Introduction of a low molecular weight agonist peptide for complement C3a receptor into soybean proglycinin A1aB1b subunit by site-directed mutagenesis.

LPLPR, a complement C3a agonist peptide, with hypocholesterolemic activity was introduced into the homologous site of soybean proglycinin A1aB1b subunit by site-directed mutagenesis. This modified proglycinin was expressed in E. coli and recovered from the insoluble fraction. LPLPR was released by the action of chymotrypsin and trypsin as expected. Furthermore, two peptides (RPSYLPLPR and PSYLPLPR) with extended sequence at the amino-terminus of LPLPR were obtained. Their ileum-contracting activity was 9 to approximately 13 times stronger than that of LPLPR. The overall yields of purified LPLPR, RPSYLPLPR and PSYLPLPR were 25%, 12%, and 0.7% respectively.     

Introduction of enterostatin (VPDPR) and a related sequence into soybean proglycinin A1aB1b subunit by site-directed mutagenesis

Enterostatin (VPDPR), having anoretic and hypocholesterolemic activities, and its homologue LPYPR, a hypocholesterolemic peptide found in the glycinin A5A4B3 subunit, were introduced into the corresponding site (TNGPQ) of the proglycinin A1aB1b subunit by site-directed mutagenesis. Modified proglycinins were expressed in E. coli and recovered from the insoluble fraction. VPDPR and LPYPR were released by the action of chymotrypsin and trypsin as expected. The overall yields of purified VPDPR and LPYPR were 40% and 62%, respectively.     

A SPECTROPHOTOMETRIC ASSAY FOR RUBISCO ACTIVITY: APPLICATION TO THE KELP LAMINARIA SACCHARINA AND IMPLICATIONS FOR RADIOMETRIC ASSAYS1

We optimized a spectrophotometric method for measuring ribulose-l,5-bisphosphate carboxylase oxygenase (Rubisco) activity in crude extracts of the kelp Laminaria saccharina Lamour. Activity exhibited a decline during the assays, such that rates determined over a 10-min period were only 70% of activity during the first minute. Activity was significantly enhanced by addition of HCO₃^? to the extraction buffer and was stable for at least 4 h. Highest activities were determined using small extract loads (1–2.5 mg fresh weight·mL^?1 of assay mixture); extract loads of 10 mg·mL^?1caused a 25% decrease in activity. Rubisco activities determined for individual kelp plants using the spectrophotometric method were significantly correlated with total protein and gross photo synthetic capacity (P_max) and were 1.5–2 times higher than P_max. Low Rubisco activities relative to photosynthetic rates reported in many previous studies of macroalgae and microalgae may have resulted at least partly from underestimation of enzyme activity by radiometric assays using relatively large volumes of un-activated crude extract and assay periods longer than 1 min.     

Improved Bile Acid-binding Ability of Soybean Glycinin A1a Polypeptide by the Introduction of a Bile Acid-binding Peptide (VAWWMY)

We have previously identified a potential bile acid-binding peptide sequence (VAWWMY) in acidic polypeptide A1a of the soybean glycinin A1aB1b subunit (Choi, S. K., et al., Biosci. Biotechnol. Biochem., 66, 2395–2401 (2002)). In this study, we introduced the nucleotide sequence encoding this peptide in the coding DNA which corresponds to amino acids between 251 and 256, and 282 and 287 into the A1a polypeptide by replacement to respectively give modified versions A1aM1 and A1aM2. A fluorescence analysis demonstrates that their bile acid-binding ability was improved compared to A1a. Moreover, modified proglycinin A1aB1b with the VAWWMY sequence at the same sites as those of A1aM1 and A1aM2 was judged to assume the correct conformation. These results suggest the possibility of developing transgenic crops to accumulate the modified glycinin.     

