Preparation and properties of highly soluble chitosan–l-glutamic acid aerogel derivative

The objective of this work is to improve the solubility of chitosan at neutral or basic pH using the supercritical carbon dioxide (sc.CO₂). A novel water-soluble chitosan–l-glutamic acid (Cl-GA) aerogel derivative was synthesized by reaction of 85% deacetylated chitosan with l-glutamic acid (l-GA) in aq.AcOH subjected to solvent exchange prior to using sc.CO₂ as a nonsolvent for the polymer. The prepared aerogel derivative and molecular conformation of modified chitosan are characterized by using UV, FTIR, ¹H NMR, and CD techniques. Some physical properties and surface morphology were analyzed by X-ray diffraction, differential scanning calorimetry (DSC), thermogravimetry (TG), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and porosimetry analysis. Overall, the sc.CO₂ assisted chitosan aerogel derivative opens new perspectives in biomedical applications.     

Efficiency of soil and fertilizer nitrogen of a sod–potato system in the humid,acid and cool environment

It was hypothesized that soil N variability, and fertilization and cropping management affect potato (Solanum tuberosum L.) growth and fertilizer N efficiency. Following a 20-year sod breakup on a loamy soil in eastern Quebec, Canada (46°37 N, 71°47 W), we conducted a 3-year (1993–1995) study to investigate the effects of soil pool N and fertilizer N management on non-irrigated potato (cv. Superior) tuber yield, fertilizer N recovery (NRE), and residual N distribution in soils under humid, cool and acid pedoclimatic conditions. The fertilizer N treatments consisted of a control, side-dress at rates of 70, 105 and 140 kg ha^–1, and split applications (at seeding and bloom) at rates of 70+70, 105+70 and 140+70 kg ha^–1, respectively. Soil acidity was corrected with limestone following the plow down of the sod. Years of cropping, main effect of N treatment, and year and fertilizer N interaction were significant on total and marketable tuber yields and N uptake, which were significantly related to soil N, and root growth. Apparent NRE ranged between 29 and 70%, depending on years and N rates. Total tuber yield, N uptake, soil N use and NRE were significantly higher in the first (sod–potato) year, but decreased by 41.8, 22.7, 21.4 and 14.7%, respectively, in the third (sod–potato–potato–potato) year. Initial soil N pool was declined by 75% following the 3-year cropping. In 2–3 years, the side-dress N (140 kg ha^–1) increased significantly tuber yields (11.4–19.8%) compared to the split N (70+70 kg ha^–1). Higher split N had no effect on tuber yield and N uptake but increased residual N at harvest. Unused fertilizer N was strongly linked (R ²=0.98) to fertilizer N rates. Time factor and N treatment had significant effects (P<0.0001) on loss of N to below the root zone. Smaller scale rate and timing of split N need to be further determined. Increasing fertilizer N use efficiency could be expected with sod breakup and 75% of regional recommendation rate under humid, cool and acid pedoclimatic conditions.     

Temporal–spatial distributions of high nitrogen concentrations in headwater areas of regions with low precipitation

Temporal–spatial distributions of nitrogen in the low-precipitation headwater areas of the Sanuki Mountain Range, Kagawa Prefecture, Japan, was studied to clarify the natural loading from forests to rivers as basic knowledge for elucidating the eutrophication in dead-water regions downstream. The survey to clarify temporal variations was carried out once every two weeks at the headwaters of five rivers in the Sanuki Mountain Range and the Shikoku Mountains on Shikoku Island, Japan, between May 6, 2002, and April 22, 2003. The survey to clarify the spatial distribution was carried out at 23 stations on the headwaters of the Sanuki Mountain Range between August 13 and 15, 2002. In the Kazuratani and Tarobei Rivers, which originate in the Sanuki Mountain Range, where precipitation is scarce, total N was consistently high – greater than 1mgl^–1 throughout the year. The maximum value of total N was 3.2mgl^–1 in the Kazuratani River. The spatial distribution of total N in the headwater areas was 0.47–3.8mgl^–1. High concentrations over 1mgl^–1 were found in the central region of the Sanuki Mountain Range and the maximum value was 3.8mgl^–1. From these results, it became clear that nitrogen concentrations in headwater areas of the Sanuki Mountain Range are high compared with those in other regions. Various factors were considered in relation to low precipitation and drying as the causes of the high nitrogen concentrations. Furthermore, It was noted that the majority of the regions with the highest NO₃ ^–-N concentrations in our study were composed of granitic rock.     

