The E5 oncoprotein target: a 16-kDa channel-forming protein with diverse functions.
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Bovine papillomavirus 1↗
Biomedical subjects
J D Pitts
Publications and source records attributed to J D Pitts.
Estradiol distribution and penetration in rat skin after topical application, studied by high resolution autoradiography.
Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography. 3H-estradiol-17 beta was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm2 to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol 3H-estradiol x cm-2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm2, a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicuous in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.
Administration, Topical↗
Tissue-specific regulation of junctional communication in the skin of mouse fetuses homozygous for the repeated epilation (Er) mutation.
Mouse embryos homozygous for the repeated epilation (Er) gene have abnormally developed skin characterised by hyper-proliferation and incomplete differentiation of the epidermis. In this report, we have studied the patterns of junctional communication in the skin of these mutants to see if the loss of control of proliferation/differentiation is associated with any altered patterns of communication. Using the dye-injection technique we have shown that, compared to normal skin, junctional communication among dermal cells of Er/Er mutants is greatly reduced and the frequency of dermal-epidermal communication is, on the other hand, increased. These results support our previously proposed model, which suggests that selective regulation of junctional communication can be a component of proliferative control in a complex tissue.
Animals↗
Tissue and species conservation of the vertebrate and arthropod forms of the low molecular weight (16-18000) proteins of gap junctions.
Gap junctions have been isolated from four murine tissues, from rat and Xenopus laevis liver, and from Nephrops norvegicus (Norway lobster) hepatopancreas. The preparations of gap junctions from each vertebrate tissue contain a single major protein, Mr 16,000, and those from Nephrops hepatopancreas a protein, Mr 18,000. Immunocytochemical studies using affinity-purified antibodies raised against gap junctions from Nephrops show the junctional origin of the 18k protein. Immunological studies using Western blotting and biochemical studies using tryptic peptide mapping show no significant differences between the 16k junctional proteins of mouse and hence provide no evidence of tissue variation. These studies also suggest that the mouse, rat, and Xenopus 16k proteins and the Nephrops 18k protein share some common structural features.
Animals↗
Effects of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate on junctional communication in intact mouse skin: persistence of homologous communication and increase of epidermal-dermal coupling.
12-O-Tetradecanoylphorbol-13-acetate (TPA) has been reported to inhibit junctional communication in some cell cultures. In view of the tumour-promoting activity of TPA in rodent skin, these results have interesting implications for the possible role of junctional communication in tumorigenesis. However, little is known about the effect of TPA on junctional communication in intact skin. Using the technique of iontophoretic injection of Lucifer Yellow CH, we have now studied the patterns of junctional communication in skin treated with TPA. In this paper we report results of such experiments showing that junctional communication in skin persists 4 h after treatment with TPA. In addition, TPA unexpectedly increases communication across the dermal-epidermal boundary. These effects of TPA can still be observed 24 h after treatment. In light of these results, we discuss the possible role of junctional communication in the control of proliferation of normal and pathological tissues.
Animals↗
Junctional communication and cellular differentiation.
Gap junctions provide pathways of direct cell to cell communication in the tissues of metazoan animals. Cells joined by gap junctions share their small ions and molecules but can maintain distinctive activities through expression of different macromolecules which are too large to pass through the junctions. The junctional channels are made of a tissue invariant, evolutionarily conserved 16-18 k protein but the formation and maintenance of active coupling also requires one or more connexins, a family of tissue-specific proteins ranging in size from 21 k to 70 k. Junctions can be isolated as complexes containing both types of protein by mild procedures using high pH but the connexins can be removed by detergent, urea and protease treatment without destroying the characteristic junctional-morphology of hexagonally packed channels in the double membrane structures. There is also some evidence for the participation in the complex of tissue-specific proteoglycans which perhaps interact with the tissue-specific connexins and account for specificity of junction formation. Such specificity in mixed cultures leads to the production of communication compartments, groups of cells joined by junctions but separated by reduced trans-boundary coupling from cells in adjacent compartments. Compartmentation also occurs in vivo resulting in specific patterns of junctional communication which have been mapped in most detail in mouse skin. These mapping data and the changes which are associated with abnormal proliferation have lead to new ideas on intercellular control.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Obstetric outcomes in a rural family practice: an eight-year experience.
