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.     

Whole cell K and Cl currents in dissociated eccrine secretory coil cells during stimulation

Using the whole-cell voltage clamp (to determine the membrane current) and current clamp (to determine membrane potential) methods in conjunction with the nystatin-perforation technique, we studied the effect of methacholine (MCh) and other secretagogues on whole cell K and Cl currents in dissociated rhesus palm eccrine sweat clear cells. Application of MCh by local superfusion induced a net outward current (at a holding potential of ?60 mV and a clamp voltage of 0 mV), and a transient hyperpolarization by 5.6 mV, suggesting the stimulation of K currents. The net outward current gradually changed to the inward (presumably Cl) currents over the next 1 to 2 min of continuous MCh stimulation. During this time the membrane potential also changed from hyperpolarization to depolarization. The inward currents were increasingly more activated than outward (presumably K) currents during repeated MCh stimulations so that a net inward current (at ?60 mV) was observed after the fourth or fifth MCh stimulation. Ionomycin (10 μm) also activated both inward and outward current. The observed effect of MCh was abolished by reducing extracellular [Ca] to below 1 nm (Ca-free + 1 mm EGTA in the bath). MCh-activated outward currents were inhibited by 5 mm Ba and by 0.1 mm quinidine, although these agents also suppressed the inward currents. Bi-ionic potential measurements indicated that the contribution of Na to the membrane potential was negligible both before and after MCh or ISO (isoproterenol) stimulations and that the observed membrane current was carried mainly by K and Cl. MCh increased the bi-ionic potential by step changes in external K and Cl concentrations, further supporting that MCh-induced outward and inward currents represent K and Cl currents, respectively. Stimulation with ISO or FK (forskolin) resulted in a depolarization by about 55 mV and a net inward (most likely Cl) current independent of external Ca. CT-cAMP mimicked the effects of FK and ISO. The bi-ionic potential, produced by step changes in the external Cl concentration, increased during ISO stimulation, whereas that of K decreased. This indicates that the ISO-induced inward current is due to Cl current and that K currents were unchanged or slightly decreased during stimulation with ISO or 10 μm FK. Both myoepithelial and dark cells responded only to MCh (but not to FK) with a marked depolarization of the membrane potential due to activation of Cl, but not K, currents. We conclude that MCh stimulates Ca-dependent K and Cl currents, whereas ISO stimulates cAMP-dependent Cl currents in eccrine clear cells.     

Small-conductance chloride channels in human peripheral T lymphocytes

During whole-cell patch-clamp recording from normal (nontransformed) human T lymphocytes a chloride current spontaneously activated in >98% of cells (n > 200) in the absence of applied osmotic or pressure gradients. However, some volume sensitivity was observed, as negative pressure pulses reduced the current. With iso-osmotic bath and pipette solutions the peak amplitude built up (time constant ≈23 sec at room temperature), a variable-duration plateau phase followed, then the current ran down spontaneously (time constant ≈280 sec). The anion permeability sequence, calculated from reversal potentials was I^?, Br^? > NO ₃ ^? , Cl^? > CH₃SO ₃ ^? , HCO ₃ ^? > CH₃COO^? > F^? > aspartate, gluconate, SO ₄ ^2? and there was no measurable monovalent cation permeability. The Cl^? current was independent of time during long voltage steps and there was no evidence of voltage-dependent gating; however, the current showed intrinsic outward rectification in symmetrical Cl^? solutions. The conductance of the channels underlying the whole-cell current was calculated from fluctuation analysis, using power-spectral density and variance-vs.-mean analysis. Both methods yielded a single channel conductance of about 0.6 pS at ?70 mV (close to the normal resting potential of T lymphocytes). The power spectral density function was best fit by the sum of two Lorentzian functions, with corner frequencies of 30 and 295 Hz, corresponding to mean open times of 0.54 and 5.13 msec. The pharmacological profile included rapid block by external application of flufenamic acid (50 μm), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 μm, [6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5-y1) oxy] acetic acid (IAA-94, 250 μm) or 100 μm 1,9-dideoxyforskolin. The stilbene derivatives DIDS (4,4′-diisothiocyano-2,2′ di-sulphonic acid stilbene, 500 μm) and SITS (4-acetamido-4′-isothiocyano-2, 2′-disulphonic acid stilbene, 500 μm) prevented buildup of Cl^? current after a 30-min preincubation at 500 μm. When tested in a mitogenic assay, DIDS, flufenamic acid, NPPB and IAA-94 all inhibited T-cell proliferation, suggesting a physiological function in addition to the observed volume sensitivity.     

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.     

