Impact of environmental pollution on cyanobacterial proline content

This study describes the toxic effects of different prominent aquatic pollutants—heavy metals (Cd & Pb), pesticides (alphamethrin and deltamethrin) and salt (NaCl)—on the intracellular proline content in the cyanobacterium, Westiellopsis prolifica–Janet strain–NCCU331. Despite a reduction in growth (measured as chlorophyll a content), the intracellular proline content increased in the presence of heavy metals, pesticides and high salt concentration. The intracellular cyanobacterial proline accumulation was more pronounced under salt stress than in the presence of pesticides and heavy metals. We have also compared whether or not anionic components influence heavy metal toxicity. It was found that the chlorides of Cd and Pb were more toxic than the NO₃ and the order of toxicity was CdCl₂ > PbCl₂ > Cd (NO₃)₂ > Pb (NO₃)₂. Among pyrethroids, deltamethrin was more toxic than alphamethrin. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

A stochastic model for the synthesis and degradation of natural organic matter part II: molecular property distributions

A stochastic biogeochemical model has been developed to simulate the transformation and degradation of natural organic matter (NOM) using an agent-based algorithm which treats each molecule as a separate and potentially unique entity. Molecules react when a pseudo-random number is lower than the calculated reaction probability in a given time step; repeated time steps simulate the transformation of precursor molecules into a complex NOM assemblage. The data for each molecule—elemental and functional group composition—can be used to calculate many properties directly and exactly for each molecule in the assemblage, e.g., molecular weight (MW), fraction of aromatic C (Ar), and charge at pH 7 (Z). Empirical quantitative structure activity relationships (QSARs) are developed which permit the estimation of thermodynamic quantities K _ow (the octanol–water partition coefficient) and pK _a (acidity) for each molecule. Root mean square errors for these QSARs are 0.39 log units for log K _ow and 0.45 log units for pK _a. Distributions of both exactly calculated (MW, Ar, Z) and estimated thermodynamic (K _ow, pK _a) properties are examined and compared with published experimental data. Molecular weight distributions from size exclusion HPLC experiments on aquatic NOM are quantitatively similar to simulation results. pH titrations and polarity distributions from reversed-phase HPLC are qualitatively similar to simulation results. This agreement suggests that the agent-based model can be used to explore hypotheses regarding both compositional and thermodynamic properties of NOM. Robert Wetzel—deceased.     

Novel iron complexes and chelators based on cis,cis-1,3,5-triaminocyclohexane: iron-mediated ligand oxidation and biochemical properties

 The interaction of Fe(II) and Fe(III) with the novel Fe(II) chelator N,N′N″-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (referred to as tachpyr) gives rise to six-coordinate, low-spin, cationic complexes of Fe(II). Tachpyr also displays a cytotoxicity toward cultured bladder cancer cells that is believed to involve coordination of intracellular iron. The anaerobic reaction of tachpyr with Fe(II) salts affords the Fe(II)-tachpyr²⁺ complex, but in presence of oxygen, oxidative dehydrogenation of one or two of the aminomethylene group(s) of the ligand occurs, with formal loss of H₂: R—N(H)—C(H)₂—(2-py) → R—N=C(H)—(2-py)+H₂. The resulting mono- and diimino Fe(II) complexes (denoted as [Fe(tachpyr-H₂)]²⁺ and [Fe(tachpyr-2H₂)]²⁺) are an inseparable mixture, but they may be fully oxidized by H₂O₂ to the known tris(imino) complex Fe(II)[cis,cis-1,3,5-tris(pyridine-2-carboxaldimino)cyclohexane]²⁺ (or [Fe(tachpyr-3H₂)]²⁺). Cyclic voltammetry of the imino complex mixture reveals an irreversible anodic wave at +0.78 V vs. NHE. Tachpyr acts as a reducing agent toward Fe(IIII) salts, affording the same two Fe(II) imino complexes as products. Tachpyr also reductively removes Fe(III) from an Fe(III)(ATP)₃ complex (which is a putative form of intracellular iron), producing the two Fe(II) imino complexes. Novel N-alkylated derivatives of tachpyr have been synthesized. N-Alkylation has two effects on tachpyr: lowering metal affinity through increased steric hindrance, and preventing Fe(III) reduction because oxidative dehydrogenation of nitrogen is blocked. The N-methyl tachpyr derivative binds Fe(II) only weakly as a high-spin complex, and no complexation or reduction of Fe(III) is observed. Corresponding to their inability to bind iron, the N-alkylated chelators are nontoxic to cultured bladder cancer cells. A tach-based chelator with three N-propyleneamino arms is also synthesized. Studies of the chemical and biochemical properties of this chelator further support a relationship between intracellular iron chelation, iron reduction, and cytotoxicity. Received: 23 March 1998 / Accepted: 1 June 1998     

