Identification and purification of calcium ion dependent modulators of actin polymerization from bovine thyroid

We describe the purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography. The 40K actin modulator has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 40 000 and an isoelectric point of 8.1. Its amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. An 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 80 000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin, and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique [Snabes, M. C., Boyd, A.E., & Bryan, J. (1981) J. Cell Biol. 90, 809-812] revealed that both the 91K and 95K proteins bind 125I-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.     

Human T-cell hybridomas producing lymphokines. II. Enhancement of lymphotoxin secretion from human T-cell hybridomas by phorbol myristate acetate

Human lymphotoxin (LT)-producing T-cell hybridomas were constructed by fusing concanavalin A-activated human peripheral blood lymphocytes with emetine-actinomycin D-pretreated human acute lymphatic leukemia cells. LT secretion from these hybridomas was considerably enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA) and concanavalin A or PMA alone. A study using cloned hybrid lines revealed that PMA/Con A acted directly on the LT-producing clones. Furthermore, PMA/Con A stimulated A-B9-24, one of the cloned hybridomas, and secreted fourfold larger amounts of LT under serum-free conditions than under serum-containing conditions. However, MIF/MAF and LT-producing cloned hybrid line E10-20 secreted rather decreased amounts of MIF/MAF when stimulated with PMA, while the LT secretion from the same hybridoma was enhanced with PMA.     

Histochemical studies on the regeneration of aminergic nerves in rat cerebral artery after superior cervical ganglionectomy

The course of regeneration of aminergic nerves in rat cerebral arteries was studied by means of histochemical methods, after uni- or bilateral cervical sympathectomy. Degeneration of aminergic nerves started on day 1 and was complete between days 3 and 7 after surgery. Between weeks 4 and 6, regenerating nerves started to appear from the proximal internal carotid artery. Regenerated aminergic nerve fibres were generally unbeaded and intensity of fluorescence was weak. The circular nerves appeared earlier than the longitudinal ones. The number of regenerating nerves reached the maximum, between months 9 and 12, at about half the normal level. AChE activity of the cerebral arteries showed no significant changes at any stage.     

The incorporation of amino acids and nucleic acid bases into the seedling,reproductive stage and young ear portion of rice plants

Summary The incorporation of C¹⁴-amino acids (aspartic acid, glutamic acid, threonine and proline) and C¹⁴-nucleic acid bases (adenine, guanine, cytosine and uracil) into the seedling, reproductive stage and young ear portion of rice plant was investigated. It was found that C¹⁴-aspartic acid was incorporated into the rice seedling more rapidly than C¹⁴-threonine or C¹⁴-proline; on the other hand C¹⁴-proline was found to be more rapidly incorporated than C¹⁴-aspartic acid into reproductive stage plant and young ear portion. Similarly C¹⁴-adenine was incorporated into the rice seedling more rapidly than other C¹⁴-labelled bases. On the other hand C¹⁴-uracil was preferentially incorporated to C¹⁴-adenine or C¹⁴-guanine into the reproductive stage plant and young ear portion. It is suggested from the results obtained that proline is polymerized into polypeptide or protein in the rice plant more rapidly at the reproductive stage than at the seedling stage and that a higher proportion of pyrimidine bases might be involved into the metabolic process at the reproductive stage of rice plant.     

Dielectric properties of polyelectrolytes. V. Dielectric dispersion of heavy meromyosin

Dielectric constant and dielectric loss of heavy meromyosin (HMM) were measured with varying pH. HMM showed a broader dispersion pattern than that with a single relaxation time especially on the high-frequencey side. The dielectric increment increased sharply with pH, above p^{H 6}, whereas the mean relaxation time and whole dispersion pattern were unchanged in the same region. The values of the increment and the mean relaxation time were much larger than those of usual globular proteins. The dispersion profile, pH dependence, and values of the increment are well explained by Oosawa's counterion fluctuation theory. Other mechanisms are more or less inadequate to our results. In the low pH region below the isoelectric precipitation region, both the increment and the mean relaxation time decreased; this is probably due to partial denaturation and suppression of the dissociation of carboxyl groups. An experiment on a urea-denatured sample supports this assumption. The biological significance of the pH dependence is discussed.     

Preparation and standardization of antigens of Histoplasma capsulatum and Coccidioides immitis

Methods have been developed for the separation and purification of various antigenically active cellular components ofH. capsulatum (34) andC. immitis (35), and chemical procedures have been employed in the characterization of these antigens. The concluding remarks illustrate the current trends in immunological studies ofH. capsulatum andC. immitis. Hopefully, these approaches will provide knowledge and insight into the basic biological properties ofH. capsulatum andC. immitis and should, in the future, culminate in the physicochemical characterization of their important antigens.