There has been debate in some quarters of whether family physicians should do obstetrics and of whether rural hospitals should provide obstetric services. Forks, Washington, is a remote logging town where family physicians and midlevel practitioners have been the sole providers of labor and delivery services. Forks offers an opportunity to evaluate the quality of an isolated rural family practice obstetric service. A retrospective audit of all labor and delivery patient charts at Forks Community Hospital from 1975 to 1983 was undertaken; 1,052 charts were abstracted with 36 factors of morbidity, mortality, and intervention examined. The results, when compared with similar studies in the literature, provide evidence of good performance. In addition, a relatively high-risk obstetric population was served with favorable outcomes. Family physicians and rural hospitals can provide high-quality obstetrical services.
Adult↗
Molecular structure of the gap junctional channel.
The proteins in various gap junctional preparations from rodent liver have been analysed by two-dimensional peptide mapping and immunoblotting. Only the protein of relative molecular mass (Mr) 16,000 (16K) is found in all gap junctional isolates, and it is unrelated to the 27K protein. The absence of the 27K protein and any of its fragments from trypsin-treated preparations suggests that this protein does not directly contribute to gap junctional structure. Peptide mapping and immunoblotting of the 16K proteins isolated from various tissues and species and of the arthropod 18K protein present in gap junctional preparations from Nephrops norvegicus show that these proteins constitute a family of related junctional proteins. A site-specific antiserum raised against the N-terminal octapeptide of the 16K protein from mouse liver cross-reacts with all 16K and 18K forms of the junctional protein so far tested, suggesting that this particular antigenic determinant is highly conserved. Immuno-localization studies show that the N-terminus is most likely located on the cytoplasmic aspect of the junction and is available to Pronase digestion.
Animals↗
Patterns of junctional communication in skin: studies on cultured keratinocytes.
Junctional communication has long been suggested to play a role in coordinating the development of multicellular tissues. A better understanding of the patterns of communication between cells in such tissues is important for the identification of areas where this process may have a role. We have investigated the patterns of communication in cultures of human epidermal keratinocytes by iontophoretic injection of Lucifer Yellow CH, using involucrin expression as a marker of cells undergoing terminal differentiation. Cells that lack involucrin (i.e., the basal, proliferating cells) transfer dye preferentially to other involucrin-negative cells, whereas involucrin-positive cells either are not coupled or transfer dye with similar frequency to involucrin-positive and involucrin-negative neighbors. This decrease in communication associated with terminal differentiation was observed in both the presence and the absence of assembled desmosomes. Our observations lead us to speculate that loss of junctional communication may influence the commitment of basal keratinocytes to terminal differentiation.
Cell Communication↗
Mechanism of inhibition of junctional communication between animal cells by phorbol ester.
The tumour promotor 4 beta-phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.
Animals↗
Some murine thymic lymphocytes can form gap junctions.
Analysis of thymic lymphocytes isolated from weanling mice has revealed a minority population able to form permeable, intercellular (gap) junctions. This population is largest in mice aged between 3 and 6 weeks, much smaller in fetal and new-born mice and undetectable in mice aged 12 weeks or more. Fractionation of the thymocytes on Percoll gradients or with peanut agglutinin (PNA) shows the cells able to form junctions are enriched in lower density fractions and agglutinated by PNA, suggesting they are among the most immature. Fractionation by complement mediated cytotoxicity (CMC) and by fluorescence activated cell sorting (FACS) using monoclonal antibodies to specific cell surface determinants shows the junction forming cells are Lyt-1+/Lyt-2- and that the phenotype is associated with both high and low Thy-1 and H-2K epitope densities.