Acetyl- and butyrylcholinesterase in normal and diabetic rat retina

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinas. Tissues were sequentially extracted with saline (S₁) and saline-detergent buffers (S₂). 50% decrease in the amphiphilic G₄ and G₁ AChE molecular forms was observed in the diabetic retina compared to the controls. Less than 5% of the cholinesterase activity was due to BChE. 60% of the BChE activity in normal retina was brought into solution and evenly distributed between S₁ and S₂. In spite of the low BChE activity in the retina it was possible to detect globular forms (G^A ₁, G^A ₂, G^A ₄, G^H ₄) and a small proportion of an asymmetric form (A₁₂) in the S₁ extract. The G^A ₄ and G^A ₁ forms were found in the S₂ extract. In the diabetic retina the activity of G^A ₄ and G^A ₁ BChE molecular forms was reduced 60% and 40% respectively. Our results indicate that diabetes caused a remarkable decrease in the activity of cholinesterase molecular forms in the retina. These decrease might participate in the alterations observed in the diabetic retina.     

Bicarbonate-Independent Sodium Conductance of Na/HCO3 Cotransporter NBCn1 Decreases NMDA Receptor Function

The sodium bicarbonate cotransporter NBCn1 is an electroneutral transporter with a channel activity that conducts Na⁺ in a HCO₃^–-independent manner. This channel activity was suggested to functionally affect other membrane proteins which permeate Na⁺ influx. We previously reported that NBCn1 is associated with the NMDA receptors (NMDARs) at the molecular and physiological levels. In this study, we examined whether NBCn1 channel activity affects NMDAR currents and whether this effect involves the interaction between the two proteins. NBCn1 and the NMDAR subunits GluN1A/GluN2A were expressed in Xenopus oocytes, and glutamate currents produced by the receptors were measured using two-electrode voltage clamp. In the absence of CO₂/HCO₃^–, NBCn1 channel activity decreased glutamate currents mediated by GluN1A/GluN2A. NBCn1 also decreased the slope of the current–voltage relationships for the glutamate current. Similar effects on the glutamate current were observed with and without PSD95, which can cluster NBCn1 and NMDARs. The channel activity was also observed in the presence of CO₂/HCO₃^–. We conclude that NBCn1 channel activity decreases NMDAR function. Given that NBCn1 knockout mice develop a downregulation of NMDARs, our results are unexpected and suggest that NBCn1 has dual effects on NMDARs. It stabilizes NMDAR expression but decreases receptor function by its Na⁺ channel activity. The dual effects may play an important role in fine-tuning the regulation of NMDARs in the brain.     

EARLY TOXIC EFFECTS OF ZINC ON PSII OF SYNECHOCYSTIS AQUATILIS F. AQUATILIS (CYANOPHYCEAE)1

Zinc toxicity on photosynthetic activity in cells of Synechocystis aquatilis f. aquatilis Sauvageau was investigated by monitoring Hill activity and fluorescence. The oxygen‐evolving activity decreased to about 80% of the initial value after exposure to 0.1 mM ZnSO₄ for 1 h. The PSII activity was inhibited by 40% in the presence of zinc concentrations ranging from 0.5 to 5.0 mM, suggesting that the metal effect is limited by zinc uptake. The fluorescence capacity (F_max–F/F_max) decreased from 0.57 to 0.35 and 0.20 in Zn‐treated cells for 15 and 60 min, respectively, thus providing evidence for rapid inactivation of electron transport at PSII. Zinc treatment promoted a rapid increase in PSII fluorescence that was counteracted by addition of 1,4‐benzoquinone, indicating that electron transfer at the reducing side of the PSII reaction center is arrested by zinc. Furthermore, a decline in the fluorescence yield could be observed after 1 h of zinc treatment as well as when Zn‐treated cells were excited in presence of 3‐(3′,4′‐dichlorophenyl)‐1,1‐dimethylurea. Under these conditions, zinc did not affect energy transfer from phycobilisomes to PSII, and the gradual quenching of PSII fluorescence may be due to a decrease in electron flow on the donor side of PSII. However, the 20% increase in the minimal fluorescence intensity (F_o) in parallel to the absence of changes in the maximal fluorescence intensity (F_max), observed in the first hour of zinc treatment, could also suggest a metal‐induced decline in the energy transfer from PSII‐chl a antenna to the PSII reaction center.     