Erwägungen über die Taxonomie und Nomenklatur der GattungActinotaenium Teil

In der botanischen Literatur werden oft neue Kombinationen und neue Namen ungültig publiziert, weil die Vorschriften des Art. 33 d. Int. Codes nicht eingehalten werden. Die Verfasser verfolgen diese Frage bei der GattungActinotaenium Teiling 1954 (Fam.Desmidiaceae, KlasseConjugatophyceae der Grünalgen), weisen auf die Unklarheiten und Unrichtigkeiton in ihrer Systematik hin und sind bestrebt, diese zu verbessern, soweit es bei heutigem Zustand der Kenntnisse möglich ist. Im Artikel ist ein neues TaxonActinotaenium messikommeri R??i?ka etPouzar beschrieben und folgende neue Kombinationen veröffentlicht;Actinotaenium adelochondrum var.kriegeri (Messik.) R??i?ka, A.angulatum (W. etG. S. West)R??i?ka etPouzar,A. angulatum var.brasiliense (Grönblad) R??i?ka etPouzar,A. cordanum (Bréb.) R??i?ka etPouzar,A. gelidum (Wittr.) R??i?ka,A. heterotaphridium (W. etG. S. West)R??i?ka etPouzar undA. hibernicum (W. West) R??i?ka.     

The trilobiteProtostygina and the composition of the Styginidae, with two new genera

The Styginidae is regarded as an exclusively Ordovician family of trilobites, separate from the Scutelluidae. The hitherto poorly known genusProtostygina Prantl &P?ibyl, 1949 is revised. It is recorded with certainty only from the Llanvirn of the Czech Republic, and the type species is a senior synonym of“Raymondaspis” rubensi rubensi P?ibyl &VANěK, 1968 and“R.” rubensi lybar ?najdr, 1976. Two new styginid genera are proposed:Cyrtocybe, with type species“Raymondaspis” turgida Whittington, 1965, is known from the upper Arenig and lower Llanvirn of Newfoundland, Maine and Norway; andPromargo, with type speciesP.forteyi n. sp., occurs in the Arenig of Newfoundland and Spitsbergen.Turgicephalus Fortey, 1980 is regarded as a junior synonym ofRaymondaspis P?ibyl inPrantl &P?ibyl, 1949. Three genera are excluded from the Styginidae:Kirkdomina Tripp, 1962,Pseudostygina Zhou inZhou et al., 1982 andStyginella P?ibyl &Vaněk, 1971.     

Observation of algal colonization on Acropora aspera killed by Acanthaster planci

Algae started colonizing branches of the coral Acropora aspera (Dana) killed by the sea star Acanthaster planci (Linnaeus) within less than 24 hours. Two blue-greens ((Microcoleus lyngbyaceus (Crouan) Ag. and Hormothamnion solutum B. & F.)) dominated the early community but became less abundant than a brown ((Giffordia indica (Sonder) Papenfuss & Chihara)) after 26 days.     

Holocene anthropogenic landscapes in the Balkans: the palaeobotanical evidence from southwestern Bulgaria

Palaeoecological reconstructions from the region of southwestern Bulgaria were used for inferring the human impact on the vegetation and landscape during the last 8 millennia. They are based on data from pollen analyses of lakes and peat-bogs, plant macrofossils, archaeobotanical finds and radiocarbon dating. During the early Holocene, after 7900?cal. b.p. (5950?cal. b.c.) the climate changed to cooler summers, milder winters and higher precipitation resulting in the formation of a coniferous belt dominated by Pinus sp. and Abies alba. These favorable environmental pre-conditions had a positive influence on the Neolithisation of the Balkans after the 8200?cal. b.p. (6250?cal. b.c.) cold event, which caused drought in the Eastern Mediterranean. Direct evidence from wood charcoal records from the Neolithic settlement layers in the study area shows a slight modification of the surrounding woodlands and an increase of the light-demanding components, probably expressed through larger forest border zones and thinning out of the wood stands. The increase in the number of settlements in the valleys of southwestern Bulgaria intensified the human activity visible in the palaeobotanical record from 6950?cal. b.p. (5000?cal. b.c.) onwards. Between ca. 5700–5100?cal. b.p. (3800–3200?cal. b.c.) signs of anthropogenic influence on the vegetation are virtually absent. The intensity of human impact increased notably after 3200?cal. b.p. (1400–1250?cal. b.c., approx. Late Bronze Age), documented by a rise of pollen anthropogenic indicators. The final transformations in the natural forest cover after 2750?cal. b.p. (800?cal. b.c. onset of the Iron Age) marked the reduction of the coniferous forests dominated by Abies alba and Pinus sp. and the expansion of Fagus sylvatica and Picea abies. These vegetation changes are contemporaneous with increase of the palaeofire activities and the next peak of anthropogenic indicators. The changes in the landscape during the Roman period and the medieval period reflect regional environmental features and were forced by the diversification of anthropogenic activity.     

