Preparation and characterization of self-assembled nanoparticles of the novel carboxymethyl pachyman-deoxycholic acid conjugates

Various novel carboxymethyl pachyman-deoxycholic acid conjugates (CMPD) were synthesized using carboxymethylated pachyman (CMP) as a hydrophilic segment and Deoxycholic Acid (DOCA) as a hydrophobic segment. The degree of DOCA substitution (DS) in CMPD conjugates, which was determined by elemental analysis, can reach to 30.0, 49.2, or 54.9 DOCA groups per hundred sugar residues of CMP. Structural characteristics of these CMPD conjugates were investigated by using ¹H NMR, dynamic light scattering, zeta potential, transmission electron microscopy (TEM) and fluorescence spectroscopy. The CMPD conjugates provided apparently smaller monodispersed self-aggregates in water, with mean diameters decreasing with increasing of DOCA DS in the range of 98–158 nm. Zeta potentials of the CMPD self-aggregated nanoparticles indicated that the nanoparticles were covered with negatively charged CMP shells. TEM images demonstrated that the nanoparticles were spherical in shape. The critical aggregation concentration (cac) of the CMPD nanoparticles (1.55 × 10⁻²–5.89 × 10⁻³ mg/mL) was comparatively low which implies that the CMPD self-assembled nanoparticles form at low concentration in aqueous solution.     

Preparation and physicochemical characteristics of self-assembled nanoparticles of deoxycholic acid modified-carboxymethyl curdlan conjugates

Various deoxycholic acid (DOCA)-modified-carboxymethylated (CM)-curdlan (DCMC) were synthesized and characterized by FTIR, ¹H NMR and XRD. The degree of DOCA substitution (DS), as spectrophotometrically determined, was 2.1, 3.2, 4.1, or 6.3 DOCA groups per hundred sugar residues of CM-curdlan. The physicochemical properties of the self-assembled nanoparticals in aqueous media were investigated using ¹H NMR, dynamic light scattering, zeta potential, transmission electron microscopy (TEM) and fluorescence spectroscopy. DCMC conjugates provided monodispersed self-assembled nanoparticles in water, with mean diameter decreasing from 192 to 347 nm with DOCA DS increasing. Moreover, the mean diameter also increased with decreasing pH in PBS. Zeta potential of DCMC self-assembled nanoparticles exhibited near −60 mV in distilled water and −26 to −36 mV in PBS, indicating these nanoparticles were covered with negatively charged CM-curdlan shells. The critical aggregation concentration (cac) of the DCMC were dependent on the degree of substitution (DS) of DOCA and were slightly lower in PBS than in distilled water. The TEM images demonstrated that these self-assembled nanoparticles were of spherical shape.     

Preparation, characterization, and antibacterial activity of oleic acid-grafted chitosan oligosaccharide nanoparticles

An oleic acid-grafted chitosan oligosaccharide (CSO-OA) with different degrees of amino substitution (DSs) was synthesized by the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. Fourier transform infrared spectroscopy (FT-IR) suggested the formation of an amide linkage between amino groups of chitosan oligosaccharide and carboxyl groups of oleic acid. The critical aggregation concentrations (CACs) of CSO-OA with 6%, 11%, and 21% DSs were 0.056, 0.042, and 0.028 mg·mL⁻¹, respectively. Nanoparticles prepared with the sonication method were characterized by means of transmission electron microscopy (TEM) and Zetasizer, and the antibacterial activity against Escherichia coli and Staphylococcus aureus was investigated. The results showed that the CSO-OA nanoparticles were in the range of 60–200 nm with satisfactory structural integrity. The particle size slightly decreased with the increase of DS of CSO-OA. The antibacterial trial showed that the nanoparticles had good antibacterial activity against E. coli and S. aureus.     

