Phorbol ester binding and protein kinase C activity in normal and transformed human keratinocytes

Normal keratinocytes, SV40-transformed keratinocytes (SVK14), and various squamous carcinoma cell (SCC) lines have been used as an in vitro model system to study the properties of phorbol ester receptor and protein kinase C expression during keratinocyte differentiation. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-4; moreover, all cell lines respond to a low external Ca2+ concentration by a decreased capacity to differentiate. Normal keratinocytes exhibited the highest number of phorbol ester receptors as compared to the other cell lines, while each individual cell line exhibited a higher number of phorbol ester receptors during growth under normal Ca2+ conditions as compared to cells grown under low Ca2+ conditions. The apparent dissociation constant (Kd) demonstrated only small variations in the various cell lines. In contrast, the cytoplasmic protein kinase C activity, was found to be higher in cells grown under low Ca2+ conditions than in cells grown under normal Ca2+ conditions, indicating the absence of a causal relationship between cytoplasmic protein kinase C activity and phorbol ester receptor expression. Therefore the properties of protein kinase C have been determined in more detail in normal keratinocytes and SCC-15 cells. These studies revealed differences between protein kinase C properties from the two cell lines grown under normal and low Ca2+ conditions. The differences included the effect of phorbol 12-myristate 13-acetate (PMA) on the redistribution pattern of protein kinase C between the cytoplasmic and particulate fractions as well as the activating effect of diolein in vitro on protein kinase C activity, partly purified from particulate or cytoplasmic fractions. These observations demonstrate that the functional protein kinase C activity of keratinocytes is determined by various endogenous and exogenous activators and that these activators are modulated differently in various cell lines, under various growth conditions (low Ca2+ versus normal Ca2+).     

Primary structure and androgen regulation of a 20-kilodalton protein specific to rat ventral prostate

Nuclear and cytosolic forms of a 20-kdalton rat ventral prostate protein were purified and partially sequenced from their N-termini. Isolated nuclei were treated with micrococcal nuclease and extracted in 0.6 M NaCl, and proteins were separated by affinity chromatography on Matrex gel green A, ammonium sulfate fractionation, and fast protein liquid chromatography on Superose 12. The 43 amino acid N-terminal sequence of the nuclear 20-kdalton protein was identical with the cytosolic protein except it lacked 7 N-terminal amino acids present in the cytosolic form. The DNA sequence of a full-length complementary DNA clone isolated from a ventral prostate gt11 library extended the N-terminal sequence of the cytosolic form by an additional nine amino acids from the predicted initiation methionine. The cDNA included the nucleotide sequence for the 43 amino acid N-terminal sequence of the purified 20-kdalton protein and predicted molecular weights of 16,686, 17,521, and 18,650, respectively, for the nuclear, cytoplasmic, and nonprocessed proteins. Northern blot analyses of reproductive tract tissue RNAs using the 20-kdalton protein cDNA as probe revealed a single mRNA species of 0.92 kb detectable only in extracts of rat ventral prostate. Expression of the 0.92-kb mRNA was androgen dependent since the mRNA was undetectable in extracts obtained 4 days after castration and was restored 16 h after restimulation with androgen.     

Properties and possible function of phosphatidylinositol-transfer proteins

It is proposed that the phosphatidylinositol-transfer protein (PI-TP) may function as a carrier of phosphatidylinositol (PI) in the cell. PI-TP occurs in all mammalian tissues examined and appears to be strongly conserved. Its intracellular distribution was studied by immunoblotting and immunofluorescence techniques. PI-TP displays a dual specificity in that it preferentially transfers PI over phosphatidylcholine (PC) between membranes. Its lipid binding site and transfer characteristics were investigated with fluorescent PI and PC analogues containing parinaroyl- and pyrenylacyl-labeled chains. PI-TP is ideally suited for maintaining PI levels in intracellular membranes, possibly the plasma membrane.     

