-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-d-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, d-fructose, and d-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter, suggesting that an invertebrate analogue of this protein may occur in shrimp.     

Selenium Modulates Oxidative Stress-Induced Cell Apoptosis in Human Myeloid HL-60 Cells Through Regulation of Calcium Release and Caspase-3 and -9 Activities

Selenium is an essential chemopreventive antioxidant element to oxidative stress, although high concentrations of selenium induce toxic and oxidative effects on the human body. However, the mechanisms behind these effects remain elusive. We investigated toxic effects of different selenium concentrations in human promyelocytic leukemia HL-60 cells by evaluating Ca²⁺ mobilization, cell viability and caspase-3 and -9 activities at different sample times. We found the toxic concentration and toxic time of H₂O₂ as 100 μm and 10 h on cell viability in the cells using four different concentrations of H₂O₂ (1 μm–1 mm) and six different incubation times (30 min, 1, 2, 5, 10, 24 h). Then, we found the therapeutic concentration of selenium to be 200 nm by cells incubated in eight different concentrations of selenium (10 nm–1 mm) for 1 h. We measured Ca²⁺ release, cell viability and caspase-3 and -9 activities in cells incubated with high and low selenium concentrations at 30 min and 1, 2, 5, 10 and 24 h. Selenium (200 nm) elicited mild endoplasmic reticulum stress and mediated cell survival by modulating Ca²⁺ release, the caspases and cell apoptosis, whereas selenium concentrations as high as 1 mm induced severe endoplasmic reticulum stress and caused cell death by activating modulating Ca²⁺ release, the caspases and cell apoptosis. In conclusion, these results explained the molecular mechanisms of the chemoprotective effect of different concentrations of selenium on oxidative stress-induced apoptosis.     

Identification of the ATP·Mg-dependent protein phosphatase activator (Fa) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules

The ATP·Mg-dependent protein phosphatase activating factor (Fa) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factorFa has further been identified as a cAMP and Ca²⁺-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factorFa could phosphorylate synapsin I with a lowK _m value of about 2 µM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factorFa specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca²⁺/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factorFa could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factorFa as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.     

l-leucine, l-methionine, and l-phenylalanine share a Na+/K+-dependent amino acid transporter in shrimp hepatopancreas

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of ³H-l-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. ³H-l-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na⁺- and K⁺-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. ³H-l-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, l-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM ³H-l-leucine in both Na⁺- and K⁺-containing incubation media. The residual ³H-l-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an l-methionine- and cation-independent transport system. ³H-l-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [l-leucine], following the carrier-mediated Michaelis–Menten equation. In NaCl, ³H-l-leucine influx displayed a low apparent K _M (high affinity) and low apparent J _max, while in KCl the transport exhibited a high apparent K _M (low affinity) and high apparent J _max. l-methionine or l-phenylalanine (7 and 20 mM) were competitive inhibitors of ³H-l-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in ³H-l-leucine influx K _M, but no significant response in ³H-l-leucine influx J _max. Potassium was a competitive inhibitor of sodium co-transport with ³H-l-leucine, significantly (p < 0.01) increasing ³H-l-leucine influx K _M in the presence of sodium, but having negligible effect on ³H-l-leucine influx J _max in the same medium. These results suggest that shrimp BBMV transport ³H-l-leucine by a single l-methionine- and l-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na⁺ or K⁺ acting as co-transport drivers binding to shared activator sites.     

Purification, characterization and kinetic properties of extracellular l-asparaginase produced by Cladosporium sp.

l-asparaginase from Cladosporium sp. grown on wheat bran by SSF was purified. Enzyme appeared to be a trimer with homodimer of 37 kDa and another 47 kDa amounting to total mass of 121 kDa as estimated by SDS-PAGE and 120 kDa on gel filtration column. The optimum temperature and pH of the enzyme were 30 °C and 6.3, respectively with Vmax of 4.44 μmol/mL/min and Km of 0.1 M. Substrate specificity studies indicated that, l-asparaginase has greater affinity towards l-asparagine with substrate hydrolysis efficiency (Vmax/Km ratio) eightfold higher than that of l-glutamine. l-asparaginase activity in presence of thiols studied showed decrease in Vmax and increase in Km, indicating nonessential mode of inactivation. Among the thiols tested, β-mercaptomethanol, exerted inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. Metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Na⁺, K⁺ and Zn²⁺ significantly affected enzyme activity whereas presence of Fe³⁺, Pb²⁺ and KI stimulated the activity. Detergents studied also enhanced l-asparaginase activity. In-vitro half-life of purified l-asparaginase in mammalian blood serum was 93.69 h. The enzyme inhibited acrylamide formation in potato chips by 96 % making it a potential candidate for food industry to reduce acrylamide content in starchy fried food commodities.     

l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP⁺ but uses also NAD⁺ as a cofactor, and showed highest catalytic efficiency (k _cat/K _m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.     

