A convenient fluorescent assay for vertebrate collagenases

A versatile, convenient assay for vertebrate collagenases has been developed using the fluorescent peptide substrate dansyl-Pro-Gln-Gly-Ile-Ala-Gly-D-Arg. This sequence resembles that of collagen at the site of cleavage but includes modifications designed to eliminate nonspecific hydrolysis by contaminating peptidases. Both human skin fibroblast and bovine corneal cell collagenases cleave the substrate specifically at the Gly-Ile bond. Plasmin, thrombin, trypsin, alpha-chymotrypsin, carboxypeptidase B, and bacterial collagenase do not cleave the substrate. Elastase and angiotensin converting enzyme display 20- and 400-fold less activity than the vertebrate collagenases, respectively, and cleave the peptide at different positions. The assay is performed by incubating a 5- to 25-microliters aliquot of trypsin-activated sample with an equal volume of 2 mM substrate overnight at 33 degrees C and pH 7.5. Thin-layer chromatography then separates the fluorescent product from the substrate in less than 20 min and allows the detection of subnanogram levels of collagenase. The assay is applicable to the screening of large numbers of samples under different conditions of pH and ionic strength and is readily adaptable for use in a variety of collagenase-dependent systems, such as assays for collagenase activating and/or inducing factors.     

Peripheral glia direct axon guidance across the CNS/PNS transition zone.

CNS glia have integral roles in directing axon migration of both vertebrates and insects. In contrast, very little is known about the roles of PNS glia in axonal pathfinding. In vertebrates and Drosophila, anatomical evidence shows that peripheral glia prefigure the transition zones through which axons migrate into and out of the CNS. Therefore, peripheral glia could guide axons at the transition zone. We used the Drosophila model system to test this hypothesis by ablating peripheral glia early in embryonic neurodevelopment via targeted overexpression of cell death genes grim and ced-3. The effects of peripheral glial loss on sensory and motor neuron development were analyzed. Motor axons initially exit the CNS in abnormal patterns in the absence of peripheral glia. However, they must use other cues within the periphery to find their correct target muscles since early pathfinding errors are largely overcome. When peripheral glia are lost, sensory axons show disrupted migration as they travel centrally. This is not a result of motor neuron defects, as determined by motor/sensory double-labeling experiments. We conclude that peripheral glia prefigure the CNS/PNS transition zone and guide axons as they traverse this region.     

Advancing mate choice studies in salmonids

Reviews in Fish Biology and Fisheries - Mate choice in most organisms is not random, but determined by a suite of interacting traits and environmental factors. While the selective pressures...     

Amide proton exchange rates of oxidized and reduced Saccharomyces cerevisiae iso-1-cytochrome c.

Proton NMR spectroscopy was used to determine the rate constant, kobs, for exchange of labile protons in both oxidized (Fe(III)) and reduced (Fe(II)) iso-1-cytochrome c. We find that slowly exchanging backbone amide protons tend to lack solvent-accessible surface area, possess backbone hydrogen bonds, and are present in regions of regular secondary structure as well as in omega-loops. Furthermore, there is no correlation between kobs and the distance from a backbone amide nitrogen to the nearest solvent-accessible atom. These observations are consistent with the local unfolding model. Comparisons of the free energy change for denaturation, delta Gd, at 298 K to the free energy change for local unfolding, delta Gop, at 298 K for the oxidized protein suggest that certain conformations possessing higher free energy than the denatured state are detected at equilibrium. Reduction of the protein results in a general increase in delta Gop. Comparisons of delta Gd to delta Gop for the reduced protein show that the most open states of the reduced protein possess more structure than its chemically denatured form. This persistent structure in high-energy conformations of the reduced form appears to involve the axially coordinated heme.     

Interaction Between Puccinia xanthii and Facultative Parasitic Fungi on Xanthium occidentale

The present study was undertaken to evaluate the possible interactions between Puccinia xanthii and facultative parasitic fungi isolated from plants of the Noogoora burr complex. Six different Colletotrichum spp., a Phomopsis sp. and Alternaria zinniae, the latter a potential bioherbicide for Xanthium weeds, were tested. Alternaria zinniae caused necrotic lesions of leaf and stem on healthy and rust-infected Noogoora burr plants and did not specifically infect rust lesions. Most Colletotrichum spp. infected Noogoora burr via leaf and stem rust lesions. The infected lesions of P. xanthii became necrotic and the pustules did not reach maturity. Colletotrichum orbiculare was the only facultative parasite able to grow beyond the rust lesions into the surrounding tissue, girdle the stem, and consequently kill the plant tissue above. Colletotrichurn orbiculare did not infect plants previously infected by A. zinniae. The possible additive or synergistic adverse effects of the rust and the facultative parasitic fungi on the Noogoora burr complex offer new avenues to explore for biological control.