Delivery of expression constructs of secreted frizzled-related protein 4 and its domains by chitosan–dextran sulfate nanoparticles enhances their expression and anti-cancer effects

In malignant mesothelioma (MM) cells, secreted frizzled-related protein 4 (SFRP4) expression is downregulated by promoter methylation. In this study, we evaluated the effect of encapsulated chitosan–dextran (CS–DS) nanoparticle formulations of SFRP4 and its cysteine-rich domain (CRD) and netrin-like domain (NLD) as means of SFRP4-GFP protein delivery and their effects in JU77 and ONE58 MM cell lines. CS–DS formulations of SFRP4, CRD, and NLD nanoparticles were prepared by a complex coacervation technique, and particle size ranged from 300 nm for empty particles to 337 nm for particles containing the proteins. Measurement of the zeta potential showed that all preparations were around 25 mV or above, suggesting stable formulation and good affinity for the DNA molecules. The CS–DS nanoparticle formulation maintained high integrity and entrapment efficiency. Gene delivery of SFRP4 and its domains showed enhanced biological effects in both JU77 and ONE58 cell lines when compared to the non-liposomal FUGENE^® HD transfection reagent. In comparison to the CRD nanoparticles, both the SFRP4 and NLD nanoparticles significantly reduced the viability of MM cells, with the NLD showing the greatest effect. The CS–DS nanoparticle effects were observed at an earlier time point and with lower DNA concentrations. Morphological changes in MM cells were characterized by the formation of membrane-associated vesicles and green fluorescent protein expression specific to SFRP4 and the NLD. The findings from our proof-of-concept study provide a stepping stone for further investigations using in vivo models.     

Conferral of enhanced natural killer cell function by KIR3DS1 in early human immunodeficiency virus type 1 infection

