Effects of Tryptophan and 5-Hydroxytryptophan on the Hepatic Cell Membrane Rigidity Due to Oxidative Stress

The ability of several indoleamines to scavenge free radicals is well documented. Our aim was to evaluate the ability of 0.01–3 mm tryptophan (Trp) and 0.1–5 mm 5-hydroxytryptophan (5-OH-Trp) to protect hepatic cell membranes against 0.1 mm FeCl₃ plus 0.1 mm ascorbic acid–induced lipid peroxidation and increases in membrane rigidity. Membrane fluidity was evaluated using fluorescence spectroscopy. Lipid and protein oxidation were estimated by quantifying malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HDA) concentrations and carbonyl group content, respectively. Exposure to FeCl₃ plus ascorbic acid increased hepatic cell membrane rigidity, MDA + 4-HDA and carbonyl content. The presence of 5-OH-Trp, but not Trp, attenuated these changes. In the absence of oxidative stress, neither indoleamine modified fluidity, MDA + 4-HDA or carbonylation. These results suggest that C5 hydroxylation determines the ability of Trp to preserve membrane fluidity in the presence of oxidative stress.     

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.     

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.     

Bacidina genus novum familiaeLecideaceae s. lat. (Ascomycetes lichenisati)

The following eleven species currently classified in the generaBacidia s. lat. andCatillaria s. lat. are transferred to the new genusBacidina Vězda gen. n. (Lecideaceae s. lat.):Bacidina apiahica (Müll. Arg.) comb. n.,B. chloroticula (Nyl.)Vězda etPoelt comb. n.,B. egenula (Nyl.) comb. n.,B. inundata (Fr.) comb. n.,B. mirabilis (Vězda) comb. n.,B. neglecta (Vězda) comb.n.,B. pallidocarnea (Müll. Arg.) comb. n.,B. phacodes (Koerb.) comb.n.,B. scutellifera (Vězda) comb.n.,B. vasakii (Vězda) comb.n., andB. ziamensis (Vězda) comb.n.     

An l-glucitol oxidizing dehydrogenase from Bradyrhizobium japonicum USDA 110 for production of d-sorbose with enzymatic or electrochemical cofactor regeneration

A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an l-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consisting of 242 amino acids with a molecular mass of 26.1 kDa. The heterologously expressed protein not only exhibited the main enantio selective activity with d-glucitol oxidation to d-fructose but also converted l-glucitol to d-sorbose with enzymatic cofactor regeneration and a yield of 90 %. The temperature stability and the apparent K _m value for l-glucitol oxidation let the enzyme appear as a promising subject for further improvement by enzyme evolution. We propose to rename the enzyme from the annotated RDH gene (locus tag bll6662) from B. japonicum USDA as a d-sorbitol dehydrogenase (EC 1.1.1.14).     

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.     

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.     

High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases

The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form d(?)-tartaric acid or l(+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[d] and CESH[l], producing d(?)- and l(+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver?CBurk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[l] showed much higher catalytic efficiency than CESH[d], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[l] and CESH[d] have different structural folds and potential active site residues. CESH[l] adopted a typical ??/??-hydrolase fold with a cap domain and a core domain, whereas CESH[d] possessed a unique TIM barrel fold composed of 8 ??-helices and 8 ??-strands, and 2 extra short ??-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[d], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.     

Untersuchungen über den PolyploidkomplexVeronica cymbalaria agg. (Scrophulariaceae)

Veronica panormitana Tin. inGuss. (sparsely distributed from Corsica and Sicily to Syria) andV. trichadena Jord. etFourr. var.freyniana M. Fisch. (Mallorca) are diploid (2n = 18), and very probably the ancestors of the allopolyploidV. cymbalaria Bod. The latter consists of tetraploid (2n = 36) and hexaploid (2n = 54) strains (confirmed on new material from Italy, Greece, and Turkey). The diploid taxa are morphologically well separated from each other and fromV. cymbalaria, whereas the tetraploids and hexaploids are not separable morphologically.—Another poorly known diploid strain from Kurdistan is described as new:Veronica cymbalaria Bod. var.baradostensis M. Fischer.—V. panormitana is reported from Greece for the first time.     

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.     

ClassAsteretea tripolium on the territory of the former USSR and Mongolia

Higher syntaxa of the classAsteretea tripolium are reviewed. This class covers plant communities of the Euro-Asian continent on low- and medium-salinized soils with medium moisture conditions where perennial herbaceous plants (hemicryptophytes) of non-succulent character prevail. On the territory of the former USSR and Mongolia, the classAsteretea tripolium is represented by the following orders:Glauco-Puccinellietalia Beeftink etWesthoff inBeeftink 1962,Cirsietalia esculenti Mirkin etV. Golub exV. Golub ord. novus,Scorzonero-Juncetalia gerardii Vicherek 1973,Halerpestetalia Mirkin et al. exV. Golub ord. novus,Artemisio santonicae-Limonietalia gmelinii V. Golub etV. Solomakha 1988,Suaedetalia corniculatae V. Golub ord. novus. Superspecies and aggregations of species are used for the diagnosis of higher syntaxa.     

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.     

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.     

Saprobitätsbestimmung nach den larven und puppen von trichoptera

On the basis of a rich material of larvae and pupae of caddis-flies collected in two south-moravian rivulets Jihlava and Bobrava, the methods of estimation of saprobity used in Czechoslovakia were compared. These were the methods of saprobic valency by Zelinka & Marvan (1961) complemented by the saprobic index Sr of Rothschein (1959), saprobic index according to Pantle & Buck (1955) and a method by Obr (1956). The locality on the Jihlava rivulet near Iváň was considered as epipotamon, the locality on the Bobrava rivulet near Želešice was taken as hyporhitron in the sense of the biocoenotical classification of Illies & Botosaneanu (1963). The most common Trichoptera are shown on Figs. 1 and 2. The procedure of estimation is presented in Tab. 1–6, that of comparison in Tab. 7. By all methods applied it was demonstrated that the Jihlava rivulet is beta-mesosaprobic and the Bobrava rivulet in the transition between oligosaprobity to beta-mesosaprobity. All methods are leading to nearly identical results, but the method of saprobic valency gives the most exact numbers and in connection with the index S_R enables a graphical presentation. The method of Pantle & Buck is less precise, but quicker with the possibility of immediate graphical presentation. The author presumes that it is possible to estimate the saprobity according to one dominant group of organisms, if the investigation is long-termed and if there are enough species with a known saprobic valency.     

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.     

l-Carnitine is essential to β-oxidation of quarried fatty acid from mitochondrial membrane by PLA2

Mitochondrial β-oxidation is an important system involved in the energy production of various cells. In this system, the function of l-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O₂ consumption without substrates, is caused by l-carnitine treatment. In this study, we investigated whether l-carnitine is essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A₂ (PLA₂) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and l-carnitine. The effect of l-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with l-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of l-carnitine. Moreover, the l-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA₂ inhibitors were treated before ADP treatment. The l-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that l-carnitine might be essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by PLA₂.     

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.