A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation

The inward rectifier K(+) channel Kir2.1 mediates the potassium I(K1) current in the heart. It is encoded by KCNJ2 gene that has been linked to Andersen's syndrome. Recently, strong evidences showed that Kir2.1 channels were associated with mouse atrial fibrillation (AF), therefore we hypothesized that KCNJ2 was associated with familial AF. Thirty Chinese AF kindreds were evaluated for mutations in KCNJ2 gene. A valine-to-isoleucine mutation at position 93 (V93I) of Kir2.1 was found in all affected members in one kindred. This valine and its flanking sequence is highly conserved in Kir2.1 proteins among different species. Functional analysis of the V93I mutant demonstrated a gain-of-function consequence on the Kir2.1 current. This effect is opposed to the loss-of-function effect of previously reported mutations in Andersen's syndrome. Kir2.1 V93I mutation may play a role in initiating and/or maintaining AF by increasing the activity of the inward rectifier K(+) channel.     

Biodeterioration of some herbal raw materials by storage fungi and aflatoxin and assessment of Cymbopogon flexuosus essential oil and its components as antifungal

Deterioration of raw materials of six medicinal plants viz. Terminalia arjuna, Acorus calamus, Rauvolfia serpentina, Holarrhena antidysenterica, Withania somnifera and Boerhaavia diffusa was examined. Some of the contaminated raw materials were found to be deteriorated by toxigenic strains of Aspergillus flavus and contain aflatoxin B₁ (41.0–95.4 μg kg⁻¹) which is above the permissible limit. Essential oil of Cymbopogon flexuosus and its components was found efficient in checking fungal growth and aflatoxin production. C. flexuosus essential oil absolutely inhibited the growth of A. flavus and aflatoxin B₁ production at 1.3 μl ml⁻¹ and 1.0 μl ml⁻¹ respectively. The individual oil components were more efficacious than the Cymbopogon oil as such which emphasizes masking of their efficacy when combined together. Eugenol exhibited potent antifungal and aflatoxin inhibitory activity at 0.3 μl ml⁻¹ and 0.1 μl ml⁻¹ respectively. Eugenol was found superior over some prevalent synthetic antimicrobials and exhibited broad fungitoxic spectrum against some biodeteriorating moulds. Prospects of exploitation of the oil and its components as acceptable plant based antimicrobials in qualitative as well as quantitative control of biodeterioration of herbal raw materials have been discussed.     

Purification and characterisation of the cardenolide-specificβ-glucohydrolase CGH II from Digitalis lanata leaves

A cardenolide-hydrolysing β-D-glucosidase was isolated from young leaves of Digitalis lanata. Since this enzyme differs from the cardenolide glucohydrolase (CGH) described and characterised previously, it was termed cardenolide glucohydrolase II (CGH II). CGH II was detected in various Digitalis tissue cultures as well as in young leaves of D. lanata. The latter source was used as the starting material for the isolation and purification of CGH II. The specific enzyme activity reached about 15 pkat·mg^–1 protein in buffered leaf extracts. Optimal CGH II activity was seen at around pH 6.0 and 50 °C. CGH II was purified about 600-fold by anion exchange chromatography, size exclusion chromatography and hydroxyapatite chromatography. The apparent molecular mass of CGH II was 65 kDa as determined by SDS-PAGE. CGH II exhibited a high substrate specificity towards cardenolide disaccharides, especially to those with a 1-4-β-linked glucose-digitoxose moiety such as glucoevatromonoside. The K_m- and V_max-values for this particular substrate were calculated to be 101 μM and 19.8 nkat·mg^–1 protein, respectively.     

