Deprotonation of β-cyclodextrin in alkaline solutions

Variable pH ¹³C NMR and ¹H NMR spectroscopic studies of the β-cyclodextrin (β-CD) in alkaline aqueous solutions revealed that β-CD does not deprotonate at pH < 12.0. Further increase in solution pH results in the deprotonation of OH-groups adjacent to C-2 and C-3 carbon atoms of β-CD glucopyranose units, whereas the deprotonation of OH-groups adjacent to C-6 carbon atoms is expressed less markedly. The pK_a values for β-CD OH-groups adjacent to C-2 and C-3 carbon atoms are rather close, pK_a1,2 being 13.5 ± 0.2 (22.5 °C).     

Large-volume sample stacking of selected drugs of forensic significance by capillary electrophoresis

Large-volume sample stacking capillary electrophoresis (LVSS-CE) and conventional capillary electrophoresis (CE) are compared for the separation of drugs of significance to forensic and clinical analyses. LVSS-CE for cations requires the use of an electroosmotic flow (EOF) modifier in conjunction with polarity switching to effect on-column concentration of an analyte and its subsequent migration in the capillary. The run buffer consists of 0.05 mol dm⁻³ disodium tetraborate adjusted to pH 2.2 with orthophosphoric acid, and the EOF modifier is 0.002 mol dm⁻³ cetyltrimethylammonium bromide. CE investigations used an identical run buffer minus the EOF modifier. LVSS-CE and CE investigations used injection times of 30 s and 3 s, respectively. Both modes of capillary electrophoresis are compared in terms of their limits of detection, efficiency, resolution and reproducibility. LVSS-CE is also applied to the analysis of a spiked urine sample.     

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K_m, k_cat and k_cat/K_m values for oNPG and lactose are 0.9 mM, 92 s⁻¹, 130 mM⁻¹ s⁻¹ and 29 mM, 98 s⁻¹, 3.3 mM⁻¹ s⁻¹, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-d-Galp-(1→6)-d-Lac, accounting for 34% of total GOS, and β-d-Galp-(1→6)-d-Glc, making up 29% of total GOS.     

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose

The BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular β-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates (μ_max) could reach 0.03 and 0.05 h⁻¹ under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobiose L⁻¹ and produced 2.3 g ethanol L⁻¹ in 48 h, while S. cerevisiae secreting β-glucosidase into culture broth used 3.6 g cellobiose L⁻¹ and produced 1.5 g ethanol L⁻¹ over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when β-glucoside permease and β-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11 h⁻¹) on cellobiose.     

Toward high-throughput monitoring of metallodrug-protein interaction using capillary electrophoresis in chemically modified capillaries

The performance of capillary electrophoresis (CE) operating with a sulfonated capillary for the separation of protein adducts of anticancer ruthenium(III)-based drugs was evaluated. The coated capillary was shown to yield improved resolution of albumin- and transferrin-bound species of ruthenium compared with that attained with the bare fused-silica capillary. The coating also showed an increased reproducibility of migration times and peak areas and allowed reasonably high efficiency separation of analytes (up to 1300 theoretical plates per meter), which display high affinity toward a fused-silica surface. In addition, due to rather high electroosmotic flow (EOF, > 45 × 10⁻⁵ cm² V⁻¹ s⁻¹) in the coated capillary, it enabled fast counter-EOF monitoring of albumin and transferrin adducts. This benefit, together with requiring only a short flush with the background electrolyte to have migration times reproducible (at < 1.5% relative standard deviation), makes this wall-modified capillary holding promise for CE examination of fast reactions such as those accompanying protein-drug interactions and biotransformations associated with drug delivery via protein binding.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins

