Prolactin-Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C-Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL-induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL-induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine, or polymyxin B blocked the PRL-induced increase in cell number with IC₅₀ values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α-phorbol 12,13-didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U-shaped dose-response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose-response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL-induced increase in cell number. Finally, TPA treatment decreased cell-surface binding of ¹²⁵I-PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.     

The amino-acid sequence of the dihydrofolate reductase of a trimethoprim-resistant strain of Escherichia coli.

The determination of the amino acid sequence of the dihydrofolate reductase from Escherichia coli RT500 is described. The sequence, comprising 159 residues, has been derived from automatic sequencing of the intact protein in conjunction with manual sequencing of lysine-blocked tryptic peptides, Staphylococcus aureus protease peptides, and alpha-lytic protease peptides. Comparison of the sequence with that of the dihydrofolate reductase from a methotrexate-resistant strain of E. coli (MB1428) shows that 145 of the residues are identical. The distribution of the differences along the length of the molecule is discussed.     

Some improvements in two-dimensional gel electrophoresis of proteins. Protein mapping of eukaryotic tissue extracts.

In the course of adapting O'Farrell's (1975, J. Biol. Chem.250, 4007–4021) two-dimensional separation technique for proteins to eukaryotic material, we have made some modifications. During sample preparation, sodium dodecyl sulfate (SDS) can be included, with a resulting enhancement in reproducibility of gel patterns. However, heating in the presence of SDS leads to artifactual spots in the gels, probably as a result of protein charge modifications. Ultracentrifugation reduces the clogging at the top of the isoelectric focussing gel. For electrophoresis, some modifications of apparatus and technique are suggested. For the analysis of gels, a simple high-efficiency method for the counting of radioactivity in spots from dried gel slabs is described. In addition, an inexpensive microdensitometer option is described for the analysis of the autoradiographs. Patterns of proteins obtained from superior cervical sympathetic ganglia of rats and from other eukaryotic tissues are illustrated. Finally, a few of the proteins commonly found in mammalian tissue are identified on the gels.     

Water-soluble (1→3), (1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure

Water-soluble (1→3),(1→4)-β-d-glucans isolated from barleys grown in Australia and the UK were depolymerised using a purified (1→3),(1→4)-β-d-glucan 4-glucanohydrolase (EC 3.2.1.73). Oligomeric products were quantitatively separated by high resolution gel filtration chromatography and their structures defined by methylation analysis. Approximately 90% (w/w) of each polysaccharide consists of cellotriosyl and cellotetraosyl residues separated by single (1→3)-linkages but blocks of 5–11 (1→4)-linked glucosyl residues are also present in significant proportions. Periodate oxidation followed by Smith degradation suggested that contiguous (1→3)-linked β-glucosyl residues are either absent, or present in very low frequency. The potential for misinterpretation of data due to incomplete Smith degradation was noted.The irregularly-spaced (1→3)-linkages interrupt the relatively rigid, ribbon-like (1→4)-β-glucan conformation and confer a flexibility and ‘irregular’ shape on the barley (1→3),(1→4)-β-d-glucan, consistent with its solubility in water. Molecular models incorporating the major structural features confirm that the polysaccharide is likely to assume a worm-like conformation in solution. Non-covalent interactions between long blocks of (1→4)-linkages in (1→3),(1→4)-β-d-glucans, or between these blocks and other polysaccharides, offer a possible explanation for the organisation of polysaccharides in the framework of the cell wall.     

Competitive exclusion,or species aggregation?

Summary There is a long-standing dispute over whether the analysis of species co-occurrence data, typically on islands in an archipelago, can disclose the forces at work in structuring a community. Here we present and utilise three scores S, C and T. S gives the mean number of islands shared by a species pair in the presence/absence data under study. The scores C and T are based on the way that a pair of species occurs on a pair of islands. When each species occurs on a different island, this adds to the checkerboard score C; if they occupy the same island, this increases the togetherness score T.In judging whether observed values of S, C and T are compatible with a null hypothesis assuming no species interaction, we follow Connor and Simberloff (1979) in generating a control group of (constrained) simulated incidence patterns.Presence/absence matrices can have paradoxical features, in combining a high mutual exclusion by species (checkerboardedness) with a degree of species aggregation that is also high. We show that this is in fact inevitable — that, given the usual contraints, C and T can differ only by a constant. This means that extreme checkerboardedness can be produced by forces making for species aggregation, just as well as by those making for avoidance.If we restrict our attention to a subset of species, the constraints are less rigid and the S, C and T scores are somewhat freer to vary. We consider the confamilial subsets in the Vanuatu archipelago as likely candidates for revealing any competition forces at work. Calculating the actual S, C and T scores for these subsets, we compare them with the corresponding scores in a sample of simulated colonization patterns.The actual species-distributions differ significantly from what we would expect if the colonization choices of different species were uncorrelated (save for some biological constraints). The confamilial species of the real world share more islands, and occur in a pattern less checkerboarded, and more aggregated, than their simulation counterparts. This suggests that competition pressures, if they exist, are overcome by countervailing factors.The method used is applicable in other ways, and to a wider class of problems, in analysing the forces behind community structure.     

Binding of iodomercurates to sulfhydryl-blocked beta-lactoglobulin-A, -B, and -C

Sulfhydryl-blocked beta-lactoglobulins (beta-LG-S-SCH2CH2OH)-A, -B, and -C bind only one iodomercurate species, HgI3-, at only one site, with a dissociation constant of 4.0 X 10(-5) M at 25 degrees, pH 5.0, 0.10 ionic strength. (Binding to native beta-LG-SH-A, -B, and -C is more complex, involving the sulfhydryl and two other sites and several iodomercurates.) The red shift of the HgI3- spectrum on binding would ordinarily suggest a hydrophobic site, but the HgI3- site is distinct from, and independent of, the alkane-binding site of native and blocked beta-LG; HgI3- may bind a group that shifts its trigonal planar structure toward the tetrahedron of HgI4(2-). Binding of HgI3- to blocked beta-LG interferes with the well-known association of beta-LG-A to octamers at pH 4.6 and low temperature. The relation of the HgI3- site to the crystallographic iodomercurate-binding sites of beta-LG-SH is examined. To facilitate these and future studies of iodomercurate binding, the 200-400 nm spectra of HgI2, HgI3-, and HgI4(2-) in aqueous solutions and the thermodynamic formation constants at 25 degrees for the equilibria HgI2 + I- = HgI3- (4.9 X 10(3) M-1) and HgI3- + I- = HgI4(2-) (0.118 X 10(3) M-1) were obtained.     

Maintenance of rat taste buds in primary culture

The differentiated taste bud is a complex end organ consisting of multiple cell types with various morphological, immunocytochemical and electrophysiological characteristics. Individual taste cells have a limited lifespan and are regularly replaced by a proliferative basal cell population. The specific factors contributing to the maintenance of a differentiated taste bud are largely unknown. Supporting isolated taste buds in culture would allow controlled investigation of factors relevant to taste bud survival. Here we describe the culture and maintenance of isolated rat taste buds at room temperature and at 37 degrees C. Differentiated taste buds can be sustained for up to 14 days at room temperature and for 3-4 days at 37 degrees C. Over these periods individual cells within the cultured buds maintain an elongated morphology. Further, the taste cells remain electrically excitable and retain various proteins indicative of a differentiated phenotype. Despite the apparent health of differentiated taste cells, cell division occurs for only a short period following plating, suggesting that proliferating cells in the taste bud are quickly affected by isolation and culture.