Mouse Acidic Chitinase Effectively Degrades Random-Type Chitosan to Chitooligosaccharides of Variable Lengths under Stomach and Lung Tissue pH Conditions.

Chitooligosaccharides exhibit several biomedical activities, such as inflammation and tumorigenesis reduction in mammals. The mechanism of the chitooligosaccharides' formation in vivo has been, however, poorly understood. Here we report that mouse acidic chitinase (Chia), which is widely expressed in mouse tissues, can produce chitooligosaccharides from deacetylated chitin (chitosan) at pH levels corresponding to stomach and lung tissues. Chia degraded chitin to produce N-acetyl-d-glucosamine (GlcNAc) dimers. The block-type chitosan (heterogenous deacetylation) is soluble at pH 2.0 (optimal condition for mouse Chia) and was degraded into chitooligosaccharides with various sizes ranging from di- to nonamers. The random-type chitosan (homogenous deacetylation) is soluble in water that enables us to examine its degradation at pH 2.0, 5.0, and 7.0. Incubation of these substrates with Chia resulted in the more efficient production of chitooligosaccharides with more variable sizes was from random-type chitosan than from the block-type form of the molecule. The data presented here indicate that Chia digests chitosan acquired by homogenous deacetylation of chitin in vitro and in vivo. The degradation products may then influence different physiological or pathological processes. Our results also suggest that bioactive chitooligosaccharides can be obtained conveniently using homogenously deacetylated chitosan and Chia for various biomedical applications.

Antibody Fragments for On-Site Testing of Cannabinoids Generated via in Vitro Affinity Maturation.

Law enforcement against illicit use of cannabis and related substances requires rapid, feasible, and reliable tools for on-site testing of cannabinoids. Notably, methods based on cannabinoid-specific antibodies enable efficient screening of multiple specimens. Antibody engineering may accelerate development of modern and robust testing systems. Here, we used in vitro affinity maturation to generate a single-chain Fv fragment (scFv) that recognizes with high affinity the psychoactive cannabinoid, Δ9-tetrahydrocannabinol (THC). A mouse monoclonal antibody against THC, Ab-THC#33, with Ka 6.2×107 M-1 (as Fab fragment) was established by the hybridoma technique. Then, a "wild-type" scFv (wt-scFv) with Ka, 1.1×107 M-1 was prepared by bacterial expression of a fusion gene combining the VH and VL genes for Ab-THC#33. Subsequently, random point mutations in VH and VL were generated separately, and the resulting products were assembled into mutant scFv genes, which were then phage-displayed. Repeated panning identified a mutant scFv (scFv#m1-36) with 10-fold enhanced affinity (Ka 1.1×108 M-1) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the VH-complementarity-determining region 2 (CDR2) sequence. In competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response curves with midpoint 0.27 ng/assay THC, which was 3-fold lower than that of wt-scFv. Even higher reactivity with a major THC metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol, indicated that the mutant scFv will be useful for testing not only THC in confiscated materials, but also the metabolite in urine. Indeed, the antibody fragment is potentially suitable for use in advanced on-site testing platforms for cannabinoids.

Prevention of postoperative pericardial adhesions with a novel regenerative collagen sheet.

BACKGROUND: Postoperative pericardial adhesions make a repeat sternotomy time-consuming and dangerous. The purpose of this study was to evaluate the efficacy of a new collagen pericardial substitute for preventing postoperative pericardial adhesions. METHODS: Our absorbable substitute consists of three layers: a middle layer of aterocollagen between two layers of sodium hyaluronic acid and aterocollagen. In experiment 1 in this study, the patch, made of 9,000 filaments of aterocollagen fibers, (group 1; n = 5) was compared with a patch made of 6,000 filaments (group 2; n = 7), an expanded polytetrafluoroethylene sheet (group 3; n = 6), and a control group (group 4; n = 4). Subsequently, in experiment 2, the patch was examined at 4 weeks (n = 5), 12 weeks (n = 5), and 24 weeks (n = 4) after the operation by light microscopy and scanning electron microscopy. RESULTS: The area of adhesion in group 1 was significantly less as compared with that in the other three groups, and the coronary vessels were clearly identifiable; on the other hand, all the animals in the control group showed moderate to severe adhesions, and the coronary vessels were completely obscured. In experiment 2, formation of a membranous tissue resembling the native pericardial membrane was observed in all animals, and the thickness of this membrane showed a marked increase by 24 weeks after the operation. Light microscopy and scanning electron microscopy also showed the formation of a mesothelium-like lining. CONCLUSIONS: The new absorbable and regenerative collagen patch seemed to be biocompatible, and its use was associated with minimal adhesion formation and preserved coronary anatomy.

