Identification and characterization of multifunctional cationic peptides from traditional Japanese fermented soybean Natto extracts.

In this study, we investigated the lipopolysaccharide (LPS)-neutralizing and angiogenic activities of cationic peptides derived from the traditional Japanese fermented product Natto, which is made by fermenting cooked soybeans using Bacillus subtilis. Initially, we prepared 20 fractions of Natto extracts with various isoelectric points (pI's) using ampholyte-free isoelectric focusing (autofocusing). Cationic peptides were then purified from fractions 19 and 20, whose pH values were greater than 12, using reversed-phase high-performance liquid chromatography, and were identified using matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among the 13 identified cationic peptides, seven (KFNKYGR, FPFPRPPHQK, GQSSRPQDRHQK, QRFDQRSPQ, ERQFPFPRPPHQK, GEIPRPRPRPQHPE, and EQPRPIPFPRPQPR) had pI's greater than 9.5, positive net charges, and differing molecular weights. These peptides were then chemically synthesized and applied to chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, and 50% effective (neutralizing) concentrations of 2.6-5.5 µM were demonstrated. In addition, tube formation assays in human umbilical vein endothelial cells revealed angiogenic activities for all but one (GEIPRPRPRPQHPE) of these seven cationic peptides, with increases in relative tube lengths of 23-31% in the presence of peptides at 10 µM. Subsequent experiments showed negligible hemolytic activity of these peptides at concentrations of up to 500 µM in mammalian red blood cells. Collectively, these data demonstrate that six cationic peptides from Natto extracts, with the exception of GEIPRPRPRPQHPE, have LPS-neutralizing and angiogenic activities but do not induce hemolysis.

The surface charge of liposomal adjuvants is decisive for their interactions with the Calu-3 and A549 airway epithelial cell culture models.

One of the main reasons for the unmet medical need for mucosal vaccines is the lack of safe and efficacious mucosal adjuvants. The cationic liposome-based adjuvant system composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6'-dibehenate (TDB) is a versatile adjuvant that has shown potential for mucosal vaccination via the airways. The purpose of this study was to investigate the importance of the liposomal surface charge on the interaction with lung epithelial cells. Thus, the cationic DDA in the liposomes was subjected to a step-wise replacement with the zwitterionic distearoylphosphatidylcholine (DSPC). The liposomes were tested with the model protein antigen ovalbumin for the mucosal deposition, the effect on cellular viability and the epithelial integrity by using the two cell lines A549 and Calu-3, representing cells from the alveolar and the bronchiolar epithelium, respectively. The Calu-3 cells were cultured under different conditions, resulting in epithelia with a low and a high mucus secretion, respectively. A significantly larger amount of lipid and ovalbumin was deposited in the epithelial cell layer and in the mucus after incubation with the cationic liposomes, as compared to incubation with the neutral liposomes, which suggests that the cationic charge is important for the delivery. The integrity and the viability of the cells without a surface-lining mucus layer were decreased upon incubation with the cationic formulations, whereas the mucus appeared to retain the integrity and viability of the mucus-covered Calu-3 cells. Our in vitro results thus indicate that DDA/TDB liposomes might be efficiently and safely used as an adjuvant system for vaccines targeting the mucus-covered epithelium of the upper respiratory tract and the conducting airways.

Kinetics of nutrient removal and expression of extracellular polymeric substances of the microalgae, Chlorella sp. and Micractinium sp., in wastewater treatment.

Two species of green algae, Chlorella sp. and Micractinium sp., were cultivated in primary effluent wastewater and high-strength wastewater (a mixture of anaerobic digestion centrate and primary effluent) to study nutrient removal and EPS (extracellular polymeric substances) expression during their growth. The high N concentration and P-limited condition in the mixed wastewater (total N=197 mg/L; N/P mass ratio=56) led to about 3 times greater specific N removal rate than the primary effluent set, indicating that algal cells growing in N-rich wastewater had N over-uptake. Both Chlorella and Micractinium grown in the high-strength wastewater also produced larger amounts of protein EPS, possibly accounting for higher N uptake in those cultivation sets. These results suggest that different types of wastewater could cause different nutrient removal kinetics and EPS expression by algae, which may subsequently influence harvesting and anaerobic digestion of their biomass.

