Coiling of beta-pleated sheets

To form strongly twisted β-sheets, strands have to be coiled as well as twisted (Nishikawa &; Scherga, 1976). I show that strands coil in the appropriate right-handed direction if their main-chain torsion angles fulfil the following conditions: ψ_i ? ?φ_{i + 1}, ψ_{i + 1} > ?φ_{i + 2}, ψ_{i + 2} ? ?φ_{i + 3}, ψ_{i + 3} > ?φ_{i + 4}…Lactate dehydrogenase, pancreatic trypsin inhibitor, thermolysin and concanavalin A contain strongly twisted β-sheets and in each case the strands are coiled by their φ, ψ values fulfilling these conditions.     

Catalytic properties of d-xylose isomerase from Streptomyces violaceoruber

The catalytic properties and stability of d-xylose isomerase from Streptomyces violaceoruber have been studied. The enzyme was activated by Mg²⁺, Co²⁺ and Mn²⁺ but Ni²⁺, Ca²⁺, Zn²⁺, Cu²⁺ and Hg were ineffective. Optimum catalytic conditions were obtained at 80°C in the pH range 7.5–9.5 and in the presence of 10 mm Mg²⁺. The specific activity of the enzyme increased after treatment with 10 mm EDTA (factor 2.4). A further increase of activity (factor 2.0–2.8) was observed after preincubation of the enzyme with Mg²⁺ or Co²⁺, the preincubation time depending on the incubation temperature. The thermal stability of the enzyme is very high. At 60°C the enzyme retained optimum activity following 30 days of storage in the presence of 1 mm Co²⁺ or 10 mm Mg²⁺. At 80°C, Co²⁺ is superior as a protector against thermal denaturation. At saturating concentrations of Mg²⁺ (35°C) the K_m-values of the EDTA-treated enzyme with respect to d-xylose and d-glucose were 2.8 and 149 mm and the dissociation constants of the enzyme-Mg²⁺ complex for xylitol and d-sorbitol were 0.455 and 4.47 mm, respectively.     

Development of a phylogenetic model for the tribe Micrarctiini (Lepidoptera, Arctiidae) by the SYNAP method

A phylogenetic model for all the 18 genera of Micrarctiini (Lepidoptera, Arctiidae) of the World fauna is discussed. The SYNAP method was used for development of the model. For these purposes, 47 characters of the general appearance, male and female genitalia, and wing pattern were used. Based on these characteristics, an evolutionary trend from the plesiomorphic to apomorphic states was revealed. The genera Apantesis and Amurrhyparia, as well as Notarctia, differ from the other genera to the greatest extent. The other genera have formed two the main clades: Grammia + (Diacrisia + (Rhyparia + Rhyparioides)). Neoarctia + (Palearctia + Holoarctia + Chelis) and all the other genera have occupied separate positions. The latter group was divided into two clades: Ebertarctia + Tancrea + Divarctia with brachypterous females and Centrarctia + (Sibirarctia + (Micrarctia + Hyperborea)).     

Characterization of microsomal ATPases from developing human placenta

Activities and some properties of microsomal ATPases have been studied in developing human placenta. The enzyme activities (Na⁺ + K⁺ + Mg²⁺, Mg²⁺, and Ca²⁺ dependent) in the placenta increase steadily with gestational age until the 18th to 21st week, and decrease in the second half of pregnancy. Mg²⁺-dependent and Na⁺ + K⁺ + Mg²⁺-dependent ATPases possess nearly the same K_m (apparent) for ATP, while the Ca²⁺-dependent enzyme shows a different one. Mg²⁺-dependent ATPase shows higher substrate affinity than Ca²⁺-dependent ATPase, although the V_max of the Mg²⁺-dependent enzyme is lower than that of the latter. However, for each enzyme, the K_m remains almost constant and V_max varies during ontogenic development. V_max of the enzymes decline at term. The enzymes are heat-labile, unaffected by amino acids, namely, l-phenylalanine, l-leucine, and l-tryptophan, and deoxycholate inhibits the enzyme activities by about 50%.     

