Lake Muzahi,Rwanda: limnological features and phytoplankton production

Lake Muhazi, a small lake of Rwanda (East Africa) was studied from 1986 to 1990. A dramatic decrease of the catch of Oreochromis niloticus (350 T y⁻¹ in the fifties vs 30 T y⁻¹ in 1982) suggested a loss of productivity or overfishing. In the same period, other ecological changes occurred: the submerged macrophytes regressed and there was a decrease in Secchi depth (0.65 m in 1987 vs 1.5 m in the fifties). Compared to other lakes of the same area, the plankton production seemed low. The results of the present study characterize lake Muhazi as a shallow lake with a rather unstable diurnal stratification and with slight differences in mixing regime between its eastern, deepest part and its western, shallowest part. Secchi disk depth does not vary seasonally to a large extent. The water has a rather high mineral content (conductivity of about 500 μS cm⁻¹ at 25 °C) and low concentrations of dissolved N and P, except in the hypolimnion, where NH _inf4 ^sup+ -N can be high. Two species, Microcystis aeruginosa and Ceratium hirundinella, account for most of the phytoplankton biomass, which is about 50–80 mg chlorophyll a m⁻² in the euphotic zone, usually with little seasonal variation. Daily gross production estimates amount to about 6 to 9.5 g O₂ m⁻² d⁻¹ with a significant difference between the two parts of the lake. Data on C:N and C:P ratio in the phytoplankton suggest that some N deficiency might occur in the eastern part. Moreover, the Zm:Zc ratio could also lead to rather low net production rates (0.21–0.25 d⁻¹ for a mixed layer of 4 m) In conclusion, the primary production of lake Muhazi is medium for African lakes and the hypothesis that decreased planktonic production could account for a reduced fish production should be discarded. Whereas the present yield of the fishery is only 20 kg ha⁻¹ y⁻¹, the yield estimated from primary production ranges between 46 and 64 kg ha⁻¹ y⁻¹. This could be reached through proper management. Finally, some hypotheses are given to explain the ecological changes which occurred in the lake.     

Limnology of Gallocanta Lake,Aragon, northeastern Spain

A limnological study was sustained from September 1980 to October 1981 to show the evolution of Gallocanta Lake (N.E. Spain) under very dry climatic conditions. It is the physical terminus within an endorheic basin of 500 km² situated 1 000 m over the sea level. In 1977 its maximum depth was 2.5 m but it decreased to 60 cm in 1981 as a consequence of the last very dry years. It is actually 6 km long and 2.5 km wide, and its area is approximately 12 km². Salt concentration was quite constant (32–43 g l^-1) since September 1980 until June 1981. The equivalent ratio Cl^-:SO _inf4 ^sup2- varied between 2.2 and 2.8. From June through October 1981 salinity increased up to 105 g l^-1 and Cl^-:SO _inf4 ^sup2- equivalent ratio varied between 2.5 and 3.4. The relative concentrations of ions were retained all year ordinated as follows: Cl^- > SO _inf4 ^sup2- > HCO _inf3 ^sup- >. CO _inf3 ^sup2- ; Na⁺ > Mg²⁺ ? Ca²⁺ > K⁺. Total reactive phosphorus was less than 1.5 µg-at l^-1 from September 1980 to January 1981. During the rest of the studied period it varied between 2 and 8 µg-at l^-1 (the maximum, in February 1981). Nitrogen oxidized forms were relatively high in winter (4–8 µg-at N-NO _inf3 ^sup- l^-1; 0.5–2.5 µg-at N-NO _inf2 ^sup- l^-1), and early May 1981 (25 µg-at N-NO _inf3 ^sup- l^-1; 1 µg-at N-NO _inf2 ^sup- l^-1). Neither reached 1 µg-at l^-1 from September through December 1980, or from June through October 1981. Planktonic algae increased in density during the period of the study from 10³ to 5 10⁵ cells ml^-1. Chromulina sp. was the main species during autumn and winter (>50% in number of cells ml^-1) while the copepod Arctodiaptomus salinus decreased its population. Lobocystis dichotoma increased its density from December 1980 to May 1981 and was dominant in spring (>90%), when a typical reproductive activity was observed in the Arctodiaptomus population. This species disappeared in late spring. In June 1981 Brachionus plicatilis reached up to near 2 000 individuals ml^-1. This species was not observed after August 1981. Then the ciliate Fabrea salina was the only zooplankter staying in the free waters of the lake and Nannochloris sp. the dominant alga (>90%). Dense macrophytic beds constituted of Lamprothamnium papulosum and Chara galioides covered the bottom of the lake in September 1980 and before that date since the observations began in 1977. They decreased in covered area since October 1980 and disappeared during summer 1981. Gallocanta Lake is in a very stressed situation produced by prolonged drought. The increased salt concentration together with the extremely cold and hot temperatures, and the lack of nitrogen relative to phosphorus are the main environmental factors that controlled the biological populations during the period of the present study.     