Metabolic responses of the body to breathing with hypoxic argon–oxygen and nitrogen–oxygen mixtures

We have studied metabolic responses of six male volunteers who were exposed to hypoxic nitrogen–oxygen and argon–oxygen respiratory mixtures under hermetic chamber conditions for a long time. We measured the values of 44 biochemical parameters of venous blood, which reflected the state of different parts of metabolism, as well as the state of organs and tissues. Under the conditions of argon–oxygen respiratory mixture with the level of oxygen of 12.8% within the first five days and 12.0% from six to ten days of the experiment, the activation of processes of anaerobic glycolysis and lipolysis with the development of metabolic acidosis was observed. Against this background, biochemical signs of unfavorable changes in the myocardium were recorded. When we used the nitrogen–oxygen respiratory mixture with the level of oxygen of 13.0% during the first five days of the experiment and 12.1% from the sixth to the tenth day, these changes were expressed much more significantly: the activation of lipolysis and a decrease in the level of serum iron were observed and the signs of damage of mitochondria, depression of the kidney function, and development of hypodynamia were recorded. Complete stabilization of metabolic processes was reached only after seven to eight days of the recovery period for both mixtures. These findings lead to the conclusion that it is physiologically more preferable to use oxygen–argon respiratory mixtures at small immersion depths.     

Effect of chitosan degradation on its interaction with β-lactoglobulin

Complexes between chitosan and β-lactoglobulin (β-Lg) were investigated, and their formation was found to depend on pH and ionic strength. The electrostatic attraction between the cationic polysaccharide and the negatively charged protein above its isoelectric point has been identified as the main driving force in the molecular recognition process. At low protein concentration, soluble complexes were shown to be formed, and their structural features were characterized by circular dichroism (CD) and steady-state fluorescence. Both the overall secondary structure of the protein and the local environment probed by its tryptophan residues are not affected by the presence of chitosan in the complex. Furthermore, the formation of the complex does not lead to a net stabilization of the native state of the protein over its denatured state due to formation of a similarly stable complex between the polyelectrolyte and the denatured state of the protein. The formation of coacervates between β-Lg and chitosan was also characterized as a function of average molecular weight of chitosan (subjected to ultrasonication for different periods of time: 0, 5, 15, and 30 min) by means of both turbidimetric and calorimetric techniques. The combination of turbidimetric as well as isothermal calorimetric titrations have allowed the deconvolution of two processes usually coupled in the formation of protein-polyelectrolyte coacervates: the formation of complex coacervates as the protein sites become saturated by polyelectrolyte molecules and the redissolution of the coacervates as the polyelectrolyte-to-protein ratio increases.     

Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignancies following vaccination with CEA peptide–pulsed dendritic cells

