Efficacy of the antagonist Aureobasidium pullulans PL5 against postharvest pathogens of peach,apple and plum and its modes of action

The efficacy of Aureobasidium pullulans PL5 against different postharvest pathogens of fruits (Monilinia laxa on plums and peaches, Botrytis cinerea and Penicillium expansum on apples) were evaluated under storage conditions when applied at 10⁸ cells ml⁻¹ and their interactions were studied in vitro and in vivo to discover the possible modes of action. Under 1.2 °C and 95% relative humidity (RH) for 28 days, the efficacy of PL5 against M. laxa on plums was 45%, reducing disease incidence from 78% to 43%. Under 1 °C and 95% RH for 21 days, the efficacy against M. laxa on peaches was 63%, reducing disease incidence from 79% to 29%. Under 4 °C and 95% RH for 45 days, the efficacy against B. cinerea and P. expansum on apples was 56% and 46%, respectively. In Lilly–Barnett minimal salt medium with the fungal cell walls of pathogens as sole carbon source, PL5 produced β-1,3-glucanase, exo-chitinase and endo-chitinase. Nutrient concentrations had significant effect on pathogen growth reduction by PL5. No attachment was observed in antagonist–pathogen interactions in vitro or in vivo. PL5 completely inhibited pathogen spore germination in PDB at 10⁸ cells ml⁻¹, whereas at 10⁶ cells ml⁻¹ the efficacy was significantly decreased. However, inactivated cells and culture filtrate of PL5 had no effect on pathogen spore germination and germ tube elongation. Our results showed that A. pullulans PL5 could be introduced in commercial formulations to control postharvest pathogens on fruits and its activity was based on secretion of lytic enzymes and competition for nutrients.     

Synthesis of novel fenfuram-diarylether hybrids as potent succinate dehydrogenase inhibitors

Twelve novel fenfuram-diarylether hybrids were designed, synthesized and characterized by ¹H NMR and MS. Their in vitro antifungal activities were evaluated against five phytopathogenic fungi by mycelial growth inhibition method. Most compounds showed significant antifungal effect on Rhizoctonia solani and Sclerotinia sclerotiorum. Compound 1c exhibited the most potent antifungal effect on R. solani with an EC₅₀ value of 0.242 mg/L, superior to the commercial fungicide boscalid (EC₅₀ = 1.758 mg/L) and the lead fungicide fenfuram (EC₅₀ = 7.691 mg/L). Molecular docking revealed that compound 1c featured a higher affinity for succinate dehydrogenase (SDH) than fenfuram. Furthermore, it was shown that the 2-chlorophenyl group of compound 1c formed a π-π stacking with D/Tyr-128 and a Cl-π interaction with B/His-249, which made compound 1c more active than fenfuram against SDH.     

Plasma levels of intermedin (adrenomedullin-2) in healthy human volunteers and patients with heart failure

Intermedin/adrenomedullin-2 (IMD) is a member of the adrenomedullin/CGRP peptide family. Less is known about the distribution of IMD than for other family members within the mammalian cardiovascular system, particularly in humans. The aim was to evaluate plasma IMD levels in healthy subjects and patients with chronic heart failure. IMD and its precursor fragments, preproIMD_25–56 and preproIMD_57–92, were measured by radioimmunoassay in 75 healthy subjects and levels of IMD were also compared to those of adrenomedullin (AM) and mid-region proadrenomedullin_45–92 (MRproAM_45–92) in 19 patients with systolic heart failure (LVEF < 45%). In healthy subjects, plasma levels (mean + SE) of IMD (6.3 + 0.6 pg ml⁻¹) were lower than, but correlated with those of AM (25.8 + 1.8 pg ml⁻¹; r = 0.49, p < 0.001). Plasma preproIMD_25–56 (39.6 + 3.1 pg ml⁻¹), preproIMD_57–92 (25.9 + 3.8 pg ml⁻¹) and MRproAM_45–92 (200.2 + 6.7 pg ml⁻¹) were greater than their respective bioactive peptides. IMD levels correlated positively with BMI but not age, and were elevated in heart failure (9.8 + 1.3 pg ml⁻¹, p < 0.05), similarly to MRproAM_45–92 (329.5 + 41.9 pg ml⁻¹, p < 0.001) and AM (56.8 + 10.9 pg ml⁻¹, p < 0.01). IMD levels were greater in heart failure patients with concomitant renal impairment (11.3 + 1.8 pg ml⁻¹) than those without (6.5 + 1.0 pg ml⁻¹; p < 0.05). IMD and AM were greater in patients receiving submaximal compared with maximal heart failure drug therapy and were decreased after 6 months of cardiac resynchronization therapy. In conclusion, IMD is present in the plasma of healthy subjects less abundantly than AM, but is similarly correlated weakly with BMI. IMD levels are elevated in heart failure, especially with concomitant renal impairment, and tend to be reduced by high intensity drug or pacing therapy.     