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.     

Chromosome counts and chromosome morphology of some selected species

In the years 1976–1981 we studied chromosome counts and karyotypic formulae of the following 29 species of plants from 41 localities (of these 6 from Bohemia, 32 from Moravia, 3 from Slovakia):Batrachium baudotii (Godron) F. W. Schultz,Chenopodium rubrum L.,C. polyspermum L.,C. murale L.,C. ficifolium Sm.,C. opulifolium Schrader ex DC. inLam. et DC.,C. strictum Roth [subsp.strictum, subsp.glaucophyllum (Aellen)Aellen inJust etAellen, subsp.striatiforme Uotila],Arenaria grandiflora L.,Illecebrum verticillatum L.,Spergula morisonii Boreau inDuchartre,Spergularia marginata (DC. inLam. et DC.)Kittel S. marina (L.)Griseb.,S. rubra (L.) J. etC. Presl,Silene conica L.,Sisymbrium loeselii L.,S. volgense Bieb. exE. Fourn.,S. orientale L. [subsp. orientale, subsp.macroloma (A. Pomel)Dvo?ák],S. officinale (L.)Scop.,Descurainia sophia (L.)Webb exPrantl inEngler etPrantl,Nasturtium officinale R. Br. inAiton,Barbarea arcuata (Opiz inPresl J. et C.)Reichenb.,Lunaria annua L.,Soldanella montana Willd.,S. carpatica Vierh. inUrban etGraebner,Lotus tenuis Waldst. etKit. exWilld.,L. uliginosus Schkuhr,Trigonella monspeliaca L.,Geranium sibiricum L.,Lactuca tatarica (L.)C. A. Meyer.     

Bemerkungen zur Variabilität vonSaxifraga marginata

Saxifraga marginata Sternb. is divided into three subspecies:S. m. subsp.marginata, S. m. subsp.bubakii (Rohlena)Chrtek etSoják,S. m. subsp.karadzicensis (Degen etKo?anin)Chrtek etSoják (environments of Skopje, Macedonia).     

Effects of l-lysine and d-lysine on ɛ-Poly-l-lysine biosynthesis and their metabolites by Streptomyces ahygroscopicus GIM8

?-Poly-l-lysine (?-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of l-lysine, which is used as a safe food preservative. In this study, the effects of l-lysine and its isomer, d-lysine, on ?-PL biosynthesis and their metabolites by the ?-PL-producing strain Streptomyces ahygroscopicus GIM8 were determined. The results indicated that l-lysine added into the fermentation medium in the production phase mainly served as a precursor for ?-PL biosynthesis during the flask culture phase, leading to greater ?-PL production. At an optimum level of 3 mM l-lysine, a ?-PL yield of 1.16 g/L was attained, with a 41.4% increment relative to the control of 0.78 g/L. Regarding d-lysine, the production of ?-PL increased by increasing its concentrations up to 6 mM in the initial fermentation medium. Interestingly, ?-PL production (1.20 g/L) with the addition of 3 mM d-lysine into the initial fermentation medium in flasks was higher than that of the initial addition of 3 mM L-lysine (1.06 g/L). The mechanism by which d-lysine improves ?-PL biosynthesis involves its utilization that leads to greater biomass. After S. ahygroscopicus GIM8 was cultivated in the defined medium with L-lysine, several key metabolites, including 5-aminovalerate, pipecolate, and l-2-aminoadipate formed in the cells, whereas only l-2-aminoadipate was observed after d-lysine metabolism. This result indicates that l-lysine and d-lysine undergo different metabolic pathways in the cells. Undoubtedly, the results of this study are expected to aid the understanding of ?-PL biosynthesis and serve as reference for the formulation of an alternative approach to improve ?-PL productivity using l-lysine as an additional substrate in the fermentation medium.     

Draba skrivanekii,eine interessante Art aus Montenegro (Jugoslawien)

In den Herbarien der botanischen Abteilung des Nationalmuseums in Pr?honice (PR) werden zwei Belege aufbewahrt, die vonRohlena alsDraba athoa Boiss. bezeichnet sind. Später ordneteRohlena selbst diese Pflanzen der ArtD. elongata Host mit Bezeichnung var.skrivanekii Rohl. zu. Durch eine Analyse des Herbarmaterials hat sich erwiesen, dass es sich um eine selbständige Art—Draba skrivanekii (Rohl.)Chrtek—handelt, die in den Gebirgslagen Montenegros (Kom Vasojevi?ki) wahrscheinlich endemisch ist. Die nächstverwandte Art istDraba athoa (Griseb.)Boiss.     