Relevance of Rheological Properties of Sodium Alginate in Solution to Calcium Alginate Gel Properties

The purpose of this study is to determine whether sodium alginate solutions’ rheological parameters are meaningful relative to sodium alginate’s use in the formulation of calcium alginate gels. Calcium alginate gels were prepared from six different grades of sodium alginate (FMC Biopolymer), one of which was available in ten batches. Cylindrical gel samples were prepared from each of the gels and subjected to compression to fracture on an Instron Universal Testing Machine, equipped with a 1-kN load cell, at a cross-head speed of 120 mm/min. Among the grades with similar % G, (grades 1, 3, and 4), there is a significant correlation between deformation work (L _E) and apparent viscosity (η _app). However, the results for the partial correlation analysis for all six grades of sodium alginate show that L _E is significantly correlated with % G, but not with the rheological properties of the sodium alginate solutions. Studies of the ten batches of one grade of sodium alginate show that η _app of their solutions did not correlate with L _E while tan δ was significantly, but minimally, correlated to L _E. These results suggest that other factors—polydispersity and the randomness of guluronic acid sequencing—are likely to influence the mechanical properties of the resultant gels. In summary, the rheological properties of solutions for different grades of sodium alginate are not indicative of the resultant gel properties. Inter-batch differences in the rheological behavior for one specific grade of sodium alginate were insufficient to predict the corresponding calcium alginate gel’s mechanical properties.     

Effect of growth regulators on microspore embryogenesis in coconut anthers

The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators at various concentrations and combinations. Three auxins (1-naphthalene acetic acid—NAA, indoleacetic acid—IAA, picloram) and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 μM NAA in combination with 100 μM 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental effects, respectively, for androgenesis induction over 100 μM 2,4-D alone. Kinetin and 2-iP enhanced the production of calli/embryos when 100 μM 2,4-D was present in the culture medium. Both cytokinins at 10 μM yielded the highest frequencies of embryos (113 and 93, respectively) whereas zeatin (1 or 2.5 μM) had no impact on microspore embryogenesis. When calli/embryos (produced from different treatments in different experiments) were sub-cultured in somatic embryo induction medium (Y₃ medium containing 66 μM 2,4-D), followed by maturation medium (Y₃ medium without growth regulators) and germination medium (Y₃ medium containing 5 μM-6-benzyladenine—BA and 0.35 μM gibberellic acid—GA₃), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%).     

CuA centers and their biosynthetic models in azurin

Cu_A is a binuclear copper center that functions as an electron transfer agent, cycling between a reduced Cu(I)Cu(I) state and an oxidized mixed-valence Cu(+1.5)···Cu(+1.5) state. The copper ions are bridged by two cysteine thiolate ligands and form a copper–copper bond, the first reported of its kind in Nature. Such a “diamond-core” Cu₂S(Cys)₂ structure allows an unpaired electron to be completely delocalized over the two copper ions and contributes to its highly efficient electron transfer properties. This review provides accounts of how the Cu_A center was structurally characterized and highlights its salient spectroscopic properties. In the process, it introduces the Cu_A center in four different systems—native protein systems, soluble protein truncates of native proteins, synthetic models using organic molecules, and biosynthetic models using proteins as ligands—with a greater emphasis on biosynthetic models of Cu_A, especially on new, deeper insights gained from their studies.     

Two-step concerted mechanism for alkane hydroxylation on the ferryl active site of methane monooxygenase

 A two-step concerted mechanism for the conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO) is discussed. We propose that the enzymatic reaction mechanism is essentially the same as that of the gas-phase methane-methanol conversion by the bare FeO⁺ complex. In the initial stage of our mechanism, the ferryl (Fe—O) "iron" active site of intermediate Q and substrate methane come into contact to form the initial Q (CH₄) complex with an OFe—CH₄ bond. The C—H bonds of methane are significantly weakened by the formation of a five-coordinate carbon species, through orbital interactions between a C _3v - or D _2d -distorted methane and the Fe—O active site. The important transition state for an H atom abstraction exhibits a four-centered structure. The generated intermediate involves an HO—Fe—CH₃ moiety, and it is then converted into the final product complex including methanol as a ligand through a methyl migration that occurs via a three-centered transition state. The two-step concerted mechanism is consistent with recent experiments on regioselectivity of enzyme-catalyzed alkane hydroxylations. Received: 15 September 1997 / Accepted: 20 December 1997     