Carcinoembryonic antigen (CEA) is strongly expressed in a vast majority of gastrointestinal carcinomas. Recently, epitope peptides of CEA were identified. We have demonstrated HLA-A24–restricted peptide, CEA652[9] (TYACFVSNL), was capable of eliciting specific cytotoxic T lymphocytes (CTLs) which could lyse tumor cells expressing HLA-A24 and CEA. HLA-A24 is the most applicable MHC class I allele in the Japanese population. In this pilot study, we have used the peptide-pulsed dendritic cells (DCs) generated from peripheral blood mononuclear cells (PBMCs) supplemented with GM-CSF and IL-4 as the source of the vaccine. Eight patients with advanced CEA-expressing gastrointestinal malignancies received subcutaneous injections every 2 or 3 weeks. Immunomonitoring was performed by ELISpot (enzyme-linked immunosorbent spot) assay to measure the precursor frequency of CTLs and their capacity to elicit antitumor CTLs in vitro. Four of seven patients have developed their CTL response after vaccinations. DTH reaction was observed in one of eight patients at the DC-injected site. Skin biopsy at the injected site showed the infiltration of the lymphocytes. Furthermore, A24/CEA peptide tetramer assay revealed an increase in peptide-specific T-cell precursor frequency in vaccinated patients. No significant toxic adverse effects were observed, except for mild diarrhea in one case after three vaccinations. Three patients have shown stabilization of the disease after vaccinations. In conclusion, our results clearly demonstrated that our vaccination protocol was safe and might develop a CEA-specific CTL response in cancer patients.     

Ab initio calculations of molecular properties of low–lying electronic states of 2–cyclopenten–1–one – link with biological activity

Geometries, vibrational frequencies, vertical and adiabatic excitation energies, dipole moments and dipole polarizabilities of the ground and the three lowest electronic excited states, S(1)(n, π (*)), T(1)(n, π (*)), and T(2)(π, π (*)) of the 2-cyclopenten-1-one molecule (2CP) were calculated at the CCSD and CCSD(T) levels of approximation. Our results indicate that two triplets T(1)(n, π (*)) and T(2)(π, π (*)) are lying very close each to other, while the singlet S(1)(n, π (*)) is well above them. There are dramatic changes in dipole moments for (n, π (*)) excited states in respect to the ground state. On the other hand the T(2)(π, π (*)) state has a similar dipole moment as the ground state. These changes can be interpreted within the MO picture using electrostatic potential maps and changes in model IR spectra. Our CCSD(T) dipole moment data for the ground state and almost isoenergetic triplets T(1)(n, π (*)) and T(2)(π, π (*)) are 1.469?a.u., 0.551?a.u., and 1.124?a.u., respectively. Dipole polarizabilities of investigated excited states are much less affected by electron excitations than dipole moments. These are the first dipole moment and polarizability data of 2CP in the literature. The changes of molecular properties upon excitation to S(1)(n, π (*)) and T(1)(n, π (*)) correlate with the experimental data on the biological activity of 2CP related to the α, β-unsaturated carbonyl group.     

Enhancement of interferon-β production with sphingomyelin from fermented milk

A fermented milk, Kefir, contains an active substance which enhances IFN- secretion of a human osteosarcoma line MG-63 treated with a chemical inducer, poly I: poly C. The active substance in the fermented milk was identified to be sphingomyelin (SpM) by a combined use of a fast atom bombardment mass spectrometry (FAB-MS) and a fast atom bombardment tandem mass spectrometry (FAB-MS/MS). SpM from fermented milk (F-SpM) was a mixture of four molecular species of SpMs having C₂₁-, C₂₂-, C₂₃- and C₂₄-fatty acids. F-SpM enhanced the IFN secretion 14 times, SpMs from other sources also enhanced moderately (2–3 times). Sphingosine and lysosphingomyelin also enhanced the activity but ceramide and cerebroside did not.Abbreviations IFN- interferon- - SpM sphingomyelin - Lyso-SpM lysosphingomyelin - SpS sphingosine - FAB-MS fast atom bombardment mass spectrometry - FAB-MS/MS fast atom bombardment tandem mass spectrometry     

Enhancement of naringenin bioavailability by complexation with hydroxypropoyl-β-cyclodextrin

