Effects of fractionated abdominal irradiation on small intestinal motility--studies in a novel in vitro animal model

Disordered small intestinal motility occurs frequently during acute radiation enteritis. However, the characteristics and time course of the motor dysfunction are poorly defined. These parameters were assessed in a novel animal model of radiation enteritis. Ileal pressures were recorded in vitro with perfused micromanometric catheter using an arterially perfused ileal loop in 22 ferrets following fractionated abdominal irradiation (9 doses 2.50 Gy thrice weekly for 3 weeks). Tissue damage was graded histologically. Studies were performed 3 to 29 days after irradiation. Tissue from 7 control animals was also studied. All treated animals developed diarrhoea. Histology showed changes consistent with mild to moderate radiation enteritis. Following irradiation, there was an initial increase in frequency followed by a non-significant reduction in the frequency, but not the amplitude of ileal pressure waves. The frequency of pressure waves showed an inverse relationship with time after radiation (r = -0.634, p < 0.002). There was no relationship between motility and histology. We conclude that abdominal irradiation is associated with a time-dependent reduction in ileal motility which does not correlate with light microscopic changes.     

Low molecular weight dermatan sulfate as an antithrombotic agent. Structure-activity relationship studies

A structure-activity relationship of low molecular weight dermatan sulfate was undertaken to understand better this new non-heparin, glycosaminoglycan-based antithrombotic agent. A dermatan sulfate prepared from bovine intestinal mucosa [average molecular weight (MWavg) 25,000], and currently in clinical trials as an antithrombotic agent, was used in this study. Dermatan sulfate was partially depolymerized using hydrogen peroxide and copper(II) as catalyst to MWavg 5600 to obtain a low molecular weight dermatan sulfate. This low molecular weight dermatan sulfate was then fractionated by gel permeation chromatography to obtain four subfractions having MWavg 7800, 5500, 4200 and 1950. The dermatan sulfate, low molecular weight dermatan sulfate and its subfractions showed substantially different optical rotations. The 1H-NMR spectroscopic analysis of dermatan sulfate samples showed some differences including increased content of GalpNAc4S6S residues and improved resolution in ring resonances for low molecular weight dermatan sulfate fractions, primarily the result of reduced molecular weight and lowered heterogeneity. Saccharide compositional analysis relied on chondroitin ABC lyase treatment followed by capillary electrophoresis. Polyacrylamide gel-based oligosaccharide mapping was also performed by treating dermatan sulfate samples with chondroitin B, AC and ABC lysases. These analyses showed increased amounts of sulfation as the MWavg decreased. In vitro bioassay showed maximum anti-Xa activity in the 4.2 kDa fraction and maximum heparin cofactor II-mediated anti-IIa activity in the 5.5 kDa fraction. The in vivo antithrombotic activity of these fractions was measured using a modified Wessler stasis thrombosis model. The 4.2 kDa fraction showed greater antithrombotic activity than the other low molecular weight dermatan sulfate fractions, dermatan sulfate, and low molecular weight dermatan sulfate. This enhanced activity may result from several structural features of the 4.2 kDa fraction including: a high content of 4,6- and 2,4-disulfated disaccharide sequences; the requirement of specific chain length; a change in the ratio of iduronic to glucuronic acid; and the presence of chondroitin ABC lyase resistant material.     

Activation of the Xenopus oocyte mitogen-activated protein kinase pathway by Mos is independent of Raf

Transgenic mice expressing the oncogenic protein-serine/threonine kinase Mos at high levels in the brain display progressive neuronal degeneration and gliosis. Gliosis developed in parallel with the onset of postnatal transgene expression and led to a dramatic increase in the number of astrocytes positive for GFAP, vimentin, and possibly tau. Interestingly, vimentin is normally expressed only in immature or neoplastic astrocytes, but appears to be induced to high levels in Mos-transgenic, mature astrocytes. Mos can activate mitogen activated protein kinase (MAPK) and MAPK has been implicated in Alzheimer-type tau phosphorylation. In the Mos-transgenic brain we found increased levels of phosphorylation at one epitope on tau containing serines 199 and 202 (numbering according to human tau), a pattern similar but not identical to that found in Alzheimer's disease. In addition, Mos-transgenic mice express a novel neurofilament-related protein that might be a proteolytic neurofilament heavy chain degradation product. These results suggest that activation of protein phosphorylation in neurons can result in changes in cytoskeletal proteins that might contribute to neuronal degeneration.     

The immunosuppressive substance 2-chloro-2-deoxyadenosine modulates lipoprotein metabolism in a murine macrophage cell line (P388 cells)

A recently developed immunosuppressive substance, 2-chloro-2-deoxyadenosine (2-CdA), was reported to inhibit monocyte functions at low concentration. Because macrophages play a key role in the formation of atherosclerotic plaques, it was of interest to study the effect of 2-CdA on cellular lipid metabolism. For this purpose we have used a macrophage cell line (P388) to perform incubation studies in the presence of acetylated low density lipoprotein (Ac-LDL) and 2-CdA. The addition of 2-CdA, in concentrations ranging from 5–20 nM, induced a dose-dependent decrease in cellular cholesterol content and in the amount of extracellular [¹⁴C]oleic acid (OA) incorporated into the cholesteryl ester (CE) fraction. The effect was maximized at 20 nM 2-CdA with an 86% reduction in cholesterol esterification compared to controls (P<0.008). To evaluate the mechanism of interaction of 2-CdA with cellular lipid metabolism, deoxycytidine (dCyt) and 3-methoxybenzamide (3-MOB), substances known to antagonize the effect of 2-CdA in different ways, were co-administered with 2-CdA. dCyt, a competitive inhibitor of dCyt kinase, which catalyzes phosphorylation to the active metabolite, antagonized the effects of 20 nM 2-CdA, producing significantly greater incorporation of extracellular [¹⁴C]OA into the CE fraction than in the presence of 2-CdA alone (P<0.0086). Co-incubation with 2-CdA and the poly-ADP-ribose synthetase inhibitor 3-MOB, which is known to render cells resistant to 2-CdA toxicity by preventing cellular nicotinamide adenine dinucleotide (NAD)- and adenosine triphosphase-depletion, also reversed the effect of 2-CdA on lipid accumulation. However, incubation of P388 cells with 20 nM 2-CdA did not result in a decrease in cellular NAD content. As 20 nM 2-CdA showed no effect on intracellular cholesterol synthesis based on measurement by 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, the decrease in cellular cholesterol content and in [¹⁴C]OA incorporation seems to be primarily due to an interference with Ac-LDL metabolism.     