Temperature and nutrient induced responses of Lake Fryxell sulfate-reducing prokaryotes and description of Desulfovibrio lacusfryxellense, sp. nov., a pervasive, cold-active, sulfate-reducing bacterium from Lake Fryxell, Antarctica

The effects of temperature and carbon substrate availability on the stimulation of sulfate reduction by indigenous populations of sulfate-reducing prokaryotes (SRP) in permanently ice-covered Lake Fryxell, Antarctica were investigated. Psychrophilic and halotolerant, lactate-degrading SRP showed significant metabolic activity throughout all sampled depths of the water column, suggesting that such organisms, possibly of marine origin, may be key contributors to carbon and sulfur cycling in Lake Fryxell. Planktonic and benthic strains of lactate-oxidizing sulfate-reducing bacteria (SRB) were isolated from samples of various depths of the anoxic water column and from surficial sediments. Phylogenetic analyses of 16S rRNA gene sequences placed the Fryxell sulfate-reducer (FSR) strains within the Deltaproteobacteria and showed them to be most closely related to the Arctic marine species of SRB Desulfovibrio frigidus and Desulfovibrio ferrireducens. Based on phylogenetic and phenotypic differences between the Antarctic FSR strains and related species of the genus Desulfovibrio, strain FSRs^T (=DSM 23315^T =ATCC BAA-2083^T) is proposed as the type strain of a novel species of cold-active SRB, Desulfovibrio lacusfryxellense, sp. nov.     

Dinoflagellate luciferases: purification of luciferases from Gonyaulax polyedra, Pyrocystis lunula, and Pyrocystis fusiformis

The isolation and purification of three luciferases from Pyrocystis lunula, Pyrocystis fusiformis, and Gonyaulax polyedra are described in this paper. The three luciferases have low molecular weights, 30,000 for G. polyedra and 40,000 for P. lunala and P. fusiformis, and each is composed of a single polypeptidic chain. The molecular weight of these luciferases is independent of both the period of the circadian rhythm and the pH of the extraction medium between pH 5.4 and pH 8. These enzymes are probably metalloproteins. Indeed, chelating agents such as EDTA, EGTA, and chlorotetracycline and also sodium azide and potassium cyanide inhibit the light emission. Three cations (Mn²⁺, Mg²⁺, and Ca²⁺) increase the flash height and the total light emitted, whereas other cations (Fe²⁺, Fe³⁺, Cu²⁺, Ni²⁺, and Zn²⁺) inhibit the light emission. The three luciferases cannot be replaced by peridoxases or oxidases as in the Balanoglossus and the Pholas systems. The pH dependence of the luciferase activities is represented by a symmetrical function with optimum near pH 7. Thus, the flashing mechanism cannot be explained by means of a switch mechanism controlled by the pH. The presence of a specific luciferin-binding protein has not been observed in the three extracts of dinoflagellates. The difference between our observations and those described in the literature may be explained by the difference of the degree of purification of these enzymes.     