Postharvest biocontrol of Monilinia laxa,Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains

The antagonistic effects of yeasts, L1 and L8, isolated from carposphere of ‘Redhaven’ peaches were tested for the first time in the same experiment against three Monilinia species (Monilinia laxa, Monilinia fructicola and Monilinia fructigena) in in vitro and in vivo trials. The two antagonists were selected after preliminary assays for their ability to reduce brown rot in peaches and nectarines, and both were identified by molecular and morphological tools as Aureobasidium pullulans. In in vivo trials, neither the autoclaved cells, nor the sterile culture filtrates of either antagonist showed any significant reduction of rot incidence produced by inocula of the three Monilinia species, while the washed cells of L1 and L8 completely inhibited M. laxa and M. fructicola rots and reduced M. fructigena infections by 70% and 90%, respectively. In other trials, nectarines treated with antagonist cells and inoculated with the pathogens were stored at 0 °C for 21 days, plus 7 days at 20 °C. The low temperature reduced brown rot development, since all fruit were free from disease symptoms on removal from cold storage. However after 7 d at 20 °C, untreated fruit were rotted over 45% depending on the Monilinia species but the antagonists completely inhibited M. laxa and M. fructicola, while M. fructigena infections were reduced by 89.8% and 91.2% by L1 and L8, respectively. For both strains, 10⁸ CFU ml^?1 was the most active concentration, although L1 showed good activity at a concentration of 10⁷ CFU ml^?1. Isolate L8 at the concentration of 10⁷ CFU ml^?1 was ineffective against M. fructicola and M. fructigena, showing no difference between treated fruit and the control, excepting the case of nectarines inoculated with M. laxa, where L8 at the concentration of 10⁷ CFU ml^?1 reduced the brown rot infections with respect to the control. The increase in population density of A. pullulans strains L1 and L8 in the wounds of nectarines stored at 0° or 20 °C was low but sufficient to control brown rot. In conclusion, the present preliminary study identified two antagonistic strains of A. pullulans as active ingredients for the development of biofungicides for postharvest application against three Monilinia species that are responsible for high economic losses in stone fruit crops.     

Comparative regional-scale soil salinity assessment with near-ground apparent electrical conductivity and remote sensing canopy reflectance

Soil salinity is recognized worldwide as a major threat to agriculture, particularly in arid and semi-arid regions. Producers and decision makers need updated and accurate maps of salinity in agronomically and environmentally relevant ranges (i.e., <20 dS m⁻¹, when salinity is measured as electrical conductivity of the saturation extract, EC_e). State-of-the-art approaches for creating accurate EC_e maps beyond field scale (i.e., 1 km²) include: (i) Analysis Of Covariance (ANOCOVA) of near-ground measurements of apparent soil electrical conductivity (EC_a) and (ii) regression modeling of multi-year remote sensing canopy reflectance and other co-variates (e.g., crop type, annual rainfall). This study presents a comparison of the two approaches to establish their viability and utility. The approaches were tested using 22 fields (total 542 ha) located in California’s western San Joaquin Valley. In 2013 EC_a-directed soil sampling resulted in the collection of 267 soil samples across the 22 fields, which were analyzed for EC_e, ranging from 0 to 38.6 dS m⁻¹. The ANOCOVA EC_a-EC_e model returned a coefficient of determination (R²) of 0.87 and root mean square prediction error (RMSPE) of 3.05 dS m⁻¹. For the remote sensing approach seven years (2007–2013) of Landsat 7 reflectance were considered. The remote sensing salinity model had R² = 0.73 and RMSPE = 3.63 dS m⁻¹. The robustness of the models was tested with a leave-one-field-out (lofo) cross-validation to assure maximum independence between training and validation datasets. For the ANOCOVA model, lofo cross-validation provided a range of scenarios in terms of RMSPE. The worst, median, and best fit scenarios provided global cross-validation R² of 0.52, 0.80, and 0.81, respectively. The lofo cross-validation for the remote sensing approach returned a R² of 0.65. The ANOCOVA approach performs particularly well at EC_e values <10 dS m⁻¹, but requires extensive field work. Field work is reduced considerably with the remote sensing approach, but due to the larger errors at low EC_e values, the methodology is less suitable for crop selection, and other practices that require accurate knowledge of salinity variation within a field, making it more useful for assessing trends in salinity across a regional scale. The two models proved to be viable solutions at large spatial scales, with the ANOCOVA approach more appropriate for multiple-field to landscape scales (1–10 km²) and the remote sensing approach best for landscape to regional scales (>10 km²).     

