Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: results of an in vitro and in vivo study

This study was designed to investigate the in vitro and in vivo transfection efficiency of chitosan nanoparticles used as vectors for gene therapy. Three types of chitosan nanoparticles [quaternized chitosan -60% trimethylated chitosan oligomer (TMCO-60%), C(43-45 KDa, 87%), and C(230 KDa, 90%)] were used to encapsulate plasmid DNA (pDNA) encoding green fluorescent protein (GFP) using the complex coacervation technique. The morphology, optimal chitosan-pDNA binding ratio and conditions for maximal in vitro transfection were studied. The in vivo transfection was conducted by feeding the chitosan/pDNA nanoparticles to 12 BALB/C-nu/nu nude mice. Both conventional and TMCO-60% could form stable nanoparticles with pDNA. The in vitro study showed the transfection efficiency to be in the following descending order: TMCO-60%>C(43-45 KDa, 87%)>C(230 KDa, 90%). TMCO-60% proved to be the most efficient and the optimal chitosan/pDNA ratio being 3.2:1. In vivo study showed most prominent GPF expression in the gastric and upper intestinal mucosa. GFP expression in the mucosa of the stomach and duodenum, jejunum, ileum, and large intestine were found, respectively, in 100%, 88.9%, 77.8% and 66.7% of the nude mice examined. TMCO-60%/pDNA nanoparticles had better in vitro and in vivo transfection activity than the other two, and with minimal toxicity, which made it a desirable non-viral vector for gene therapy via oral administration.     

Apelin activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas

Apelin was recently found to be an inotropic polypeptide in isolated rat hearts, and intravenous injection of apelin can induce a transient decrease in blood pressure. To illustrate the mechanism of apelin-induced vasodilation, we observed the in vitro effects of apelin on the L-arginine (L-Arg)/nitric oxide (NO) pathway in the incubated, isolated rat aorta. Apelin stimulated vascular NO(2)(-) product and NOS activation in a concentration- and time-dependent manner. Compared with no apelin treatment, incubation with apelin (10(-9), 10(-8), and 10(-7)mol/L) increased NO(2)(-) product by 33%, 46%, and 69% (all p<0.01), respectively, and Ca(2+)-dependent constitutive NOS (cNOS) activity by 200%, 460%, and 550% (all p<0.01), respectively. However, Ca(2+)-independent NOS (iNOS) activity was not significantly altered (p>0.05). Apelin incubation (10(-9), 10(-8), and 10(-7)mol/L) increased L-Arg uptake by 130%, 180%, and 240% (all p<0.01), respectively. The mRNA level of cationic amino acid transporters, CAT-1 and CAT-2B, in rat aortic tissues treated with 10(-7)mol/L apelin was increased by 110% and 128%, respectively (both p<0.01). Incubation with 10(-7)mol/L apelin elevated eNOS mRNA and protein levels, by 53% (p<0.05) and 319% (p<0.01), respectively. Collectively, these results demonstrate that apelin directly activated the vascular L-Arg/NOS/NO pathway, which could be one of the important mechanisms of apelin-regulated vascular function.     

Effect of vagotomy on dynamics of mesenteric lymphatic vessels in the rat

The functional modulation of lymphatic vessels may be closely associated with intact structures of the vagus nerve. In the present study, the vagotomy was done in Wistar rat to investigate the effect of vagus nerves on dynamic changes of mesenteric lymphatic vessels. After denervation, the mesenteric lymphatics showed significant decreases in contraction rate, diameter in the static state and overall contractile activity under a microscopic observation. The lymphatic contraction rhythm and valve movement became irregular and inconsistent. These findings indicated that the lymphatic innervation might be an important factor for active lymph formation and transportation.     

Nanoparticle-based detection and quantification of DNA with single nucleotide polymorphism (SNP) discrimination selectivity

Sequence-specific DNA detection is important in various biomedical applications such as gene expression profiling, disease diagnosis and treatment, drug discovery and forensic analysis. Here we report a gold nanoparticle-based method that allows DNA detection and quantification and is capable of single nucleotide polymorphism (SNP) discrimination. The precise quantification of single-stranded DNA is due to the formation of defined nanoparticle-DNA conjugate groupings in the presence of target/linker DNA. Conjugate groupings were characterized and quantified by gel electrophoresis. A linear correlation between the amount of target DNA and conjugate groupings was found. For SNP detection, single base mismatch discrimination was achieved for both the end- and center-base mismatch. The method described here may be useful for the development of a simple and quantitative DNA detection assay.     

Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance

A biosensor based on bovine serum albumin (BSA) for the detection of lead (Pb(2+)) ion was developed and characterized. BSA was immobilized onto a colloidal Au-modified piezoelectric quartz crystal (PQC) as a biosensor for the detection of Pb(2+) ion by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb(2+) ion showed excellent linearity throughout the concentration range from 1.0 x 10(-7) to 3.0 x 10(-9)mol/L. The interaction between the Pb(2+) ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 x 10(-9)mol/L for Pb(2+) was obtained. Kinetic parameters of the Pb(2+)-BSA interactions were also determined by using this chip. The sensor chip could be regenerated for use by dipping in the ethylenediaminetetraacetic acid (EDTA) solution for approximately 2h, and the chip was used to detect Pb(2+) ion for eight times without obvious signal attenuation.     

Crystal structure of homoserine O-acetyltransferase from Leptospira interrogans

Homoserine O-acetyltransferase (HTA, EC 2.3.1.31) initiates methionine biosynthesis pathway by catalyzing the transfer of acetyl group from acetyl-CoA to homoserine. This study reports the crystal structure of HTA from Leptospira interrogans determined at 2.2 Å resolution using selenomethionyl single-wavelength anomalous diffraction method. HTA is modular and consists of two structurally distinct domains—a core α/β domain containing the catalytic site and a helical bundle called the lid domain. Overall, the structure fold belongs to α/β hydrolase superfamily with the characteristic ‘catalytic triad’ residues in the active site. Detailed structure analysis showed that the catalytic histidine and serine are both present in two conformations, which may be involved in the catalytic mechanism for acetyl transfer.