Calmodulin-activated cyclic nucleotide phosphodiesterase from brain. Relationship of subunit structure to activity assessed by radiation inactivation

The apparent target sizes of the basal and calmodulin-dependent activities of calmodulin-activated phosphodiesterase from bovine brain were estimated using target theory analysis of data from radiation inactivation experiments. Whether crude or highly purified samples were irradiated, the following results were obtained. Low doses of radiation caused a 10 to 15% increase in basal activity, which, with further irradiation, decayed with an apparent target size of approximately 60,000 daltons. Calmodulin-dependent activity decayed with an apparent target size of approximately 105,000 daltons. The percentage stimulation of enzyme activity by calmodulin decreased markedly as a function of radiation dosage. These observations are consistent with results predicted by computer-assisted modeling based on the assumptions that: 1) the calmodulin-activated phosphodiesterase exists as a mixture of monomers which are fully active in the absence of calmodulin and dimers which are inactive in the absence of calmodulin; 2) in the presence of calmodulin, a dimer exhibits activity equal to that of two monomers; 3) on radiations destruction of a dimer, an active monomer is generated. This monomer-dimer hypothesis provides a plausible explanation for and definition of basal and calmodulin-dependent phosphodiesterase activity.     

Elbow joint biomechanics for preclinical evaluation of total elbow prostheses

Total elbow arthroplasty is a clinically successful procedure, yet long-term implant survival rates have historically lagged behind those reported for total hips and knees. Clinical complications associated with implant wear, osteolysis, stem loosening and device fracture have been implicated as reasons for limited long-term survivorship. Unfortunately, there is little published information on the biomechanics and method(s) for preclinical evaluation of total elbow prostheses that could provide insight into the mechanisms of failure. Additionally, there are no consensus testing standards or summaries of loading profiles of the humero-ulnar joint associated with a range of activities of daily living. Such data would facilitate the standardized preclinical assessment of total elbow devices such is commonplace for other large joints. The objective of the work here is therefore to provide a comprehensive review of elbow joint biomechanics as it relates to preclinical evaluation of total elbow implants. This summary includes a review of elbow joint forces, kinematics, the types and frequency of humero-ulnar joint motions associated with activities of daily living and clinical outcomes, as well as proposing a methodology for deriving humero-ulnar joint reaction force magnitudes and vector orientations as a function of a known mass/force at the hand. From these data, a scalable, bi-axial loading profile is proposed as a foundation for the development of clinically relevant, laboratory simulations for assessment of total elbow prostheses performance.     

The salt-extractable fraction of dynein from sea urchin sperm flagella: An analysis by gel electrophoresis and by adenosine triphosphatase activity

Previous work has shown that the dynein from axonemes of sea urchin sperm consists of two distinct fractions which differ substantially in their extractability by salt. Upon gel electrophoresis of whole demembranated axonemes solubilized with sodium dodecyl sulfate, the dynein fraction shows two closely spaced bands with apparent molecular weights of 520,000 and 460,000; the proteins in these bands are termed the A and B components of the dynein. Similar electrophoresis of the soluble fraction obtained by extracting the axonemes with 0.5 M NaCl shows a single prominent band containing approximately half of the A component of the dynein (A₁ component). The residue of extracted axonemes contain the other half of the A component of the dynein (A₂ component) and all the B component. Densitometry of the bands indicates that the A₁, A₂ and B components of the dynein are present in approximately equal molar quantity. Electron microscopic studies show that the A₁ component of the dynein constitutes the outer arms on the doublet tubules. Assay of ATPase activity in 0.05 M KCl and l mM ATP indicates about 65% of the total ATPase activity becomes soluble when the A₁ component of the dynein is extracted with salt.     

Polymer and sprinkler droplet energy effects on sugar beet emergence, soil penetration resistance, and aggregate stability

The rapid development of agricultural biotechnology and release of new transgenic plants for agriculture has provided many economic benefits, but has also raised concern over the potential impact of transgenic plants on the environment. Considerable research has now been conducted on the effects of transgenic plants on soil microorganisms. These effects include unintentional changes in the chemical compositions of root exudates, and the direct effects of transgenic proteins on nontarget species of soil microorganisms. Most studies to date suggest that transgenic plants that have been released cause minor changes in microbial community structures that are often transient in duration. However, due to our limited knowledge of the linkage between microbial community structure and function, more work needs to be done on a case-by-case basis to further evaluate the effects of transgenic plants on soil microorganisms and soil ecosystem functions. This review summarizes the results of a variety of experiments that have been conducted to specifically test the effects of transgenic plants on soil microorganisms, and particularly examines the types of methods that are being used to study microbial interactions with transgenic plants.     

Community development following gamma radiation at a pine-oak forest, Brookhaven National Laboratory, Long Island, New York1

We investigated a unique source of forest disturbance: gamma radiation. While the temporal patterns of ecological succession are well understood for the forests of eastern North America, this is not the case for massively irradiated forests. Our objective was to compare vascular plant community change after irradiation at the five vegetation zones described in 1962 by Woodwell at Brookhaven National Laboratory, Long Island, New York. No follow-up studies have been done since the gamma radiation experiments were terminated in 1978. Ecological successional theory (e.g., Bormann and Likens, 1994, Likens and Bormann, 1995) does not explain long-term forest recovery after radiation damage. Our null hypothesis was that 47 yr after initial gamma ray exposure, the sites would have recovered such that floristic composition would be the same as the pine-oak forest control. This hypothesis was rejected statistically. In 2007/2008, the five concentric zones of vegetation centered about the gamma source retained their floristic heterogeneity as measured by Jaccard coefficients.     

Rapid purification of calmodulin and S-100 protein by affinity chromatography with melittin immobilized to sepharose

Melittin-Sepharose was prepared for Ca2+-dependent affinity chromatography of calmodulin and S-100 protein. This matrix exhibits extremely high capacity (approximately 10 mg calmodulin/ml gel), low nonspecific binding, and excellent recovery (greater than 90%) under optimal conditions. Recovery of calmodulin from melittin-Sepharose was related to the degree of saturation of column capacity with lower yields when only partial saturation was achieved. Large-scale, simultaneous purification of calmodulin and S-100 protein from brain was carried out using selective adsorption to organomercurial agarose followed by melittin-Sepharose chromatography; yields were 250-300 mg of calmodulin and 200-300 mg of S-100 per kg tissue. Calmodulin also was purified in a single step from bovine testis supernatant using melittin-Sepharose in yields comparable to those from brain.     

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A. nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.