Interpopulational comparative cytogenetics analysis among three Astyanax (Characiformes: Incertae Sedis) species of two streams of upper Paraná River basin,Brazil

Populations of Astyanax paranae, Astyanax fasciatus and Astyanax altiparanae collected from the Pindorama and Lopei streams (upper Paraná river basin, Brazil) were cytogenetically studied, checking for chromosomal differentiation and the evolutionary trend in the three species. Forty-eight chromosomes (8m + 20sm + 10st + 10a) were observed in both populations of Astyanax paranae. Astyanax fasciatus exhibited 50 chromosomes (8m + 18sm + 10st + 14a, Pindorama stream; 8m + 14sm + 12st + 16a, Lopei stream). The diploid number for A. altiparanae was 50 chromosomes in both populations (6m + 30sm + 4st + 10a). The three studied species exhibited multiple AgNORs with differences among the populations of A. paranae and A. fasciatus. Regarding the heterochromatin distribution, interpopulational differences were observed in the three species. The data of the present study demonstrate that both A. paranae and A. fasciatus exhibit karyotypical interpopulational differences more conspicuous than A. altiparanae populations, probably due to the characteristics that restricted both the A. paranae and the A. fasciatus to the upper portion of the streams, leading to the fixation of different chromosomal rearrangements in the populations. However, the absence of conspicuos interpopulational karyotypical differences in A. altiparanae suggests a gene flow between both populations. In the present study, the role of the streams as an environment which maintains the diversity of fish with different genetic pools became evident.     

Proton relaxation rate and fluorometric studies of manganese and rare earth binding to concanavalin A

The binding of Mn²⁺, Ca²⁺, and rare earth ions to apoconcanavalin A has been studied by water proton relaxation enhancement, electron paramagnetic resonance spectroscopy, and fluorescence spectroscopy. An electron paramagnetic resonance and water proton relaxation rate study of the titration of apoconcanavalin A with Mn²⁺ gives evidence of two equivalent binding sites per monomer with K_D = 50 μm ± 4 μm. When a similar Mn²⁺ titration of apoconcanavalin A is performed in the presence of Ca²⁺ ion, very little free Mn²⁺ is detected by electron paramagnetic resonance until the two Mn²⁺ binding sites per monomer are filled. The substitution of a rare earth ion for Ca²⁺ ion in the above experiment often resulted in a slight displacement of Mn²⁺ from the transition metal site as detected by electron paramagnetic resonance. A water proton relaxation rate study of the titration of apoconcanavalin A with Gd³⁺ reflects two binding sites with a K_D = 40 μm ± 4 μm and two with a K_D = 200 μm ± 50 μm. The fluorescence emission spectrum of concanavalin A (λ_em = 340 nm) is slightly quenched by the addition of Tb³⁺ while Tb³⁺ fluorescence is greatly enhanced. A fluorometric titration of apoconcanavalin A with Tb³⁺ also reflects two sites with a K_D = 40 μm ± 15 μm and two with a K_D = 270 μm ± 50 μm.     

Ca2+ Efflux Is Involved in Cinnamaldehyde-Induced Growth Inhibition of Phytophthora capsici

As a destructive fungus-like plant pathogen, the oomycete Phytophthoracapsici is unable to synthesize its own ergosterol as the potential target of fungicide cinnamaldehyde (CA). In this study, CA exerted efficient inhibitory effects on both mycelial growth (EC50=0.75 mM) and zoospore germination (MIC=0.4 mM) of P . capsici . CA-induced immediate Ca²⁺ efflux from zoospores could be confirmed by the rapid decrease in intracellular Ca²⁺ content determined by using Fluo-3 AM and the increase in extracellular Ca²⁺ concentration determined by using ICP-AES (inductively coupled plasma atomic emission spectrometry). Blocking Ca²⁺ influx with ruthenium red and verapamil led to a higher level of CA-induced Ca²⁺ efflux, suggesting the simultaneous occurrence of Ca²⁺ influx along with the Ca²⁺ efflux under CA exposure. Further results showed that EGTA-induced decrease in intracellular Ca²⁺ gave rise to the impaired vitality of P . capsici while the addition of exogenous Ca²⁺ could suppress the growth inhibitory effect of CA. These results suggested that Ca²⁺ efflux played an important role in CA-induced growth inhibition of P . capsici . The application of 3-phenyl-1-propanal, a CA analog without α,β- unsaturated bond, resulted in a marked Ca²⁺ influx in zoospores but did not show any growth inhibitory effects. In addition, exogenous cysteine, an antagonist against the Michael addition (the nucleophilic addition of a carbanion or another nucleophile) between CA and its targets, could attenuate CA-induced growth inhibition of P . capsici by suppressing Ca²⁺ efflux. Our results suggest that CA inhibits the growth of P . capsici by stimulating a transient Ca²⁺ efflux via Michael addition, which provides important new insights into the antimicrobial action of CA.     

Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a₃, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa₃-type cytochrome c oxidase consists of overlapping bands of the hemes a²⁺ and a₃²⁺. It shows a peak at ～444 nm and a distinct shoulder at ～425 nm. However, attribution of individual spectral lineshapes to hemes a²⁺ and a₃²⁺ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a²⁺ and a₃²⁺ using two approaches. First, we reconstructed bovine CcO heme a²⁺ spectrum using a selective Ca²⁺-induced spectral shift of the heme a²⁺. Second, we investigated photobleaching of the reduced Thermus thermophilus ba₃- and bovine aa₃-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B_0x-, B_0y-transitions of both reduced hemes. The heme a²⁺ spectrum is shifted to the red relative to heme a₃²⁺ spectrum. The ～425 nm shoulder is mostly attributed to heme a₃²⁺.     

Ca2+-mobility in the sarcoplasmic reticulum of ventricular myocytes is low

The sarcoplasmic reticulum (SR) in ventricular myocytes contains releasable Ca²⁺ for activating cellular contraction. Recent measurements of intra-SR (luminal) Ca²⁺ suggest a high diffusive Ca²⁺-mobility constant (D_CaSR). This could help spatially to unify SR Ca²⁺-content ([Ca²⁺]_SRT) and standardize Ca²⁺-release throughout the cell. But measurements of localized depletions of luminal Ca²⁺ (Ca²⁺-blinks), associated with local Ca²⁺-release (Ca²⁺-sparks), suggest D_CaSR may actually be low. Here we describe a novel method for measuring D_CaSR. Using a cytoplasmic Ca²⁺-fluorophore, we estimate regional [Ca²⁺]_SRT from localized, caffeine-induced SR Ca²⁺-release. Caffeine microperfusion of one end of a guinea pig or rat myocyte diffusively empties the whole SR at a rate indicating D_CaSR is 8-9 μm²/s, up to tenfold lower than previous estimates. Ignoring background SR Ca²⁺-leakage in our measurement protocol produces an artifactually high D_CaSR (>40 μm²/s), which may also explain the previous high values. Diffusion-reaction modeling suggests that a low D_CaSR would be sufficient to support local SR Ca²⁺-signaling within sarcomeres during excitation-contraction coupling. Low D_CaSR also implies that [Ca²⁺]_SRT may readily become spatially nonuniform, particularly under pathological conditions of spatially nonuniform Ca²⁺-release. Local control of luminal Ca²⁺, imposed by low D_CaSR, may complement the well-established local control of SR Ca²⁺-release by Ca²⁺-channel/ryanodine receptor couplons.     

The effects of calcium and lanthanide ions on the activity of bovine heart nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase

(i) The activity of purified NAD-specific isocitrate dehydrogenase from bovine heart was stimulated by free Ca²⁺ in the presence of ADP and subsaturating levels of magnesium isocitrate, but not in absence of ADP. However, Ca²⁺ was not absolutely required for ADP activation. This was particularly apparent when free Mg²⁺ was kept low (0.0024–0.020 mm) and the substrate magnesium dl-isocitrate ranged from 0.07–0.25 mm. When kinetic constants were determined at pH 7.4 under these conditions and in the absence of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetate, Ca²⁺ had little or no effect on K_m (app) for ADP; the stimulation of rate by Ca²⁺ was mainly due to increased V (app). With subsaturating ADP, there was an interdependence in the interaction of the enzyme with substrate and Ca²⁺. Thus, with ADP constant (0.30 mm) the values of K_m (app) for magnesium dl-isocitrate declined from 0.35 mm at zero Ca²⁺ to 0.19 mm with saturating Ca²⁺ without affecting V; K_m (app) for free Ca²⁺ declined with increasing magnesium isocitrate to a limiting K_m of 0.3 μm. (ii) Ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetate, frequently used as a calcium buffer, inhibited enzyme activity with and without ADP. (iii) The enzyme was not inhibited by the calmodulin inhibitors trifluoperazine and chlorpromazine. Inhibition by lanthanide ions of the isocitrate dehydrogenase was competitive with magnesium isocitrate and not with respect to Ca²⁺. The values of K_is (1.8 to 3.1 μm) for La³⁺, Yb³⁺, Gd³⁺, Eu³⁺, Tb³⁺, and Er³⁺ were about two orders of magnitude smaller than K_m for magnesium dl-isocitrate.     

