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.     

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Fort Greely, Alaska has an extensive complex of weapon training and testing areas located on lands withdrawn from the public domain under the Military Lands Withdrawal Act (PL106-65).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.     

Effects of urbanization on streams of the Melbourne region, Victoria, Australia. I. Benthic macroinvertebrate communities

1. Macroinvertebrate community composition was assessed in small streams of the Melbourne region to test the effects of (a) urban density (catchment imperviousness 0–51%) and (b) stormwater drainage intensity (comparing the intensively drained metropolitan area with urban areas of the hinterland, which had open drains and some localized stormwater drainage).
2. Hinterland communities separated into two groups of sites correlating strongly with patterns of electrical conductivity (EC), basalt geology and annual rainfall. Community composition varied little in the high-EC, western group (imperviousness 0.2–1.2%), but in the eastern group it was strongly correlated with catchment imperviousness (0–12%), with lower taxon richness in more impervious catchments.
3. Metropolitan communities (imperviousness 1–51%) were all severely degraded, with high abundances of a few tolerant taxa. Community composition was poorly correlated with patterns of geology, rainfall or imperviousness. Differences between metropolitan and hinterland communities were well explained by patterns of biochemical oxygen demand and electrical conductivity, which were postulated to indicate the more efficient transport of pollutants to receiving streams by the metropolitan stormwater drainage system.
4. Degradation of macroinvertebrate community composition was well explained by urban density but intensive urban drainage increased degradation severely at even low urban densities. Quantification of relationships between imperviousness, drainage intensity and stream degradation can better inform the assessment, conservation and restoration of urban streams.     

Improving the clinical efficiency of T2 mapping of ligament integrity

Current MR methods use T₂^? relaxation time as a surrogate measure of ligament strength. Currently, a multi-echo voxel-wise least squares fit is the gold standard to create T₂^? maps; however, the post-processing is time-intensive and serves as a stopgap for clinical use. The study objective was to determine if an alternative method could improve post-processing time without sacrificing fidelity of T₂^? values for eventual translational use in the clinic. Using a 6 echo FLASH sequence, three different methods were used to determine intact posterior cruciate ligament (PCL) median T₂^? Two of these methods utilized a voxel-wise method to establish T₂^? maps: (1) a current “gold standard” method using a voxel-wise 6 echo least-squares fit (6LS) and (2) a voxel-wise 2 echo point T₂^? determination (2MM). The third method used median ligament signal intensity and a single nonlinear least-squares fit (6LS_ROI) instead of a voxel-wise basis. The resulting median T₂^? values of the PCL and computational time were compared. The median T₂^? values were 42% higher using the 2MM compared to the 6LS method (p<0.0001). However, a strong correlation was found for the median T₂^? values between the 2MM and 6LS methods (R²=0.80). The median T₂^? values were not significantly different between the 6LS and 6LS_ROI methods (p=0.519). Using the 2MM (which provides a regional map) and the 6LS_ROI (which efficiently provides the median T₂^? value) methods in tandem would take only minutes of post-processing computational time compared to the 6LS method (~540 min), and hence would facilitate clinical application of T₂^? maps to predict ligament structural properties as a patient outcome measure.     

Diethyl phthalate, a chemotactic factor secreted by Helicobacter pylori.

The structure of a small-molecule, non-peptide chemotactic factor has been determined from activity purified to apparent homogeneity from Helicobacter pylori supernatants. H. pylori was grown in brucella broth media until one liter of solution had 0.9 absorbance units. The culture was centrifuged, and the bacteria re-suspended in physiological saline and incubated at 37 degrees C for 4 h. A monocyte migration bioassay revealed the presence of a single active chemotactic factor in the supernatant from this incubation. The chemotactic factor was concentrated by solid phase chromatography and purified by reverse phase high pressure liquid chromatography. The factor was shown to be indistinguishable from diethyl phthalate (DEP) on the basis of multiple criteria including nuclear magnetic resonance spectroscopy, electron impact mass spectroscopy, UV visible absorption spectrometry, GC and high pressure liquid chromatography retention times, and chemotactic activity toward monocytes. Control experiments with incubated culture media without detectable bacteria did not yield detectable DEP, suggesting it is bacterially derived. It is not known if the bacteria produce diethyl phthalate de novo or if it is a metabolic product of a precursor molecule present in culture media. DEP produced by H. pylori in addition to DEP present in man-made products may contribute to the high levels of DEP metabolites observed in human urine. DEP represents a new class of chemotactic factor.