The Interaction of Caveolin 3 Protein with the Potassium Inward Rectifier Channel Kir2.1: PHYSIOLOGY AND PATHOLOGY RELATED TO LONG QT SYNDROME 9 (LQT9)*

Mutations in CAV3 cause LQT syndrome 9 (LQT9). A previously reported LQT9 patient had prominent U waves on ECG, a feature that has been correlated with Kir2.1 loss of function. Our objective was to determine whether caveolin 3 (Cav3) associates with Kir2.1 and whether LQT9-associated CAV3 mutations affect the biophysical properties of Kir2.1. Kir2.1 current (I_K1) density was measured using the whole-cell voltage clamp technique. WT-Cav3 did not affect I_K1. However, F97C-Cav3 and T78M-Cav3 decreased I_K1 density significantly by ∼60%, and P104L-Cav3 decreased I_K1 density significantly by ∼30% at −60 mV. Immunostained rat heart cryosections and HEK293 cells cotransfected with Kir2.1 and WT-Cav3 both demonstrated colocalization of Kir2.1 and WT-Cav3 by confocal imaging. Cav3 coimmunoprecipitated with Kir2.1 in human ventricular myocytes and in heterologous expression systems. Additionally, FRET efficiency was highly specific, with a molecular distance of 5.6 ± 0.4 nm, indicating close protein location. Colocalization experiments found that Cav3 and Kir2.1 accumulated in the Golgi compartment. On-cell Western blot analysis showed decreased Kir2.1 cell surface expression by 60% when expressed with F97C-Cav3 and by 20% when expressed with P104L-Cav3 compared with WT-Cav3. This is the first report of an association between Cav3 and Kir2.1. The Cav3 mutations F97C-Cav3, P104L-Cav3, and T78M-Cav3 decreased I_K1 density significantly. This effect was related to a reduced cell surface expression of Kir2.1. Kir2.1 loss of function is additive to the increase described previously in late I_Na, prolonging repolarization and leading to arrhythmia generation in Cav3-mediated LQT9.     

V1 receptor in ENS mediates the excitatory effect of vasopressin on circular muscle strips of gastric body in vitro in rats

The purpose of this study was to localize vasopressin (VP) V_1a receptor in stomach and to characterize the role of VP in the regulation of gastric motility in rats. Double staining was used to locate the V_1a receptor in the gastric body of the rat. The contraction of the circular muscle strips of gastric body was monitored by a polygraph. V_1a receptor was expressed on the neurons of myenteric plexus of the gastric body. VP (10^− 10–10^− 6 M) caused a concentration-dependent contractile effect on the circular muscle strips of gastric body in vitro. V-1880 ([deamino-Pen¹, O-Me-Tyr², Arg⁸]-Vasopressin, 10^− 7 M), a V₁ receptor antagonist, inhibited the spontaneous contraction of the strips. Tetradotoxin (TTX, 10^− 6 M) and V-1880 (10^− 7 M) abolished the excitatory effect of VP. Atropine (10^− 6 M) partially inhibited VP-induced excitatory effect on the muscle strips but hexamethonium (10^− 4 M) did not influence it. These results suggest that V_1a receptor was expressed on the neurons of myenteric nerves. The cholinergic nerve was involved in the excitatory effect of VP on the contraction of gastric body.     

Properties of a novel β-glucosidase from Fusarium proliferatum ECU2042 that converts ginsenoside Rg3 into Rh2

A novel β-glucosidase from Fusarium proliferatum ECU2042 (FPG) was successfully purified to homogeneity with a 506-fold increase in specific activity. The molecular mass of the native purified enzyme (FPG) was estimated to be approximately 78.7 kDa, with two homogeneous subunits of 39.1 kDa, and the pI of this enzyme was 4.4, as measured by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50 °C, respectively. The enzyme was stable at pH 4.0–6.5 and temperatures below 60 °C, and the deactivation energy (E_d) for FPG was 88.6 kJ mo1⁻¹. Moreover, it was interesting to find that although the purified enzyme exhibited a very low activity towards p-nitrophenyl β-d-glucoside (pNPG), and almost no activity towards cellobiose, a relatively high activity was observed on ginsenoside Rg₃. The enzyme hydrolyzed the 3-C, β-(1 → 2)-glucoside of ginsenoside Rg₃ to produce ginsenoside Rh₂, but did not sequentially hydrolyze the β-d-glucosidic bond of Rh₂. The K_m and V_max values of FPG for ginsenoside Rg₃ were 2.37 mM and 0.568 μmol (h mg protein)⁻¹, respectively. In addition, this enzyme also exhibited significant activities towards various alkyl glucosides, aryl glucosides and several natural glycosides.     

