The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

Determination of the distribution coefficient of rifabutin by the fluorescence study in the system liposome-water

The distribution coefficient (Kd) of tuberculostatic rifabutin in a liposome/water system at pH 6.4 and 7.4 has been determined by the fluorescence quenching method. Large unilamellar vesicles composed of phosphatidylcholine alone or its combination with cholesterol or cardiolipin were used. The fluorescent probe anthrylphosphatidylcholine, which contains the anthryl moiety in the hydrophobic part, was incorporated into large unilamellar vesicles. The Kd values calculated with the use of dynamic quenching theory (Stern-Volmer model), were comparable for phosphatidylcholine and its combination with cholesterol at both pH. The value of lgKd (24-2.6) demonstrates the hydrophobicity of the rifabutin molecule. After the introduction of negatively charged cholesterol, Kd increases more than tenfold at pH 6.4. At pH 7.4, a second phosphate group of cholesterol undergoes ionization, and Kd of rifabutin gains an additional increase. The results obtained demonstrate a strong influence of electrostatic forces on rifabutin-model membranes interaction.     

Interaction of the synthetic immunomodulatory dipeptide bestim with murine macrophages and thymocytes

The tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by high-temperature solid-state catalytic isotope exchange. It was found that [³H]bestim binds with a high affinity to murine peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM), as well as with plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM, respectively). The specific binding of [³H]bestim to macrophages and thymocytes was inhibited by the unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM, respectively). After treatment with trypsin, macrophages and thymocytes lost the ability to bind [³H]bestim. Bestim in the concentration range of 10^?10 to 10^?6 M reduced the adenylate cyclase activity in the membranes of murine macrophages and thymocytes.     

Impacts on Micro- and Macro-Structure of Thermally Stabilised Whey Protein-Pectin Complexes: A Fluorescence Approach

Stability of whey protein-pectin complexes is an essential criterion for their application in different food matrices. The impact of process parameters on micro- and macro-structural characteristics of thermally stabilised whey protein-pectin complexes was investigated using fluorescence spectroscopy, ζ-potential measurements, dynamic light scattering and phase separation. Complexes prepared from whey protein isolate (WPI) and pectins with different degrees of esterification (HMP, LMP) were generated at different biopolymer concentrations (WPI + pectin: 5.0 % + 1.0 %, c _{h i g h} ; 2.75 % + 0.55 %, c _{m e d} ; 0.5 % + 0.1 %, c _{l o w} ), heating temperatures (80-90^°C) and pH levels (6.1-4.0). Micro- and macro-structural characteristics of the complexes depended on concentration level and degree of esterification, with complexes being more sensitive towards environmental changes at c _{l o w} than at c _{m e d} and c _{h i g h} . WPI-LMP complexes exhibited sizes <1 μm suitable for micro-encapsulation, whereas WPI-HMP complexes at c _{m e d} achieved sizes from 1-10 μm and at c _{h i g h} from 10-200 μm underlining their potential as fat-replacers and structuring agents, respectively. Slopes and intercepts derived from intensity ratios of fluorescence spectra gave insights into the state of unfolding of β-lactoglobulin within the complexes and thus about the protective effect of pectin addition.     

Kinetic characterization of a novel acid ectophosphatase from <Emphasis Type="Italic">Enterobacter asburiae</Emphasis>

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10⁸ cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4°C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows “Michaelis-Menten” kinetics with V = 61.2 U/mg and K_0.5 = 60 μM, while ATP hydrolysis showed V = 19.7 U/mg, K_0.5 = 110 μM, and n_H = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K_0.5 = 84 μM, and n_H = 2.3. Arsenate and phosphate were competitive inhibitors with Ki = 0.6 mM and K_i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K_0.5 = 2.2 mM and n_H = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.     

