Purification of smooth-muscle myosin free of calmodulin and myosin light-chain kinase. Susceptibility to oxidation.

Smooth-muscle myosin purified as described by Persechini & Hartshorne [(1983) Biochemistry 22, 470-476] contains trace amounts of calmodulin and myosin light-chain kinase, which can be removed by Ca2+-dependent hydrophobic-interaction chromatography followed by calmodulin-Sepharose affinity chromatography. The resultant column-purified myosin exhibits properties similar to those of the non-purified myosin, e.g. actin activation of the Mg2+-ATPase requires Ca2+/calmodulin-dependent phosphorylation of the two 20 kDa light chains. However, unlike the non-purified myosin, the column-purified myosin undergoes a time-dependent transition to a form which no longer requires phosphorylation for actin activation of the myosin Mg2+-ATPase. This transition is identified as a time-dependent change in conformation of the column-purified myosin from a 10 S to 6 S form and is caused by slow oxidation of the column-purified myosin, since it could be prevented by storage under N2 and reversed by 5 mM-dithiothreitol.     

Interacting Effects of Discharge and Channel Morphology on Transport of Semibuoyant Fish Eggs in Large,Altered River Systems

Habitat fragmentation and flow regulation are significant factors related to the decline and extinction of freshwater biota. Pelagic-broadcast spawning cyprinids require moving water and some length of unfragmented stream to complete their life cycle. However, it is unknown how discharge and habitat features interact at multiple spatial scales to alter the transport of semi-buoyant fish eggs. Our objective was to assess the relationship between downstream drift of semi-buoyant egg surrogates (gellan beads) and discharge and habitat complexity. We quantified transport time of a known quantity of beads using 2–3 sampling devices at each of seven locations on the North Canadian and Canadian rivers. Transport time was assessed based on median capture time (time at which 50% of beads were captured) and sampling period (time period when 2.5% and 97.5% of beads were captured). Habitat complexity was assessed by calculating width∶depth ratios at each site, and several habitat metrics determined using analyses of aerial photographs. Median time of egg capture was negatively correlated to site discharge. The temporal extent of the sampling period at each site was negatively correlated to both site discharge and habitat-patch dispersion. Our results highlight the role of discharge in driving transport times, but also indicate that higher dispersion of habitat patches relates to increased retention of beads within the river. These results could be used to target restoration activities or prioritize water use to create and maintain habitat complexity within large, fragmented river systems.     

Sampling for Explosives-Residues at Fort Greely,Alaska

Fort Greely, Alaska has an extensive complex of weapon training and testing areas located on lands withdrawn from the public domain under the Military Lands Withdrawal Act (PL106-65). The Army has pledged to implement a program to identify possible munitions contamination. Because of the large size (344,165,000 m²) of the high hazard impact areas, characterization of these constituents will be difficult. We used an authoritative sampling design to find locations most likely to contain explosives-residues on three impact areas. We focused our sampling on surface soils and collected multi-increment and discrete samples at locations of known firing events and from areas on the range that had craters, pieces of munitions, targets, or a designation as a firing point. In the two impact areas used primarily by the Army, RDX was the most frequently detected explosive. In the impact area that was also used by the Air Force, TNT was the most frequently detected explosive. Where detected, the explosives concentrations generally were low (<0.05 mg/kg) except in soils near low-order detonations, where the explosive-filler was in contact with the soil surface. These low-order detonations potentially can serve as localized sources for groundwater contamination if positioned in recharge areas.     

Factor 390 chromophores: phosphodiester between AMP or GMP and methanogen factor 420

Two chromophores with absorbance maxima at 390 nm (factors 390) have been isolated from oxidized cells of Methanobacterium thermoautotrophicum delta H. The isolation procedure included anion-exchange chromatography of the soluble cofactor pool followed by reverse-phase chromatography. The factor 390 species are novel derivatives of methanogen coenzyme factor 420 in which the 5-deazaflavin 8-hydroxy group is in a phosphodiester linkage to adenosine 5'-phosphate or guanosine 5'-phosphate. The structural assignments were based, in part, on the UV-visible and 1H NMR spectra. In addition, the results from amino acid analysis, phosphate determination, 31P NMR spectroscopy, and fast atom bombardment mass spectrometry were consistent with the proposed structures. Confirmation of the factor 390 structures was made following phosphodiesterase release of the nucleotide monophosphates from factor 420. The nucleotide monophosphates were identified as AMP and GMP by UV-visible spectra and based on elution position by using reverse-phase and anion-exchange high-performance liquid chromatography. The presence of AMP was further demonstrated by using adenylate-5'-phosphate kinase which induced a spectral shift during conversion of the sample to IMP. In addition, the presence of GMP was established by a specific enzymatic assay.     