Barley limit dextrinase [Hordeum vulgare limit dextrinase (HvLD)] catalyzes the hydrolysis of α-1,6 glucosidic linkages in limit dextrins. This activity plays a role in starch degradation during germination and presumably in starch biosynthesis during grain filling. The crystal structures of HvLD in complex with the competitive inhibitors α-cyclodextrin (CD) and β-CD are solved and refined to 2.5 Å and 2.1 Å, respectively, and are the first structures of a limit dextrinase. HvLD belongs to glycoside hydrolase 13 family and is composed of four domains: an immunoglobulin-like N-terminal eight-stranded β-sandwich domain, a six-stranded β-sandwich domain belonging to the carbohydrate binding module 48 family, a catalytic (β/α)₈-like barrel domain that lacks α-helix 5, and a C-terminal eight-stranded β-sandwich domain of unknown function. The CDs are bound at the active site occupying carbohydrate binding subsites + 1 and + 2. A glycerol and three water molecules mimic a glucose residue at subsite − 1, thereby identifying residues involved in catalysis. The bulky Met440, a unique residue at its position among α-1,6 acting enzymes, obstructs subsite − 4. The steric hindrance observed is proposed to affect substrate specificity and to cause a low activity of HvLD towards amylopectin. An extended loop (Asp513-Asn520) between β5 and β6 of the catalytic domain also seems to influence substrate specificity and to give HvLD a higher affinity for α-CD than pullulanases. The crystal structures additionally provide new insight into cation sites and the concerted action of the battery of hydrolytic enzymes in starch degradation.     

Evaluation of the accessible inclusion sites in copolymer materials containing β-cyclodextrin

The accessible inclusion sites of insoluble copolymers containing β-cyclodextrin (β-CD) were studied in aqueous solutions by measuring the absorbance changes (decolourization) of phenolphthalein (phth) at pH 10.5. The various copolymers were reacted at different β-CD:crosslinker mole ratios with five individual types of crosslinker agents (epichlorohydrin (EP), sebacoyl chloride (SCL), terephthaloyl chloride (TCL), glutaraldehyde (GLU), and poly(acrylic) acid (PAA), respectively). The decolourization provided estimates of the 1:1 binding constants (K₁) for the β-CD monomer/phth complex. Comparable values of K₁ were measured for copolymer/phth complexes with highly accessible β-CD inclusion sites as compared with the 1:1 β-CD/phth complex. The surface accessibility of the β-CD inclusion binding sites for the polymers ranged from ∼10 to 72%. The observed variability of the inclusion sites was attributed to: (i) steric effects in the annular hydroxyl region of β-CD, (ii) the degree of crosslinking of the copolymer and (iii) the accessibility of the micropore sites within the copolymers. The Gibbs free energy (ΔG°) and site occupancy (θ) of phth adsorbed to the copolymer materials was estimated independently using the Sips isotherm model. The ΔG° values ranged between −27.6 and −30.9 kJ mol⁻¹ for the copolymers and are in close agreement with the value for the 1:1 β-CD/phth complexes (ΔG° = −27 kJ mol⁻¹) in aqueous solution.     

Quantification of carnosine-related peptides by microchip electrophoresis with chemiluminescence detection

A microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of carnosine-related peptides, including carnosine, homocarnosine, and anserine, in biological samples. A simple integrated MCE-CL system was built to perform the assays. The highly sensitive CL detection was achieved by means of the CL reaction between hydrogen peroxide and N-(4-aminobutyl)-N-ethylisoluminol-tagged peptides in the presence of adenine as a CL enhancer and Co²⁺ as a catalyst. Experimental conditions for analyte labeling, MCE separation, and CL detection were studied. MCE separation of the above-mentioned three peptides took less than 120 s. Detection limits (signal/noise ratio [S/N] = 3) of 3.0 × 10⁻⁸, 2.8 × 10⁻⁸, and 3.4 × 10⁻⁸ M were obtained for carnosine, anserine, and homocarnosine, respectively. The current MCE-CL method was applied for the determination of carnosine, anserine, and homocarnosine in human cerebrospinal fluid (CSF) and canine plasma. Homocarnosine was detected at the micromolar (μM) level in the CSF samples analyzed, whereas the levels of carnosine and anserine in these samples were below the detection limit of the assay. Interestingly, both carnosine and anserine were detected in the canine plasma samples, whereas homocarnosine was not.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Inter-population variability of leaf morpho-anatomical and terpenoid patterns of Pistacia atlantica Desf. ssp. atlantica growing along an aridity gradient in Algeria