Characterization of an α-amino-ɛ-caprolactam racemase with broad substrate specificity from <Emphasis Type="Italic">Citreicella</Emphasis> sp. SE45

α-Amino-ε-caprolactam (ACL) racemizing activity was detected in a putative dialkylglycine decarboxylase (EC 4.1.1.64) from Citreicella sp. SE45. The encoding gene of the enzyme was cloned and transformed in Escherichia coli BL21 (DE3). The molecular mass of the enzyme was shown to be 47.4 kDa on SDS–polyacrylamide gel electrophoresis. The enzymatic properties including pH and thermal optimum and stabilities were determined. This enzyme acted on a broad range of amino acid amides, particularly unbranched amino acid amides including l-alanine amide and l-serine amide with a specific activity of 17.5 and 21.6 U/mg, respectively. The K _m and V _max values for d- and l-ACL were 5.3 and 2.17 mM, and 769 and 558 μmol/min.mg protein, respectively. Moreover, the turn over number (K _cat) and catalytic efficiency (K _cat/K _m ) of purified ACL racemase from Citreicella sp. SE45 using l-ACL as a substrate were 465 S^?1 and 214 S^?1mM^?1, respectively. The new ACL racemase from Citreicella sp. SE45 has a potential to be used as the biocatalytic application.     

Suspended-sediment responses after strip thinning in headwater catchments

We examined changes in suspended-sediment yields (SSY) after a 50 % strip thinning in headwater streams draining a Japanese cedar (Cryptomeria japonica) and cypress (Chamaecyparis obtusa) plantation forest. We applied a paired-catchment analysis to treated (K _T : 17.1 ha) and control (K _C : 8.9 ha) catchments. Annual suspended-sediment yield (SSY_an) in the prethinning period in the K _T and K _C catchments was 110.0 and 142.1 kg/ha per year. For the postthinning period, SSY_an in the K _T catchment became 5055.6 kg/ha per year, whereas that in the K _C catchment increased 893.2 kg/ha per year. The paired-catchment analysis revealed that SSY in the K _T catchment increased 17.0-fold compared with the prethinning period. However, the dominant hysteresis pattern remained clockwise in both pre- and postthinning periods. Sequences of large storm events in the postthinning period elevated SSY in both catchments. Increase in suspended sediment in the K _T catchment was associated with the combined impacts of thinning and sequences of storm events during the period of thinning operation.     

A new recombinant <Emphasis Type="Italic">endo-</Emphasis>1,3-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanase from the marine bacterium <Emphasis Type="Italic">Formosa algae</Emphasis> KMM 3553: enzyme characteristics and transglycosylation products analysis

A specific endo-1,3-β-d-glucanase (GFA) gene was found in genome of marine bacterium Formosa algae KMM 3553. For today this is the only characterized endo-1,3-β-d-glucanase (EC 3.2.1.39) in Formosa genus and the only bacterial EC 3.2.1.39 GH16 endo-1,3-β-d-glucanase with described transglycosylation activity. It was expressed in E. coli and isolated in homogeneous state. Investigating the products of polysaccharides digestion with GFA allowed to establish it’s substrate specificity and classify this enzyme as glucan endo-1,3-β-d-glucosidase (EC 3.2.1.39). The amino-acid sequence of GFA consists of 556 residues and shows sequence similarity of 45–85% to β-1,3-glucanases of bacteria belonging to the CAZy 16th structural family of glycoside hydrolases GH16. Enzyme has molecular weight 61 kDa, exhibits maximum of catalytic activity at 45?°C, pH 5.5. Half-life period at 45 °С is 20 min, complete inactivation happens at 55?°C within 10 min. K_m for hydrolysis of laminarin is 0.388 mM. GFA glucanase from marine bacteria F. algae is one of rare enzymes capable to catalyze reactions of transglycosylation. It catalyzed transfer of glyconic part of substrate molecule on methyl-β-d-xylopyranoside, glycerol and methyl-α-d-glucopyranoside. The enzyme can be used in structure determination of β-1,3-glucans (or mixed 1,3;1,4- and 1,3;1,6-β-d-glucans) and enzymatic synthesis of new carbohydrate-containing compounds.     