Antioxidant activity of aqueous methanol extracts from the lucanid beetle,Serrognathus platymelus castanicolor Motschulsky (Coleoptera: Lucanidae)

The objective of this study was to evaluate the antioxidant properties of extracts of the lucanid beetle, Serrognathus platymelus castanicolor Motschulsky, obtained at different growth stages. The antioxidant activities of six different extracts were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and singlet oxygen (¹O₂). The activity level of pupal methanol extracts (PME) was higher in the DPPH and ABTS radical scavenging assays, whereas that of the water extracts was weaker in all assays. The ¹O₂ quenching ability of the PME was comparable to that of ascorbic acid (effective concentration of 50% ¹O₂ quenching: EC₅₀ = 0.184 mg/ml^-1 and 0.167 mg/ml^-1, respectively). The free radical scavenging antioxidant ability of the extracts significantly altered phenolic contents, important factors in the potency of antioxidant capacity. Our results suggest that these extracts may reduce oxidative stress in living organisms and reduce oxidative damage in insects under unfavorable conditions.     

Estimation of gross primary production of irrigated maize using Landsat-8 imagery and Eddy Covariance data

A study was conducted to understand the potential of Landsat-8 in the estimation of gross primary production (GPP) and to quantify the productivity of maize crop cultivated under hyper-arid conditions of Saudi Arabia. The GPP of maize crop was estimated by using the Vegetation Photosynthesis Model (VPM) utilizing remote sensing data from Landsat-8 reflectance (GPP_VPM) as well as the meteorological data provided by Eddy Covariance (EC) system (GPP_EC), for the period from August to November 2015. Results revealed that the cumulative GPP_EC for the entire growth period of maize crop was 1871 g C m⁻². However, the cumulative GPP determined as a function of the enhanced vegetation index – EVI (GPP_EVI) was 1979 g C m⁻², and that determined as a function of the normalized difference vegetation index – NDVI (GPP_NDVI) was 1754 g C m⁻². These results indicated that the GPP_EVI was significantly higher than the GPP_EC (R² = 0.96, P = 0.0241 and RMSE = 12.6%). While, the GPP_NDVI was significantly lower than the GPP_EC (R² = 0.93, P = 0.0384 and RMSE = 19.7%). However, the recorded relative error between the GPP_EC and both the GPP_EVI and the GPP_NDVI was −6.22% and 5.76%, respectively. These results demonstrated the potential of the landsat-8 driven VPM model for the estimation of GPP, which is relevant to the productivity and carbon fluxes.     

Glucocorticoid receptors on and in a unicellular organism,Cryptobia salmositica

This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50 ng ml⁻¹ of RU486 was more effective in inhibiting parasite replication in cultures with 7,000 parasites ml⁻¹ than in cultures with 14,000 parasites ml⁻¹. Also, 100 ng ml⁻¹ of RU486/ml was more effective than 50 ng ml⁻¹ in inhibiting parasite multiplication in the 14,000 parasites ml^-1 cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand–receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host–parasite interaction.     

Effects of Yucca schidigera extract on fermentation and degradation of steroidal saponins in the rumen simulation technique (RUSITEC)