Phytoremediation affects microbial development on a limestone quarry

Phytoremediation was used to regenerate a limestone quarry area. Plant growth mixed medium added over the quarry surface, consisting of a mixture of pyrolusite byproducts, natural soil, sand, and rice husk. Three different plant species: pine, cypress, and broom were planted at 9 randomized plots in order to assess the effects of vegetation on the microbial development, which was measured for the following 3 years. Substrate samples were analyzed for organic carbon content (C_org), microbial biomass (C_mic), basal CO₂ respiration activity (BR), alkaline phosphatase (ALP), and acid phosphatase activities at each plant specie and year. Furthermore, the ratio C_mic/C_org, the metabolic quotient (qCO₂), and the C mineralization quotient (qM) were determined. The highest survival rates occurred for broom (93.52%), followed by cypress and pine (82.41%) at the final year, while the content of C_mic, BR, and ALP was increased significantly under plants (pine, cypress, and broom) compared with control. C_mic content and BR was plant dependent. Cypress sites had the highest values of C_mic (214.9 μgCg^?1) and BR (112.8 μgCO₂-Cg^?1d^?1) at the 3^rd year. The plant root environment clearly enhances and regulates the microbial community, in correspondence to the species used. Below ground enhanced activity could fulfill the scope of phytoremediation strategies.     

Enhancement of the hydrolysis activity of F0F1‐ATPases using 60 Hz magnetic fields

The effects of extremely low frequency (ELF) magnetic fields on membrane F₀F₁‐ATPase activity have been studied. When the F₀F₁‐ATPase was exposed to 60 Hz magnetic fields of different magnetic intensities, 0.3 and 0.5 mT magnetic fields enhanced the hydrolysis activity, whereas 0.1 mT exposure caused no significant changes. Even if the F₀F₁‐ATPase was inhibited by N,N‐dicyclohexylcarbodiimide, its hydrolysis activity was enhanced by a 0.5 mT 60 Hz magnetic field. Moreover, when the chromatophores which were labeled with F‐DHPE were exposed to a 0.5 mT, 60 Hz magnetic field, it was found that the pH of the outer membrane of the chromatophore was unchanged, which suggested that the magnetic fields used in this work did not affect the activity of F0. Taken together, our results show that the effects of magnetic fields on the hydrolysis activity of the membrane F₀F₁‐ATPases were dependent on magnetic intensity and the threshold intensity is between 0.1 and 0.3 mT, and suggested that the F1 part of F₀F₁‐ATPase may be an end‐point affected by magnetic fields. Bioelectromagnetics 30:663–668, 2009. © 2009 Wiley‐Liss, Inc.     

Impact of a Streptomyces (AS1) strain and its metabolites on control of Aspergillus flavus and aflatoxin B1 contamination in vitro and in stored peanuts

Six actinomycetes were isolated from peanuts in Egypt. Of these, a Streptomyces strain (AS1) was found in in vitro assays to inhibit directly or via secondary metabolites both germination and growth of Aspergillus flavus. Tests of the AS1 cells for direct control of A. flavus populations or aflatoxin B₁ (AFB₁) production on stored peanuts was unsuccessful over 14-day storage periods. However, crude extracts of AS1 metabolites at 50 and 100 ppm completely inhibited spore germination of conidia of A. flavus in vitro over 48 h. Comparison of solvents for extracting the metabolites showed that the ethyl acetate extract was most effective. This gave greater than 85% inhibition of mycelial growth at these concentrations at different water availabilities (water activity; a _w; 0.95, 0.92, and 0.89) and 25°C. Doses of 50, 200, and 500 ppm of AS1 metabolites significantly inhibited populations of A. flavus on stored peanuts at two water stress levels (0.90, 0.93 a _w) at 25°C over 14-day storage periods. The amounts of AFB₁ produced by A. flavus on peanuts stored at 0.90 a _w were significantly decreased by AS1 metabolites for only 7 days. However, at 0.93 a _w doses of 200 and 500 ppm significantly controlled AFB₁ accumulation in peanuts for 14 days.     