A community of submerged aquatic CAM plants in Lake Kalgaard,Denmark

The CO₂ exchange pattern of leaves of dominant Isoetes lacustris L., Littorella uniflora (L.) Aschers. and Lobelia dortmanna L. from oligotrophic, carbon-poor Lake Kalgaard, Denmark was examined by gas-exchange experiments and by following the diurnal acidity rhythm. Both variables suggest Crassulacean Acid Metabolism (CAM) in Littorella and Isoetes. During the dark, both species had a continuous, but declining CO₂ uptake. The maximum uptake in Littorella was 28 μmol CO₂ g^?1 DW h^?1 or about 36% of the light CO₂ assimilation rate. In Isoetes, corresponding figures were 19 μmol CO₂ g^?1 DW h^?1 and 22%.A peak of 49.5 μEq g^?1 FW titratable acidity (Littorella) and 41.9 μEq g^?1 FW (Isoetes) was found at the end of the dark period, reflecting an increase of 11.6 μEq g^?1 FW (9 h)^?1 (Littorella) and 16.3 μEq g^?1 FW (9 h)^?1 (Isoetes). These results agreed with the cumulative CO₂ assimilation during the dark.The assimilation of CO₂ in the dark is important, apparently, for growth of Littorella and Isoetes in Lake Kalgaard. Lobelia, which exhibits little dark CO₂ assimilation, is also the least abundant species, at less than 1% of the total cover.     

Characterization of Amylolytic Enzymes, Having Both α-1,4 and α-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90°C) and Pyrococcus furiosus (optimal growth temperature, 98°C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca²⁺, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140°C. The addition of Ca²⁺ also affected substrate binding, as evidenced by a decrease in K_m for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the α-1,6 linkages in pullulan, α-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller α-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.     

Localization, solubilization and characterization of plant membrane-associated calcium-dependent protein kinases

Membrane fractions from mature silver beet (Beta vulgaris) deveined leaf and leaf stem homogenates have associated Ca²⁺ -dependent protein kinase. The Ca²⁺ -dependent protein kinase activity is associated with plasma membranes (density 1.14-1.18 grams per cubic centimeter) as determined from copurification on isopycnic centrifugation with plasma membrane markers such as β-glucan synthetase, eosin-5-maleimidelabeling, and specific naphthylphthalamic acid-binding. The Ca²⁺ -dependent protein kinase is not specifically associated with chloroplasts or mitochondria. The membrane-bound Ca²⁺ -dependent protein kinases were solubilized with 0.8% (volume/volume) Nonidet P40. The solubilized enzymes were extensively purified by a protocol involving binding to diethylaminoethyl-cellulose (Whatman DE-52), Ca²⁺ -dependent binding to phenyl-Sepharose CL-4B, gradient elution from diethylaminoethyl-Sephacel (resolving two distinct Ca²⁺ -dependent protein kinases), and gel filtration on Ultrogel AcA 44. These two membrane-derived enzymes have similar molecular weights but differ in protein substrate specificity, in K_m values for ATP, and in Ca²⁺ -independent activation by unsaturated fatty acids. The membrane-bound enzymes correspond closely in these properties to two Ca²⁺ -dependent protein kinases present in the soluble phase.     

Conjugation of the linoleic acid oxidation product, 13-oxooctadeca-9,11-dienoic acid,a bioactive endogenous substrate for mammalian glutathione transferase

The oxidation of linoleic acid leads to the generation of several products with biological activity, including 13-oxooctadeca-9,11-dienoic acid (13-OXO), a bioactive 2,4-dienone that has been linked to cell differentiation. In the current work, the conjugation of 13-OXO by human glutathione transferases (GSTs) of the alpha (A1–1, A4–4), mu (M1–1, M2–2) and pi (the allelic variants P1–1/ile, and P1–1/val) classes, and a rat theta (rT2–2) class enzyme has been evaluated. The kinetics and stereoselectivity of the production of the 13-OXO-glutathione conjugate (13-OXO-SG) have been examined. In contrast to many xenobiotic substrates, the endogenous substrate 13-OXO does not exhibit an appreciable non-enzymatic rate of conjugation under physiological conditions. Therefore, the GST-catalyzed conjugation takes on greater significance as it provides the only realistic means for formation of 13-OXO-SG in most biological systems. Alpha class enzymes are most efficient at catalyzing the formation of 13-OXO-SG with k_cat/K_m values of 8.9 mM⁻¹ s⁻¹ for GST A1–1 and 2.14 mM⁻¹ s⁻¹ for GST A4–4. In comparison, enzymes from the mu and pi classes exhibit specificity constants from 0.4 to 0.8 mM⁻¹ s⁻¹. Conjugation of 13-OXO with glutathione at C-9 of the substrate can yield a pair of diastereomers that can be resolved by chiral HPLC. GSTs from the mu and pi classes are the most stereoselective enzymes and there is no apparent relationship between catalytic efficiency and stereoselectivity. The role of GST in the metabolic disposition of the bioactive oxidation products of linoleic acid has implications for the regulation of normal cellular functions by these versatile enzymes.     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