The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefruits, has been shown to possess hypolipidemic and anti-inflammatory effects both in vitro and in vivo. Recently, our group demonstrated that naringenin inhibits hepatitis C virus (HCV) production, while others demonstrated its potential in the treatment of hyperlipidemia and diabetes. However, naringenin suffers from low oral bioavailability critically limiting its clinical potential. In this study, we demonstrate that the solubility of naringenin is enhanced by complexation with β-cyclodextrin, an FDA approved excipient. Hydroxypropoyl-β-cyclodextrin (HPβCD), specifically, increased the solubility of naringenin by over 400-fold, and its transport across a Caco-2 model of the gut epithelium by 11-fold. Complexation of naringenin with HPβCD increased its plasma concentrations when fed to rats, with AUC values increasing by 7.4-fold and C(max) increasing 14.6-fold. Moreover, when the complex was administered just prior to a meal it decreased VLDL levels by 42% and increased the rate of glucose clearance by 64% compared to naringenin alone. These effects correlated with increased expression of the PPAR co-activator, PGC1α in both liver and skeletal muscle. Histology and blood chemistry analysis indicated this route of administration was not associated with damage to the intestine, kidney, or liver. These results suggest that the complexation of naringenin with HPβCD is a viable option for the oral delivery of naringenin as a therapeutic entity with applications in the treatment of dyslipidemia, diabetes, and HCV infection.     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

Effect of platelet activating factor on leukocytes II. Enhancement of eosinophil chemotactic factor and β-glucuronidase release

Synthetic 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) and 1-O-alkyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF) have previously been shown to induce chemotaxis and chemokinesis of human neutrophils. We present here data showing that these agents are inactive by themselves, but that they enhance neutrophil secretion once it has been initiated by a calcium ionosphore or by zymosan. Two substances, the lipid eosinophil chemotactic factor (ECF) and the lysosomal enzyme β-glucuronidase, are used as markers for neutrophil release. PAF augments secretion of both substances in a dose-dependent fashion, with lyso-PAF being less potent. The kinetics of enhancement are very rapid (<2 min) and are not reversible by washing of the cells. A pyrazoline derivative that inhibits arachidonate cyclo-oxygenation and lipoxygenation, reduces the enhancing effect of PAF and lyso-PAF. PAF, and less so lyso-PAF, are thus potentially important modulators of neutrophil secretion during inflammatory processes.     

Design and characterization of alcalase–chitosan conjugates as potential biocatalysts

Bioprocess and Biosystems Engineering - In this study, alcalase (protease from Bacillus licheniformis) immobilization by adsorption, enzyme crosslinking and covalent enzyme binding to activated...     

Rapid immobilization of applied nitrogen in saline–alkaline soils

The dynamics of inorganic N are important in soil, and this applies particularly to the saline–alkaline soils of the former lake Texcoco in Mexico with high pH and salinity where a forestation program was started in the 1970s. In soils of lake Texcoco, in Mexico, more than 50% of applied N could not be accounted for one day after application of 200 mg kg^–1 soil along with glucose amendment. It was not clear whether this was due to abiotic or biotic processes, the form of inorganic N applied or the result of applying an easily decomposable substrate. We investigated this by adding glucose and 200 mg kg^–1 soil as (NH₄)₂SO₄-N or KNO₃-N to sterilized and unsterilized soil. The changes in inorganic and ninhydrin N, microbial biomass C and production of CO₂ were then monitored. Between the time of applying N and extraction with 0.5 M K₂SO₄, i.e., after ca 2 h, approximately 110 mg NH₄ ⁺-N kg^–1 dry soil could not be accounted for in the unsterilized and sterilized soil and that remained so for the entire incubation in the sterilized soil. After 1 day this increased to 140 mg NH₄ ⁺-N kg^–1 dry soil in the unsterilized control and 170 mg NH₄ ⁺-N kg^–1 dry soil in C amended soil. Volatilization of NH₃ accounted for 56 mg NH₄ ⁺-N kg^–1 so the rest appeared to be adsorbed on the soil matrix. The NH₃ volatilization and NH₄ ⁺ fixed in the soil matrix remained constant over time and no oxidation to NO₂ ^– or NO₃ ^– had occurred, so unaccounted N in unsterilized soil was probably incorporated into the microbial biomass in excess of what was required for metabolic activity. The unaccounted N was ca 70 mg NO₃ ^––N in nitrate amended soil after 3 days and 138 NO₃ ^––N when glucose was additionally added. Losses through abiotic processes were absent as inferred from changes in sterilized soil and the aerobic incubation inhibited possible losses through denitrification. It was inferred that NO₃ ^– that could not be accounted for was taken up by micro-organisms in excess of what was required for metabolic activity.     