Use of Raphanus sativus L. press cake, a solid residue from biodiesel processing, in the production of adsorbents by microwave activation.

The objective of this study was to evaluate the performance of an adsorbent, based on a solid residue arising from biodiesel production, for the removal of cationic dyes from wastewaters. The adsorbent was produced by microwave thermal activation. The Freundlich model provided the best fit for equilibrium data, indicating heterogeneous adsorption. The adsorption capacity increased in comparison to the adsorbent obtained by thermal processing of the same residue in a conventional oven, showing that microwave processing is an attractive alternative for adsorbent production, given the significant reduction in processing time (decreased from 60 min to 3 min). Both the removal efficiency and the removal capacity decreased with an increase in temperature, pointing towards the exothermic nature of the removal process.

Separation and determination of cations in beverage products by capillary zone electrophoresis.

Cation determination is important for quality control of beverage products. To determine a large group simultaneously, a capillary electrophoresis procedure is developed with indirect UV at 214 nm in a three-complex buffer system (10 mM N,N-dimethylbenzylamine (DBA), 8 mM lactic acid and 2 mM 18-crown-6) with good mobility matching with desired cations. Under optimized conditions with pH adjusted to 4.65, a baseline separation is achieved for 14 cations (Rb(+), NH(4)(+), K(+), Ca(2+), Na(+), Mg(2+), Mn(2+), Co(2+), Fe(2+), Cd(2+), Cr(3+), Ni(2+), Zn(2+) and Cu(2+)) within 7 min using an uncoated silica column. To cover ng/l to mug/l range, both hydrostatic and electrokinetic sampling are studied, showing working ranges within (0.05-50)/(0.005-2) microg/l and detection limits (13-78)/(1.4-10) ng/l, respectively with satisfactory repeatability (RSD 0.31-0.47% for migration time, and 3.0-4.0% for peak height measurement). Agreeable results with established inductively coupled plasma-atomic emission spectrometry method have been obtained for orange juice and tea samples.

A simple and effective method for the removal of trace metal cations from a mammalian culture medium supplemented with 10% fetal calf serum.

Direct batch addition of sterile Chelex ion-exchange resin to Dubecco's modified Eagle's medium supplemented with 10% fetal calf serum with gentle stirring removed a very wide variety of trace metal ions from the medium to varying extents dependent upon Chelex content (between 0.01 and 4% w/v), exposure time (between 5 min and 10 days) and temperature (4, 25 and 37 degrees C). Prolonged treatment (10 days) with 4% w/v Chelex at 4 degrees C reduced the concentration of zinc, strontium, aluminum, copper, manganese, nickel and chromium from 100 to 2.7, 12.1, 7.7, 22.6, 13.0, 14.7 and 53.3% of their original concentrations, respectively. Re-supplementation of the metal depleted medium with a defined cocktail of metals restored the growth potential of the medium which was then capable of supporting growth over at least three subcultures without a decrease in fibroblast cell yield, demonstrating its suitability in cell culture studies on trace metal ions.

Anion-cation separations on a mixed bed alumina-silica column.

Mixed bed ion-exchange (MBIE) columns containing alumina and silica were evaluated for the simultaneous separation of anion and cation analytes. At the mobile phase pH used alumina provides anion exchange sites while silica provides cation exchange sites. Since alumina and silica exhibit weak acid and base properties, their anion and/or cation exchange properties are pH dependent. Ion exchange capacities, rates of exchange and analyte ion exchange selectivities are also pH dependent. The major mobile phase parameters affecting analyte anion and cation resolution and elution order are pH and type and concentration of counter anion and counter cation, respectively. The weight ratio of the two exchangers and/or the exchange capacities of the two in the column can also be used to alter resolution and elution order. Several examples of the simultaneous separation of inorganic mono- and divalent anions and cations using a single sample injection, a single column and a single detector (conductivity) illustrate the parameters and scope of the alumina-silica MBIE column.