The variability of critical species ofLotus L. in Iran and in the neighbouring countries II

The paper deals with some problems of distribution and variability of the speciesL. angustissimus L.,L. halophilus Boiss. etSprun.,L. laricus Rech. fil.,Aellen etEsfandiari, L.,schimperi Steud.,L. compactus Chrtková-?ertová,L. garcinii DC.     

Engineered Bacillus subtilis 168 produces l-malate by heterologous biosynthesis pathway construction and lactate dehydrogenase deletion

In the present work, Bacillus subtilis was engineered to produce l-malate. Initially, the study revealed that the slight fumarase activity under anaerobic conditions is extremely favourable for l-malate one-step fermentation accumulation. Subsequently, an efficient heterologous biosynthesis pathway formed by Escherichia coli phosphoenolpyruvate carboxylase and Saccharomyces cerevisiae malate dehydrogenase was introduced into B. subtilis, which led to 6.04?±?0.19?mM l-malate production. Finally, the l-malate production was increased 1.5-fold to 9.18?±?0.22?mM by the deletion of lactate dehydrogenase. Under two-stage fermentation conditions, the engineered B. subtilis produced up to 15.65?±?0.13?mM l-malate, which was 86.3?% higher than that under anaerobic fermentation conditions. Though the l-malate production by the recombinant was low, this is the first attempt to produce l-malate in engineered B. subtilis and paves the way for further improving l-malate production in B. subtilis.     

The Effects of Magnesium, l-Carnitine, and Concurrent Magnesium–l-Carnitine Supplementation in Migraine Prophylaxis

Given the conflicting results about the positive effects of magnesium and l-carnitine and as there is no report concerning concurrent supplementation of magnesium and l-carnitine on migraine prophylaxis, the effects of magnesium, l-carnitine, and concurrent magnesium?Cl-carnitine supplementation on migraine indicators were assessed. In this clinical trial, 133 migrainous patients were randomly assigned into three intervention groups: magnesium oxide (500?mg/day), l-carnitine (500?mg/day), and Mg?Cl-carnitine (500?mg/day magnesium and 500?mg/day?l-carnitine), and a control group. After 12?weeks of supplementation, the checklist of migraine indicators including migraine attacks/month, migraine days/month, and headache severity was completed, and serum concentrations of magnesium and l-carnitine were measured by atomic absorption spectrophotometry and enzymatic UV test, respectively. The results showed a significant reduction in all migraine indicators in all studied groups (p?<?0.05). The ANOVA results showed a significant reduction in migraine frequency across various supplemented and control groups (p?=?0.008). By separating the effects of magnesium supplementation from other confounding factors such as routine treatments using the repeated measures and nested model, it was clarified that magnesium supplementation had a significant effect on all migraine indicators. Oral supplementation with magnesium oxide and l-carnitine and concurrent supplementation of Mg?Cl-carnitine besides routine treatments could be effective in migraine prophylaxis; however, larger trials are needed to confirm these preliminary findings.     

Staudengesellschaften mitGeranium sanguineum undTrifolium medium in der (sub)montanen Stufe des Walliser Rhônetals (Schweiz)

Die Arbeit beschreibt die synsystematische Stellung und die primären Standortsfaktoren der xerothermen Staudengesellschaften im Wallis und in angrenzender Waadt (Schweizer Zentral-Alpen). Diese Gesellschaften sind alsBrachypodio pinnati-Geranion sanguinei Tx. inMüller 1962 emvan Gils etKoz?owska 1977,Brachypodietalia Korneck 1974 undFestuco-Brometea Br.-Bl. etTx. 1943 klassifiziert.     

New and critical species ofVicia in Iran and neighbouring countries 1. TheVicia variegata group

In Iran and the neighbouring regions of Turkey, Iraq, U.S.S.R. and Afghanistan, eight already known species belonging to the subsectionVariegatae Radzhi, occur:V. persica Boiss.,V. armena Boiss.,V. variegata Willd.,V. akhmaganica Kazar.,V. gregaria Boiss. etHeldr.,V. aucheri Jaub. etSpach and the two new species described here,V. rechingeri Chrtková-?ertová andV. afghanica Chrtková-?ertová. The occurrence of most species is restricted to a limited area which may be one of the evolutionary centres of this subsection.     

Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.