Impact of anti-inflammatories, beta-blockers and antibiotics on leaf litter breakdown in freshwaters.

Pharmaceuticals are now recognised as important pollutants in freshwater systems, but a shortcoming of effects studies is that they have focused on structural endpoints and impacts on ecosystem functioning are poorly understood. The decomposition of organic matter is an important functional process in aquatic systems, and it is known that this can be impacted by the presence of pollutants. Previous studies on leaf litter breakdown have only considered the effects of antibiotics and not other groups of drugs though. The current study investigated the effects of anti-inflammatories, a beta-blocker and an antibiotic on microbially mediated breakdown of leaf litter in the laboratory; colonisation of leaf packs by benthic macroinvertebrates when placed in a stream; and shredding of leaf litter by these organisms. Furthermore, the effects of single compounds relative to their mixture were assessed. It was found that exposure of leaf litter to the study compounds did not influence its breakdown by microbes in the laboratory or macroinvertebrates in a stream. Exposure of leaf litter to pharmaceuticals also had no effect on its colonisation by macroinvertebrates in this study. Many unknowns remain, however, and further studies of the effects of pharmaceuticals on structural and functional endpoints are needed to aid aquatic conservation.

Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*.

AIMS: A Lactobacillus buchneri strain NRRL B-30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B-30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B-30929 for fermenting mixed substrates from biomass hydrolysates. METHODS AND RESULTS: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B-30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B-30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1.98-2.35 g l(-1) ethanol from corn stover hydrolysates and 2.92-3.01 g l(-1) ethanol from wheat straw hydrolysates when supplemented with either 0.25x MRS plus 1% corn steep liquor or 0.5x MRS. CONCLUSIONS: The L. buchneri NRRL B-30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are valuable for future research in engineering L. buchneri NRRL B-30929 for fermentative production of ethanol and chemicals from biomass.

Alpha2-macroglobulin associates with beta-amyloid peptide and prevents fibril formation.

We have used the yeast two-hybrid system to isolate cDNAs encoding proteins that specifically interact with the 42-aa beta-amyloid peptide (Abeta), a major constituent of senile plaques in Alzheimer's disease. The carboxy terminus of alpha2-macroglobulin (alpha2M), a proteinase inhibitor released in response to inflammatory stimuli, was identified as a strong and specific interactor of Abeta, utilizing this system. Direct evidence for this interaction was obtained by co-immunoprecipitation of alpha2M with Abeta from the yeast cell, and by formation of SDS-resistant Abeta complexes in polyacrylamide gels by using synthetic Abeta and purified alpha2M. The association of Abeta with alpha2M and various purified amyloid binding proteins was assessed by employing a method measuring protein-protein interactions in liquid phase. The dissociation constant by this technique for the alpha2M-Abeta association using labeled purified proteins was measured (Kd = 350 nM). Electron microscopy showed that a 1:8 ratio of alpha2M to Abeta prevented fibril formation in solution; the same ratio to Abeta of another acute phase protein, alpha1-antichymotrypsin, was not active in preventing fibril formation in vitro. These results were corroborated by data obtained from an in vitro aggregation assay employing Thioflavine T. The interaction of alpha2M with Abeta suggests new pathway(s) for the clearance of the soluble amyloid peptide.

The effect of flow arrest on distal embolic events during arterial occlusion with detachable coils: a canine study.

PURPOSE: To determine the effect of proximal flow arrest on the frequency and timing of distal embolic events during occlusion of the common femoral artery with detachable coils. METHODS: Twenty-three complex fibered platinum coils were delivered into 10 common femoral arteries without proximal flow arrest. The arteries were continuously monitored for flow and embolic events by Doppler sonography during delivery and for at least 10 minutes after delivery of each coil. Thirty-four coils were delivered into 6 arteries after proximal flow arrest by inflation of a nondetachable balloon. After balloon deflation, each artery was monitored by Doppler sonography for 10 minutes. RESULTS: In the 10 arteries occluded without flow arrest, 87 events (8.7 per artery) occurred, of which 47 were embolic and 40 were indeterminate. In the 6 arteries with flow arrest, the number of emboli detected was 3 (0.5 per artery). Embolic events occurred only if there was residual flow. In those arteries that were occluded when the flow-arrest balloon was deflated, no emboli were detected. CONCLUSIONS: Proximal flow arrest virtually eliminates the risk of distal emboli during arterial occlusion with detachable fibered coils. The use of fibered coils, in conjunction with proximal flow arrest, allows for safe arterial occlusion when detachable balloons are not available or their use is not feasible.

Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.

Nitric oxide-dependent release of vasodilator quantities of calcitonin gene-related peptide from capsaicin-sensitive nerves in rabbit skin.

1. Calcitonin gene-related peptide (CGRP) is a potent and long lasting vasodilator in the cutaneous microvasculature of many species including the rabbit. In this study we have investigated the role of nitric oxide in the release of endogenous CGRP, in response to capsaicin, in rabbit skin. 2. Cutaneous blood flow was measured in response to intradermally-injected agents by a multiple site 133Xenon clearance technique. 3. The increased blood flow induced by capsaicin (100 nmol/site) and CGRP (3 pmol/site) was totally inhibited by the CGRP antagonist CGRP(8-37) (1 nmol/site), whilst the increased blood flow induced by sodium nitroprusside (0.3, 1 and 3 nmol/site) was unaffected by CGRP(8-37). 4. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 30 nmol/site) had no effect on the vasodilator response induced by CGRP, but significantly inhibited capsaicin-induced blood flow. The inhibitory effect of L-NAME on capsaicin-induced blood flow was reversed by intradermal L-arginine (300 nmol/site), whilst the inactive enantiomer D-NAME (30 nmol/site) and the alpha-adrenoceptor agonist phenylephrine (10 pmol/site), at a dose which had a similar effect to L-NAME on basal blood flow, had no effect on capsaicin-induced blood flow. 5. These results suggest that CGRP is the important vasodilator which is released from capsaicin-sensitive sensory nerves in rabbit skin and that the release of CGRP, but not its mechanism of vasodilator action, is nitric oxide-dependent in the rabbit cutaneous microvasculature.

Using the mission statement to craft a least-restraint policy.

St. Peter's Hospital used the Mission Statement as a foundation upon which to build a new "Least Restraint Policy." The Mission Statement guided the change process by assisting us to 1) identify the need for change, 2) identify the values and beliefs on which a new policy should be based, 3) identify the need for interdisciplinary planning, and 4) identify the need for staff and family education before implementation of the new policy.

The contribution of calcitonin gene-related peptide (CGRP) to neurogenic vasodilator responses.

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator in the microcirculation of many tissues including the skin and joint. In order to elucidate the mechanism of endogenous CGRP release, we have used a multiple site 133Xe clearance technique to measure local blood flow changes in response to agents injected intradermally in the rabbit. Capsaicin (100 nmol/site) and human alpha CGRP (3 pmol/site) stimulated similar increases in blood flow and, in both cases, the effect was totally abolished by the CGRP antagonist, CGRP8-37 (1 nmol/site). By contrast, the nitric oxide synthase inhibitor L-nitro arginine methyl ester (L-NAME, 30 nmol/site) had little effect on human alpha CGRP-induced vasodilation, but caused significant inhibition of the response to capsaicin (p < 0.05). These results show that increased blood flow in rabbit skin caused by exogenous CGRP is independent of nitric oxide. In addition, however, they suggest that nitric oxide is required for either the release of endogenous CGRP from capsaicin-sensitive nerves or its subsequent activity.

Olvanil: more potent than capsaicin at stimulating the efferent function of sensory nerves.

The capsaicin analogue olvanil stimulated an increase in cutaneous blood flow when injected intradermally into the anaesthetised rabbit, as measured using a 133Xenon clearance method. Olvanil was found to be a 10-fold more potent vasodilator (on a molar basis) than capsaicin. The effect of both vasodilators was significantly inhibited by the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37). These findings suggest that olvanil stimulates the efferent function of cutaneous sensory nerves in a more potent manner than capsaicin. We therefore suggest that olvanil is a useful pharmacological tool for studying the activity of neuropeptides released from sensory nerves.

Evidence that calcitonin gene-related peptide contributes to inflammation in the skin and joint.

