18O-Labeling of guanosine monophosphate upon hydrolysis of cyclic guanosine 3':5'-monophosphate by phosphodiesterase

The hydrolysis of cGMP by phosphodiesterase was conducted in [18O]water to determine the site of bond cleavage and the stoichiometry of 18O incorporation into 5'-GMP. Three different forms of phosphodiesterase including a calmodulin-calcium-dependent enzyme in its basal and activated states were examined. The hydrolysis of cGMP catalyzed by each of the forms of phosphodiesterase proceeded with incorporation of 1 18O atom recoverable in the phosphate moiety of each molecule of 5'-GMP generated. No molecular species of phosphate deriving from the 5'-GMP generated containing two or three 18O were detectable. These results indicate that the phosphodiesterase-catalyzed hydrolysis of cGMP proceeds by nucleophilic substitution at phosphorus resulting in P-O bond cleavage. The stoichiometry of 18O incorporation indicates that the reaction proceeds without phosphate-water oxygen exchange when the hydrolytic reaction is catalyzed by diverse forms of phosphodiesterase in the basal or activated state. These considerations of the phosphodiesterase reaction help to establish the validity of monitoring the rate of enzyme-catalyzed hydrolysis of cGMP as a function of the rate of 18O-labeling of the phosphate of 5'-GMP when the reaction proceeds in a medium of predetermined 18O enrichment.     

On the structure and chromosome location of the 72- and 92-kDa human type IV collagenase genes.

The 72- and 92-kDa type IV collagenases are members of a group of secreted zinc metalloproteases. Two members of this family, collagenase and stromelysin, have previously been localized to the long arm of chromosome 11. Here we assign both of the two type IV collagenase genes to human chromosome 16. By sequencing, the 72-kDa gene is shown to consist of 13 exons, 3 more than have been reported for the other members of this gene family. The extra exons encode the amino acids of the fibronectin-like domain which has so far been found in only the 72- and 92-kDa type IV collagenase. The evolutionary relationship among the members of this gene family is discussed.     

Carbonic Anhydrases in Chick Extra-embryonic Structures: A Role for CA in Bicarbonate Reabsorption Through the Chorioallantoic Membrane

The villus cavity cells, a specific cell type of the chick chorioallantoic membrane, express both cytosolic carbonic anhydrase in their cytoplasm and [Formula: See Text] anion exchangers at their basolateral membranes. By immunohistochemical analysis, we show here that villus cavity cells specifically react with antibodies directed against the membrane-associated form of carbonic anhydrase, CAIV. Staining is restricted to the apical cell membranes, characteristically invaginated toward the shell membrane, as well as to endothelia of blood vessels present in the mesodermal layer. The occurrence of a membrane-associated CA form at the apical pole of villus cavity cells, when definitively confirmed, would be fairly consistent with the role proposed for these cells in bicarbonate reabsorption from the eggshell so to prevent metabolic acidosis in the embryo during development.     

Occupancy in dynamic systems: accounting for multiple scales and false positives using environmental DNA to inform monitoring

Occupancy is an important metric to understand current and future trends in populations that have declined globally. In addition, occupancy can be an efficient tool for conducting landscape-scale and long-term monitoring. A challenge for occupancy monitoring programs is to determine the appropriate spatial scale of analysis and to obtain precise occupancy estimates for elusive species. We used a multi-scale occupancy model to assess occupancy of Columbia spotted frogs in the Great Basin, USA, based on environmental DNA (eDNA) detections. We collected three replicate eDNA samples at 220 sites across the Great Basin. We estimated and modeled ecological factors that described watershed and site occupancy at multiple spatial scales simultaneously while accounting for imperfect detection. Additionally, we conducted visual and dipnet surveys at all sites and used our paired detections to estimate the probability of a false positive detection for our eDNA sampling. We applied the estimated false positive rate to our multi-scale occupancy dataset and assessed changes in model selection. We had higher naïve occupancy estimates for eDNA (0.37) than for traditional survey methods (0.20). We estimated our false positive detection rate per qPCR replicate at 0.023 (95% CI: 0.016–0.033). When the false positive rate was applied to the multi-scale dataset, we did not observe substantial changes in model selection or parameter estimates. Conservation and resource managers have an increasing need to understand species occupancy in highly variable landscapes where the spatial distribution of habitat changes significantly over time due to climate change and human impact. A multi-scale occupancy approach can be used to obtain regional occupancy estimates that can account for spatially dynamic differences in availability over time, especially when assessing potential declines. Additionally, this study demonstrates how eDNA can be used as an effective tool for improved occupancy estimates across broad geographic scales for long-term monitoring.     

Synthesis of a "free" form of hyaluronic acid by articular chondrocytes in monolayer culture

Primary and first passage rabbit chondrocyte cultures synthesized a "free" form of hyaluronic acid (HA-f) previously characterized in rabbit cartilage. HA-f was isolated from the [3H] glcN/35SO4-labelled cell-associated-fraction (CAF) and from the culture medium by successive equilibrium centrifugations in Cs2SO4/CsCl/Cs2SO4 under low salt conditions. The culture medium HA-f appeared in the void volume of Sepharose CL-2B eluted with low salt, (0.5M sodium acetate), and was susceptible to digestion with Streptomyces hyaluronidase. HA-f aggregated purified rabbit cartilage proteoglycan monomer. These results indicated that HA-f probably subserves hyaluronic acid already complexed with proteoglycan monomer. Newly synthesized HA-f may be required for the continual formation of proteoglycan aggregates.