Denaturation of DNA in the presence of chromous (Cr2+) ions

The effect of Cr²⁺ ions on the T_m (melting temperature) of DNA has been investigated under appropriate conditions for the stabilization of DNA by Mg²⁺ ions. A significant lowering of T_m, analogous to that observed for Cu²⁺ under normal conditions, was found, for Cr²⁺ at pH = 4.2 and [Mg²⁺] = 5.3 mol per mole of DNA base pair. Cu²⁺ also lowers T_m under similar conditions. The similarity of the effects of Cr²⁺ and Cu²⁺ under comparable conditions may be related to similarities in their coordination properties. It is proposed that Cr²⁺ and Cu²⁺ ions facilitate denaturation by holding together groups on the DNA chains in such a manner that base pairing and base stacking are inhibited. Comparative results for Cr³⁺ and Co²⁺ are also given for these low pH/Mg²⁺ ion conditions.     

Identification and characterization of zinc-starvation-induced ZIP transporters from barley roots

Zinc (Zn) is an essential element for plants but limited information is currently available on the molecular basis for Zn²⁺ transport in crop species. To expand the knowledge on Zn²⁺ transport in barley (Hordeum vulgare L.), a cDNA library prepared from barley roots was expressed in the yeast (Saccharomyces cerevisiae) mutant strain Δzrt1/Δzrt2, defective in Zn²⁺ uptake. This strategy resulted in isolation and identification of three new Zn²⁺ transporters from barley. All of the predicted proteins have a high similarity to the ZIP protein family, and are designated HvZIP3, HvZIP5 and HvZIP8, respectively. Complementation studies in Δzrt1/Δzrt2 showed restored growth of the yeast cells transformed with the different HvZIPs, although with different efficiency. Transformation into Fe²⁺ and Mn²⁺ uptake defective yeast mutants showed that the HvZIPs were unable to restore the growth on Fe²⁺ and Mn²⁺ limited media, respectively, indicating a specific role in Zn²⁺ transport. In intact barley roots, HvZIP8 was constitutively expressed whereas HvZIP3 and HvZIP5 were mainly expressed in ?Zn plants. These results suggest that HvZIP3, HvZIP5 and HvZIP8 are Zn²⁺ transporters involved in Zn²⁺ homeostasis in barley roots. The new transporters may facilitate breeding of barley genotypes with improved Zn efficiency and Zn content.     

Resistance and bioaccumulation of Cd2+, Cu2+, Co2+ and Mn2+ by thermophilic bacteria, Geobacillus thermantarcticus and Anoxybacillus amylolyticus

In this study, bioaccumulation and heavy metal resistance of Cd²⁺, Cu²⁺, Co²⁺ and Mn²⁺ ions by thermophilic Geobacillus thermantarcticus and Anoxybacillus amylolyticus was investigated. The bacteria, in an order with respect to metal resistance from the most resistant to the most sensitive, was found to be Mn²⁺ > Co²⁺ > Cu²⁺ > Cd²⁺ for both G. thermantarcticus and A. amylolyticus. It was determined that the highest metal bioaccumulation was performed by A. amylolyticus in Mn²⁺ (28,566 μg/g dry weight), and the lowest metal bioaccumulation was performed by A. amylolyticus in Co²⁺ (327.3 μg/g dry weight). The highest Cd²⁺ capacities of dried cell membrane was found to be 36.07 and 39.55 mg/g membrane for G. thermantarticus and A. amylolyticus, respectively, and the highest Cd²⁺ capacities of wet cell membrane was found to be 14.36 and 12.39 mg/g membrane for G. thermantarcticus and A. amylolyticus, respectively.     