A flurry of recent reports on the role of activating and inhibitory forms of the killer cell immunoglobulin-like receptors (KIR) in natural killer (NK) cell activity against human immunodeficiency virus type 1 (HIV-1) have yielded widely divergent results. The role of the activating NK receptor encoded by the KIR3DS1 allele and its putative ligands, members of the HLA class I Bw4Ile80 cluster, in early HIV-1 disease is controversial. We selected 60 treatment-naïve adults for study from the OPTIONS cohort of individuals with early HIV-1 infection in San Francisco. We performed NK cell functional assays measuring gamma interferon (IFN-γ) and CD107a expression by NK cells in the unstimulated state and after stimulation by the major histocompatibility complex class I-deficient 721.221 B-lymphoblastoid cell line. In addition, we measured CD38 expression (a T-cell activation marker) on T and NK cells. Persons who have at least one copy of the KIR3DS1 gene had higher IFN-γ and CD107a expression in the unstimulated state compared to those who do not possess this gene. After stimulation, both groups experienced a large induction of IFN-γ and CD107a, with KIR3DS1 carriers achieving a greater amount of IFN-γ expression. Differences in effector activity correlating with KIR3DS1 were not attributable to joint carriage of HLA Bw4Ile80 and KIR3DS1. We detected a partial but not complete dependence of KIR3DS1 on the members of B*58 supertype (B*57 and B*58) leading to higher NK cell function. Possessing KIR3DS1 was associated with lower expression of CD38 on both CD8⁺ T and NK cells and with a loss or weakening of the known strong associations between CD8⁺ T-cell expression of CD38 mean fluorescence intensity and the HIV-1 viral load. We observed that possessing KIR3DS1 was associated with higher NK cell effector functions in early HIV-1 disease, despite the absence of HLA Bw4Ile80, a putative ligand of KIR3DS1. Carriage of KIR3DS1 was associated with diminished CD8⁺ T-cell activation, as determined by expression of CD38, and a disruption of the traditional relationship between viral load and activation in HIV-1 disease, which may lead to better clinical outcomes for these individuals.NK cell function is regulated by a family of receptors encoded by the killer cell immunoglobulin-like receptor (KIR) genes (18, 33). Within the KIR family, certain genes encode inhibitory receptors that recognize HLA class I ligands (i.e., HLA-Bw4 or HLA-C), whereas other KIR genes encode activating receptors which are not completely known. Studies on the role of KIRs in human immunodeficiency virus (HIV) disease have focused on the activating receptor encoded by the KIR3DS1 allele. However, recent genetic association and functional studies of KIR and HIV disease have yielded widely disparate results on the role of KIR3DS1 and its putative ligands, a subset of HLA class I-B alleles referred to as Bw4Ile80. The Bw4Ile80 cluster is a subset of HLA-B alleles that bear an isoleucine at position 80 in the α-1 helix, on the rim of the peptide-binding cleft. The inhibitory receptors encoded by KIR3DL1 alleles, which are highly related in the extracellular domains to the activating receptor encoded by KIR3DS1, specifically recognize HLA-Bw4 ligands (5). Because of this similarity, KIR3DS1 has been assumed to also recognize Bw4Ile80 ligands. In 2002, Martin et al. reported that HIV-infected individuals in the Multicenter AIDS Cohort Study possessing the KIR3DS1 allele demonstrated significantly delayed progression to AIDS, provided that the individuals also expressed a Bw4Ile80 allele (20).In 2005, Gaudieri et al. reported on the association of the entire KIR gene cluster and HLA class I in HIV disease progression in an Australian HIV cohort (8, 9). These authors observed a trend toward slowed CD4⁺ T-cell percent loss among those who carried both Bw4Ile80 and KIR3DS1 (8). However, this trend was not statistically significant, and Gaudieri et al. simultaneously observed an acceleration of time to AIDS (1987 definition) among joint KIR3DS1 and Bw4Ile80 carriers. In 2006, Qi et al. published a follow-up report from the Multicenter AIDS Cohort Study cohort documenting an association between the coexpression of KIR3DS1 and Bw4Ile80 and enhanced protection against certain opportunistic infections in HIV-infected individuals (26), an effect partially attributed to very modest differences in viral load. In 2007, our group observed that KIR3DS1 gene carriage was associated with higher CD4⁺ T-cell counts and hence protection against HIV type 1 (HIV-1) progression in early disease (4); however, we observed that this effect was not attributable to differences in the viral load and further was independent of Bw4Ile80. In other words, our analyses suggested that the KIR3DS1 and Bw4Ile80 genes were each associated with protection against HIV disease but via different mechanisms.Until recently, it was not clear if KIR3DS1 was expressed on the surface of NK cells; however, two recent reports have conclusively established that KIR3DS1 is expressed on NK cells (6, 24) and that expression is dose dependent, with higher expression for homozygotes. These studies also demonstrated that KIR3DS1 recognizes neither HLA-Bw4 nor HLA-Bw6 ligands, at least when these major histocompatibility complex (MHC) class I molecules are expressed on Epstein-Barr virus-transformed B-lymphoblastoid cell lines. Similarly, an independent study by another group reported that KIR3DS1 fails to bind to soluble Bw4Ile80 tetrameric complexes (10). In contrast, Alter et al. have presented results from in vitro cytotoxicity assays suggesting that target cells possessing HLA-Bw4Ile80 are better targets for NK cells possessing KIR3DS1 (1); however, no evidence was provided to confirm a physical interaction between the KIR3DS1 and HLA-Bw4 proteins.Here, we present a study of the NK cell phenotype and function in 60 treatment-naïve, recently HIV-1-infected persons with defined HLA-B and KIR3DS1/KIR3DL1 allotypes. We also measured the expression of CD38 on NK cells and CD8⁺ T cells, a widely used marker of disease progression and virulence in HIV research and a marker of immune activation. The expression of CD38, as measured by flow cytometry, is known to be elevated on CD8⁺ T cells in HIV disease, reaching steady-state levels in early HIV-1 infection (7), and predicts disease progression independently of the viral load (19). The individuals studied were selected from our recent genetic association study of KIR and HLA among 255 recently HIV-1-infected persons (4), in which KIR3DS1 carriage alone was associated with higher CD4⁺ T-cell counts, despite the absence of a difference in the viral loads. On the basis of these clinical findings, we performed this study to determine whether persons who carried the KIR3DS1 gene had enhanced NK cell phenotypic and functional profiles and if these profiles were further enhanced by carriage of the putative KIR3DS1 ligands encoded by HLA-Bw4Ile80 alleles. Flow cytometry-based detection of KIR3DS1 has been hampered by the absence of a monoclonal antibody that can bind to KIR3DS1 specifically and not cross-react with the related KIR3DL1 proteins (25). Hence, we used genotypic KIR assignments for our analyses rather than flow cytometry-based methods.     