Characterization of the ATPase activity of a novel chimeric fusion protein consisting of the two nucleotide binding domains of MRP1

Nucleotide Binding Domains (NBDs) are responsible for the ATPase activity of the multidrug resistance protein 1 (MRP1). A series of NBD1-linker-NBD2 chimeric fusion proteins were constructed, expressed and purified, and their ATPase activities were analyzed. We report here that a GST linked NBD1_642-890-GST-NBD2_1286-1531 was able to hydrolyze ATP at a rate of about 4.6 nmol/mg/min (K_m = 2.17 mM, V_max = 12.36 nmol/mg/min), which was comparable to the purified and reconstituted MRP1. In contrast, neither a mixture of NBD1 and GST-NBD2 nor the NBD1-GST-NBD1 fusion protein showed detectable ATPase activity. Additionally, the E1455Q mutant was found to be nonfunctional. Measurements by both MIANS labeling and circular dichroism spectroscopy revealed significant conformational differences in the NBD1-GST-NBD2 chimeric fusion protein compared to the mixture of NBD1 and GST-NBD2. The results suggest a direct interaction mediated by GST between the two NBDs of MRP1 leading to conformational changes which would enhance its ATPase activity.     

Design,synthesis and biological activity evaluation of desloratadine analogues as H1 receptor antagonists

A series of N-substituted desloratadine analogues were designed and synthesized. They were tested for H₁ antihistamine activity by inhibiting histamine-induced contraction of isolated ileum muscles of guinea-pigs in vitro and inhibiting histamine-induced asthmatic reaction in guinea-pigs in vivo. All the evaluated compounds exhibited significant antihistamine activity compared with desloratadine. Five active compounds induced no sedative effects on mouse and four of them exhibited lower anticholinergic side effects than desloratadine. Among these analogues, compound 10, (1S,4S)-4-chlorocyclohexyl desloratadine displayed the highest activity and best safety profile. And it was believed to be a potential candidate as the 3rd generation antihistamine.     

EFFECT OF TEMPERATURE ON THE SENSITIVITY OF NITROGENASE TO OXYGEN IN TWO HETEROCYSTOUS CYANOBACTERIA1

The effect of temperature and oxygen on nitrogenase activity in two heterocystous cyanobacteria, Anabaena variabilis Kütz. ATCC29413 and Nostoc sp. PCC7120, was investigated. The cyanobacteria were grown under a 12:12 light:dark (L:D) cycle at 27°C and were subsequently exposed to different temperatures (27, 36, 39, and 42°C) at different steady‐state O₂ concentrations (20, 10, 5, 0%). Light response curves of nitrogenase activity were recorded under each of these conditions using an online acetylene reduction assay combined with a sensitive laser photoacoustic ethylene detection method. The light response curves were fitted with the rectangular hyperbola model from which the model parameters N_m, N_d, and α were derived. In both strains, nitrogenase activity (N_tot = N_m + N_d) was the highest at 39°C–42°C and at 0% O₂. The ratio N_tot/N_d was 4.1 and 3.1 for Anabaena and Nostoc, respectively, indicating that respectively 25% and 33% of nitrogenase activity was supported by respiration (N_d). N_tot/N_d increased with decreasing O₂ concentration and with increasing temperature. Hence, each of these factors caused a relative increase in the light‐driven nitrogenase activity (N_m). These results demonstrate that photosynthesis and respiration both contribute to nitrogenase activity in Anabaena and Nostoc and that their individual contributions depend on both O₂ concentration and temperature as the latter may dynamically alter the flux of O₂ into the heterocyst.     