Regulation of Cardiac Inward Rectifier Potassium Current (IK1) by Synapse-associated Protein-97

Synapse-associated protein-97 (SAP97) is a membrane-associated guanylate kinase scaffolding protein expressed in cardiomyocytes. SAP97 has been shown to associate and modulate voltage-gated potassium (Kv) channel function. In contrast to Kv channels, little information is available on interactions involving SAP97 and inward rectifier potassium (Kir2.x) channels that underlie the classical inward rectifier current, I_K1. To investigate the functional effects of silencing SAP97 on I_K1 in adult rat ventricular myocytes, SAP97 was silenced using an adenoviral short hairpin RNA vector. Western blot analysis showed that SAP97 was silenced by ∼85% on day 3 post-infection. Immunostaining showed that Kir2.1 and Kir2.2 co-localize with SAP97. Co-immunoprecipitation (co-IP) results demonstrated that Kir2.x channels associate with SAP97. Voltage clamp experiments showed that silencing SAP97 reduced I_K1 whole cell density by ∼55%. I_K1 density at −100 mV was −1.45 ± 0.15 pA/picofarads (n = 6) in SAP97-silenced cells as compared with −3.03 ± 0.37 pA/picofarads (n = 5) in control cells. Unitary conductance properties of I_K1 were unaffected by SAP97 silencing. The major mechanism for the reduction of I_K1 density appears to be a decrease in Kir2.x channel abundance. Furthermore, SAP97 silencing impaired I_K1 regulation by β₁-adrenergic receptor (β1-AR) stimulation. In control, isoproterenol reduced I_K1 amplitude by ∼75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that β1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and β1-AR. SAP97 immunolocalizes with protein kinase A and β1-AR in the cardiac myocytes. Our results suggest that in cardiac myocytes SAP97 regulates surface expression of channels underlying I_K1, as well as assembles a signaling complex involved in β1-AR regulation of I_K1.     

SO2-induced decrease in photosynthetic activity in two barley cultivars. Evidence against specific damage at the protein-pigment complex level

This research investigated the SO₂-induced effects on photosynthetic apparatus in two barley (Hordeum vulgare L.) cultivars (cv), Panda and Express. Following a chronic treatment with SO₂ (80 ppb, 75 d) neither cv showed any visible signs of injury or chlorosis on leaf surfaces, while a significant reduction in A_max and G_w was detected in both cvs, although to different extents. Thylakoids of SO₂-treated plants showed a decrease in the electron transport activity at the whole chain, photosystem II (PSII) and photosystem I (PSI) level in both cvs. The high performance liquid chromatography (HPLC) analysis of leaf pigments revealed a significant decrease in both cvs, more pronounced in Panda than in Express. Deriphat-polyacrilamide gel electrophoresis (Deriphat-PAGE) and two dimensional (2-D) electrophoretic analyses of the pigment-protein complexes did not show differences in SO₂-treated samples of either cv. HPLC analysis of the green bands also showed no differences in the pigment content of fumigated samples of either cv, except for a decrease in β-carotene content and xanthophyll cycle pigment (VAZ) content respectively at band 1 (PSI) and band 5 (minor light-harvesting polypeptides of PSII) level in cv Panda, where the de-epoxidation index (DEP) significantly increased, while in Express, an increase in VAZ content and DEP value of band 5 was observed. These results suggest that the decrease in the photosynthetic activity can be ascribed, in addition to stomata closure induced by SO₂, to a generalised, rather than specific, pollutant effect on photosynthetic apparatus, which could be interpreted as an adaptation to the adverse environment.     