Generation of Rat-Induced Pluripotent Stem Cells from a New Model of Metabolic Syndrome

We recently characterized DahlS.Z-Lepr^fa/Lepr^fa (DS/obese) rats, derived from a cross between Dahl salt-sensitive rats and Zucker rats, as a new animal model of metabolic syndrome (MetS). Although the phenotype of DS/obese rats is similar to that of humans with MetS, the pathophysiological and metabolic characteristics in each cell type remain to be clarified. Hence, the establishment of induced pluripotent stem cells (iPSCs) derived from MetS rats is essential for investigations of MetS in vitro. Reports of rat iPSCs (riPSCs), however, are few because of the difficulty of comparing to other rodents such as mouse. Recently, the advantage of using mesenchymal stromal cells (MSCs) as a cell source for generating iPSCs was described. We aimed to establish riPSCs from MSCs in adipose tissues of both DS/obese rats and their lean littermates, DahlS.Z-Lepr⁺/Lepr⁺ (DS/lean) rats using lentivirus vectors with only three factors Oct4, Klf4, and Sox2 without c-Myc. The morphology, gene expression profiles, and protein expression of established colonies showed embryonic stem cell (ESCs)-like properties, and the differentiation potential into cells from all three germ layers both in vitro and in vivo (teratomas). Both riPSCs became adipocytes after induction of adipogenesis by insulin, T3, and dexamethasone. Real-time PCR analysis also revealed that both riPSCs and the adipose tissue from DS/obese and DS/lean rats possess similar expression patterns of adipocyte differentiation-related genes. We succeeded in generating riPSCs effectively from MSCs of both DS/obese and DS/lean rats. These riPSCs may well serve as highly effective tools for the investigation of MetS pathophysiology in vitro.     

Acetylation and characterization of xylan from hardwood kraft pulp

Alkaline treatment of eucalyptus hardwood kraft pulp with 10% NaOH yielded 6-8% xylan. The acetylation of the extracted xylan was carried in DMAC/LiCl/pyridine system to obtain a series of xylan acetates with different degrees of substitution (DS). Structure elucidation of xylan and xylan acetate was obtained by ¹H and ¹³C NMR spectroscopy and other homonuclear and heteronuclear 2D-NMR techniques. Inverse-gated ¹³C NMR was employed to determine the DS of xylan acetate. Furthermore, results also revealed equal reactivities at the C-2 and C-3 positions of xylan towards acetylation. Thermal stability, solubility behavior and nanofiber formation of xylan acetate were influenced by its DS values. The mechanical properties of xylan acetate propionate were also investigated.     

Preparation, characterization, and in vitro drug release behavior of biodegradable chitosan-graft-poly(1, 4-dioxan-2-one) copolymer

A novel copolymer of chitosan-g-poly(p-dioxanone) (CGP) was synthesized in bulk by ring-opening polymerization of p-dioxanone (PDO) initiated by the hydroxyl group or amino group of chitosan using SnOct₂ as catalyst. The chemical structure was determined by ¹H NMR. It was found that the feed ratio of chitosan to PDO had a great effect on the degree of polymerization (DP) and the substitution (DS) of PDO. The thermal stability and crystallization behavior of graft copolymer CGP were closely related to the values of DP and DS. When the resulting copolymer was used as Ibuprofen carrier, the release rate of Ibuprofen decreased compared with that of pure chitosan carrier. The drug release behavior was also influenced by the structure of graft copolymers.     

Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling

To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [³H]myristic acid or [³H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus.     

New dextrin-vinylacrylate hydrogel: Studies on protein diffusion and release

New dextrin hydrogels with degrees of substitution (DS) from ca. 10% (DS 10) to 70% (DS 70) were prepared by radical polymerization of aqueous solutions of vinylacrylate (VA)-derivatized dextrin. A preliminary analysis on the potential of these hydrogels for the controlled release of bioactive molecules was carried out. The protein (bovine serum albumin) diffusion coefficients on the hydrogels were calculated using the lag-time analysis. Values in range 10^?7 cm²/s were obtained for DS 20 and DS 40 and a smaller value of 10^?8 cm²/s arised upon DS increasing to 70%, revealing the dependence of the diffusivity on the crosslinking density. The release of BSA from dextrin-VA hydrogels, in the presence of amyloglucosidase was shown to be mainly dependent on the diffusion and, to a smaller extent, on the degradation kinetics. The protein release can be tailored from days to months by varying the DS.     