Three Algerian populations of female Pistacia atlantica shrubs were investigated in order to check whether their terpenoid contents and morpho-anatomical parameters may characterize the infraspecific variability. The populations were sampled along a gradient of increasing aridity from the Atlas mountains into the northwestern Central Sahara.As evidenced by Scanning Electron Microscopy, tufted hairs could be found only on seedling leaves from the low aridity site as a population-specific trait preserved also in culture. Under common garden cultivation seedlings of the high aridity site showed a three times higher density of glandular trichomes compared to the low aridity site. Increased aridity resulted also in reduction of leaf sizes while their thickness increased. Palisade parenchyma thickness also increases with aridity, being the best variable that discriminates the three populations of P. atlantica.Analysis of terpenoids from the leaves carried out by GC-MS reveals the presence of 65 compounds. The major compounds identified were spathulenol (23 μg g⁻¹ dw), α-pinene (10 μg g⁻¹ dw), verbenone (7 μg g⁻¹ dw) and β-pinene (6 μg g⁻¹ dw) in leaves from the low aridity site; spathulenol (73 μg g⁻¹ dw), α-pinene (25 μg g⁻¹ dw), β-pinene (18 μg g⁻¹ dw) and γ-amorphene (16 μg g⁻¹ dw) in those from medium aridity and spathulenol (114 μg g⁻¹ dw), α-pinene (49 μg g⁻¹ dw), germacrene D (29 μg g⁻¹ dw) and camphene (23 μg g⁻¹ dw) in leaves from the high aridity site. Terpene concentrations increased with the degree of aridity: the highest mean concentration of monoterpenes (136 μg g⁻¹ dw), sesquiterpenes (290 μg g⁻¹ dw) and total terpenes (427 μg g⁻¹ dw) were observed in the highest arid site and are, respectively, 3-, 5- and 4-fold higher compared to the lower arid site. Spathulenol and α-pinene can be taken as chemical markers of aridity. Drought discriminating compounds in low, but detectable concentrations are δ-cadinene and β-copaene. The functional roles of the terpenoids found in P. atlantica leaves and principles of their biosynthesis are discussed with emphasis on the mechanisms of plant resistance to drought conditions.     

Structural characterization and protective effect against murine sepsis of fucogalactans from Agaricus bisporus and Lactarius rufus

Fucogalactans from edible Agaricus bisporus (RFP-Ab) and wild Lactarius rufus (RFP-Lr) mushrooms were obtained on aqueous extraction followed by purification. RFP-Ab had M_w 43.8 × 10⁴ g mol⁻¹ and RFP-Lr M_w 1.4 × 10⁴ g mol⁻¹. RFP-Lr had a (1 → 6)-linked α-d-Galp main-chain partially substituted at O-2 by nonreducing end-units of α-l-Fucp (29%). While RFP-Ab had a similar main chain, it was partially substituted at O-2 by nonreducing end-units of α-l-Fucp (2.8%) and β-d-Galp (14.5%), and partially methylated at HO-3. Both RFP-Lr and RFP-Ab were tested in mice against polymicrobial sepsis. Lethality rate, myeloperoxidase (MPO) activity and cytokine levels were determined. It was observed a reduction in late mortality rate by 62.5% and 50%, respectively, prevention of neutrophil accumulation in ileum and decreasing in TNF-α and IL-1β serum levels.     

Bicarbonate use in three aquatic plants

Our study aimed to test the ability of aquatic plants to use bicarbonate when acclimated to three different bicarbonate concentrations. To this end, we performed experiments with the three species Ceratophyllum demersum, Egeria densa, Lagarosiphon major to determine photosynthetic rates under varying bicarbonate concentrations. We measured bicarbonate use efficiency, photosynthetic performance and respiration. For all species, our results revealed that photosynthetic rates were highest in replicates grown at low alkalinity. Thus, E. densa had approx. five times higher rates at low (264 ± 15 μmol O₂ g⁻¹ DW h⁻¹) than at high alkalinity (50 ± 27 μmol O₂ g⁻¹ DW h⁻¹), C. demersum had three times higher rates (336 ± 95 and 120 ± 31 μmol O₂ g⁻¹ DW h⁻¹), and L. major doubled its rates at low alkalinity (634 ± 114 and 322 ± 119 μmol O₂ g⁻¹ DW h⁻¹). Similar results were obtained for bicarbonate use efficiency by E. densa (136 ± 44 and 43 ± 10 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹) and L. major (244 ± 29 and 82 ± 24 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹). As to C. demersum, efficiency was high but unaffected by alkalinity, indicating high adaptation ability to varied alkalinities. A pH drift experiment supported these results. Overall, our results suggest that the three globally widespread worldwide species of our study adapt to low inorganic carbon availability by increasing their efficiency of bicarbonate use.     