Antagonism of Aeromonas hydrophila by propolis and its effect on the performance of Nile tilapia, Oreochromis niloticus

Propolis, a resinous substance collected by Apis mellifera bees from various plant sources and mixed with secreted beeswax, is a multifunctional material used by bees in the construction, maintenance, and protection of their hives. The collected propolis sample, from High Egypt, was dark-green with olive-odor. The minimal inhibition concentration (MIC) of propolis-ethanolic-extract, against Aeromonas hydrophila, was 80 μg Propolis-ethanolic-extract and crude propolis (1%) were added to artificial basal diet with (30% crude protein) to evaluate their efficacy on the fish growth-performance, immunostimulation and resistance to A. hydrophila. Two hundred and twenty-five Oreochromis niloticus (8 ± 0.45 g/fish) were divided into three equal treatments (T) of triplet replicates. The fish of T₁ were fed on basal diet (control). The fish of T₂ were given the basal diet, containing propolis-ethanolic-extract. The fish of T₃ were given the basal diet containing crude propolis for 28 day. The fish were intraperitoneally challenged by A. hydrophila (0.2 × 10⁷ cells ml⁻¹) at the end of the feeding period and kept for 15 more days.The best growth rate and feed conversion ratio were obtained with T_2. The increase in the average daily gain, specific growth rate and feed efficiency ratio were highly significances in T₂ followed by T₃ when compared with the control group. The HCT-level and monocyte-counts were increased (T₂). No significant change, in the large lymphocytic-count was found among the three treatments (28–27–28%), while the neutrophil-count was significantly decreased (7%) with T₂ and increased (13.11%) with the control. A significant increase in serum lysozyme and serum bactericidal activities was found with T₂. The RLP against A. hydrophila was high with T₂ and T₃.The propolis-ethanolic-extract enhanced the growth, immunity and resistance of O. niloticus against A. hydrophila more than the crude propolis.     

Ascaris suum NADH-methemo(myo)globin reductase systems recovering differential functions of hemoglobin and myoglobin, adapting to environmental hypoxia

We reported previously that Ascaris suum cytochrome b₅, specifically expressed in this nematode at the adult stage and dually localized in extracellular perienteric fluid and hypodermis, is involved in both perienteric NADH-methemoglobin and cytosolic NADH-metmyoglobin reduction, where cytochrome b₅ functions as an electron carrier between NADH-mediated cytochrome b₅ reductase and substrates, methemo(myo)globins to reduce the nonfunctional globins back to functional ferrous hemo(myo)globins. To further characterize NADH-methemo(myo)globin reductase systems, the midpoint potentials of A. suum perienteric hemoglobin and body wall myoglobin, as well as the affinities of Ascaris methemoglobin and metmyoglobin toward cytochrome b₅, were evaluated using potentiometric titration and surface plasmon resonance techniques, respectively. Midpoint potentials of + 7.2 mV and + 19.5 mV were obtained for Ascaris perienteric hemoglobin and body wall myoglobin, respectively. The affinities of Ascaris perienteric methemoglobin and body wall metmyoglobin toward the nematode cytochrome b₅ were comparable to that for mammalian hemoglobin and cytochrome b₅; association constants were 0.585 × 10³ M^− 1 and 2.32 × 10³ M^− 1, respectively, with rapid equilibration kinetics. These observations highlight the physiological importance of A. suum perienteric NADH-methemoglobin and cytosolic metmyoglobin reductase systems. Differential roles of A. suum perienteric hemoglobin and body wall myoglobin are also discussed from the viewpoint of oxygen homeostasis under hypoxic conditions.     

Identifying populations useful for improving parents of a single cross based on net transfer of alleles

Summary Theory and methods for identifying populations (P _y ) with the highest frequency of favorable dominant alleles not present in an elite single cross (I ₁× I ₂) have been developed recently. During selection, new favorable alleles can be transferred from P _y to either I ₁ or I ₂ only at the risk of losing favorable alleles already present in the single cross. A net improvement (NI) statistic, which estimates the relative number of favorable alleles that can be gained from P _y minus the relative number of favorable alleles that can be lost from I ₁ or I ₂, is presented. NI is calculated as maximum [(I ₁×P _y –I ₁×I ₂)/2,(I ₂×P _y –I ₁×I ₂)/2]. Because I ₁ × I ₂ is constant in an experiment, the method reduces to choosing P _y populations with the best mean performance in combination with either I ₁ or I ₂. For a set of maize (Zea mays L.) grain yield data, NI was highly correlated to three other statistics proposed for choosing populations, namely: (1) minimally biased estimate (l ) of the relative number of favorable dominant alleles present in P _y but not in I ₁ and I ₂; (2) minimum upper bound on l ; and (3) predicted performance of the three-way cross [P _y (I ₁× I ₂)]. While l estimates potential improvement likely to be achieved only through long-term recurrent selection, NI is probably a better predictor of short-term improvement in single-cross performance.A contribution from Lifaco Genetics, a subsidiary of Groupe Limagrain     