A rumen simulation technique (RUSITEC) apparatus with eight 940 ml fermentation vessels was used to study the effects of the steroidal saponins in Yucca schidigera extract (YE) on ruminal microbial activity and saponin degradation. The YE contained approximately 4.4% (w/w) saponin, as smilagenin equivalents, and was included at 0 (control) or 0.5 mg ml⁻¹ (n=4) in the McDougall's buffer infused continuously into the vessels (dilution rate=0.75 day⁻¹). Each vessel received 5 g chopped alfalfa hay and 5 g concentrate (as-fed basis) daily for 22 days. Ammonia concentrations were lower (P<0.05) in effluent from vessels receiving YE than from controls for the first half of the study, but did not differ thereafter. Total amounts of VFA in effluent were not affected (P>0.05) by YE, but molar proportions of iso-butyric and iso-valeric acids were lower (P<0.05) in the YE vessels than in the controls in the first half of the experiment. Yucca extract at 0.5 mg ml⁻¹ did not affect (P>0.05) dry matter disappearance (DMD) from hay or from concentrate, nor did it affect total gas or methane production, or bacterial numbers (total or cellulolytic populations) in homogenates prepared from fermenter vessel liquid and feed particles. Protozoal numbers in the homogenates were substantially reduced (P<0.01) by YE (at 0.5 mg ml⁻¹), protease activity was increased (P<0.05), deaminase activity and activity against Ala₂ were unaffected (P>0.05) and activity against Ala₅ was reduced by 25% (P>0.05). When the homogenates from control and YE-supplemented (0.5 mg ml⁻¹) vessels were used to inoculate roll tubes containing 0 or 5 mg ml⁻¹ of YE, fewer colonies developed (P<0.01) in roll tubes containing YE than in those without YE, irrespective of the source of inoculum. Homogenates were also assayed for saponin degradation and for protease, peptidase and deaminase activities. Inoculum from the vessels receiving YE degraded saponin slightly during a 2 h incubation. Yucca extract at 0.5 mg ml⁻¹ altered proteolytic activity and reduced protozoal numbers, but did not affect DMD or bacterial activity, and did not induce resistance to YE at a concentration of 5 mg ml⁻¹.     

Design,synthesis, antiviral and cytostatic activity of ω-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues

The efficient synthesis of a new series of polyhydroxylated dibenzyl ω-(1H-1,2,3-triazol-1-yl)alkylphosphonates as acyclic nucleotide analogues is described starting from dibenzyl ω-azido(polyhydroxy)alkylphosphonates and selected alkynes under microwave irradiation. Selected O,O-dibenzylphosphonate acyclonucleotides were transformed into the respective phosphonic acids. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses and for cytostatic activity against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells. Compound (1S,2S)-16b exhibited antiviral activity against Influenza A H3N2 subtype (EC₅₀ = 20 μM—visual CPE score; EC₅₀ = 18 μM—MTS method; MCC >100 μM, CC₅₀ >100 μM) in Madin Darby canine kidney cell cultures (MDCK), and (1S,2S)-16k was active against vesicular stomatitis virus and respiratory syncytial virus in HeLa cells (EC₅₀ = 9 and 12 μM, respectively). Moreover, compound (1R,2S)-16l showed activity against both herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC₅₀ = 2.9 and 4 μM, respectively) and feline herpes virus in CRFK cells (EC₅₀ = 4 μM) but at the same time it exhibited cytotoxicity toward uninfected cell (MCC ⩾ 4 μM). Several other compounds have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC₅₀ in the 4–50 μM range. Among them compounds (1S,2S)- and (1R,2S)-16l were the most active (IC₅₀ in the 4–7 μM range).     

Thieno[2,3-b]pyridines—A new class of multidrug resistance (MDR) modulators

To identify new potent multidrug resistance modulators, we have synthesized a series of novel thieno[2,3-b]pyridines and furo[2,3-b]pyridines, and examined their stucture–activity relationships. All synthesized compounds were tested to determine BCRP1, P-gp, and MRP1 inhibitor activity, and most potent MDR modulators were also screened for their toxicity, cytotoxicity and Ca²⁺ channel antagonist activity. Among these compounds, thieno[2,3-b]pyridine (6r) was found to exhibit a potent P-gp inhibitory action with EC₅₀ = 0.3 ± 0.2 μM, MRP1 inhibitory action with EC₅₀ = 1.1 ± 0.1 μM and BCRP1 inhibitory action with EC₅₀ = 0.2 ± 0.05 μM and may represent suitable candidate for further pharmacological studies.     