Detergent binding in trigonal crystals of OmpF porin from Escherichia coli

The structure of the detergent, ocytyl hydroxyethylsufoxide (C8(HE)SO), bound to the OmpF porin from E coli (in the trigonal crystal form) has been determined by neutron crystallography. Due to a dynamic exchange of detergent molecules with their environment they are not ordered on an atomic scale. The structure reported here is therefore at a resolution of approximately 16 A. The X-ray crystallographically determined structure of the protein provides a starting point for the neutron analysis in which the detergent is visualized primarily thanks to its high contrast against D2O. The structure shows the detergent to be located mainly in two areas. It forms toroidal annuli around each OmpF trimer, these annuli fusing to form a detergent belt surrounding a solvent filled column traversing the crystal. Those areas of the protein to which the detergent binds are formed almost exclusively of hydrophobic residues and form a band about 30 A high around the trimer. Its upper and lower bounds are defined by two bands of aromatic residues, tyrosines pointing away from the detergent belt and interacting with the polar headgroups while phenylalanines point inwards. This strongly suggests that the same areas define, in vivo, the location at which protein interacts with lipid. The hydrophobic moiety of detergent is also found mediating the hydrophobic protein-protein interactions at the interface between two trimers on the crystallographic two-fold axis.     

Mechanism of heparin activation of antithrombin. Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.     

Phase Separation in La2CuO4+δ by electrical transport measurements

The dc conductivity and the high-frequency capacitance of a La₂CuO_4+δ single crystal are determined by impedance spectroscopy in the frequency range 20 Hz to 1 MHz at liquid helium temperature. Using isochronal annealing, the effect of the phase separation on these quantitites is studied for E parallel and perpendicular to the CuO₂ planes as well as for different oxygen doping levels,δ, of the sample. The results are consistent with the model of a diffusion-controlled formation of a metallic—and belowT _c superconducting—percolation network in an insulating background.     

Interplay between oxygen demand reactions and kinetic gas-water transfer in porous media

Gas-water phase transfer associated with the dissolution of trapped gas in porous media is a key process that occurs during pulsed gas sparging operations in contaminated aquifers. Recently, we applied a numerical model that was experimentally validated for abiotic situations, where multi-species kinetic inter-phase mass transfer and dissolved gas transport occurred during pulsed gas penetration-dissolution events [Balcke, G.U., Meenken, S., Hoefer, C. and Oswald, S.E., 2007. Kinetic gas-water transfer and gas accumulation in porous media during pulsed oxygen sparging. Environmental Science & Technology 41(12), 4428-4434]. Here we extend the model by using a reactive term to describe dissolved oxygen demand reactions via the formation of a reaction product, and to study the effects of such an aerobic degradation process on gas-water mass transfer and dissolution of trapped gas in porous media. As a surrogate for microbial oxygen reduction, first-order oxygen demand reactions were based on the measured oxidation of alkaline pyrogallol in column experiments. This reaction allows for adjusting the rate to values close to expected biodegradation rates and detection of the reaction product. The experiments and model consistently demonstrated accelerated oxygen gas-water mass transfer with increasing oxygen demand rates associated with an influence on the partitioning of other gases. Thus, as the oxygen demand accelerates, less gas phase residues, consisting mainly of nitrogen, are observed, which is in general beneficial to the performance of field biosparging operations. Model results additionally predict how oxygen demand influences oxygen mass transfer for a range of biodegradation rates. A typical field case scenario was simulated to illustrate the observed coupling of oxygen consumption and gas bubble dissolution. The model provides a tool to improve understanding of trapped gas behavior in porous media and contributes to a model-assisted biosparging.     

The effect of parboiled rice on glycemia in Wistar rats

Starch is an important energy source and can represent more than 60% of the calories of the human diet. The starch fraction resistant to enzymatic digestion is called resistant starch. When rice is parboiled, the starch retrogrades with the formation of type 3-resistant starch (retrograded), which presents beneficial effects on the health, since it acts as a prebiotic. In the present study three types of rice were selected, with high, medium and low amylose contents, with the objective of evaluating the effects of conventional and parboiled rice on glycemia in Wistar rats. The samples with high and medium amylose contents were soaked for 6 h at 65 degrees C, and the low amylose sample for 7 h at 70 degrees C. The samples were subsequently autoclaved for 10 minutes at 0.7kgf x cm(-2). Six male Wistar rats were used for each treatment. Seven experimental diets were elaborated, formulated according to AIN-93M, the control diet and diets substituting the carbohydrate source with conventional or parboiled rice. Resistant starch was determined in the diets and glycemia monitored using glucose paper strips, the sample being blood obtained from the distal part of the rat's tail. For the glycemic curve, glycemia was measured in the fasting state and during 90 minutes post-prandial. The results indicated there were no significant differences between the diets formulated with high, medium and low amylose, parboiled or conventionally prepared, with respect to fasting or post-prandial glycemia in Wistar rats.