Glycan-binding profile of a D-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata

A lectin recognizing D-galactose was purified from the pacific annelid Perinereis nuntia ver. vallata (Polychaeta) by affinity chromatography. Hemagglutinating activity, with a very low titer suggesting the presence of lectin appeared in the supernatant from the homogenization of body with Tris-buffered saline. However, dialyzed supernatant from the precipitate homogenized by galactose in the buffer revealed strong hemagglutinating activity against human erythrocytes. The crude supernatant was applied onto lactosyl–agarose column, and only the supernatant eluted from precipitate with galactose was obtained a galactose-binding lectin with 32 kDa polypeptide was obtained from the supernatant of the precipitate, extracted in presence of galactose. It suggests that the lectin tightly binds with glycoconjugate as endogenous ligand(s) in the tissue. Hemagglutinating activity against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and asialofetuin. Glycan-binding profile of the lectin analyzed by frontal affinity chromatography shows that the lectin recognizes branched complex type N-linked oligosaccharides and both type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) lactosamine. The surface plasmon resonance study of the lectin against asialofetuin showed the k_ass and k_diss values are 5.14 × 10⁴ M^− 1 s^− 1 and 2.9 × 10⁻³ s^− 1, respectively. The partial primary structure of the lectin reveals 182 amino acids with novel sequence.     

Biosorption of acid violet dye from aqueous solutions using native biomass of a new isolate of Penicillium sp.

Laboratory investigation of the potential use of Penicillium sp. as biosorbent for the removal of acid violet dye from aqueous solution was studied with respect to pH, temperature, biosorbent, initial dye concentrations. Penicillium sp. decolourizes acid violet (30 mg l⁻¹) within 12 h agitation of 150 rpm at pH 5.7 and temperature of 35 °C. The pellets exhibited a high dye adsorption capacity (5.88 mg g⁻¹) for acid violet dye over a pH range (4–9); the maximum adsorption was obtained at pH 5.7. The increase of temperature favored biosorption for acid violet, but the optimum temperature was 35 °C. Adsorption kinetic data were tested using pseudo-first-order, pseudo-second-order and kinetic studies showed that the biosorption process follows pseudo-first-order rate kinetics with an average rate constant of 0.312 min⁻¹. Isotherm experiments were conducted to determine the sorbent–desorption behavior of examined dye from aqueous solutions using Langmuir and Freundlich equations. Langmuir parameter indicated a maximum adsorption capacity of 4.32 mg g⁻¹ for acid violet and R_L value of 0.377. Linear plot of log q_e vs log C_e shows that applicability of Freundlich adsorption isotherm model. These results suggest that this fungus can be used in biotreatment process as biosorbent for acid dyes.     

Inward-rectifier K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript> Current in Guinea-pig Ventricular Myocytes Exposed to Hyperosmotic Solutions