Evaluation of the adverse effects of two commonly used fertilizers,DAP and urea,on motility and orientation of the green flagellate Euglena gracilis

The effect of two commonly used fertilizers, DAP (diammonium phosphate) and urea was studied on the freshwater flagellate Euglena gracilis using the automatic biotest ECOTOX. NOEC and EC₅₀ values for various parameters like motility, velocity, cell shape and gravitaxis were calculated. The NOEC and EC₅₀ values obtained for DAP were much lower than those for urea; i.e. DAP showed a stronger inhibitory effect as compared to urea. The inhibition caused by DAP increased with increasing exposure time over 24 h but urea showed no augmentation with increasing exposure time. Application of DAP resulted in an increased pH and high concentrations of ammonia but urea did neither affect the pH nor affect the ammonia concentration. Recovery experiments in deionized water after urea application showed a reconstitution of motility after 72 h. After an application of 1.35 g L⁻¹ (24 h EC₅₀ for motility) DAP motility recovered after 72 h but motility did not recover when the concentration was doubled (2.7 g L⁻¹). The EC₅₀ values obtained were compared with the EC₅₀/LC₅₀ values reported for other aquatic organisms and were found to be comparable with the reported values.     

Synthesis and biological evaluation of imidazole thioacetanilides as novel non-nucleoside HIV-1 reverse transcriptase inhibitors

A series of 2-(1-aryl-1H-imidazol-2-ylthio)acetamide [imidazole thioacetanilide (ITA)] derivatives were synthesized and evaluated as potent inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 4a5 (EC₅₀ = 0.18 μM), and 4a2 (EC₅₀ = 0.20 μM), which were more effective than the lead compound L1 (EC₅₀ = 2.053 μM) and the reference drugs nevirapine and delavirdine. The preliminary structure–activity relationship (SAR) of the newly synthesized congeners is discussed.     

Isolation,structural characterization and neuraminidase inhibitory activities of polyphenolic constituents from Flos caryophylli

Neuraminidase (NA) is one of the key surface proteins of the influenza virus, which is an important target for anti-influenza therapy. In the present study, bioassay-guided fractionation led to isolation of two new compounds, rhamnetin-3-O-β-d-glucuronide-6″-methyl ester (1) and rhamnazin-3-O-β-d-glucuronide-6″-methyl ester (2), along with seventeen known compounds (3-19), from the MeOH extract of Flos Caryophylli using in vitro NA inhibition assay. These isolated compounds exhibited significantly inhibitory effects on the NA with IC₅₀ values ranging from 8.4 to 94.1 μM and were found to protect MDCK cells from A (H1N1) influenza infections (EC₅₀ = 1.5–84.7 μM) with very low cytotoxicity to the host cells (CC₅₀ = 374.3–1266.9 μM)), with selective index (SI) ranging from 7 to 297. The primary structure-relationships of these isolates were also discussed.     

Biocontrol of major postharvest pathogens on apple using Rhodotorula glutinis and its effects on postharvest quality parameters

The biocontrol activity of Rhodotorula glutinis on gray mold decay and blue mold decay of apple caused by Botrytis cinerea and Penicillium expansum, respectively, was investigated, as well as its effects on postharvest quality of apple fruits. The results show there was a significant negative correlation between concentrations of the yeast cells and the disease incidence of the pathogens. The higher concentration of the R. glutinis, the better effect of the biocontrol capacity. At concentrations of R. glutinis 1 × 10⁸ CFU ml^?1, the amount of gray mold decay was completely inhibited after 5 days incubation at 20 °C, after challenge with B. cinerea spores suspension of 1 × 10⁵ spores ml^?1; While the blue mold decay was completely inhibited at concentrations of 5 × 10⁸ CFU ml^?1, at challenged with P. expansum spores suspension of 5 × 10⁴ spores ml^?1_. These results demonstrated that the efficacy of R. glutinis in controlling of gray mold decay of apples was better than the efficacy of controlling blue mold. R. glutinis within inoculated wounds on apples increased in numbers at 20 °C from an initial level of 9.5 × 10⁵ CFU per wound to 2.24 × 10⁷ CFU at 20 °C after 1 day. The highest population of the yeast was recovered 4 days after inoculation, the yeast population in wounds increased by 56.9 times. After that, the population of the yeast began to decline very slowly. R. glutinis significantly reduced the incidence of natural infections on intact fruit from 75% in the control fruit to 28.3% after 5 days at 20 °C, and from 58.3 to 6.7% after 30 days at 4 °C followed by 4 days at 20 °C. R. glutinis treatment had no deleterious effect on quality parameters after 5 days at 20 °C or after 30 days at 4 °C followed by 4 days at 20 °C.     