An S-Curve-Based Approach of Identifying Biological Sequences

The main idea of S-curve diagram is to assign different angle values (from 0° to 180°) to different nucleotide acid residues or to different protein amino acids, and then according to cos α _j and sin α _j , the values are accumulated to construct an S-curve diagram, which is in strict one-to-one correspondence with the biological sequence. In addition, the S-curve diagram proves to be without the degeneracy phenomenon, so that both the degeneracy problem represented by diagrams and the problem of visualization for biological sequence data are solved. Meanwhile, a new approach to differentiate the similarity of biological sequences—the degree of similarity—is put forward on the basis of the S-curve diagram. To put it in detail, the least square approach is first adopted to obtain a straight line equation according to the S-curve diagram, then according to the distance formula of the point to the straight line, the average ratio of square sum for the distance between the S-curve and the straight line is calculated, and finally, the similarity of the biological sequences is presented by the new standard—the degree of similarity. As is shown by the experimental results, the S-curve diagram can better represent biological sequences (such as protein’s) within Cartesian coordinate system, and the mutation point of biological sequence. Thus, it turns out that the new standard—the degree of similarity is of obviously great advantage.     

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

Archaea utilize a branched modification of the classical Entner–Doudoroff (ED) pathway for sugar degradation. The semi-phosphorylative branch merges at the level of glyceraldehyde 3-phosphate (GAP) with the lower common shunt of the Emden-Meyerhof-Parnas pathway. In Sulfolobus solfataricus two different GAP converting enzymes—classical phosphorylating GAP dehydrogenase (GAPDH) and the non-phosphorylating GAPDH (GAPN)—were identified. In Sulfolobales the GAPN encoding gene is found adjacent to the ED gene cluster suggesting a function in the regulation of the semi-phosphorylative ED branch. The biochemical characterization of the recombinant GAPN of S. solfataricus revealed that—like the well-characterized GAPN from Thermoproteus tenax—the enzyme of S. solfataricus exhibits allosteric properties. However, both enzymes show some unexpected differences in co-substrate specificity as well as regulatory fine-tuning, which seem to reflect an adaptation to the different lifestyles of both organisms. Phylogenetic analyses and database searches in Archaea indicated a preferred distribution of GAPN (and/or GAP oxidoreductase) in hyperthermophilic Archaea supporting the previously suggested role of GAPN in metabolic thermoadaptation. This work suggests an important role of GAPN in the regulation of carbon degradation via modifications of the EMP and the branched ED pathway in hyperthermophilic Archaea. Thijs J. G. Ettema and Hatim Ahmed have contributed equally to this work.     

Effect of aluminum on the binding properties of α-chymotrypsin

 The effect of aluminum ions on the binding properties of α-chymotrypsin has been studied. The results show that aluminum does not affect the catalytic rate constant k _cat, but it acts as an enzyme activator favoring the binding of the substrate to the catalytic site (i.e. decreasing K _m). Furthermore, aluminum binding to α-chymotrypsin displays about a threefold decrease in its affinity for the macromolecular inhibitor bovine pancreatic trypsin inhibitor (BPTI). Altogether, the different effect of aluminum on the binding of synthetic substrates (e.g. N-α-benzoyl-l-tyrosine ethyl ester, BTEE) and macromolecular inhibitors (e.g. BPTI) to α-chymotrypsin suggests the occurrence of an aluminum-linked conformational change in the enzyme molecule which brings about a marked structural change at the primary and secondary recognition sites for substrates and inhibitors. The modulative effect exerted by aluminum on the enzyme hydrolytic activity has been investigated also as a function of pH. The ion-linked effect appears to be dependent on the pH in a complex fashion, which suggests that aluminum binding is controlled by the protonation of at least two classes of residues on the enzyme molecule. Received: 5 December 1996 / Accepted: 11 March 1997     

ABA may affect the dormancy in triticale caryopses by inhibition of embryo class I RNases,enzymes involved in P<Subscript>i</Subscript> release