Superfusion of heart cells with hyperosmotic solution causes cell shrinkage and inhibition of membrane ionic currents, including delayed-rectifer K⁺ currents. To determine whether osmotic shrinkage also inhibits inwardly-rectifying K⁺ current (I_K1), guinea-pig ventricular myocytes in the perforated-patch or ruptured-patch configuration were superfused with a Tyrodes solution whose osmolarity (T) relative to isosmotic (1T) solution was increased to 1.3–2.2T by addition of sucrose. Hyperosmotic superfusate caused a rapid shrinkage that was accompanied by a negative shift in the reversal potential of Ba²⁺-sensitive I_K1, an increase in the amplitude of outward I_K1, and a steepening of the slope of the inward I_K1-voltage (V) relation. The magnitude of these effects increased with external osmolarity. To evaluate the underlying changes in chord conductance (G_K1) and rectification, G_K1-V data were fitted with Boltzmann functions to determine maximal G_K1 (G_K1max) and voltage at one-half G_K1max (V_0.5). Superfusion with hyperosmotic sucrose solutions led to significant increases in G_K1max (e.g., 28±2% with 1.8T), and significant negative shifts in V_0.5 (e.g., –6.7±0.6 mV with 1.8T). Data from myocytes investigated under hyperosmotic conditions that do not induce shrinkage indicate that G_K1max and V_0.5 were insensitive to hyperosmotic stress per se but sensitive to elevation of intracellular K⁺. We conclude that the effects of hyperosmotic sucrose solutions on I_K1 are related to shrinkage-induced concentrating of intracellular K⁺.     

Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis

Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding α1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative α1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM–CSF in Kloch1 mutant, contained oligosaccharide Man_13–14GlcNAc₂, and in Kloch1 mnn1 mutant, contained oligosaccharide Man_9–11GlcNAc₂, whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man_>30GlcNAc₂. Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes α1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.     

α2-macroglobulin ‘fast’ forms inhibit superoxide production by activated macrophages

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human α₂-macroglobulin converted to its ‘fast’ form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both α₂-macroglobulin-trypsin and α₂-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O₂⁻) while native α₂-macroglobulin had little effect except at high concentration. The α₂-macroglobulin ‘fast’ forms, which bind with a K_d of about 8 nM, inhibited 50% generation of O₂⁻(ID₅₀) at a concentration of 7 nM while α₂-macroglobulin inhibited O₂⁻ production with an ID₅₀ of 141 nM. The ‘fast’ forms of α₂-macroglobulin may play a role in the feedback regulation of inflammatory reactions.     

Almost Pure Iα Cellulose in the Cell Wall of Glaucocystis

Crystalline features of cellulose microfibrils in the cell walls of Glaucocystis (Glaucophyta) were studied by combined spectroscopy and diffraction techniques, and the results were compared with those of Oocystis (Chlorophyta). Although these algae are grouped into two different classes, by the composition of their chloroplasts for instance, their cell walls are quite similar in size and morphology. The most striking features of their cellulose crystallites are that they have the highest cellulose I_α contents reported to date. In particular, the I_α fraction of cellulose from Glaucocystis was found to be as high as 90% from ¹³C NMR analysis. The mode of preferential orientation of cellulose crystallites in their cell walls is also interesting; equatorial 0.53-nm lattice planes were oriented parallel to the cell surface in the case of Glaucocystis, while the 0.62-nm planes were parallel to the Oocystis cell surface. Such a structural variation provides another link to the evolution of cellulose structure, biosynthesis, and its biocrystallization mechanism.     

Effect of oxygen on the growth (yield) of Chlorella vulgaris

The growth yield of Chlorella vulgaris, Y _kJ defined as g cells harvested per kJ of light energy absorbed by the cells, was assessed in a turbidostat culture by varying CO₂ and O₂ partial pressures ( and ). The value of Y _kJ ranged from 3.1×10^-3 to 5.0×10^-3 g cells/kJ under light-limited conditions [ = 1.02.4%, = 065%; total pressure of gas (composed of CO₂, O₂ and N₂)=1 atm]. In the light-limited environment, the algal specific growth rate deteriorated appreciably with the increase of . The deterioration accounts for the above range of Y _kJ observed. The growth inhibition due to oxygen that was defined by subtracting from 1.0 the ratio of at given values of to that at = 0% extended from 0.07–0.30 (7–30%). However, glycolate could not be detected in the turbidostat culture. Isotopic experiments on the specific rate of ¹⁴CO₂ uptake also revealed that the inhibition due to oxygen was from 22–38% when was varied from 0 to nearly 100%. These effects of oxygen were discussed, referring to the activity of ribulose-1,5-bisphosphate carboxylase that is inhibited competitively by oxygen.Non-Standard Abbreviations INH isonicotinic acid hydrazide - PPO 2,5-diphenyloxazole - DCMU 3-(-3,4-dichlorophenyl)-1,1-dimetylurea - CA carbonic anhydrase - RuP₂ ribulose-1,5-bisphosphate     