A susceptibility gene for alveolar lung tumors in the mouse maps between Hsp70.3 and G7 within the H2 complex

Lung tumor susceptibility (LTS) in the mouse is influenced by multiple loci within the H2 complex. We compared the LTS of two H2 congenic strains with intra H2 recombinations, B10.A(1R) and B10.A(2R), whose genetic difference has been reduced to a region of approximately 50 kilobases within the C4-H2D interval, between Hsp70.3 and G7. After transplacental induction with N-ethyl-N-nitrosourea the load of alveolar lung tumors in strain B10.A(2R) is significantly higher than in strain B10.A(1R) (P <0.001). For papillary tumors no significant differences were observed. We conclude that the alveolar lung tumor load is influenced by an LTS gene located within the Hsp70.3-G7 interval.     

Cloning and characterization of a sialidase from the murine histocompatibility-2 complex: low levels of mRNA and a single amino acid mutation are responsible for reduced sialidase activity in mice carrying the Neula allele

The Neu1 locus, in the S region of the murine histocompatibility-2complex, regulates the sialic acid content of several liverlysosomal enzymes. Three alleles, Neu1^a, Neu1^b, and Neu1^c, havebeen described on the basis of differential sialylation of theenzyme liver acid phosphatase. The Neu1^a allele occurs in asmall number of mouse strains, e.g., SM/J and is associatedwith sialidase deficiency. We recently described G9, a sialidasegene in the human major histocompatibility complex (Milner etal. (1997) J. Biol. Chem., 272, 4549–4558), and we nowreport the characterization of the equivalent gene in mouse.The protein product of the murine G9 gene is 409 amino acidsin length and is 83% identical to its human orthologue. Expressionof the murine G9 protein in insect cells has confirmed thatit is a sialidase, with optimal activity at pH 5. To elucidatethe basis of sialidase deficiency in mouse strains carryingthe Neu1^a allele, we have sequenced the G9 coding regions frommice carrying the three Neu1 alleles and hence defined the aminoacid sequence characteristic of each allotype. Of particularinterest is a Leu-209 to Ile mutation that is unique to theNeu1^a allotype and is associated with reductions in sialidaseactivity of 68% and 88% compared to the Neu1^b and Neu1^c allotypes,respectively, when these three protein variants are expressedin insect cells. Additional factors, such as differential expression,may also influence the activities of the Nen1 allotypes in vivo.We have observed that the level of G9 mRNA is substantiallyreduced in mice carrying the Neu1^a allele compared to the Neu1^b(85–95% reduction) and Neu1^c (70% reduction) alleles. H2 complex MHC Neu1 sialidase     

Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF)β-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGFβ-like activities at an apparent molecular weight of 6–10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal expiants. The relation between this activity and a recently described TGFβ-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.     

A new H-2.1-PositiveD region allele,D dx,controlling two molecules,H-2Ddx and H-2Ldx

The inbred strains GRS/A and LIS/A carry the haplotypeH-2 ^dx , which had earlier been shown to have theK ^d ,I ^f ,S ^f , andG ^f alleles and a previously unknownD region allele,D ^dx . We show here that theD ^dx allele determines a new private specificity, H-2.63, is H-2.28 negative, and determines at least one public specificity of the H-2.1 family. It is thus a second example (afterD ^k ) of a H-2.1-positive H-2.28-negativeD region allele. Capping experiments show that the D^dx product comprises two molecules: H-2D^dx bearing the private specificity H-2.63, and H-2L^dx, which is H-2.63-negative but reacts with sera against the H-2.1 family of specificities. SDS gel electrophoresis of detergent-solubilized immunoprecipitated D^dx products shows that the H-2L^dx antigen has a molecular weight of approximately 45,000 daltons and is associated with a smaller polypeptide (mol. wt. 12,000).     

Lipopolysaccharide-binding protein is vectorially secreted and transported by cultured intestinal epithelial cells and is present in the intestinal mucus of mice

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host's response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.