Response surface methodology: Synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus

A transferase was isolated, purified and characterised from Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60 °C, respectively and under such conditions remained stable with no decrease in activity after 5 h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1 U mg^?1 after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85 kDa and K_m and V_max values of 272.3 mM and 166.7 μmol min^?1 ml^?1. The influence of pH, temperature, reaction time, and enzyme and sucrose concentration on the formation of short-chain fructooligosaccharides (FOS) was examined by statistical response surface methodology (RSM). The enzyme showed both transfructosylation and hydrolytic activity with the transfructosylation ratio increasing to 88% at a sucrose concentration of 600 mg ml^?1. Sucrose concentration (400 mg ml^?1) temperature (60 °C), and pH (5.6) favoured the synthesis of high levels of GF₃ and GF₄. Incubation time had a critical effect on the yield of FOS as the major products were GF₂ after 4 h and GF₄ after 8 h. A prolonged incubation of 16 h resulted in the conversion of GF₄ into GF₂ as a result of self hydrolase activity.     

Application of experimental designs to optimize medium composition for production of thermostable lipase/esterase by Geobacillus thermodenitrificans AZ1

Thirty two morphologically different bacterial were isolated from different soil samples and screened for their ability to produce lipolytic enzymes. Among all isolates, the isolate coded AZ1 was selected due to its high potency to produce lipase at elevated temperature up to 65 °C. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Geobacillus thermodenitrificans. The effect of ten culture variable on lipase production was evaluated by implementing Plackett–Burman statistical design. d-sucrose, peptone and soy bean flour were the most significant variables affecting lipase production. A pre-optimized medium based on this experiment yielded an enzyme activity of 260 U min^?1 ml^?1. For further optimization, a fourteen trials’ multi-factorial Box–Behnken experimental design was applied to find out the optimum level of each of the significant variables. The tested variables, namely: d-sucrose (X₁); peptone (X₂) and soy bean flour (X₃) were examined, each at three different levels coded ?1, 0, +1. The optimal levels of the three components were founded to be (g/L): d-sucrose, 6.56; peptone, 6.35; and soy bean flour, 6.92, with a predicted activity of approximately 610 U min^?1 ml^?1. According to the results of the Plackett–Burman and Box–Behnken designs the following medium composition is expected to be optimum (g/L): d-sucrose 6.56, peptone 6.35, soy bean flour 6.92, CaCl₂ 0.02, Y.E. 2.5, K₂HPO₄ 1.0, MgSO₄.7H₂O 0.2 and Fe₂ (SO₄)₃ 0.02; pH, 8; cultivation temperature 55 °C and incubation time 24 h, the enzyme activity measured in the medium was approximately 593 U min^?1 ml^?1.     

Endotoxin-neutralizing activity and mechanism of action of a cationic α-helical antimicrobial octadecapeptide derived from α-amylase of rice

We have previously reported that AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice, is a novel cationic α-helical peptide that exhibited antimicrobial activity against human pathogens, including Porphyromonas gingivalis, Pseudomonas aeruginosa, Propionibacterium acnes, Streptococcus mutans, and Candida albicans. In this study, to further investigate the potential functions of AmyI-1-18, we examined its inhibitory ability against the endotoxic activities of lipopolysaccharides (LPSs, smooth and Rc types) and lipid A from Escherichia coli. AmyI-1-18 inhibited the production of endotoxin-induced nitric oxide (NO), an inflammatory mediator, in mouse macrophages (RAW264) in a concentration-dependent manner. The results of a chromogenic Limulus amebocyte lysate assay illustrated that the ability [50% effective concentration (EC₅₀): 0.17 μM] of AmyI-1-18 to neutralize lipid A was similar to its ability (EC₅₀: 0.26 μM) to neutralize LPS, suggesting that AmyI-1-18 specifically binds to the lipid A moiety of LPS. Surface plasmon resonance analysis of the interaction between AmyI-1-18 and LPS or lipid A also suggested that AmyI-1-18 directly binds to the lipid A moiety of LPS because the dissociation constant (K_D) of AmyI-1-18 with lipid A is 5.6 × 10⁻¹⁰ M, which is similar to that (4.3 × 10⁻¹⁰ M) of AmyI-1-18 with LPS. In addition, AmyI-1-18 could block the binding of LPS-binding protein to LPS, although its ability was less than that of polymyxin B. These results suggest that AmyI-1-18 expressing antimicrobial and endotoxin-neutralizing activities is useful as a safe and potent host defense peptide against pathogenic Gram-negative bacteria in many fields of healthcare.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.