Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro

Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.05 mM Ca2+ medium, and terminal differentiation is induced when the Ca2+ concentration is increased to 1 mM. However, only a small fraction of the cells express the markers K1, K10, CE, or filaggrin in the higher Ca2+ medium. To explore the factors required for marker expression, cultured epidermal cells were exposed to intermediate Ca2+ concentrations and extracts were analyzed using specific antibody and nucleic acid probes for the four markers of interest. These studies revealed that marker expression was enhanced at a restricted concentration of Ca2+ in the medium of 0.10-0.16 mM. At this Ca2+ concentration, both protein and mRNA levels for each marker were substantially increased, whereas at higher or lower Ca2+ concentrations they were diminished or undetected. The percentage of cells expressing each marker was increased two- to threefold in the permissive Ca2+ medium as determined by immunofluorescence analysis. This optimal level of Ca2+ was required both to initiate and sustain marker expression. At the permissive Ca2+ concentration, expression of the markers was sequential and similar to the order of appearance in vivo. K1 was expressed within 8-12 h and K10 was expressed in the ensuing 12-24-h period. CE and filaggrin were expressed in the subsequent 24 h. Inhibition of K1 expression by cycloheximide suggested that an inducible protein was involved. Other investigators have determined that a shallow Ca2+ gradient exists in epidermis, where the basal cells and spinous cells are in a Ca2+ environment substantially below serum Ca2+ levels. These in vitro results suggest that the Ca2+ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of the Ca2+ gradient in vivo.     

Employment of a noninvasive magnetic method for evaluation of gastrointestinal transit in rats

ABSTRACT: AC Biosusceptometry (ACB) was previously employed towards recording gastrointestinal motility. Our data show a reliable and successful evaluation of gastrointestinal transit of liquid and solid meals in rats, considering the methods scarcity and number of experiments needed to endorsement of drugs and medicinal plants. ACB permits real time and simultaneous experiments using the same animal, preserving the physiological conditions employing both meals with simplicity and accuracy.     

Reproductive success in varying light environments: direct and indirect effects of light on plants and pollinators

Plant populations often exist in spatially heterogeneous environments. Light level can directly affect plant reproductive success through resource availability or by altering pollinator behavior. It can also indirectly influence reproductive success by determining floral display size which may in turn influence pollinator attraction. We evaluated direct and indirect effects of light availability and measured phenotypic selection on phenological traits that may enhance pollen receipt in the insect-pollinated herb Campanulastrum americanum. In a natural population, plants in the sun had larger displays and received 7 times more visits than plants in the shade. Using experimental arrays to separate the direct effects of irradiance on insects from their response to display size, we found more visits to plants in the sun than in the shade, but no association between number of visits each flower received and display size. Plants in the sun were not pollen limited but pollen-augmented shade flowers produced 50% more seeds than open-pollinated flowers. Phenological traits, which may influence pollen receipt, were not under direct selection in the sun. However, earlier initiation and a longer duration of flowering were favored in the shade, which may enhance visitation in this pollen-limited habitat.     

Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins

Monospecific antibodies to mouse epidermal keratins were generated in rabbits and guinea pigs by injecting synthetic peptides of unique keratin sequences. The sequences were deduced from nucleotide sequences of cDNA clones representing basal (K14) and suprabasal (K1 and K10) cell-specific and hyperproliferative (K6) keratins of both the type-I and type-II subclasses. By applying single-and double-label immunofluorescence analysis, the expression of keratin peptides was analyzed in cultured keratinocytes maintained in the basal or suprabasal cell phenotypes. These cell types were selected by growth in medium containing 0.05 mM Ca2+ (basal cell) or 1.4 mM Ca2+ (suprabasal cell). The cultured basal cells expressed K6 and K14, but less than 1% expressed K1 and K10. Within a few hours after being placed in 1.4 mM Ca2+, K1 expression was observed, and by 24 h, 10%-17% of the cells expressed K1. K10 expression appeared to lag behind K1 expression, with only 5%-10% of cells in 1.4 mM Ca2+ exhibiting K10 immunoreactivity. Double-labeling studies indicated that virtually all K10-positive cells also expressed K1, while only about one-half of the K1-positive cells expressed K10. The treatment of basal cells with retinoic acid at pharmacological concentrations prevented the expression of K1 and K10 when cells were challenged by 1.4 mM Ca2+. Similarly, the introduction of the v-rasH oncogene into basal cells by a defective retroviral vector prevented the expression of suprabasal keratins in 1.4 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibroblast growth factor receptor-1 interacts with the T-cell receptor signalling pathway