Homogeneous modification of cellulose with succinic anhydride in ionic liquid using 4-dimethylaminopyridine as a catalyst

Cellulose, extracted from sugarcane bagasse, was successfully succinylated in ionic liquid 1-buty-3-methylimidazolium (BMIMCl) using 4-dimethylaminopyridine (DMAP) as a catalyst. Parameters investigated included the mass ratio of DMAP/succinic anhydride in a range from 0% to 15%, reaction time (from 30 to 120 min), reaction temperature (from 60 to 110 °C). The succinylated cellulosic derivatives had a degree of substitution (DS) ranging from 0.24 to 2.34. It was found that the DS of succinylated cellulosic derivatives using DMAP as a catalyst was higher than that without any catalyst under the same reaction conditions. The products were characterized by FT-IR, solid-state CP/MAS ¹³C NMR, and thermal analysis. FT-IR and solid-state CP/MAS ¹³C NMR spectra showed that succinoylation occurred at C-6, C-2 and C-3 positions. The thermal stability of the succinylated cellulose decreased upon chemical modification.     

Increased N-acylphosphatidylethanolamine biosynthesis in elicitor-treated tobacco cells

Previous studies with tobacco (Nicotiana tabacum L.) cell suspensions indicated that elicitation of defense response (production of phytoalexins) with xylanase (1,4-β-D-xylanxylanohydrolase: EC 3.2.1.8) resulted in a dramatic acylation of phytosterols (Moreau et al. 1994). N-acylphosphatidylethanolamine (NAPE), an acylated derivative of phosphatidylethanolamine (PE), was recently demonstrated to be synthesized in vivo in plant tissues (Chapman and Moore 1993a). Here we report that acylation of PE was increased in elicitor-treated cells. NAPE levels increased 3-fold (from 1.6 to 4.8 mol% of total phospholipids) after a 2-h treatment of cell suspensions with xylanase (1 δg ml^?1). Specific activity of NAPE synthase increased in parallel with NAPE levels. Levels of NAPE and NAPE synthase activity declined during the period of 2–4 h after elicitation while levels of acylated sterolglycosides (ASG) continued to increase. Radiolabeling studies with [2^?14C]-ethanolamine confirmed that three times as much NAPE was synthesized in elicitor-treated cells compared to that in unelicited cells. Patterns of incorporation of [1-¹⁴C]-palmitic acid into membrane phospholipids in elicitor-treated cells suggested that increased acylation of lipids may be a result of changes in the acyl-coenzyme A pool. Treatment of cells with purified ethylene biosynthesis-inducing xylanase (EIX; 1 δg ml^?1 cells) resulted in increased levels of NAPE synthase activity comparable to those observed with the commercial preparations of xylanase. Boiled xylanase did not elicit an increase in the specific activity of NAPE synthase. Collectively our results demonstrate that the accumulation of NAPE in tobacco cells is attributable to increased activity of NAPE synthase. This suggests that NAPE may be specifically synthesized to play a protective role in membranes of plant cells as has been suggested for membranes of damaged animal cells.     

Acetylation of α-chitin in ionic liquids

Acetylation of α-chitin using acetic anhydride in an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), was performed. First, a mixture of chitin and AMIMBr (2% w/w) was heated at 100 °C for 24 h for dissolution. Then, acetic anhydride (5–20 equiv) was added to the solution and the mixture was heated with stirring at desired temperatures for 24 h. The product was precipitated by the addition of the reaction mixture into methanol. The IR spectrum of the product indicated the progress of acetylation. The degrees of substitution (DS), which were determined from the IR spectra, increased with increasing the amounts of acetic anhydride used for the reaction. The highest DS was 1.86, which was obtained by the reaction using 20 equiv of acetic anhydride at 100 °C. The product with this DS value was soluble in DMSO, and thus the structure of the product was further confirmed by ¹H NMR spectroscopy in DMSO-d₆. The DS value estimated by the integrated ratio of signals due to acetyl protons to a signal due to anomeric protons was in good agreement with that determined from the IR spectrum.     