Assessing biodegradation of furfuryl alcohol using <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MTCC 1194 and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 1034 and its kinetics

The biodegradation of furfuryl alcohol (FA) in shake flask experiments using a pure culture of Pseudomonas putida (MTCC 1194) and Pseudomonas aeruginosa (MTCC 1034) was studied at 30 °C and pH 7.0. Experiments were performed at different FA concentrations ranging from 50 to 500 mg/l. Before carrying out the biodegradation studies, the bacterial strains were acclimatized to the concentration of 500 mg/l of FA by gradually raising 100 mg/l of FA in each step. The well acclimatized culture of P. putida and P. aeruginosa degraded about 80 and 66% of 50 mg/l FA, respectively. At higher concentration of FA, the percentage of FA degradation decreased. The purpose of this study was to determine the kinetics of biodegradation of FA by measuring biomass growth rates and concentration of FA as a function of time. Substrate inhibition was calculated from experimental growth parameters using the Haldane equation. Data for P. putida were determined as µ _max ?=?0.23 h^?1, K _s ?=?23.93 mg/l and K _i ?=?217.1 mg/l and for P. aeruginosa were determined as µ _max ?=?0.13 h^?1, K _s ?=?21.3 mg/l and K _i ?=?284.9 mg/l. The experimental data were fitted in Haldane, Aiba and Edwards inhibition models.     

Ion-exchange properties of the cell walls isolated from suspension-cultured plant cells

Ion-exchange capacity of the cell walls isolated from suspension-cultured Panax japonicus, Polyscias filicifolia and Dioscorea deltoidea cells was analyzed at pH 2.8–12 and constant ionic strength (100 mM). The cell walls of all cultures contain three types of ion-exchange groups: primary amino groups (pK _a < 3), carboxyl groups of polygalacturonic acid (pK _a 3.71), and carboxyl groups of hydroxycinnamic acids (pK _a 7.62). Amount of primary amino groups ranges from 500 (D. deltoidea) to 710 (P. japonicus) µmol/g cell wall dry weight, carboxyl groups with pK _a 3.71—from 570 (D. deltoidea) to 670 (P. filicifolia), carboxyl groups with pK _a 7.62—from 270 (P. filicifolia) to 370 (P. japonicus) µmol/g cell wall dry weight. The comparison of the data obtained by elemental and functional analyses demonstrated that the cell walls of all cultures are characterized by high content of pectins (~40% by weight) and structural proteins (~17–30% by weight), but do not contain phenolic OH–groups, which presumably signifies the absence of lignin in them.     

Expression,purification, and characterization of a membrane-bound <Emphasis Type="SmallCaps">d</Emphasis>-amino acid dehydrogenase from <Emphasis Type="Italic">Proteus mirabilis</Emphasis> JN458

Objectives

To characterize a novel membrane-bound d -amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD).

Results

The recombinant PmDAD protein, encoding a peptide of 434 amino acids with a MW of 47.7 kDa, exhibited broad substrate specificity with d -alanine the most preferred substrate. The K _m and V _max values for d -alanine were 9 mM and 20 μmol min^?1 mg^?1, respectively. Optimal activity was at pH 8 and 45 °C. Additionally, this PmDAD generated H₂O₂ and exhibited 68 and 60% similarity with E. coli K12 DAD and Pseudomonas aeruginosa DAD, respectively, with low degrees of sequence similarity with other bacterial DADs.

Conclusions

d-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase.

     

Stomatal regulation in Douglas fir following a fungal-mediated chronic reduction in leaf area

