28,000-dalton polypeptide (p28) of adult T-cell leukemia-associated antigen encoded by 24 S mRNA of human T-cell leukemia virus has an associated protein kinase activity

Human T-cell leukemia virus producer cell line MT-2 was labeled with [32P]phosphoric acid, and its cell extracts were immunoprecipitated with mouse monoclonal antibodies (GIN-7, and KK-1) and rabbit sera (anti-p24, and anti-gp68). Analysis of the immunocomplexes on sodium dodecyl sulfate-polyacrylamide gell electrophoresis revealed that p53, p28, and p19 of adult T-cell leukemia-associated antigens were phosphorylated in vivo. Immunocomplexes of MT-2 cell extract with monoclonal antibody KK-1 were incubated with [gamma-32P]ATP in vitro and it was revealed that the phosphokinase activity was associated with p28. The phosphokinase activity of p28 was specific to the serine residue but was not to the tyrosine residue.     

Phagocytosis of different matrix components by different cell types at bone-forming sites in cultured mouse calvariae

Summary Sites of bone formation on fragments of parietal bone of fetal-mice cultured for 10 days were examined by electron microscopy after addition of either ruthenium red or ferrocyanide to the postfixation fluid. Osteoclasts, osteoblast-like cells, and macrophages were the principal active cells at these formation sites. The mononuclear cells (osteoblast-like cells and macrophages) in the osteoid tissue showed evidence of having incorporated elements of calcified tissue. Osteoblast-like cells had phagocytized collagen fibrils and calcified bone matrix. This occurred more frequently in the calcifying area. Mononuclear macrophages showed not only phagocytosis and digestion of cellular debris and bone spicules in the osteoid, but also active incorporation of calcified bone matrix that had been detached from its surroundings by its pseudopod-like projections from long cytoplasmic processes. Collagen fibrils were seldom observed within the macrophages. These observations suggest that in our culture system osteoblast-like cells and macrophages at bone formation sites have a phagocytic role in bone remodeling.This study was supported in part by a grant from the Ministry of Education. Science and Culture of Japan (No. 59771321)     

Video microscopy of colloidal gold particles and immuno-gold labelled microtubules in improved rectified DIC and epi-illumination

Modification of rectified Nomarski differential interference contrast optics (Nikon) and the epi-illumination system (Nikon IGS-cube) improved the detection of colloidal gold particles with analog video enhanced microscopy. Immuno-gold labelled microtubules of Haemanthus endosperm are visualized at a level of detection unmatched in conventional light microscopy. Single gold, or gold silver enhanced particles in suspension viewed with the modified epi-illumination after pressure injection into cells, are well distinguished from other granular cell components. Immuno-gold has also been detected on the surface of chromosomes and the nuclear envelopes in cells during the rapid experimental disassembly of microtubules. Thus, under certain conditions tubulin in a form other than microtubules may be detected. Practical applications of this "optical stain" for non fading immuno-gold 5-40 nm markers are discussed.     

Characterization of alpha-actinin from Acanthamoeba

Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666-7670, 1981] has shown that it resembles the actin filament cross-linking protein, alpha-actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 mm is 1.8 X 10(5)M-1 X cm-1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha-actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha-actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross-links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross-linking proteins, amoeba alpha-actinin inhibits the actin-activated ATPase of muscle myosin subfragment-1. Although Acanthamoeba alpha-actinin resembles the alpha-actinin from other cells in shape and ability to cross-link actin filaments, antibodies to amoeba and smooth muscle alpha-actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.     

Population of K-lymphocytes in various kinds of thyroid disease

We have measured the population of Killer (K) lymphocytes in the peripheral blood of 108 patients with various kinds of thyroid disease. In the patients with Hashimoto's thyroiditis and Graves' disease, the relative and absolute numbers of K-lymphocytes were significantly lower than those seen in healthy controls (p less than 0.001), and the longer the duration of medication, the lower the K-lymphocyte population. However, there was no apparent correlation between the serum titers of thyroid autoantibodies and the K-lymphocyte population. In the patients with malignant and benign thyroid tumors, the relative and absolute numbers of K-lymphocytes significantly decreased when compared with those of controls (p less than 0.001), the decrease was more prominent after surgical operation than before operation. A prominent decrease in the K-lymphocyte population was evoked to maximum 2 to 4 weeks after surgical operation. The patients with both malignant and benign tumors having abundant lymphocytic infiltration in their surgical specimens generally revealed a lower K-lymphocyte population than those having no lymphocytic infiltration.     

Effect of septum-initiation mutations on sporulation and competent cell formation in Bacillus subtilis

Summary Sporulation and competent cell formation have been studied in four Bacillus subtilis strains, carrying septum-initiation mutations of different loci, div-31, div-341, div-12 and div-355 which exhibit filamentous growth at 45° C. The div-31 mutant was found to be defective in competence development at 30°–40°C whereas the div-12 mutant was affected only slightly. The div-341 and div-355 mutants showed lower competence, particularly at the higher temperatures. The four div mutant strains all showed poor sporulation at higher temperatures compared to the wild-type strain. We propose that some of the initial steps of septation are involved both in sporulation (possibly in forespore septum formation) and in competent cell formation and that these two processes share certain common features distinct from those in vegetative cell division.     

Peripheral resistance and red cell LiNa countertransport in borderline hypertensives

We have studied ouabain-resistant, external sodium-stimulated, lithium efflux (LiNa countertransport) in red blood cells from 21 borderline hypertensives with at least one hypertensive first degree relative (BH-F), 19 borderline hypertensives without family history of essential hypertension (BH-NF), and 35 age-matched normotensive subjects. The data indicate the finding of an increased LiNa countertransport in all BH (F+NF), but with a significant overlap between BH values and control ones: LiNa countertransport is significantly higher only in BH-F but it is normal in BH-NF. Moreover, there is a significant correlation of LiNa countertransport to total peripheral resistance but not to mean blood pressure in all hypertensive patients. It is suggested that in BH the increase of erythrocyte Na flux is mediated by the NaNa exchange diffusion, and its abnormality may be associated to the hereditary trait of essential hypertension rather than the high blood pressure per se, probably resulting in the development of hypertension, through the increased vascular smooth muscle tone.