Dermatan sulfate and bone marrow mononuclear cells used as a new therapeutic strategy after arterial injury in mice

Background aimsPreviously, we have demonstrated that administration of dermatan sulfate (DS) suppresses neointima formation in the mouse carotid artery by activating heparin co-factor II. A similar suppressive effect was observed by increasing the number of progenitor cells in circulation. In this study, we investigated the combination of DS and bone marrow mononuclear cells (MNC), which includes potential endothelial progenitors, in neointima formation after arterial injury.MethodsArterial injury was induced by mechanical dilation of the left common carotid artery. We analyzed the extension of endothelial lesion, thrombus formation, P-selectin expression and CD45⁺ cell accumulation 1 and 3 days post-injury, and neointima formation 21 days post-injury. Animals were injected with MNC with or without DS during the first 48 h after injury.ResultsThe extension of endothelial lesion was similar in all groups 1 day after surgery; however, in injured animals treated with MNC and DS the endothelium recovery seemed to be more efficient 21 days after lesion. Treatment with DS inhibited thrombosis, decreased CD45⁺ cell accumulation and P-selectin expression at the site of injury, and reduced the neointimal area by 56%. Treatment with MNC reduced the neointimal area by 54%. The combination of DS and MNC reduced neointima formation by more than 91%. In addition, DS promoted a greater accumulation of MNC at the site of injury.ConclusionsDS inhibits the initial thrombotic and inflammatory processes after arterial injury and promotes migration of MNC to the site of the lesion, where they may assist in the recovery of the injured endothelium.     

Simple organocatalytic route for the synthesis of starch esters

Starch acetates and starch butyrates with degree of substitution (DS) in the range of 0.06–1.54 were prepared by a simple direct solvent-free organocatalytic methodology of starch acylation. The starch esters synthesized have important applications in the food and pharmaceutical industries, among others. The acylation methodology used involves a non-toxic biobased α-hydroxycarboxylic acid as catalyst, and proceeds with high efficiency in absence of solvents. The effect of reaction time on the advance of starch modification was studied as a simple way to control the level of substitution achieved, when all other reaction parameters were kept constant. Starch esters were characterized by means of Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). FTIR spectroscopy qualitatively confirmed the esterification of starch by the appearance of bands which are associated with esters groups. Scanning electron microscopy showed that the granular structure of the polysaccharide was preserved upon acylation, although acylated granules had rougher surfaces; and wrinkles, grooves and deformed zones appeared in some granules at high DS. Thermogravimetric analysis showed a gradual reduction in the water content of acylated starches, as well as noticeable changes in their thermal properties at increasing DS. X-ray diffraction analysis showed that the acetylation treatment led to lower crystallinity at increasing DS, although characteristic corn starch A-type patterns could be identified even at the highest DS achieved (DS = 1.23). Specific bands and weight losses derived from FTIR and TGA data could be very well correlated with the substitution degree achieved in acetylated starches at DS lower/equal than 0.6. The organocatalytic methodology described for the synthesis of starch acetates and butyrates has the potential to be easily extended to the synthesis of other starch esters using a variety of anhydrides or carboxylic acids as acylating agents     

Synthesis and characterization of phosphorylcholine-substituted chitosans soluble in physiological pH conditions

A polymer analogous synthesis involving the reductive amination of phosphorylcholine (PC)-glyceraldehyde with primary amines of deacetylated chitosan (M(w) approximately 57000 g mol(-1)) was used to prepare phosphorylcholine-substituted chitosans (PC-CH) with a degree of substitution (DS) ranging from approximately 11 to approximately 53 mol % PC-substituted glucosamine residues. The PC-CH derivatives were characterized by (1)H NMR spectroscopy, FTIR spectroscopy, and multiangle laser light scattering gel permeation chromatography (MALLS-GPC). The pK(a) of the PC-substituted amine groups (pK(a) approximately 7.20) was determined by (1)H NMR titration. The PC-CH samples (1.0 g L(-1)) were shown to be nontoxic using an MTT assay performed with human KB cells. Aqueous solutions of PC-CH samples (4.0 g L(-1)) of DS >or= 22 mol % PC-substituted glucosamine residues remained clear, independently of pH (4.0 < pH < 11.0). The remarkable water solubility and nontoxicity displayed by the new PC-CH samples open up new opportunities in the design of chitosan-based biomaterials and nanoparticles.     