Methionine-oxidized horse heart cytochromes c. I. Reaction with Chloramine-T,products, and their oxidoreduction properties

Spectroscopically, the modification of horse heart ferricytochrome c with N-chloro-4-toluolsul-fonamide (Chloramine-T, CT) occurs through a two-step process, the disruption of the methionine-80 sulfur-iron linkage and a reagent-independent change, an intramolecular rearrangement. Chromatographic purification of the preparation at a 2.5:1 reagent-to-protein ratio, pH 8.0–8.5, yields two major products, the F^II and F^III CT-cytochromes c. Both products contain modification of only the methionines, 80 and 65, to sulfoxides; both are monomeric, reduced by ascorbate, and the ferrous forms are oxidized by molecular oxygen and bind carbon monoxide. The redox potentials of F^II and F^III are 135 and 175±15 mV. The F^III is indistinguishable from the native protein in its binding and the electron donor property toward mammalian cytochrome c oxidase. It also binds nearly as effectively as the native protein to yeast cytochrome c peroxidase, but is a less efficient donor. It is, however, a poor electron acceptor from both mammalian cytochrome c reductase and chicken liver sulfite oxidase. F^II lacks cytochrome c oxidase activity and is also a poorer substrate for the other three enzymes. Both the derivatives are consistently better electron donors than acceptors. It is concluded that the binding of cytochrome c to cytochrome c oxidase and to cytochrome c peroxidase does not require the integrity of the methionine-80 sulfur linkage and that the complexation process has a finite degree of freedom with regard to the state of the heme crevice opening. The alterations of the oxidoreduction function have been analyzed in light of both prevailing models of cytochrome c function, the two-site model (one site for oxidizing and the other for reducing enzymes) and the single-site model (the same site for the oxidizing and reducing enzymes). These observations can be accommodated by either model, given the latitude that the binding domains for the oxidizing and the reducing enzymes have finite overlapping and nonoverlapping regions.     

Completing the series of BVMOs involved in camphor metabolism of Pseudomonas putida NCIMB 10007 by identification of the two missing genes, their functional expression in E. coli, and biochemical characterization

The camphor-degrading Baeyer?CVilliger monooxygenases (BVMOs) from Pseudomonas putida NCIMB 10007 have been of interest for over 40?years. So far the FMN- and NADH-dependent type II BVMO 3,6-diketocamphane 1,6-monooxygenase (3,6-DKCMO) and the FAD- and NADPH-dependent type I BVMO 2-oxo-?³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) have not been entirely studied, since it was not possible to produce those enzymes in satisfactory amounts and purity. In this study, we were able to clone and recombinantly express both enzymes and subsequently use them as biocatalysts for various mono- and bicyclic ketones. Full conversion could be reached with both enzymes towards (±)-cis-bicyclo[3.2.0]hept-2-en-6-one and with 3,6-DKCMO towards (?)-camphor. Further OTEMO gave full conversion with norcamphor. OTEMO was found to have a pH optimum of 9 and a temperature optimum of 20?°C and converted (±)-cis-bicyclo[3.2.0]hept-2-en-6-one with a k _cat/K _M value of 49.3?mM^?1?s^?1.     

tRNA methylases from HeLa cells: purification and properties of an adenine-1-methylase and a guanine-N2-methylase

