L-arginine supplementation improves responses to injury and inflammation in dextran sulfate sodium colitis

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y(+) cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS(-/-) mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.     

N-methylpyridoxamine: novel canine vitamin B6 urine metabolite

Cation-exchange HPLC analysis of urine from dogs given large daily doses of pyridoxamine revealed an unidentified metabolite hypothesized to be N-methylpyridoxamine. Identity was established by N-methylpyridoxamine synthesis and HPLC comparison to the canine metabolite. Compound synthesis was confirmed by IR, NMR, UV-vis and emission spectroscopy. It seems to have less fluorescent character than other routinely-measured vitamin B(6) metabolites. Upon administration of substantial pyridoxamine doses, N-methylpyridoxamine appears to be a quantifiable canine urine metabolite, although, at either pharmacological or dietary pyridoxamine intakes, its relevance to vitamin B(6) metabolism in other species, including humans, is not yet determined.     

Does the frequency content of the surface mechanomyographic signal reflect motor unit firing rates? A brief review.

The purpose of this review is to examine the literature that has investigated the potential relationship between mechanomyographic (MMG) frequency and motor unit firing rates. Several different experimental designs/methodologies have been used to address this issue, including: repetitive electrical stimulation, voluntary muscle actions in muscles with different fiber type compositions, fatiguing and non-fatiguing isometric or dynamic muscle actions, and voluntary muscle actions in young versus elderly subjects and healthy individuals versus subjects with a neuromuscular disease(s). Generally speaking, the results from these investigations have suggested that MMG frequency is related to the rate of motor unit activation and the contractile properties (contraction and relaxation times) of the muscle fibers. Other studies, however, have reported that MMG mean power frequency (MPF) does not always follow the expected pattern of firing rate modulation (e.g. motor unit firing rates generally increase with torque during isometric muscle actions, but MMG MPF may remain stable or even decrease). In addition, there are several factors that may affect the frequency content of the MMG signal during a voluntary muscle action (i.e. muscle stiffness, intramuscular fluid pressure, etc.), independent of changes in motor unit firing rates. Despite the potential influences of these factors, most of the evidence has suggested that the frequency domain of the MMG signal contains some information regarding motor unit firing rates. It is likely, however, that this information is qualitative, rather than quantitative in nature, and reflects the global motor unit firing rate, rather than the firing rates of a particular group of motor units.     

Membrane proteins implicated in long-chain fatty acid uptake by mammalian cells: CD36, FATP and FABPm

Long-chain fatty acids can transfer passively across mammalian cell membranes. However, under physiological conditions of low fatty acid to albumin ratios in the circulation, the major fraction of uptake appears to be mediated by a saturable, protein-facilitated component. A simple diffusion process becomes significant at high molar ratios of fatty acid to albumin as the concentration of free fatty acid in solution is increased. Identification of the mammalian membrane fatty acid transporter(s) has been the focus of active investigation by several research groups. In this review we discuss three candidate proteins: FABPm, FAT/CD36 and FATP which have been cloned and are currently being characterized. Recent evidence arguing for an important role of the fatty acid transport step in general metabolism and linking these proteins to physiologic or metabolic abnormalities is described.     

Trehalose Increases Freeze-Thaw Damage in Liposomes Containing Chloroplast Glycolipids

Chloroplast thylakoids contain three classes of glycolipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG). We have investigated the stability of large unilamellar vesicles made from egg phosphatidylcholine (EPC) and different chloroplast glycolipids during freezing to -18 degreesC, as a function of the presence of three sugars: glucose, sucrose, or trehalose. Contrary to the situation in thylakoids, where cryoprotection increases from glucose < sucrose < trehalose, liposomes containing 50% DGDG showed the opposite behavior. In fact, carboxyfluorescein leakage increased over the control values (freezing in the absence of sugar) in the presence of trehalose. This effect was not seen in vesicles made from pure EPC, or a mixture of EPC and MGDG, or EPC and SQDG. Liposomes made from mixtures of all three glycolipids, however, showed even more leakage in the presence of trehalose than liposomes containing only DGDG and EPC. Copyright 1998 Academic Press.     

