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)     

Transposon-like properties of the major, long repetitive sequence family in the genome of Physarum polycephalum

A family of long, highly-repetitive sequences, referred to previously as `HpaII-repeats', dominates the genome of the eukaryotic slime mould Physarum polycephalum. These sequences are found exclusively in scrambled clusters. They account for about one-half of the total complement of repetitive DNA in Physarum, and represent the major sequence component found in hypermethylated, 20-50 kb segments of Physarum genomic DNA that fail to be cleaved using the restriction endonuclease HpaII. The structure of this abundant repetitive element was investigated by analysing cloned segments derived from the hypermethylated genomic DNA compartment. We show that the `HpaII-repeat' forms part of a larger repetitive DNA structure, ~8.6 kb in length, with several structural features in common with recognised eukaryotic transposable genetic elements. Scrambled clusters of the sequence probably arise as a result of transposition-like events, during which the element preferentially recombines in either orientation with target sites located in other copies of the same repeated sequence. The target sites for transposition/recombination are not related in sequence but in all cases studied they are potentially capable of promoting the formation of small `cruciforms' or `Z-DNA' structures which might be recognised during the recombination process.     

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.     

Inflammatory cytokines inhibit Kaposi's sarcoma-associated herpesvirus lytic gene transcription in in vitro-infected endothelial cells

The response of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) to inflammatory cytokine treatment of experimentally infected endothelial cells was investigated. The cytokines inhibited spontaneous KSHV lytic gene expression but not the level of infection. The data suggest that if inflammatory cytokines present in KS lesions contribute to KSHV pathogenesis, they do so in part by promoting latent KSHV infection of the endothelial cells.     

Root exudation from Hordeum vulgare in response to localized nitrate supply

Root proliferation as a response to exploit zones of nutrient enrichment in soil has been demonstrated for a wide range of plant species. However, the effectiveness of this as a strategy to acquire nutrients is also dependent on interactions with the soil microbial community. Specifically, C-flow from roots modifies microbial activity and probably the balance between nutrient mineralization and immobilization processes in the rhizosphere. In this study, near-natural abundance 13C-labelling and gene-reporter methods were applied to determine the effects of uneven nitrate supply to roots of Hordeum vulgare on assimilate partitioning and root exudation. Plants were initially grown in uniform nitrate supply in split-root, sand microcosms after which one treatment continued to receive uniform supply, and the other received nitrate to one root compartment only. At the time of imposing the treatments, the CO2 supplied to the plants was switched to a cylinder source, providing a distinct delta13C-signature and allowing the fate of new assimilate within the plants to be determined. The labelling approach allowed quantification of the expected preferential allocation of new C-assimilate to roots in enriched nitrate, prior to any measurable effect on whole biomass or root architecture. Biosensor (lux-marked Pseudomonas fluorescens 10586 pUCD607) bioluminescence, quantified spatially by CCD imaging, demonstrated that root exudation was significantly increased for roots in enriched nitrate. This response of root exudation, being primarily associated with root apices and concurrent with enhanced assimilate supply, strongly suggests that C-flow from roots is an integral component of the proliferation response to nitrate.     

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.