DieFestucetalia valesiacae-Gesellschaften m Ostteil des Gebirges České středohoří (Böhmisches Mittelgebirge) 2. Synökologie,Sukzession und syntaxonomische Ergänzungen

Die vorliegende Arbeit befasst sich mit zwei, die Vegetation felsiger vulkanischer Substrate einschliessenden Verbänden:Alysso-Festucion pallentis Moravec 1967 undFestucion valesiacae Klika 1933, in deren Rahmen acht Assoziationen untersucht wurden:Alysso-Festucetum Klika ex?e?ovský 1949,Asperulo-Festucetum Preis inKlika 1939,Allio-Sedetum Klika 1939,Melico-Sempervivetum Preis inKlika 1939,Alysso-Potentilletum Preis 1939,Carici-Festucetum Klika 1951,Erysimo-Festucetum Klika 1933 undFestuco-Stipetum Sillinger 1931. Im ökologischen Abschnitt wurde auf den Vergleich von chemischen eigenschaften der Böden (Azidität und ihr Saisonverlauf, Gehalt an Austauschionen—H⁺, Al³⁺, Ca²⁺, Mg²⁺ und an C und N) und deren morphologischen Eigenschaften (Tiefe und Stratigraphie der Bodenhorizonte, ihre Beschreibung) Nachdruck gelegt. Ebenso wurde dem Standort und den Beziehungen der Gesellschaft zu den anschliessend benachbarten Gesellschaften an der Lokalität Aufmerksamkeit gewidmet. In der Arbeit werden auch einige selten vorkommende oder noch nicht beschriebene Syntaxa erwähnt, die mit den oben erwähnten Assoziationen in Kontakt stehen:Calluno vulgaris-Cytisetum nigricantis Prsg. 1953 em.Oberd. 1957,Polytricho piliferi-Scleranthetum perennis Moravec 1967,Geranio sanguinei-Dictamnetum albae Wendelberger 1954,Geranio sanguinei-Trifolietum alpestris Th. Müller 1961,Dictamno albae-Sorbetum Knapp 1942; weiter die neubeschriebenen Syntaxa:Alysso saxatilis-Festucetum duriusculae cotoneasteretosum integerrimae, Potentillo opacae-Festucetum sulcatae pulsatilletosum patentis mit Var.Vaccinium myrtillus und Var.Pimpinella saxifraga und Subass.eryngietosum campestris, Pulsatillo pratensis-Avenochloetum pratensis, Koelerio macranthae-Stipetum joannis mit fünf Subassoziationen:verbascetosum lychnitis, stipetosum pulcherrimae, stipetosum smirnovii, stipetosum glabratae undstipetosum tirsae undErysimo crepidifolii-Festucetum valesiacae dictamnetosum albae. Alle Syntaxa sind mit Aufnahmematerial belegt und ihre Sukzessions- und topographischen Beziehungen werden beschrieben.     

D2O-induced ion channel activation in Characeae at low ionic strength

Effects of D₂O were studied on internodal cells of the freshwater alga Nitellopsis obtusa under plasmalemma perfusion (tonoplast-free cells) with voltage clamp, and on Ca²⁺ channels isolated from the alga and reconstituted in bilayer lipid membranes (BLM). External application of artificial pond water (APW) with D₂O as the solvent to the perfused plasmalemma preparation led to an abrupt drop of membrane resistance (R _m = 0.12 ±0.03 kΩ · cm²), thus preventing further voltage clamping. APW with 25% D₂O caused a two-step reduction of R _m : first, down to 2.0 ± 0.8 kΩ · cm², and then further to 200 Ω · cm², in 2 min. It was shown that in the first stage, Ca²⁺ channels are activated, and then, Ca²⁺ ions entering through them activate the Cl^? channels. The Ca²⁺ channels are activated irreversibly. If 100 mm CsCl was substituted for 200 mm sucrose (introduced for isoosmoticity), no effect of D₂O on R _m was observed. Intracellular H₂O/D₂O substitution also did not change R _m . In experiments on single Ca²⁺ channels in BLM H₂O/ D₂O substitution in a solution containing 100 mm KCl (trans side) produced no effect on channel activity, while in 10 mm KCl, at negative voltage, the open channel probability sharply increased. This effect was irreversible. The single channel conductance was not altered after the H₂O/D₂O substitution. The discussion of the possible mechanism of D₂O action on Ca²⁺ and Cl^? channels was based on an osmotic-like stress effect and the phenomenon of higher D-bond energy compared to the H-bond.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

Overexpression of d-psicose 3-epimerase from Ruminococcus sp. in Escherichia coli and its potential application in d-psicose production

The d-psicose 3-epimerase (DPE) gene from Ruminococcus sp. was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and characterized. It was optimally active at pH 7.5–8.0 and 60?°C. Activity was not dependent on the presence of metal ions; however, it became more thermostable with added Mn²⁺. The K _m of the enzyme for d-psicose (48?mM) was lower than that for d-tagatose (230?mM), suggesting that d-psicose is the optimum substrate. More importantly, the thermostability of the novel DPE from Ruminococcus is the strongest among all of the d-psicose and d-tagatose 3-epimerases and may be suitable for the industrial production of d-psicose from fructose.