Pathogens can cause chronic premature needle abscission in coniferous species. To assess the potential impacts on tree productivity, stomatal regulation was investigated in Douglas fir with chronic stomatal occlusion and defoliation from varying levels of the Swiss needle cast (SNC) fungus, Phaeocryptopus gaeumannii. Levels of SNC disease and subsequent defoliation were manipulated by choosing six sites with varying levels of disease and by foliar applications of fungicides on six trees per site. Diurnal measurements of leaf water potential (Ψ_leaf), stomatal conductance (g _s) and vapor pressure deficit (D) were made on six fungicide treated and six control trees per site. In addition, leaf specific hydraulic conductance was calculated on a single branch (K _{L_B}) from three trees per treatment per site. Stomatal conductance at D=1 kPa (g _sref) was negatively correlated with fungal colonization (number of fruiting bodies present in needle stomata) and positively correlated with K _{L_B}. Despite reduced needle retention in diseased trees, K _L declined due to a reduction in sapwood area and permeability (i.e., increasing presence of latewood in functional sapwood). In general, stomatal sensitivity to D for all foliage was consistent with stomatal regulation based on a simple hydraulic model [g _s=K _L(Ψ_soil?Ψ_leaf)/ D], which assumes strict stomatal regulation of Ψ_leaf. However, when fungal presence reduced maximum g _s below the potential maximum supported by hydraulic architecture, stomatal sensitivity was lower than expected based on the theoretical relationship: dg _s/dlnD=0.6·g _sref. The results indicate that losses in productivity associated with physical blockage of stomata and defoliation are compounded by additional losses in K _L and a reduction in g _s in remaining functional stomata.     

The Effect of Five Biomass Cropping Systems on Soil-Saturated Hydraulic Conductivity Across a Topographic Gradient

Understanding the environmental impact of bioenergy crops is needed to inform bioenergy policy development. We determined the effects of five biomass cropping systems—continuous maize (Zea mays), soybean (Glycine max)-triticale (Triticosecale ×)/soybean-maize, maize-switchgrass (Panicum virgatum), triticale/sorghum (Sorghum bicolor), and triticale-aspen (Populus alba × P. grandidentata)—on soil-saturated hydraulic conductivity (K _S ) across a toposequence in central Iowa, USA. We compared data from the time of cropping system establishment in 2009 to 4 years post-establishment. Both our 2009 and 2013 data confirmed that cropping system impacts on K _S vary by landscape position. We found that differences in cropping system impacts were more likely to occur at lower landscape positions, specifically, within footslope and floodplain positions. Previous research on cropping system impacts suggested that grass and woody systems were associated with a general increase in K _S over time, with greater changes likely occurring at landscape positions with a higher erosive potential or lower SOC content. Our results confirmed that the triticale-aspen woody system was associated with a significant increase in K _S across all landscape positions. In contrast, we did not observe an increase in K _S under maize-switchgrass, which we attributed to the high density of switchgrass roots by the fourth year of study, but expect an increase in K _S under switchgrass under longer measurement periods. We also found a significant increase in K _S in the annual systems, likely due to the conversion to no-till soil management with cropping system establishment. We expect such differences to become more apparent over longer time scales as cropping systems continue to impact soil hydraulic properties.     

Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) hydraulic conductivity links to leaf venation architecture under well-watered condition rather than PEG-induced water deficit

Higher plant hydraulic conductivity (K _plant) is vital for plant growth, especially under PEG-induced water deficit stress (PEG-IWDS). Leaf venation architecture is a key determinant of leaf hydraulic conductivity (K _leaf) and K _leaf is a major component of K _plant across different plant species. However, there is little information about (1) varietal difference in leaf vein development in cereal crops, such as rice plants; (2) the effects of PEG-IWDS on leaf vein development; (3) the coordination between leaf venation architecture and K _plant as well as K _leaf under PEG-IWDS. In the present study, widely cultivated eight rice cultivars were grown hydroponically under well-watered condition (WWC) and PEG-IWDS, simulated by adding 15 % (w/v) PEG6000. Leaf venation architecture, including total longitudinal leaf vein number, leaf vein numbers per unit width (LVNW), vein thickness and leaf mass per area, as well as K _plant and K _leaf were measured to address above-mentioned questions. The results showed that leaf venation architecture exhibited significant varietal differences and PEG-IWDS significantly increased LVNW while decreased vein thickness. PEG-IWDS suppressed both K _plant and K _leaf but the decrease was much higher in K _plant than K _leaf. There was a significant and positive correlation observed between LVNW and K _leaf under both WWC and PEG-IWDS but the correlation between LVNW and K _plant was only significant under WWC. K _leaf was significantly and positively correlated with K _plant under WWC but not under PEG-IWDS. It is concluded that K _leaf is a major determinant for K _plant under WWC but not under PEG-IWDS; therefore, breeding or selecting rice cultivars with high LVNW can improve shoot water supplement under WWC but not under PEG-IWDS condition.     