Two enzymes (methylases) that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to tRNA (prepared from Escherichia coli) have been partially purified from extracts of HeLa cells. One catalyzes the methylation of adenine residues of the tRNA to give 1-methyladenine units and the other is responsible for the conversion of guanine residues to N²-methylguanine and N²,N²-dimethylguanine (and may be a mixture of two enzymes). Activities of these relatively unstable enzymes could be maintained by storage at ?20 °C in the presence of 50% glycerol. Substrate specificity studies have revealed that bacterial tRNA (E. coli, Bacillus subtilis) can be used as substrate, whereas tRNA of animal origin (HeLa cells, rat liver) cannot be used. Of the specific tRNA's tested, E. coli tRNA^fMet was used as substrate by both enzymes. E. coli tRNA^Tyr was used by the adenine-1-methylase but not by the guanine-N²-methylase. The adenine-1-methylase catalyzed the transfer of approximately one methyl group per mole of either tRNA^fMet or tRNA^Tyr offered as substrate; in the presence of the guanine-N²-methylase 1 mole of E. coli tRNA^fMet accepted 1 mole of methyl. Studies with the use of both enzymes established that enzymic methylation of the guanine site of E. coli tRNA^fMet did not interfere with subsequent methylation of an adenine residue and neither did prior methylation of adenine interfere with the subsequent methylation of a guanine residue. In the presence of both enzymes, approximately 2 moles of methyl groups were accepted by 1 mole of the E. coli tRNA^fMet.     

Abundance and toxicity of Planktothrix rubescens in the pre-alpine Lake Ammersee,Germany

Regular occurrences of the cyanobacterium Planktothrix rubescens have been observed in several lakes that have undergone recent re-oligotrophication, e.g. Lake Ammersee. Planktothrix species are known to produce microcystins, potent phosphatase inhibitors that have been associated with morbidities and mortalities in humans and animals. The aim of this study was to characterise the temporal and spatial abundance and toxicity of P. rubescens in Lake Ammersee.P. rubescens cell densities and biovolumes were calculated via fluorescence image analyses. P. rubescens was present during the entire observation period from 1999 to 2004, albeit at different cell densities. Maximum biovolumes of 45 cm³ m^?2 were observed in May 2001. Filaments were regularly distributed over the entire water column during winter and stratified in distinct metalimnic layers during summer, reaching maximum cell densities of ≤15,000 (winter) and ≤77,000 cells ml^?1 (summer). The results demonstrate that P. rubescens abundance is strongly influenced by water transparency, i.e. illumination in the metalimnion. Moreover, the P. rubescens abundance appears to result from regular phosphate depletion in the epilimnion, possibly additionally benefiting from high nitrogen loads.Microcystin (MC) was detectable in 27 and 38 of 54 seston samples via HPLC and Adda-ELISA measurements, respectively. The main microcystin congeners in the seston samples were [Asp³]-MC-RR and [Asp³,Dhb⁷]-MC-RR. Microcystin concentrations correlated significantly with the respective phycoerythrin (PE)-concentrations. The variation in the MC/PE-ratios was low suggesting that the microcystin production of P. rubescens in Lake Ammersee is consistent and indicates that the appearance of P. rubescens coincides with measurable microcystin levels. Moreover, the observation of pronounced metalimnic oxygen depletions appears to be causally related to recurring high P. rubescens abundance.In conclusion the results suggest that aquatic organisms such as indigenous fish populations (e.g. coregonids) are regularly confronted with potentially adverse P. rubescens densities, which might provide a possible explanation for the often observed impaired health and growth retardation of coregonid populations in P. rubescens containing pre-alpine lakes.     

Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: Heterologous expression,purification and properties