Calcitonin gene-related peptide produces dose-related vasodilatation after intradermal injection in several species. In the present study, CGRP increased blood flow in rabbit skin but had no direct effect on edema formation in rat or rabbit skin or in the rat knee joint. However, CGRP produced significant potentiation of edema formation when co-injected with histamine, a potent mediator of increased vascular permeability. Therefore, release of CGRP from stimulated C-fiber nerves may contribute to the vascular changes that are an integral part of the inflammatory process. The activity of the putative CGRP antagonist CGRP8-37 (300 pmol) against CGRP was also investigated in rabbit and rat skin. Whereas it was found to selectively antagonize the effects of CGRP in rabbit skin, the antagonist produced edema in rat skin at the same dose. Thus, CGRP8-37 may be used in the rabbit to study the effects of endogenously released CGRP, but caution is required when this antagonist is used in the rat.

A calcitonin gene-related peptide (CGRP) antagonist (CGRP8-37) inhibits microvascular responses induced by CGRP and capsaicin in skin.

1. The effect of the calcitonin gene-related peptide (CGRP) antagonist CGRP8-37 on responses to CGRP and other mediators was investigated in rabbit dorsal skin. 2. Blood flow changes at intradermally-injected sites were measured by a multiple site 133xenon clearance technique. CGRP8-37 had little effect on blood flow at doses up to 0.3 nmol/site, when injected alone, although a significant increase in blood flow was observed at the highest dose tested (1 nmol/site). 3. CGRP8-37 dose-dependently inhibited the increased blood flow induced by human alpha CGRP and human beta CGRP, but had no effect on equivalent vasodilator responses induced by vasoactive intestinal peptide (VIP) and prostaglandin E1 (PGE1). CGRP8-37 showed a preferential ability to inhibit alpha CGRP (IC50 0.04 nmol), when compared with beta CGRP (IC50 greater than or equal to 0.3 nmol). 4. Capsaicin, which selectively activates sensory nerves, caused a dose-dependent increase in blood flow when injected intradermally into rabbit skin. The effects of capsaicin (0.01-0.1 mumol/site) were inhibited by CGRP8-37 (0.3 nmol/site), with a partial but significant attenuation of blood flow induced by the highest dose of capsaicin. 5. Oedema formation, induced by intradermal histamine injection (3 nmol/site), was measured in rabbit skin by the local accumulation of intravenously-injected 125I-labelled albumin. Vasodilator doses of CGRP, PGE1 and capsaicin potentiated, in a synergistic manner, oedema formation induced by histamine. GRP8-37 totally inhibited the potentiating effect of CGRP, partially inhibited the synergistic effect of capsaicin, but did not affect PGE1-induced responses.6. The results suggest that capsaicin acts to release a rabbit form of CGRP in skin and that CGRP8 37 is a useful antagonist for investigating the potential of CGRP as a neurogenic mediator of inflammation.

Developmentally regulated cDNA expressed exclusively in neural tissue.

A cDNA clone, labeled Cl-13, isolated from an adult rat brain cDNA library, has been characterized and found by Northern blot and S1 nuclease mapping experiments to be solely expressed in neuronal tissue, principally, but not exclusively, in the brain. The associated mRNA is first detected in embryonic life, reaches maximum levels of expression at birth, and remains expressed in the adult. Northern blot analysis shows the transcript is not localized to one particular area of the brain, but is present in numerous regions. Low message levels of this transcript are also found in the peripheral nervous system, demonstrating that the expression of the associated gene is not restricted to the central nervous system. In addition, results indicate expression is limited to neuronal cells, and is not detected in glia. The identification of the cDNA clone Cl-13, which possesses limited nucleotide homology to tropomyosin, is exciting, particularly considering the neural-specific expression that it manifests and the unique cytoskeletal and motile properties exhibited by neurons.

Evidence that endogenous nitric oxide modulates oedema formation induced by substance P.

The possibility that nitric oxide (NO) could have a role in the modulation of inflammatory oedema formation was investigated in rat skin using selective inhibitors of NO synthesis. Intradermally injected substance P (0.03-1 nmol) induced oedema which was inhibited by concurrent administration of the inhibitor of NO synthesis L-NG-nitro arginine methyl ester (L-NAME), but not by the enantiomer D-NAME. L-Arginine reversed the inhibitory effect of L-NAME. A second inhibitor of NO formation, L-NG-monomethyl arginine (L-NMMA), had a similar inhibitory effect on substance P-induced oedema. The results suggest that endogenous NO has a modulatory role in oedema formation induced by mediators of increased microvascular permeability.