Enzymatic ethylene formation from 1-aminocyclopropane-1-carboxylic acid by manganese,a protein fraction and a cofactor of etiolated pea shoots

The cofactor of enzymatic, 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation was concentrated on cation exchange columns. When chelators of cations were added to the homogenates, cofactor activity was lost. Cofactor fractions were partly resistant to oxidation at 600° C. Mn²⁺ substituted for the cofactor in ethylene formation from 1-aminocyclopropane-1-carboxylic acid by a protein fraction isolated from etiolated pea shoots. In addition, Mn²⁺ enhanced the stimulatory effect of the concentrated cofactor. The elution volume for the cofactor on a Sephadex G-25 column was lower than that of MnCl₂. In paper electrophoresis the cofactor migrated to the cathode at pH 10.8 and 2.2. The R_F of cofactor on cellulose plates developed in butanol: acetic acid: H₂O was 0.4. After cellulose chromatography, cofactor activity had to be reconstituted by the addition of MnCl₂. Chelators, anti-oxidants, and catalase were inhibitors of Mn²⁺-cofactor-dependent ethylene formation. The protein necessary for 1-aminocyclopropane-1-carboxylic acid dependent ethylene formation in vitro was seperated from 95–98% of the total protein in homogenates by DE-52 cellulose chromatography and (NH₄)₂SO₄-fractionation.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EDTA ethylenediaminetetraacetic acid - DDTC diethyldithiocarbamate     

Influence of salinity on the biological and biochemical activity of a calciorthird soil

Irrigation of agricultural land with saline waters can lead to soil degradation. In this paper the effect of the irrigation with water containing different concentrations of NaCl or Na₂SO₄ (0.1 M, 0.3 M, 0.6 M, 0.8 M, 1 M, and 1.3 M) on the biological and biochemical characteristics of a calciorthid soil was studied. In general, the increase of scil electrical conductivity caused by the addition of saline solutions had a negative effect on soil's biological and biochemical fertility, (that related directly with the soil's microbiological activity) this effect being more noticeable with NaCl than with Na₂SO₄. Soil microbial respiration was inhibited as much as 57% by a 1.3 M solution of NaCl. The activity of hydrolases such as protease, -glucosidase and phosphatase was more negatively affected by salinity than that of oxidoreductases (dehydrogenase and catalase). Soil NO₃ ^- content decreased with salinity while NH₄ ⁺ content increased. Carbohydrate content, which is closely connected with soil aggregate stability, was also negatively affected by salinity.     

Isocostic Acid,a Promising Bioactive Agent from the Essential Oil of Inula viscosa (L.): Insights from Drug Likeness Properties,Molecular Docking and SAR Analysis

The chemical composition of the essential oil (LEO) and its volatile fractions (V₁–V₁₀) collected during the hydrodistillation process every 15 min from the fresh leaves of I. viscosa (L.), growing in Tunisia, were analyzed by GC‐FID and GC/MS. Eighty‐two compounds, representing 90.9–99.4 % of the total samples, were identified. The crude essential oil (LEO) and its fractions (V₁–V₁₀) were characterized by the presence of a high amount of oxygenated sesquiterpenes (82.7–95.8 %). Isocostic acid ( 1 ) was found to be the most abundant component (37.4–83.9 %) and was isolated from the same essential oil over silica gel column chromatography and identified by spectroscopic methods (¹H, ¹³C, DEPT 135 NMR and EI‐MS) and by comparison with literature data. Furthermore, the fresh leaves essential oil (LEO), its volatile fractions (V₁–V₁₀) as well as compound 1 were screened for their antibacterial, antityrosinase, anticholinesterase and anti‐5‐lipoxygenase activities. It was found that the isolated compound 1 exhibited an interesting antibacterial activity against Staphylococcus aureus ATCC 25923 (MIC=32 μg/mL) and Enterococcus faecalis ATCC 29212 (MIC=32 μg/mL) and the highest antityrosinase activity (IC₅₀=13.82±0.87 μg/mL). Compound 1 was also found to be able to strongly inhibit 5‐lipoxygenase with an IC₅₀ value of 59.21±0.85 μg/mL. The bioactivity and drug likeness scores of compound 1 were calculated using Molinspiration software and interpreted, and the structure‐activity relationship (SAR) was discussed with the help of molecular docking analysis.     