A simple method for preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca²⁺]_o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca²⁺]_o or amino acids elicits intracellular Ca²⁺ ([Ca²⁺]_i) oscillations. Here, we report the central role of predicted Ca²⁺-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca²⁺-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca²⁺]_o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca²⁺]_o and l-Phe in eliciting CaSR-mediated [Ca²⁺]_i oscillations. The heterocommunication between Ca²⁺ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca²⁺]_o signaling by positively impacting multiple [Ca²⁺]_o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca²⁺]_o and amino acids into intracellular signaling events.     

Cadmium ion stimulation of growth and versicolorin synthesis in a mutant strain ofAspergillus parasiticus

Cultures ofAspergillus parasiticus produce the polyketide versicolorin A in response to elevation of the Zn²⁺ content of the growth medium. With suboptimal Zn²⁺ (0.8 μM) mycelial growth is about half maximal, and versicolorin synthesis is essentially zero. Inclusion of Cd²⁺ (1–100 μM) in the Zn²⁺-limiting growth medium allows optimal growth and stimulates full versicolorin synthesis. Cd²⁺, like Zn²⁺, will stimulate versicolorin sysnthesis only when added within the first 30 h after conidial inoculation. The transport system for Cd²⁺ uptake may be the same as that for Zn²⁺, as judged byin vivo competition studies. Cd²⁺ is a competitive inhibitor of Zn²⁺ uptake, with K_i = 20 μM.     

EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a²⁺₃-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a²⁺₃-NO and recombination of NO with cytochrome a²⁺₃ (in the 30–70 K region) revealed no differences in structure between cytochrome a²⁺₃ in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a²⁺₃-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a²⁺₃-NO. Photodissociation of cytochrome a²⁺₃-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a²⁺₃ to cytochrome a³⁺. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20–40 K region) which differ completely from those observed in cytochrome a²⁺₃-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a²⁺₃-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu²⁺_B-NO compound. The triplet species with Δm_s = 2 EPR signals at g 4 and Δm_s = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.     

Mechanism of hexavalent chromium detoxification by microorganisms and bioremediation application potential: A review

Chromium has been widely used in various industries. Hexavalent chromium (Cr⁶⁺) is a priority toxic, mutagenic and carcinogenic chemical, whereas its reduced trivalent form (Cr³⁺) is much less toxic and insoluble. Hence, the basic process for chromium detoxification is the transformation of Cr⁶⁺ to Cr³⁺. A number of aerobic and anaerobic microorganisms are capable of reducing Cr⁶⁺. In the presence of oxygen, microbial reduction of Cr⁶⁺ is commonly catalyzed by soluble enzymes, except in Pseudomonas maltophilia O-2 and Bacillus megaterium TKW3, which utilize membrane-associated reductases. Recently, two soluble Cr⁶⁺ reductases, ChrR and YieF, have been purified from Pseudomonas putida MK1 and Escherichia coli, respectively. ChrR catalyzes an initially one-electron shuttle followed by a two-electron transfer to Cr⁶⁺, with the formation of intermediate(s) Cr⁵⁺ and/or Cr⁴⁺ before further reduction to Cr³⁺. YieF displays a four-electron transfer that reduces Cr⁶⁺ directly to Cr³⁺. The membrane-associated Cr⁶⁺ reductase of B. megaterium TKW3 was isolated, but its reduction kinetics is as yet uncharacterized. Under anaerobic conditions, both soluble and membrane-associated enzymes of the electron transfer system were reported to mediate Cr⁶⁺ reduction as a fortuitous process coupled to the oxidation of an electron donor substrate. In this process, Cr⁶⁺ serves as the terminal electron acceptor of an electron transfer chain that frequently involves cytochromes (e.g., b and c). An expanding array of Cr⁶⁺ reductases allows the selection of enzymes with higher reductive activity, which genetic and/or protein engineering may further enhance their efficiencies. With the advancement in technology for enzyme immobilization, it is speculated that the direct application of Cr⁶⁺ reductases may be a promising approach for bioremediation of Cr⁶⁺ in a wide range of environments.