Shift in soil–plant nitrogen dynamics of an alpine–nival ecotone

We investigated the nitrogen (N) dynamics of an alpine–nival ecotone on Mt. Schrankogel, Tyrol, Austria, in relation to temperature. Natural abundance of ¹⁵N was used as a tool to elucidate differences in N cycling along an altitudinal transect ranging from 2,906 to 3,079 m, corresponding to a gradient in mean annual temperature of 2.4 °C. The amount of total soil N, of plant available N and soil C/N ratio decreased significantly with increasing altitude, whereas soil pH increased. Soil δ ¹⁵N decreased with increasing altitude from +2.2 to −2.1‰ and δ ¹⁵N of plant tissues (roots and leaves) decreased from −3.7 to −5.5‰. The large shift in soil δ ¹⁵N of 4.3‰ from the lowest to the highest site suggested substantial differences in N cycling in alpine and nival ecosystems in the alpine nival ecotone investigated. We concluded that N cycling at the alpine–nival ecotone is likely to be controlled by various factors: temperature, soil age and development, atmospheric N deposition and plant competition. Our results furthermore demonstrate that the alpine–nival ecotone may serve as a sensitive indicator of global change.     

Impact of magnetic nanofillers in the swelling and release properties of κ-carrageenan hydrogel nanocomposites

Natural polysaccharides such as κ-carrageenan are an important class of biomaterials for drug delivery. The incorporation of magnetic nanoparticles in polysaccharide hydrogels is currently being explored as a strategy to confer to the hydrogels novel functionalities valuable for specific bio-applications. Within this context, κ-carrageenan magnetic hydrogel nanocomposites have been prepared and the effect of magnetic (Fe₃O₄) nanofillers in the swelling of the hydrogels and in the release kinetics and mechanism of a model drug (methylene blue) has been investigated. In vitro release studies demonstrated the applicability of the composites in sustained drug release. The mechanism controlling the release seems to be determined by the strength of the gel network and the extent of gel swelling, both being affected by the incorporation of nanofillers. Furthermore, it was demonstrated that the release rate and profile could be tailored using variable Fe₃O₄ nanoparticles load. Thus, this seems to be a promising strategy for the development of drug delivery systems with tailored released behavior.     

Self-aggregates formation and mucoadhesive property of water-soluble β-cyclodextrin grafted with chitosan

Water-soluble β-cyclodextrin grafted with chitosan (CD-g-CS) was carried out by quaternizing the CD-g-CS with glycidyltrimethyl ammonium chloride (GTMAC) under mild acidic condition, corresponding to the quaternized CD-g-CS (QCD-g-CS). The degrees of substitution (DS) and quaternization (DQ), ranging from 5% to 23% and 66% to 80%, respectively, were determined by (1)H NMR spectroscopy. Self-aggregates formation of all QCD-g-CSs were investigated in water using dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM) techniques. The result revealed that all QCD-g-CSs are able to form self-aggregates in water. Large particle sizes ranged from 800 to 3000nm were obtained by DLS while zeta-potentials were ranging from 25 to 40mV. AFM and TEM depicted a spherical shape with particle sizes ranging from 100 to 900nm. Mucoadhesive and cytotoxic properties of all QCD-g-CSs were evaluated using a mucin particle method and MTT assay compared to quaternized chitosan (QCS). It was found that the mucoadhesive property increased with decreasing DS due to less quaternary ammonium moiety into the chitosan backbone. On the other hand, the cytotoxicity increased with increasing DS even though the DQ is decreased.