Fibroblast growth factor receptors are expressed by some T cells, and provide costimulation for these cells. Such receptors allow T cells to respond to fibroblast growth factors expressed in response to injury and inflammation and may provide a mechanism for 'context-dependent' responses to antigens within the local microenvironment. The mechanisms by which fibroblast growth factor receptors might interact with the TCR signalling pathway are not defined. Here we show that the TCR and fibroblast growth factor receptors co-localize during combined stimulation. Signalling via fibroblast growth factor receptors alone results in phosphorylation of Lck and induces nuclear translocation of nuclear factors of activated T cells. Combined stimulation via fibroblast growth factor receptors and the TCR synergistically enhances the activation of nuclear factors of activated T cells. The results suggest that peptide growth factors produced at sites of injury and inflammation can contribute to the outcome of T-cell encounters with antigen.     

Subsets of the major tyrosine phosphorylation sites in Crk-associated substrate (CAS) are sufficient to promote cell migration

Crk-associated substrate (p130(CAS) or CAS) is a major integrin-associated Src substrate that undergoes tyrosine phosphorylation at multiple YXXP motifs in its substrate domain (SD) to create docking sites for SH2-containing signaling effectors. Notably, recruitment of Crk adaptor proteins to the CAS SD sites is implicated in promoting cell migration. However, it is unclear which or how many of the 15 CAS SD YXXP tyrosines are critically involved. To gain a better understanding of CAS SD function, we assessed the signaling capacity of individual YXXP motifs. Using site-directed mutagenesis combined with tryptic phosphopeptide mapping, we determined that the ten tyrosines in YXXP motifs 6-15 are the major sites of CAS SD phosphorylation by Src. Phosphopeptide binding assays showed that all of these sites are capable of binding the Crk SH2 domain. To evaluate the requirement for CAS YXXP sites in stimulating cell migration, a series of phenylalanine substitution variants were expressed in CAS -/- mouse embryo fibroblasts. CAS expression enhanced the rate of cell migration into a monolayer wound in a manner dependent on the major sites of Src phosphorylation. Effective wound healing was achieved by CAS variants containing as few as four of the major sites, indicating sufficiency of partial SD signaling function in this cell migration response.     

Structure of an archaeal PCNA1-PCNA2-FEN1 complex: elucidating PCNA subunit and client enzyme specificity

The archaeal/eukaryotic proliferating cell nuclear antigen (PCNA) toroidal clamp interacts with a host of DNA modifying enzymes, providing a stable anchorage and enhancing their respective processivities. Given the broad range of enzymes with which PCNA has been shown to interact, relatively little is known about the mode of assembly of functionally meaningful combinations of enzymes on the PCNA clamp. We have determined the X-ray crystal structure of the Sulfolobus solfataricus PCNA1–PCNA2 heterodimer, bound to a single copy of the flap endonuclease FEN1 at 2.9 Å resolution. We demonstrate the specificity of interaction of the PCNA subunits to form the PCNA1–PCNA2–PCNA3 heterotrimer, as well as providing a rationale for the specific interaction of the C-terminal PIP-box motif of FEN1 for the PCNA1 subunit. The structure explains the specificity of the individual archaeal PCNA subunits for selected repair enzyme ‘clients’, and provides insights into the co-ordinated assembly of sequential enzymatic steps in PCNA-scaffolded DNA repair cascades.     

A conserved motif in the C-terminal tail of DNA polymerase α tethers primase to the eukaryotic replisome

The DNA polymerase α-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol α synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol α remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol α that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452-1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe(1463). Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol α binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol α allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol α is critical for the normal action of DNA replication forks in eukaryotic cells.