The cannabinoid 1 receptor antagonist, AM251, prolongs the survival of rats with severe acute pancreatitis.

It has recently been recognized that anandamide (arachidonylethanolamide), which is an endogeneous-cannabinoid (endocannabinoid), mediates septic shock. Cannabinoid means a mind-active material in cannabis (marijuana). Anandamide is mainly produced by macrophages. Cannabinoid 1 (CB1) receptor, which is one of the cannabiniod receptors, is also known to mediate hypotensive shock. The role of endocannabinoids in the progression of acute pancreatitis is unclear. The aims of this study are to clarify their relationship and to find a new therapeutic strategy by regulating the endocannabinoid signaling in acute pancreatitis. Male Wistar rats were injected with caerulein intravenously to induce mild edematous pancreatitis or injected with 5% sodium taurocholate to the bilio-pancreatic duct to induce severe necrotizing pancreatitis. The animals in the latter group were also injected with a CB1 receptor antagonist, AM251, or vehicle solution to see if the inhibition of endocannabinoids improves their survival. Plasma anandamide level was measured by the liquid chromatography/tandem mass spectrometry method. In both models of acute pancreatitis, the plasma anandamide levels were increased, and the levels were significantly higher in rats with severe necrotizing pancreatitis than those in rats with mild edematous pancreatitis. The mean arterial pressure and survival rate were significantly improved by the treatment with AM251, despite that the local inflammatory changes in the pancreas and various parameters (white blood cells, hematocrit, serum amylase, and serum interleukin-6) were similar. This is the first report to show that endocannabinoids are involved in the deterioration of acute pancreatitis and that the down-regulation of endocannabinoid signaling may be a new therapeutic strategy for severe acute pancreatitis.

Rat experimental model of continuous regional arterial infusion of protease inhibitor and its effects on severe acute pancreatitis.

OBJECTIVES: The rat experimental model of continuous regional arterial infusion of protease inhibitor (CRAI) on acute pancreatitis has yet to be established. Therefore, the aims of this study were (1) to establish the rat experimental model of CRAI and (2) to evaluate the effects of nafamostat on rat severe acute pancreatitis via different routes of administration. METHODS: The rat internal jugular vein or the celiac artery was infused with nafamostat, and the concentration of nafamostat in the lung and pancreas was measured. After the induction of severe acute pancreatitis, rats received intravenous or regional intraarterial infusion of nafamostat and then concentrations of trypsinogen activated peptide (TAP) and serum interleukin (IL-6), and histologic sections of the pancreas were examined and the 96-hour survival rate was evaluated. RESULTS: CRAI rats had higher concentrations of nafamostat in the pancreas than those infused intravenously. However, CRAI rats had lower concentrations of nafamostat in the lung that those infused intravenously. CRAI significantly reduced the levels of TAP and pancreatic necrosis. Moreover, the levels of serum IL-6 and the mortality rate were significantly reduced after CRAI compared with the intravenous infusion of nafamostat. CONCLUSION: The effectiveness of the rat experimental model of CRAI on acute pancreatitis was clearly demonstrated. The concentration of nafamostat in the lung and pancreas and the effects of nafamostat differ according to the route of administration.

Magnetic properties of Heusler alloys Ru2-xFexCrSi.

We report on the structural and magnetic properties of newly synthesized Heusler alloys, Ru2-xFexCrSi, which have quite recently been shown to be candidates for ferromagnetic metals with high spin polarization from band structure calculations. Polycrystalline samples of Heusler alloys Ru2-xFexCrSi were prepared for 0.5≤x≤1.8. They were found to have L21 structures for 0.5≤x≤1.5 and B2 for x = 1.8. Magnetic measurements showed that they are ferromagnets. The Curie temperature for x = 1.0 was found to be 370 K. The Curie temperature tends to increase with increasing Fe concentration x. The saturation magnetic moment increases almost linearly as x increases. For higher Fe concentration the saturation magnetic moment is close to 2 µB per formula unit, which is theoretically expected.

Theoretical and experimental examination of near-field acoustic levitation.

A planar object can be levitated stably close to a piston sound source by making use of acoustic radiation pressure. This phenomenon is called near-field acoustic levitation [Y. Hashimoto et al., J. Acoust. Soc. Am. 100, 2057-2061 (1996)]. In the present article, the levitation distance is predicted theoretically by numerically solving basic equations in a compressible viscous fluid subject to the appropriate initial and boundary conditions. Additionally, experiments are carried out using a 19.5-kHz piston source with a 40-mm aperture and various aluminum disks of different sizes. The measured levitation distance agrees well with the theory, which is different from a conventional theory, and the levitation distance is not inversely proportional to the square root of the surface density of the levitated disk in a strict sense.