Effect of NaCl on photosynthesis,salt accumulation and ion compartmentation in two mangrove species, Kandelia candel and Bruguiera gymnorhiza

In a 4-week study, we investigated the effects of increasing soil NaCl (100–400 mM) on photosynthesis, salt uptake and transport, and intracellular compartmentation of Na⁺ and Cl⁻ in 1-year-old seedlings of Kandelia candel (L.) Druce and Bruguiera gymnorhiza (L.) Savigny. Increasing NaCl stress significantly elevated Na⁺ and Cl⁻ in root and shoot tissues (stem + leaf) of both species, but B. gymnorhiza showed a rapid Na⁺ accumulation upon the initiation of salt stress and leaves contained 90% more Na⁺ and 40% more Cl⁻ than K. candel at the end of experiment. Net photosynthetic rate (Pn) declined with increasing salinity, and the most marked reduction occurred after exposure of mangrove seedlings to a severe salinity, 400 mM NaCl. However, the inhibitory effects of severe stress varied with species: Pn decreased by 80% in K. candel whereas in B. gymnorhiza the decline was 60%. The quantum yield (AQY) and carboxylation efficiency (CE) response to severe salinity showed a trend similar to Pn, in which a lesser reduction of AQY and CE was observed in B. gymnorhiza (33–35%), as compared to K. candel (43–52%). X-ray microanalysis of leaf mesophyll cells showed evidence of distinct vacuolar compartmentation of Na⁺ in K. candel but Cl⁻ in B. gymnorhiza after seedlings were subjected to 100 mM NaCl for 7 d. Moreover, Na⁺ within cell wall, cytoplasm, vacuole and chloroplast remained 23–72% lower in stressed B. gymnorhiza as compared to K. candel. In conclusion, B. gymnorhiza exhibited effective salt exclusion from chloroplasts although increasing salt stress caused a rapid and higher build up of Na⁺ and Cl⁻ in the leaves. We suggest that the salt-induced Pn reduction in the two mangrove species is correlated with the ability to exclude Na⁺ and Cl⁻ from the chloroplast, rather than with the bulk leaf salt concentration.     

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10⁵-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k_cat′ for desulfination of l-cysteine sulfinate increased to 0.5 s^− 1 (from 0.05 s^− 1 in wild-type enzyme), whereas k_cat′ for transamination of the same substrate was reduced from 510 s^− 1 to 0.05 s^− 1. Similarly, k_cat′ for β-decarboxylation of l-aspartate increased from < 0.0001 s^− 1 to 0.07 s^− 1, whereas k_cat′ for transamination was reduced from 530 s^− 1 to 0.13 s^− 1. l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.     

A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase

Protoporphyrinogen oxidase (PPO) is a flavin adenine dinucleotide (FAD)-containing enzyme in the tetrapyrrole biosynthetic pathway that leads to the formation of both heme and chlorophylls, which has been identified as one of the most important action targets of commercial herbicides. The literature reports gave different PPO-catalytic kinetic parameters for the substrate protoporphyrinogen IX (K_m of 0.1 to 10.4 μM) with different sources of PPO using fluorescent or HPLC methods. Herein we assayed the enzymatic activity of recombinant Bacillus subtilis PPO by using capillary electrophoresis (CE), a method with high separation efficiency, easy automation, and low sample consumption. The Michaelis constant and maximum reaction velocity were determined as 7.0 ± 0.6 μM and 0.38 ± 0.02 μmol min^-1 μg⁻¹, respectively. The interaction between PPO and acifluorfen, a commercial PPO-inhibiting herbicide, was measured as the inhibition constant 186.9 ± 9.3 μМ. The relationship between cofactor FAD and PPO activity can also be quantitatively studied by this CE method. The CE method used here should also be a convenient, reliable method for PPO study.     