The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N⁶-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytokinin oxidase activity, but molecular oxygen is a comparatively poor electron acceptor. The CKX gene family of Arabidopsis thaliana comprises seven members. Four code for proteins secreted to the apoplast, the remainder are not secreted. Two are targeted to the vacuoles and one is restricted to the cytosol. This study presents the purification and characterization of each of these non-secreted CKX enzymes and substrate specificities are discussed with respect to their compartmentation. Vacuolar enzymes AtCKX1 and AtCKX3 were produced in Pichia pastoris and cytosolic enzyme AtCKX7 was expressed in Escherichia coli. The recombinant proteins were purified by column chromatography. All enzymes preferred synthetic electron acceptors over oxygen, namely potassium ferricyanide and 2,3-dimetoxy-5-methyl-1,4-benzoquinone (Q₀). In slightly acidic conditions (pH 5.0), N⁶-(2-isopentenyl)adenine 9-glucoside (iP9G) was the best substrate for AtCKX1 and AtCKX7, whereas AtCKX3 preferentially degraded N⁶-(2-isopentenyl)adenine 9-riboside-5′-monophosphate (iPMP). Moreover, vacuolar AtCKX enzymes in certain conditions degraded N⁶-(2-isopentenyl)adenine di- and triphosphates two to five times more effectively than its monophosphate.     

The effect of Hg2+ on rabbit hepatic and pulmonary solubilized,partially purified N,N-dimethylaniline N-oxidases

Solubilization and partial purification of the rabbit pulmonary and hepatic N,N-dimethylaniline N-oxidases were carried out in order to study the effect of Hg²⁺ in vitro observed previously in the microsomal enzymes. Rabbit lung microsomal N,N-dimethylaniline (DMA) N-oxidase activity was stimulated 1.5–2 times by 0.1 mM Hg²⁺ added in vitro. This concentration of mercury inhibited hepatic microsomal N-oxidase by 50%. Upon solubilization and partial purification of the lung N-oxidase enzyme, stimulation of the N-oxidase activity by 0.1 mM Hg²⁺ was lost. It was found that the concentration of Hg²⁺ that would stimulate the partially purified pulmonary N-oxidases was 25 μM or less. Stimulation by 0.1 mM Hg²⁺ of the partially purified N-oxidase from lung was restored by addition of flavins (FMN or FAD) or a heat-stable (NH₄)₂SO₄ precipitated fraction obtained during the purification of the N-oxidase from solubilized pulmonary or hepatic microsomes. However, addition of the flavins or the solubilized, heat-stable fraction from liver or lung microsomes did not reverse inhibition by 0.1 mM Hg²⁺ of the N-oxidase in hepatic microsomes or in partially purified preparations from these hepatic microsomes. Kinetic data suggest that flavins and the heatstable factor isolated from microsomes lower the concentration of free Hg²⁺.The determination of kinetics of Hg²⁺ inhibition (liver) and activation (lung) with the partially purified N-oxidases showed that the pulmonary and hepatic DMA N-oxidase enzymes are markedly different with respect to their in vitro response to Hg²⁺. This suggests that the N-oxidases from liver and lung may be different enzymes.     

Arabidopsis thaliana Contains Both Ni2+ and Zn2+ Dependent Glyoxalase I Enzymes and Ectopic Expression of the Latter Contributes More towards Abiotic Stress Tolerance in E. coli

The glyoxalase pathway is ubiquitously found in all the organisms ranging from prokaryotes to eukaryotes. It acts as a major pathway for detoxification of methylglyoxal (MG), which deleteriously affects the biological system in stress conditions. The first important enzyme of this system is Glyoxalase I (GLYI). It is a metalloenzyme which requires divalent metal ions for its activity. This divalent metal ion can be either Zn²⁺ as found in most of eukaryotes or Ni²⁺ as seen in prokaryotes. In the present study, we have found three active GLYI enzymes (AtGLYI2, AtGLYI3 and AtGLYI6) belonging to different metal activation classes coexisting in Arabidopsis thaliana. These enzymes have been found to efficiently complement the GLYI yeast mutants. These three enzymes have been characterized in terms of their activity, metal dependency, kinetic parameters and their role in conferring tolerance to multiple abiotic stresses in E. coli and yeast. AtGLYI2 was found to be Zn²⁺ dependent whereas AtGLYI3 and AtGLYI6 were Ni²⁺ dependent. Enzyme activity of Zn²⁺ dependent enzyme, AtGLYI2, was observed to be exceptionally high (~250–670 fold) as compared to Ni²⁺ dependent enzymes, AtGLYI3 and AtGLYI6. The activity of these GLYI enzymes correlated well to their role in stress tolerance. Heterologous expression of these enzymes in E. coli led to better tolerance against various stress conditions. This is the first report of a higher eukaryotic species having multiple active GLYI enzymes belonging to different metal activation classes.     