Novel BACE1 inhibitors with a non-acidic heterocycle at the P1′ position

We have reported potent peptidic and non-peptidic BACE1 inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. However, our potent inhibitors possess a tetrazole ring at the P₁′ position. It is desirable that central nervous system (CNS) drugs do not possess an acidic moiety. In this study, we synthesized non-acidic BACE1 inhibitors with heterocyclic derivatives at the P₁′ position. KMI-1764 (27) exhibited potent inhibitory activity (IC₅₀ = 27 nM). Interestingly, these non-acidic inhibitors tended to follow the quantitative structure–activity relationship (QSAR) equation and interacted with BACE1-Arg235 in the binding model.     

Zinc-aggravated M1 microglia regulate astrocytic engulfment via P2×7 receptors

BackgroundGlial cells such as astrocytes and microglia play an important role in the central nervous system via communication between these glial cells. Activated microglia can exhibit either the inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which influences astrocytic neuroprotective functions, including engulfment of cell debris. Recently, extracellular zinc has been shown to promote the inflammatory M1 phenotype in microglia through intracellular zinc accumulation and reactive oxygen species (ROS) generation.PurposeHere, we investigated whether the zinc-enhanced inflammatory M1 phenotype of microglia affects the astrocytic engulfing activity.MethodsEngulfing activity was assessed in astrocytes treated with microglial-conditioned medium (MCM) from lipopolysaccharide (LPS)-activated or from ZnCl₂-pretreated LPS-activated M1 microglia. The effect of zinc on microglia phenotype was also validated using the zinc chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the ROS scavenger Trolox.ResultsAlthough treatment of astrocytes with LPS showed no significant effect on the engulfing activity, MCM from LPS-induced M1 microglia increased the beads uptake by astrocytes. This increased uptake activity was suppressed when MCM from LPS-induced M1 microglia pretreated with ZnCl₂ was applied to astrocytes, which was further abolished by the intracellular zinc chelator TPEN and the ROS scavenger Trolox. In addition, expression of P2×7 receptors (P2×7R) was increased in astrocytes treated with MCM derived from M1 microglia but not in the M1 microglia pretreated with ZnCl₂.ConclusionThese findings suggest that zinc pre-treatment abolishes the ability of LPS-induced M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2×7R.     

Rubimetide,humanin, and MMK1 exert anxiolytic-like activities via the formyl peptide receptor 2 in mice followed by the successive activation of DP1, A2A,and GABAA receptors

Rubimetide (Met-Arg-Trp), which had been isolated as an antihypertensive peptide from an enzymatic digest of spinach ribulose-bisphosphate carboxylase/oxygenase (Rubisco), showed anxiolytic-like activity prostaglandin (PG) D₂-dependent manner in the elevated plus-maze test after administration at a dose of 0.1 mg/kg (ip.) or 1 mg/kg (p.o.) in male mice of ddY strain. In this study, we found that rubimetide has weak affinities for the FPR1 and FPR2, subtypes of formyl peptide receptor (FPR). The anxiolytic-like activity of rubimetide (0.1 mg/kg, ip.) was blocked by WRW4, an antagonist of FPR2, but not by Boc-FLFLF, an antagonist of FPR1, suggesting that the anxiolytic-like activity was mediated by the FPR2. Humanin, an endogenous agonist peptide of the FPR2, exerted an anxiolytic-like activity after intracerebroventricular (icv) administration, which was also blocked by WRW4. MMK1, a synthetic agonist peptide of the FPR2, also exerted anxiolytic-like activity. Thus, FPR2 proved to mediate anxiolytic-like effect as the first example of central effect exerted by FPR agonists. As well as the anxiolytic-like activity of rubimetide, that of MMK1 was blocked by BW A868C, an antagonist of the DP₁-receptor. Furthermore, anxiolytic-like activity of rubimetide was blocked by SCH58251 and bicuculline, antagonists for adenosine A_2A and GABA_A receptors, respectively. From these results, it is concluded that the anxiolytic-like activities of rubimetide and typical agonist peptides of the FPR2 were mediated successively by the PGD₂-DP₁ receptor, adenosine-A_2A receptor, and GABA-GABA_A receptor systems downstream of the FPR2.