Treatment of industrial wastewater with two-stage constructed wetlands planted with Typha latifolia and Phragmites australis

Industrial wastewater treatment comprises several processes to fulfill the discharge permits or to enable the reuse of wastewater. For tannery wastewater, constructed wetlands (CWs) may be an interesting treatment option. Two-stage series of horizontal subsurface flow CWs with Phragmites australis (UP series) and Typha latifolia (UT series) provided high removal of organics from tannery wastewater, up to 88% of biochemical oxygen demand (BOD₅) (from an inlet of 420 to 1000 mg L⁻¹) and 92% of chemical oxygen demand (COD) (from an inlet of 808 to 2449 mg L⁻¹), and of other contaminants, such as nitrogen, operating at hydraulic retention times of 2, 5 and 7 days. No significant (P < 0.05) differences in performance were found between both the series. Overall mass removals of up to 1294 kg COD ha⁻¹ d⁻¹ and 529 kg BOD₅ ha⁻¹ d⁻¹ were achieved for a loading ranging from 242 to 1925 kg COD ha⁻¹ d⁻¹ and from 126 to 900 kg BOD₅ ha⁻¹ d⁻¹. Plants were resilient to the conditions imposed, however P. australis exceeded T. latifolia in terms of propagation.     

Sorgoleone,a sorghum root exudate: Algicidal activity and acute toxicity to the ricefish Oryzias latipes

The discovery of natural and natural-based compounds has resulted in its application as an alternative to synthetic algicides to control harmful algae in aquatic systems. Of the many natural-product-based algicides, sorgoleone, a natural plant product from Sorghum bicolor root exudates has been investigated for its controlling effect on different algal species and its acute fish toxicity. Growth of the blue green algal species Microcystis aeruginosa Kützing was completely inhibited by the crude methanol extract of sorghum root at 20 μg mL⁻¹. The most noticeable inhibition was observed in the bioassay of n-hexane soluble extract, where 98% growth inhibition occurred in M. aeruginosa at the concentration of 1.25 μg mL⁻¹. Sorgoleone very effectively controlled blue green algae inhibiting 97% of M. aeruginosa at 0.5 μg mL⁻¹ and 99% of Anabaena affinis Lemmermann at 4 μg mL⁻¹. In contrast, inhibition of the green algae species Chlorella vulgaris Beijerinck and Scenedensmus spp. at 16 μg mL⁻¹ sorgoleone was 87 and 68%, respectively. There were no mortalities or adverse effects observed in any of the fish exposed to water control, solvent control, and a nominal concentration of 1 μg mL⁻¹ during the test period. The no observed effect concentration (NOEC) value was 1.5 μg mL⁻¹ for the tested fish (O. latipes). Sorgoleone can be considered as an effective and an ecologically and environmentally sustainable approach to controlling harmful algae.     

The effects of bee (Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria

The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2 h and 12 h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml⁻¹ bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2 h after treatment but viability decreased up to 8 h. Even very low concentrations of bvPLA2 (10⁻¹² mg ml⁻¹) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2 h bacterial cultures but bacteriostatic to 12 h ones. Minimum bactericidal concentrations were 10⁻⁵-10⁻⁶ mg ml⁻¹. The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.     

Inhibition studies with anions and small molecules of two novel β-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium

Two new β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were characterized kinetically. The two enzymes possess appreciable activity as catalysts for the hydration of CO₂ to bicarbonate, with k_cat of 0.79 × 10⁶ s⁻¹ and 1.0 × 10⁶ s⁻¹, and k_cat/K_m of 5.2 × 10⁷ M⁻¹ s⁻¹ and of 8.3 × 10⁷ M⁻¹ s⁻¹, respectively. A large number of simple/complex inorganic anions as well as other small molecules (sulfamide, sulfamic acid, phenylboronic acid, phenylarsonic acid, dialkyldithiocarbamates) showed interesting inhibitory properties towards the two new enzymes, with several low micromolar inhibitors discovered. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the β-CAs investigated here may be useful for developing lead compounds for novel types of antibacterials.