The main objective of this study was to analyze the differences in profiles of RNase activities from triticale embryos (Triticosecale, cv. Ugo) between dormant and non-dormant caryopses and to determine the influence of exogenous abscisic acid (ABA) on the activities of these enzymes. The major RNase from the examined tissue was detected following SDS-PAGE, with substrate-based gel assay, described by Yen and Green (Plant Physiol 97:1487–1493, 1991). The activities of enzymes were characterized according to their pH optima, ion dependence, EDTA sensitivity and DNase activity. In embryos with arrested growth (in a natural way by dormancy or artificially by ABA treatment), the activity of two enzymes—24 and 27 kDa—belonging to class I RNases was completely inhibited, whereas that of two other RNases of this family—23 and 25 kDa—was detectable. However, the activity of the class I ribonucleases (enzymes responsible for cellular P_i release) was very low. Moreover, in contrast with non-dormant caryopses, imbibing embryos of dormant or ABA-treated seeds contained 13- and 14-kDa enzymes. These enzymes have not been classified so far, and their specific properties are different from the generally accepted properties of ribonucleolytic enzymes. In addition to the above results, the P_i content in the analyzed samples was determined by the Ames (Methods Enzymol 8:115–118, 1966) method. The results suggest a very low and constant level of inorganic phosphate in dormant samples as well as an evidently decreasing P_i content in embryos under the influence of ABA treatment. The inhibition of the class I RNases activity induced by abscisic acid implies that one of the roles of ABA in seed dormancy may consist in arresting the catabolic release of P_i, which results in retarding the embryo’s growth.     

Influence of intramolecular interactions on conformational and dynamic properties of analogs of heptapeptide AFP<Subscript>14–20</Subscript>

Conformational and dynamic properties of proteins and peptides play an important role in their functioning. However, mechanisms that underlie this influence have not been fully elucidated. In the present work we computationally constructed analogs of heptapeptide AFP_14–20 (LDSYQCT) — one of the biologically active sites of human α-fetoprotein (AFP) — to study their conformational and dynamic properties using molecular dynamics simulation. Analogs were obtained by point substitutions of amino acid residues taking into account differences in their physicochemical properties and also on the basis of analysis of amino acid substitutions in the AFP_14–20-like motifs revealed in different physiologically active proteins. It is shown that changes in conformational mobility of amino acid residues of analogs are due to disruption or arising of intramolecular interactions that, in turn, determine existence of steric restrictions during rotation around covalent bonds of the peptide backbone. Substitution of an amino acid by another one with significant difference in physicochemical properties may not lead to remarkable changes in conformational and dynamic properties of the peptide if intramolecular interactions remain unchanged.     

Impact of different pH control agents on biopesticidal activity of<Emphasis Type="Italic"> Bacillus thuringiensis</Emphasis> during the fermentation of starch industry wastewater

Different pH control agents (NaOH/H₂SO₄—SodSulp, NaOH/CH₃COOH—SodAcet, NH₄OH/CH₃COOH—AmmoAcet and NH₄OH/H₂SO₄—AmmoSulp) were used to investigate their effects on growth, enzyme production (alkaline protease and amylase), and entomotoxicity of Bacillus thuringiensis var. kurstaki HD-1 (Btk) against eastern spruce budworm larvae (Choristoneura fumiferana) using starch industry wastewater (SIW) as a raw material in a 15-l fermentor. AmmoSulp and SodSulp were found to be the best pH control agents for alkaline protease and amylase production, respectively; whereas, the fermented broth obtained by using SodAcet as pH control agents recorded the highest delta-endotoxin production of 1043.0 mg/l and entomotoxicity value 18.4 × 10⁹ SBU/l. Entomotoxicity of re-suspended centrifuged pellet in one-tenth of original volume in case of SodAcet as pH control agents was 26.7 × 10⁹ SBU/l and was the highest value compared to three other pH control agents.     

The effects of methionine and cobalt ions on sex expression and development of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

One of the prime precursor for ethylene synthesis — L-methionine and the inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) — Co²⁺-were tested for their effects on sex expression and development of Anemia phyllitidis fern gametophytes. Five concentrations of both chemicals (0, 10, 25, 50, 100 μM) were analysed with reference to antheridia and archegonia formation, number and size of cells as well as thalli length using the three-zone model of gametophyte structure. Both substances, however at different concentrations, enhanced the number of GA₃-induced antheridia and similarly stimulated the cell number and inhibited thalli length. Both of them at 100 μM concentrations without GA₃ induced meristematic area formation while methionine also induced archegonia in the apical parts of gametophytes. These findings correspond with the previous observations concerning the important role of ethylene synthesis precursor (ACC) in controlling gibberellic acid-induced male sex expression in ferns and broaden the knowledge about the mechanisms of fern gametophyte development.     

Effect of the synthetic growth regulator Cytodef and heavy metals on oxidative status in cucumber plants

We studied the influence of a synthetic cytokinin-like growth regulator (Cytodef) and heavy metal ions—Pb²⁺, Sr²⁺, Zn²⁺, and Ni²⁺—on generation of superoxide anion (O₂^⊙−), lipid peroxidation, and carotenoids content in leaves of 7-day-old cucumber plants (Cucumis sativus L., cv. Izyashchnyi). In some instances Cytodef reduced the toxicity of heavy metals: it mitigated the negative effect of metals on oxidative processes and elevated the concentration of antioxidants (carotenoids).     