OGR1/GPR68 Modulates the Severity of Experimental Autoimmune Encephalomyelitis and Regulates Nitric Oxide Production by Macrophages

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a proton-sensing molecule that can detect decreases in extracellular pH that occur during inflammation. Although OGR1 has been shown to have pro-inflammatory functions in various diseases, its role in autoimmunity has not been examined. We therefore sought to determine whether OGR1 has a role in the development of T cell autoimmunity by contrasting the development of experimental autoimmune encephalomyelitis between wild type and OGR1-knockout mice. OGR1-knockout mice showed a drastically attenuated clinical course of disease that was associated with a profound reduction in the expansion of myelin oligodendrocyte glycoprotein 35-55-reactive T helper 1 (Th1) and Th17 cells in the periphery and a reduced accumulation of Th1 and Th17 effectors in the central nervous system. We determined that these impaired T cell responses in OGR1-knockout mice associated with a reduced frequency and number of dendritic cells in draining lymph nodes during EAE and a higher production of nitric oxide by macrophages. Our studies suggest that OGR1 plays a key role in regulating T cell responses during autoimmunity.     

CGS8216 noncompetitively antagonizes the discriminative effects of diazepam in rats

The benzodiazepine antagonist properties of CGS8216 were evaluated in rats trained to discriminate between saline and 1.0 mg/kg of diazepam in a two-choice, stimulus-shock termination procedure. CGS8216 (0.3 to 100 mg/kg) administered alone, either s.c., p.o. or i.p., occasioned only saline-appropriate responding. When administered concomitantly with a constant 1.0 mg/kg dose of diazepam, CGS8216 produced dose-related decreases in drug-appropriate responding. CGS8216 was most potent by the i.p. route, and approximately tenfold less potent by the oral route. CGS8216 was dermatotoxic after s.c. administration. CGS8216 i.p. had a long duration of action. A dose of 30 mg/kg completely antagonized the discriminative effects of the 1.0 mg/kg training dose of diazepam when the antagonist was administered 8 hr before the start of the test session. In order to determine the type of antagonism by CGS8216, the dose-effect curve for diazepam was redetermined in the presence of varying doses of CGS8216 (0.3 to 3.0 mg/kg, i.p.). CGS8216 produced a dose-related rightward shift in the diazepam dose-effect curve, but also decreased the slope and appeared to decrease the maximal effect. These results are consistent with the interpretation that CGS8216 antagonizes diazepam in a noncompetitive manner. It may do so because either it interacts with a subpopulation of benzodiazepine receptors, it functions as a pseudo-irreversible antagonist due to its high affinity, or because it is an antagonist with agonist properties.     

Novel methoxy-carotenoids from the burgundy-colored plumage of the Pompadour Cotinga Xipholena punicea

Recent advances in the fields of chromatography, mass spectrometry, and chemical analysis have greatly improved the efficiency with which carotenoids can be extracted and analyzed from avian plumage. Prior to these technological developments, Brush (1968) [1] concluded that the burgundy-colored plumage of the male pompadour Cotinga Xipholena punicea is produced by a combination of blue structural color and red carotenoids, including astaxanthin, canthaxanthin, isozeaxanthin, and a fourth unidentified, polar carotenoid. However, X. punicea does not in fact exhibit any structural coloration. This work aims to elucidate the carotenoid pigments of the burgundy color of X. punicea plumage using advanced analytical methodology. Feathers were collected from two burgundy male specimens and from a third aberrant orange-colored specimen. Pigments were extracted using a previously published technique (McGraw et al. (2005) [2]), separated by high-performance liquid chromatography (HPLC), and analyzed by UV/Vis absorption spectroscopy, chemical analysis, mass spectrometry, nuclear magnetic resonance (NMR), and comparison with direct synthetic products. Our investigation revealed the presence of eight ketocarotenoids, including astaxanthin and canthaxanthin as reported previously by Brush (1968) [1]. Six of the ketocarotenoids contained methoxyl groups, which is rare for naturally-occurring carotenoids and a novel finding in birds. Interestingly, the carotenoid composition was the same in both the burgundy and orange feathers, indicating that feather coloration in X. punicea is determined not only by the presence of carotenoids, but also by interactions between the bound carotenoid pigments and their protein environment in the barb rami and barbules. This paper presents the first evidence of metabolically-derived methoxy-carotenoids in birds.