Molecular cloning and characterization of Mn-superoxide dismutase from disk abalone (Haliotis discus discus)

The mitochondrial enzyme manganese superoxide dismutase (mitMn-SOD) is one of the antioxidant enzymes involved in cellular defense against oxidative stress and catalyzes the conversion of O₂⁻ into the stabler H₂O₂. In this study, a putative gene encoding Mn-SOD from disk abalone (Haliotis discus discus, aMn-SOD) was cloned, sequenced, expressed in Escherichia coli K12(TB1) and the protein was purified using pMAL protein purification system. Sequencing resulted ORF of 681 bp, which corresponded to 226 amino acids. The protein was expressed in soluble form with molecular weight of 68 kDa including maltose binding protein and pI value of 6.5. The fusion protein had 2781 U/mg activity. The optimum temperature of the enzyme was 37 °C and it was active in a range of acidic pH (from 3.5 to 6.5). The enzyme activity was reduced to 50% at 50 °C and completely heat inactivated at 80 °C. The alignment of aMn-SOD amino acid sequence with Mn-SODs available in NCBI revealed that the enzyme is conserved among animals with higher than 30% identity. In comparison with human mitMn-SOD, all manganese-binding sites are also conserved in aMn-SOD (H28, H100, D185 and H189). aMn-SOD amino acid sequence was closer to that of Biomphalaria glabrata in phylogenetic analysis.     

Peculiarities of the antioxidant and radioprotective effects of hydrated C60 fullerene nanostuctures in vitro and in vivo

Aqueous solutions of highly stable supramolecular donor–acceptor complexes of chemically nonmodified pristine C₆₀ fullerene molecules with H₂O molecules (hydrated C₆₀ fullerene–C₆₀HyFn) and their labile nano-sized clusters were examined for their antioxidant effects on removal of hydroxyl radicals (OH) and protecting DNA against oxidative damage induced by ionizing radiation in vitro. The suppressing influence of C₆₀HyFn on the formation of OH-radicals in water exposed to X-rays at doses of 1–7 Gy was assessed by determination of oxidation levels of coumarin-3-carboxylic acid. C₆₀HyFn demonstrates apparent antiradical activity in vitro in the range of concentrations of 10⁻¹¹–10⁻⁶ M. Paradoxically, the OH-removing efficacy of C₆₀HyFn was in reverse correlation with fullerene concentration. It was hypothesized that the antiradical action of C₆₀HyFn in water medium generally is due to a “nonstoichiometric” mechanism, supposedly to a hydrated free radical recombination (self-neutralization), which is catalyzed by specific water structures ordered by C₆₀HyFn. With the use of 8-oxoguanine as a marker of oxidative damage to DNA, it has been demonstrated that C₆₀HyFn in concentrations of 10⁻⁷–10⁻⁶ M protects nucleic acids against radical-induced damage. The second part of the present study was aimed to evaluate the overall radioprotective efficacy of C₆₀HyFn in doses of 0.1 or 1 mg/kg b.w. injected intraperitoneally to mice either 1 h before or 15 min after lethal dose exposure of the X-ray (7 Gy) irradiation. Survival rate of the mice was observed at 30 day intervals after irradiation, while the weight gains of experimental animals were monitored as well. The most significant protective effect was demonstrated when 1 mg/kg dosage of C₆₀HyFn was administered before irradiation. The outcome of the substance testing is 15% survival rate of irradiated animals at 30 days of observation, and prevention of noticeable weight loss characteristic for radiation impact, versus unprotected control animals. In conclusion, results of the study obviate that the apparent protective action of C₆₀HyFn in vivo is determined by its considerable ability to decrease X-ray-generated reactive oxygen species. Based on the results and that neat C₆₀ is nontoxic, actually in the hydrated form, without side effects and with sufficient radioprotective effects in low doses, C₆₀HyFn may be considered as a novel antioxidant agent, which substantially diminishes the harmful effects of ionizing radiation.