Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.

The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.     

Immunological and molecular characterization of the cAMP-dependent protein kinases in AtT20 cells

The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.     

Temporal and tissue-specific expression of myosin heavy chain isoforms in developing and adult avian muscle

We have raised monoclonal antibodies (Mabs) to myosin heavy chain isoforms (MHCs) that have specific patterns of temporal expression during the development of quail pectoral muscle and that are expressed in very restricted, tissue-specific patterns in adult birds. We find that an early embryonic, a perinatal, and an adult-specific, fast myosin heavy chain are co-expressed at different levels in the pectoral muscle of 8-12 day quail embryos. The early embryonic MHC disappears from the pectoral muscle at approximately 14 days in ovo, whereas the perinatal MHC persists until 26 days post-hatching. The adult-specific MHC accumulates preferentially and eventually completely replaces the other isoforms. These Mabs cross-react with the homologous isoforms of the chick and detect a similar pattern of MHC expression in the pectoral muscle of developing chicks. Although the early embryonic and perinatal MHC isoforms recognized by our Mabs are expressed in the pectoral muscle only during distinct developmental stages, our Mabs also recognize MHC isoforms present in the heart and extraocular muscle of adult quail. Immunofingerprinting using Staphylococcus aureus protease V8 suggests that the early embryonic and perinatal MHC isoforms that we see are strongly homologous with the adult ventricular and extraocular muscle isoforms, respectively. These observations suggest that at least three distinct MHC isoforms, which are normally expressed in adult muscles, are co-expressed during the early development of the pectoral muscle in birds. In this respect, the pattern of expression of the MHCs recognized by our Mabs in developing, fast muscle is very similar to the patterns described for other muscle contractile proteins.     

Tolerance of extremely halophilic archaebacteria towards bromide

The tolerance of halophilic archaebacteria towards bromide was tested in view of the fact that bromide occurs in natural brines in concentrations of up to 66 mM. It was found that, while concentrations of up to 0.8–1M are tolerated well by all halobacterial types examined, great differences exist between species with respect to bromide tolerance. WhileHalobacterium (H. salinarium, H. halobium, andH. sodomense) andNatronobacterium species are only moderately tolerant,Haloarcula (H. vallismortis, H. marismortui), andHaloferax species (H. mediterranei, H. gibbonsii) tolerate higher concentrations.Haloferax volcanii proved extremely tolerant and showed growth in bromide media at very low chloride concentrations (below 50 mM). No correlation was found between bromide tolerance and the bromide concentration in the habitat from which the strains were isolated. Iodide proved much more toxic than bromide. Bromide-tolerant strains also proved relatively resistant to growth inhibition by iodide.     

Myosin light chain phosphorylation in 32P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.     

Kinetics of in vivo bacteriochlorophyll fluorescence yield and the state of photosynthetic apparatus of purple bacteria

The light-induced electron transport in purple bacterium Rhodobacter sphaeroides was studied in vivo by means of kinetic difference absorption spectroscopy and kinetics of bacteriochlorophyll fluorescence yield. Measurements of redox state of the oxidised primary donor and cytochrome c and the membrane potential revealed a complex pattern of changes of the electron flow. Effects of the membrane potential on the fluorescence yield were also analysed, and a model for the fluorescence induction curve is presented. The data indicate substantial positive effect of the membrane potential on the fluorescence emission in vivo. Moreover, light-induced changes in light scattering were observed, which suggests occurrence of structural changes on the level of the photosynthetic membrane.     

The pollen harvest

At least three million people in the United States suffer from seasonal allergy caused by inhalation of airborne pollens of many species. Each affected person is a potential ultimate consumer of refined pollen products, and the annual collecting of pollen for this purpose amounts to $80,000 to $100,000 for the collectors.