Carboxymethyl Chinese yam starch: synthesis, characterization, and influence of reaction parameters

Carboxymethyl starch (CMS) was obtained as a product of the reaction of starch and monochloroacetic acid (MCA) in the presence of sodium hydroxide. The influence of the molar ratio of NaOH/AGU, the molar ratio of MCA/AGU, the reaction time, reaction temperature, and the water content on the degree of substitution (DS) was studied. The optimal molar ratio of NaOH/AGU and MCA/AGU is 2.4 and 1.0, respectively. Increase of the ratio of NaOH/AGU or MCA/AGU leads to an increase in DS, but only to certain extent. The highest values of the DS were obtained when the carboxymethylation was performed at 60 °C for 2.5 h. The water content in the reaction media ethanol was optimal at 20% (v/v). The scanning electron micrographs (SEMs) revealed that the carboxymethylation affected the structural arrangement of the starch and caused granular disintegration. The particle size distribution (PSD) also displayed that the average particle diameter increased greatly after modification from 37.37 μm to 72.88 μm. Wide angle X-ray diffractometry (XRD) revealed that starch crystallinity was obviously reduced after carboxymethylation. The new bands at 1600 cm⁻¹ and 1426 cm⁻¹ in FT-IR indicated that the starch granules were substituted.     

Thy.1lowCD3− Cells Sorted from Nylon Wool-Passed Bone Marrow Cells Can Augment the H-2 Identical but Not Non-Identical Cytotoxic T Lymphocyte Precursors in Mixed Lymphocyte Cultures

Thy. 1^lowCD3^– cells obtained from nylon wool-passed murine bone marrow (NW-BM) cells by cell sorting did not express CD4, CD8, or T cell receptor-α/β and -γ/δ on their cell surfaces. An extremely limited number of B10.BR (H-2^k) responder lymph node (LN) cells were stimulated with B10. D2 (H-2^d) stimulator spleen cells in cultures containing the minimum required dose of rat T cell growth factor (TCGF). In these cultures, the generation of cytotoxic T lymphocytes (CTL) was very low. B10.BR Thy.1^lowCD3^– NW-BM cells, added to these cultures, could augment the CTL generation vigorously, but neither B10 (H-2^b) nor B10.D2 cells could. When B10 LN cells were used as responder cells in these cultures, B10 Thy. 1^lowCD3^– NW-BM cells could augment the CTL generation, but neither B10.BR nor B10.D2 cells could. Similar findings were obtained when Lyt-2⁺ cells or Thy.1⁺ L3T4^– (CTL precursor) cells sorted from spleen cells were used as responder cells. Both elements, rat-TCGF and Thy.1^low CD3^– NW-BM cells, were essential for this augmentation of the CTL generation in this culture system because neither one alone could augment generation, and rat-TCGF could be replaced by Thy.1⁺ Lyt-2^– helper T (Th) cells sorted from spleen cells. These findings showed that NW-BM cells could augment CTL precursors in a self-major histocompatibility complex (self-MHC)-antigen restricted manner, and further that both NW-BM cells and Th cells had different and independent functions to induce CTL.     

Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C₁ cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C₁ was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10) (m) increased furosemideinsensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanideinsensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloridedepleted cells. When cells with normal chloride content were depolarized (135 mm medium potassium + 10 m valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl^–Br^–>NO ₃ ^– >I^–. The electrogenic transport pathway was inhibited in chloridedepleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.The technical assistance of Dwight Derr is gratefully acknowledged. We also thank Dr. Chahrzad Montrose-Rafizadeh for help in performance of the chloride efflux experiments. This work was supported by National Institutes of Health grants RO1-DK42457 and PO1-DK44484.