Smooth muscle raft-like membranes

We developed a method for extracting raft-like, liquid-ordered membranes from the particulate fraction prepared from porcine trachealis smooth muscle. This fraction, which contains most of the plasma membrane in this tissue, was homogenized in the presence of cold 0.5% Triton X-100. After centrifugation, membranes containing high contents of sphingomyelin (SM) and cholesterol and low phosphatidylcholine (PC) contents remained in the pellet. Thirty-five millimolar octyl glucoside (OG) extracted 75% of these membranes from the Triton X-100-resistant pellet. These membranes had low buoyant densities and accounted for 28% of the particulate fraction lipid. Their lipid composition, 22% SM, 60% cholesterol, 11% phosphatidylethanolamine, 8% PC, <1% phosphatidylinositol, and coisolation with 5'-nucleotidase and caveolin-1 suggest that they are liquid-ordered membranes. We compared characteristics of OG and Triton X-100 extractions of the particulate fraction. In contrast to Triton X-100 extractions, membranes released from the particulate fraction by OG were mainly collected in low buoyant fractions at densities ranging from 1.05 to 1.11 g/ml and had phospholipid and cholesterol contents consistent with a mixture of liquid-ordered and liquid-disordered membranes. Thus, OG extraction of apparent liquid-ordered membranes from Triton X-100-resistant pellets was not due to selective extraction of these membranes. Low buoyant density appears not to be unique for liquid-ordered membranes.     

Development of an oxazolopyridine series of dual thrombin/factor Xa inhibitors via structure-guided lead optimization

Thrombin-inhibitor X-ray crystal structures, in combination with the installation of binding elements optimized within the pyrazinone series of thrombin inhibitors, were utilized to transform a weak triazolopyrimidine lead into a series of potent oxazolopyridines. A modification intended to attenuate plasma protein binding (i.e., conversion of the P3 pyridine to a piperidine) conferred significant factor Xa activity to this series. Ultimately, these dual thrombin/factor Xa inhibitors demonstrated excellent in vitro and in vivo anticoagulant efficacy.     

Role of gelsolin in actin depolymerization of adherent human neutrophils.

Human neutrophils generally function adherent to an extracellular matrix. We have previously reported that upon adhesion to laminin- or fibronectin-coated, but not uncoated, plastic there is a depolymerization of actin in neutrophils. This phenomenon was not affected by inhibitors of the more well-studied components of the signal transduction pathway, specifically, pertussis toxin, an inhibitor of G-proteins, H-7 or staurosporine, inhibitors of protein kinase C, or herbimycin A, an inhibitor of nonreceptor tyrosine kinase. We therefore focused our attention on actin-binding proteins and measured the changes in the partitioning of gelsolin between the Triton X-100-soluble and -insoluble cellular fractions which occur upon neutrophil adhesion by means of quantitating anti-gelsolin antibody binding to aliquots of these fractions. It was found that approximately 90% of the total cellular gelsolin was found in the Triton X-100-soluble fraction in suspended cells, but that upon adherence to either fibronectin- or laminin-coated plastic about 40% of the soluble gelsolin could be detected in the insoluble fraction. This effect was not observed in cells adherent to uncoated plastic, wherein more than 90% of the gelsolin was found in the soluble fraction. Results of immunofluorescence microscopy of these cell preparations was consistent with this data. A gelsolin translocation to the insoluble cellular actin network may account for a part of the observed actin depolymerization.     

Impact of highway construction on leaf processing in aquatic habitats of eastern Tennessee

Rates of leaf litter processing at eight sites were used in conjunction with other methods to evaluate the impact of highway construction on aquatic habitats. Monthly processing of white oak leaves from four different mesh size bags at unimpacted reference sites indicated that the three larger mesh sizes were useful for comparing sites, as they did not restrict invertebrate colonization and provided similar rates of processing within sites. Small mesh size (0.12 mm) prevented leaf shredding macroinvertebrates from colonizing bags, and caused significantly slower leaf processing in a riffle.Leaf processing was fastest in a reference riffle above an area of highway construction but was significantly slower in the reference pool due to low current velocity and the absence of shredders. Leaf processing in a riffle below the highway was slower than the reference riffle, and the number of shredders was reduced. Removal of streamside vegetation during highway construction caused increased stream temperatures and reduced the amount of natural leaf accumulations, thereby reducing shredder habitat. At other sites highway construction caused less of an impact than preexisting environmental influences. Leaf processing in an riffle of a lower pH stream was significantly slower than in the reference riffle. Shredders were absent from the low pH stream, and rates of leaf processing in the acid impacted riffle and pool were similar to those of the reference pool. Comparisons of the low pH stream with the reference stream indicated that physical processes were less important than biological processes of leaf decay. Leaf processing in ponds was similar to that of stream pools. Processing was faster in a small versus large pond, and a high density of invertebrates was associated with leaf bags in the small pond after one year.     

Bicyclic pyridones as potent, efficacious and orally bioavailable thrombin inhibitors

A new class of conformationally constrained thrombin inhibitors is described. These compounds contain a unique bicyclic pyridone scaffold which serves as a P3P2 dipeptide surrogate. The synthesis and antithrombotic activity of these inhibitors is reported.