Synthetic constructs functionally mimicking ribonuclease A

The mechanism of hydrolysis of RNA substrates—diribonucleoside monophosphate CpA and decaribonucleotide UUCAUGUAAA—by chemical constructs functionally mimicking ribonuclease A was studied. It is shown that RNA cleavage by chemical RNases 2L2 and 2D3 proceeds similar to the RNase A-induced RNA hydrolysis through 2′,3′-cyclophosphate as an intermediate product. A comparison of hydrolyses of CpA in water and D₂O revealed an isotope effect (K _H/K _D=2.28), which implies acid-base catalysis at the limiting stage of the reaction. Two feasible mechanisms of RNA hydrolysis by chemical RNases (linear and adjacent) are discussed.     

Do parafluvial zones have an impact in regulating river pollution? Spatial and temporal dynamics of nutrients,carbon, and bacteria in a large gravel bar of the Doubs River (France)

The changes in both physical and chemical properties of interstitial water were investigated within a large gravel bar to determine if the parafluvial zone was a hotspot for nutrient transformations in a lowland eutrophic river, the Doubs (Eastern France). Interstitial water was sampled in 30 piezometers along five transects across the gravel bar, surface water was sampled in main and chute channels. Five campaigns of sampling were performed among summer and winter. In both interstitial and surface samples, water chemistry (electrical conductivity, pH, chloride, dissolved oxygen concentrations—DO) and nutrient concentrations (nitrate—NO₃ ⁻, ammonium—NH₄ ⁺, soluble reactive phosphorus—SRP, dissolved organic carbon—DOC) were measured. Moreover, temperature, water level, sediment grain size distribution and total bacterial abundance were assessed along a flowpath through a lateral gravel bar of the Doubs River. Measurements of water table elevation in the bar, main and chute channels indicated that the parafluvial flowpath was perpendicular to the main channel. Very low changes in chloride concentration and electrical conductivity showed minor groundwater input along the flowpath. The parafluvial zone was 0.9 m thick under the mean piezometric level, hydraulic gradients along the flowpath were 0.3%, and the discharge of interstitial water through the bar calculated at low flow was 40.6 m³ day⁻¹. Most changes in interstitial nutrients occurred during the warm season, suggesting that biotic mechanisms occurred. Along the flowpath, DO, DOC, and bacterial abundance declined, while phosphate increased. Temporal trends of nitrate were less clear, exhibiting a slight increase during spring (organic matter biodegradation) and a decrease during summer (denitrification). Using the parafluvial discharge and nutrient concentrations of water infiltrating into and seeping from the gravel bar, the retention capacity of the bar was 171 gC day⁻¹ for DOC and 48.3 gN day⁻¹ for nitrate. Phosphate production ranged from 0.65 to 2.3 gP day⁻¹. These values were low compared to the nutrient fluxes in the river Doubs, suggesting a minimal local impact of the parafluvial zone in regulating of river pollution. Handling editor: J. Padisak     

Serotonergic Agonists Inhibit Calcium-Activated Potassium and Voltage-Dependent Sodium Currents in Rat Taste Receptor Cells

Recently we reported that rat taste receptor cells respond to the neurotransmitter serotonin with an inhibition of a calcium-activated potassium current [17]. In the present study, this observation is confirmed and extended by studying the effects of an array of serotonergic agonists on membrane properties, calcium-activated potassium current, and voltage-dependent sodium current in taste receptor cells using the patch-clamp recording technique in the whole-cell configuration. Serotonergic inhibition of calcium-activated potassium current was mimicked by the agonists N-(3-trifluoromethylphenyl)piperazine and by (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Both produced reversible inhibition of K _Ca as well as significantly increasing the input resistance of the cell. The agonists 1-(1-naphthyl)piperazine and buspirone (both serotonin receptor 1A agonists) were similarly effective in reducing K _Ca . Outward current was unaffected by application of phenylbiguanide, a serotonin receptor 3 agonist, though current was affected by subsequent application of (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Two agonists—N-(3-trifluoromethylphenyl)piperazine and (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene—were also tested on voltage-dependent sodium currents; both were effective and reversible in reducing its magnitude at a variety of applied potentials. These data are consistent with the notion that serotonin effects in rat taste receptor cells are mediated by serotonin 1A receptors, though other receptor subtypes may be additionally expressed. Serotonin may affect the taste cell electrical properties during active stimulation in a paracrine fashion. Received: 10 May 1999/Revised: 27 September 1999