Purification and characterization of a β-1,4-glucosidase from a newly isolated strain of <Emphasis Type="Italic">Fomitopsis pinicola</Emphasis>

An efficient ß-1,4-glucosidase (BGL) producing strain, Fomitopsis pinicola KMJ812, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular BGL was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The relative molecular weight of F. pinicola BGL was determined to be 105 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, or 110 kDa by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 4.5 and a temperature optimum of 50°C. The enzyme showed high substrate specificity and high catalytic efficiency (k _cat?=?2,990 s^?1, K _m?=?1.76 mM, k _cat/K _m?=?1,700 mM^?1 s^?1) for p-nitrophenyl-β-d-glucopyranoside. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 3, indicating that the F. pinicola BGL is a member of glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, F. pinicola BGL is distinguished from other BGLs by its high catalytic efficiency and strict substrate specificity.     

Characterization and fine mapping of <Emphasis Type="Italic">osh15</Emphasis>(<Emphasis Type="Italic">t</Emphasis>), a novel dwarf mutant gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plant height is one of the most important agronomic traits of plant architecture, and also affects grain yield in rice. In this study, we obtained a novel dwarf rice mutant of japonica variety Shennong9816, designated Shennong9816d. Compared with wild-type, the Shennong9816d plant height was significantly reduced, and the tiller number significantly increased. Additionally, the mutant yield component, and the number of large and small vascular bundles were significantly decreased compared with wild-type. Genetic analysis indicated that the Shennong9816d dwarf phenotype was controlled by a recessive nuclear gene, while the plant was shown to be sensitive to gibberellic acid. Using a large F₂ population derived from a cross between Shennong9816d and the indica rice variety Habataki, the osh15(t) gene was fine mapped between RM20891 and RM20898, within a physical distance of 73.78 kb. Sequencing analysis showed that Shennong9816d carries a 1 bp mutation and a 30 bp insertion in the OSH15 region. These results suggest that osh15(t) is a novel allelic mutant originally derived from japonica variety Shennong9816, which may be useful for introducing the semi-dwarf phenotype to improve plant architecture in rice breeding practice.     

Modeling the effects of extracellular potassium on bursting properties in pre-Bötzinger complex neurons

There are many types of neurons that intrinsically generate rhythmic bursting activity, even when isolated, and these neurons underlie several specific motor behaviors. Rhythmic neurons that drive the inspiratory phase of respiration are located in the medullary pre-Bötzinger Complex (pre-BötC). However, it is not known if their rhythmic bursting is the result of intrinsic mechanisms or synaptic interactions. In many cases, for bursting to occur, the excitability of these neurons needs to be elevated. This excitation is provided in vitro (e.g. in slices), by increasing extracellular potassium concentration (K _out ) well beyond physiologic levels. Elevated K _out shifts the reversal potentials for all potassium currents including the potassium component of leakage to higher values. However, how an increase in K _out , and the resultant changes in potassium currents, induce bursting activity, have yet to be established. Moreover, it is not known if the endogenous bursting induced in vitro is representative of neural behavior in vivo. Our modeling study examines the interplay between K _out , excitability, and selected currents, as they relate to endogenous rhythmic bursting. Starting with a Hodgkin-Huxley formalization of a pre-BötC neuron, a potassium ion component was incorporated into the leakage current, and model behaviors were investigated at varying concentrations of K _out . Our simulations show that endogenous bursting activity, evoked in vitro by elevation of K _out , is the result of a specific relationship between the leakage and voltage-dependent, delayed rectifier potassium currents, which may not be observed at physiological levels of extracellular potassium.     

