A genome-wide analysis of biomineralization-related proteins in the sea urchin Strongylocentrotus purpuratus.

Biomineralization, the biologically controlled formation of mineral deposits, is of widespread importance in biology, medicine, and engineering. Mineralized structures are found in most metazoan phyla and often have supportive, protective, or feeding functions. Among deuterostomes, only echinoderms and vertebrates produce extensive biomineralized structures. Although skeletons appeared independently in these two groups, ancestors of the vertebrates and echinoderms may have utilized similar components of a shared genetic "toolkit" to carry out biomineralization. The present study had two goals. First, we sought to expand our understanding of the proteins involved in biomineralization in the sea urchin, a powerful model system for analyzing the basic cellular and molecular mechanisms that underlie this process. Second, we sought to shed light on the possible evolutionary relationships between biomineralization in echinoderms and vertebrates. We used several computational methods to survey the genome of the purple sea urchin Strongylocentrotus purpuratus for gene products involved in biomineralization. Our analysis has greatly expanded the collection of biomineralization-related proteins. We have found that these proteins are often members of small families encoded by genes that are clustered in the genome. Most of the proteins are sea urchin-specific; that is, they have no apparent homologues in other invertebrate deuterostomes or vertebrates. Similarly, many of the vertebrate proteins that mediate mineral deposition do not have counterparts in the S. purpuratus genome. Our findings therefore reveal substantial differences in the primary sequences of proteins that mediate biomineral formation in echinoderms and vertebrates, possibly reflecting loose constraints on the primary structures of the proteins involved. On the other hand, certain cellular and molecular processes associated with earlier events in skeletogenesis appear similar in echinoderms and vertebrates, leaving open the possibility of deeper evolutionary relationships.

Teaching responsibility through sport education: prospects of a coalition.

The purpose of this study was to provide a database analysis of a combined sport model and from this to develop a theory from practice. In particular, the original concern was to examine the extent to which the teaching of personal and social responsibility (TPSR) could form a coalition with the Sport Education model. Forty-five 6th-grade boys, who had a previous history of struggling with the fair play requirements of Sport Education, participated in a 26-lesson season of Xball, an invasion game designed by those students. As a general summary, it was found that some of the features of TPSR strengthened the foundation of Sport Education, but due to the need to introduce new tasks and problems for students, a hybrid model (Empowering Sport) was developed. This hybrid, with a predominately ecological integration perspective, presents a curriculum model that allows for achievement within a powerful triangle of goals--sport skill competence, social responsibility, and personal empowerment.

Actinomyces hordeovulneris, a canine pathogen that produces L-phase variants spontaneously with coincident calcium deposition.

The spontaneous production of cell-wall-deficient filaments and protoplasts by a strain of Actinomyces hordeovulneris (UCD 81-332-9) in 10% sucrose L-form media is reported. Multiple mineral deposits were present within the variants at 48 hours. Electron microscopy revealed that these deposits were amorphous, dense, and at the inner face of the cytoplasmic membrane in wall-less protoplasts and also in filaments which had a thin wall of 10 nm. These cell-wall-deficient variants produced L-form colonies when cultured for an additional 48 hours on 10% sucrose-BYE L-form plates. The colonies were composed of only a few filaments and many vesicles which were negative with Dienes' stain. Silver substitution stains of UCD 81-332-9 cells that had been grown in L-form broth for 5 days revealed heavy calcification of all cells including protoplasts. Gram stains of L-form grown cells revealed the presence of long-beaded, infrequently branched gram-positive filaments similar to those observed in clinical specimens. The formation of cell wall-deficient variants with coincident mineralization is believed to be related to the phenomenon of sulfur granule formation in vivo.

Nocardia asteroides recovery from a dog with steroid- and antibiotic-unresponsive idiopathic polyarthritis.

This report describes a fatal case of idiopathic polyarthritis in a dog that was partially responsive to vigorous immunosuppressive treatment. Synovial fluids were cultured for L-forms at the following stages of disease: (i) acute arthritic relapse, (ii) incomplete remission, and (iii) death. Nocardia asteroides UCD 1-581 was recovered from the L-form broth culture of the specimen taken during acute relapse, 5 weeks after inoculation, but not at any other stage of disease. Numerous conventional microbiological cultures were unproductive during all phases. Changes occurring in L-form plates included the formation of large irregular mineral deposits and many transferable bodies resembling pseudocolonies. Microscopic examination revealed the presence of many intracellular golden-brown granules and acid-fast bodies in macrophages of the lung and bronchial lymph node tissues. The granules are believed to be the variants embedded in calcium deposits similar to those which developed in the L-form cultures in vitro. Fluorescence of these acid-fast bodies with antibody specific for superoxide dismutase of N. asteroides GUH-2 and labeled anti-immunoglobulin G established their relationship to the isolate. The unrelenting course of disease and the persistence of N. asteroides as an L-form in this animal despite vigorous immunosuppression suggest that this organism plays a direct role in the etiology of this disease.