Involvement of a Novel Enzyme, MdpA, in Methyl tert-Butyl Ether Degradation in Methylibium petroleiphilum PM1

Methylibium petroleiphilum PM1 is a well-characterized environmental strain capable of complete metabolism of the fuel oxygenate methyl tert-butyl ether (MTBE). Using a molecular genetic system which we established to study MTBE metabolism by PM1, we demonstrated that the enzyme MdpA is involved in MTBE removal, based on insertional inactivation and complementation studies. MdpA is constitutively expressed at low levels but is strongly induced by MTBE. MdpA is also involved in the regulation of tert-butyl alcohol (TBA) removal under certain conditions but is not directly responsible for TBA degradation. Phylogenetic comparison of MdpA to related enzymes indicates close homology to the short-chain hydrolyzing alkane hydroxylases (AH1), a group that appears to be a distinct subfamily of the AHs. The unique, substrate-size-determining residue Thr⁵⁹ distinguishes MdpA from the AH1 subfamily as well as from AlkB enzymes linked to MTBE degradation in Mycobacterium austroafricanum.     

Spatiotemporal distribution of bacteriochlorophylls in the meromictic Lake Suigetsu, Japan

The spatiotemporal distribution of chlorophyll pigments (chloropigments) in the water column of a meromictic lake, Lake Suigetsu (Fukui, Japan), was investigated. Water samples were collected from the central basin of Lake Suigetsu bimonthly between May 2008 and March 2010 at appropriate depths, including the oxic surface, oxic–anoxic interface, and anoxic bottom layers. Chlorophyll a, related to cyanobacteria and eukaryotic phytoplankton, was detected throughout the water column during the years of the study, whereas bacteriochlorophyll e, related to brown-colored green sulfur bacteria, was detected in the anoxic layers below the chemocline at a maximum concentration of 825 μg L^?1. The concentration of bacteriochlorophyll e was generally maximal at or just below the chemocline of the lake. The cellular content of bacteriochlorophyll e was estimated to be low in the upper part of the chemocline and tended to increase with increasing water depth. Bacteriochlorophyll a, which was presumably related to purple sulfur bacteria, was only detected at the chemocline during summer and autumn at concentrations of 5.4–16.3 μg L^?1. Our analysis of the chloropigment distribution for the two years of the study suggested that brown-colored green sulfur bacteria are the predominant phototroph in the anoxic layers of Lake Suigetsu, and that these play a significant role in the carbon and sulfur cycling of the lake, especially from spring to summer.     

Carbon isotope ratios of amylose,amylopectin and mutant starches

Carbon isotope ratios (expressed as δ¹³C values) were determined for various sources of starch and the starch fractions amylose and amylopectin. The δ¹³C values of amylose were consistently less negative, 0.4–2.3 ‰, than those of amylopectin in kernal starch from maize (Zea mays) and barley (Hordeum vulgare) and in tuber starch from potato (Solanum tuberosum). Kernel starch isolated from the maize mutants wx1 and ae1, with known genetic lesions in the starch biosynthetic pathway, also showed significant differences in δ¹³C values. Collectively, these results suggest that variation in carbon isotope ratios in the amylose and amylopectin components of starch may be attributed to isotopic discrimination by the enzymes involved in starch biosynthesis.     

Plasma membranes from fibroblastic cells in culture isolation,morphological and enzymatic identification

Plasma membranes were isolated from murine plasmocytoma cells in culture, by a procedure involving lysis in hypoosmotic medium leaving the nuclei intact, and separation of surface membranes from the lysate constituents on a discontinuous sucrose gradient.The purity of the fractions was assessed by electron microscopy and by assaying enzymes for cross-contaminants. Phosphohydrolases, including the (Na⁺ + K⁺)-stimulated Mg²⁺-ATPase (EC 3.6.1.3) and 5′-nucleotidase (EC 3.1.3.5), were concentrated in the plasma membrane-rich fractions. These fractions were essentially free from NADH: cytochrome c reductase, lysosomes and mitochondrial membrane enzymes.