Overexpression and characterization of a novel cold-adapted and salt-tolerant GH1 β-glucosidase from the marine bacterium <Emphasis Type="Italic">Alteromonas</Emphasis> sp. L82

A novel gene (bgl) encoding a cold-adapted β-glucosidase was cloned from the marine bacterium Alteromonas sp. L82. Based on sequence analysis and its putative catalytic conserved region, Bgl belonged to the glycoside hydrolase family 1. Bgl was overexpressed in E. coli and purified by Ni²⁺ affinity chromatography. The purified recombinant β-glucosidase showed maximum activity at temperatures between 25°C to 45°C and over the pH range 6 to 8. The enzyme lost activity quickly after incubation at 40°C. Therefore, recombinant β-glucosidase appears to be a cold-adapted enzyme. The addition of reducing agent doubled its activity and 2 M NaCl did not influence its activity. Recombinant β-glucosidase was also tolerant of 700 mM glucose and some organic solvents. Bgl had a K_m of 0.55 mM, a V_max of 83.6 U/mg, a k_cat of 74.3 s^-1 and k_cat/K_m of 135.1 at 40°C, pH 7 with 4-nitrophenyl-β-D-glucopyranoside as a substrate. These properties indicate Bgl may be an interesting candidate for biotechnological and industrial applications.     

Influence of Daily Short-Term Temperature Drops on Respiration to Photosynthesis Ratio in Chilling-Sensitive Plants

Cucumber (Cucumis sativus L.), tomato (Solanum lycopersicum L.), and sweet pepper (Capsicum annuum L.) plants were subjected daily over 13 days to short-term (2 h) temperature drops to 12, 8, 4, and 1°C (DROP treatments) at the end of night periods, and effects of these chilling treatments on the ratio of dark respiration in leaves (R_d) to gross photosynthesis (A_g) were examined. The results showed that DROP treatments affected the R_d/A_g ratio in leaves: this ratio increased significantly in cucumber and tomato plants and was slightly affected in pepper plants. When the temperature drops to 12°C were applied, the increase in R_d/A_g ratio in cucumber and tomato plants was entirely due to the rise in R_d. In the case of temperature drops to 8°C and below, the increase in R_d/A_g was determined by both elevation of R_d and the concurrent decrease in Ag. In cucumber plants, the extent of Ag and R_d changes increased with the DROP severity, i.e., with lowering the temperature of DROP treatment. The inhibition of photosynthesis by DROP treatment in cucumber plants was accompanied by the diminished efficiency of light energy use for photosynthesis and by the increase in the light compensation point. The elevation in R_d/A_g ratio in cucumber plants was accompanied by the decline in growth characteristics, such as accumulation of aboveground biomass, plant height, and leaf area. It was concluded that the R/A ratio is an important indicator characterizing the adaptive potential of chilling-sensitive plant species and their response to daily short-term temperature drops.     

Modulation of alfa-adrenoceptor activity by beta-adrenoceptor agonists and antagonists in rat brain cortex membranes

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosin and [³H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α₁- and α₂-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [³H]prazosin binding to α₁-adrenoceptors were: K _d = 1.85 ± 0.16 nM, B _max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.57 ± 0.27 nM, B _max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α₁- and α₂-adrenoceptors occurred according to the same model. The affinity to [³H]prazosin and the concentration of active α₁-adrenoceptors increased by 27% (K _d = 1.36 ± 0.03 nM) and 84% (B _max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α₂-adrenoceptors to [³H]RX821002 decreased by 56% (K _d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [³H]prazosin and [³H]RX821002, two pools of receptors were detected with the following parameters: K _d1 = 1.13 ± 0.09, K _d2 = 6.07 ± 1.06 nM, B _m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K _d1 = 0.61 ± 0.02, K _d2 = 3.41 ± 0.13 nM, B _m1 = 1.88 ± 0.028, B _m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B _max) increased twofold for both ligands. It was suggested that α₁- and α₂-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α₁- and α₂- adrenoceptors was revealed, which was manifested in the activating effect on the [³H]prazosin binding parameters, in the inhibitory effect on the [³H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.