Recovery of microorganisms from synovial and pleural fluids of animals using hyperosmolar media.

L-phase (CWD) broth and plate media were used in parallel with conventional microbiological media during a 3-year period for culturing synovial and pleural fluids of animals. Two kinds of recoveries were obtained where parallel conventional methods were negative: (1) parent or normal bacteria, in very low numbers; and (2) Type B CWD variants in equally low numbers. Organisms in group 1 were: Streptococcus zooepidemicus from horses (2x); beta-hemolytic streptococci, Lancefield Gp. G (2x); Staphylococcus aureus; Actinobacillus, and Actinomyces viscosus. Group 2 consisted of Bacteroides sp., Propionibacterium acnes, and three "Nocardia-like" sp. Catalase + Actinomyces was not recovered equally well on CWD plates as on conventional media with fluids obtained during ampicillin treatment. This occurred in spite of the fact that the CWD media was shown to support growth and reversion of laboratory induced L-phase variants of Nocardia caviae and N. asteroides, and had facilitated recovery of a Bacteroides L-phase variant from a pleural fluid. The nature of this fault in the media is under investigation in this laboratory.

Possible involvement of phospholipase A2 in A23187-induced histamine release from purified rat mast cells.

A23187-induced histamine release from purified rat mast cells is accompanied by degradation of mast cell phosphatidylcholine. When inhibitors of histamine release such as ethacrynic acid, eicosatetraynoic acid and phenylmethylsulfonylfluoride are added to the mast cell system, the phosphatidylcholine hydrolysis is inhibited along with the release of histamine. A simultaneous striking increase in prostaglandin D2 synthesis by the A23187-treated mast cells suggests that the degradation of phosphatidylcholine is associated with the activation of phospholipase A2. Arachidonic acid, one of the major products formed as a result of this activation, inhibits A23187 and antigen-induced histamine release when added 5 min prior to the histamine release inducers. Lysophosphatidylcholine, the other major product, demonstrates a biphasic effect, stimulating histamine release at low concentrations and inhibiting release at slightly higher concentrations. These findings suggest a histamine release regulating function for arachidonic acid and lysophosphatidylcholine.

Stimulation of Bacillus subtilis membrane adenosine triphosphatase by cationic bactericidal agents.

The environmental Mg2+ used in preparation of Bacillus subtilis membranes was found to influence the responses of the associated ATPase to cetyltrimethylammonium bromide (CTAB). Membranes prepared using fluids containing higher Mg2+ levels exhibited lower control activity than was seen with low Mg2+ membranes. Increased environmental Mg2+ resulted in higher stimulations with lower doses of the agent. ATPase of all three membrane types was stimulated in two concentration ranges, but in the doses tested, CTAB inhibited the ATPase of only those membranes obtained using fluids containing high Mg2+ for every stage of the isolation. Sonication of membranes for 25 s at half maximum output yielded three fractions, consisting of a soluble form which was sensitive to CTAB stimulation at 25 microg/ml of assay mixture; small, 95-110 nm, vesicles, which were resistant to CTAB at 25, 75, and 150 microg/ml, and large vesicles, similar to untreated membranes both in morphology and responses to detergent. Combinations of detergent and protein (beta-lysin or arginine-rich histone) produced activity appearing to be additive when the protein level was present in a high concentration and the detergent was present at levels corresponding to the minimum influence. Mixtures of a maximally stimulating dose (75 or 100 microg/ ml) of detergent and a small amount of protein produced activities that were at least 92% or more of the expected sums of individual stimulations. Interference occurred with the following mixtures: high amounts of detergent and protein; high protein and 10 or 15 microg/ml CTAB; and beta-lysin and arginine-rich histone, both at high levels. These data are consistent with a hypothesis that the two peaks in CTAB stimulation reflect two adjacent ATPase sites, one of which is also susceptible to stimulation by cationic protein.