Animals↗
Retinoic acid inhibits junctional communication between animal cells.
Retinoic acid inhibits junctional communication between a variety of vertebrate cell types in culture. It reduces the intercellular transfer of 3H-nucleotides between Syrian hamster kidney fibroblasts (BHK 21/13), Chinese hamster lung fibroblasts (V79), rat liver epithelial cells (BRL), Swiss mouse embryo fibroblasts (3T3), rainbow trout gonadal fibroblasts (RTG2) and Xenopus embryo fibroblasts (Xen). It also reduces metabolic cooperation between hypoxanthine-guanine phosphoribosyl transferase deficient mutant and wild-type BHK cells. The inhibition is rapid (intercellular transfer of iontophoretically injected Lucifer Yellow CH between BRL cells is completely blocked after the cells have been exposed to 10(-4) M retinoic acid for 5 min), and is fully reversed when the drug is removed. Based on these results and the observation that the amount of gap junctional protein isolated from cells grown in the presence of retinoic acid for 1 h is the same and after 24 h is increased (1.3- to 3.1-fold) compared with the amount isolated from untreated cells, we suggest that the inhibitory effect is mediated by the reversible closure of junctional channels.
1-Octanol↗
Patterns of junctional communication in skin.
The patterns of junctional communication in whole skin have been studied by iontophoretic injection of the fluorescent dye Lucifer Yellow CH into excised strips of tissue from newborn (less than 48-h) mice. Sections of injected specimens embedded in resin show that: coupling in the dermis is extensive, the injected dye spreads to more than 500 cells in 5 min; coupling in the epidermis is more restricted, dye spreads to less than 25 cells in 5 min; cells of the epidermis are not detectably dye-coupled to the cells of the dermis except at occasional restricted sites, probably related to hair follicle development; immature sebaceous gland cells are coupled but appear to be isolated from other cell types; and endothelial cells in the dermis are coupled to surrounding fibroblasts.
Animals↗
Communication compartments in mixed cell cultures.
Mixed cultures of epithelial (BRL) cells and fibroblasts (BHK), which sort themselves out into separate domains of each cell type, form communication compartments. Electrical coupling, dye coupling and metabolic coupling measurements have been used to show that small ions and molecules can move freely via intercellular junctions between all the cells in a domain, while their movement across the boundaries between domains is severely restricted. Metabolic coupling is the most sensitive method for detecting trans-boundary communication but the results obtained from all three methods are compatible. The data suggest the reduced transfer across the boundaries is due to fewer channels, resulting from a lower frequency of junction formation between heterologous cells, rather than to channels of smaller diameter. Concentration gradients of small cytoplasmic molecules can be established within these communication compartments which are similar to those predicted to explain pattern formation in developing systems. It is suggested that the cell surface features which cause this sorting out are also responsible for the reduced frequency of heterologous junction formation and hence for compartmentalization.
Animals↗
Computer-assisted grain counting for autoradiography.
The two programs described here are designed to be used on the System III Image Analyser which partially automate the counting process of autoradiography and present the data in histogram form. The system has the advantages of low cost, accuracy and reproducibility. Although the programs are developed for grain counting, they can be easily adapted to perform other particle counting processes using the same system.
Autoradiography↗
Growth inhibition of normal, tumour, and transformed cells by antibodies to a cell-surface proteinase.
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Antibodies↗
Inhibition and recovery of DNA synthesis in PHA-stimulated u.v.-irradiated lymphocytes from patients with actinic keratosis.
The recovery, after inhibition by u.v.-irradiation, of phytohaemagglutinin-stimulated DNA synthesis is impaired in lymphocytes from patients with multiple actinic keratoses (AK) compared to the recovery in lymphocytes from age-matched, control individuals. This shows that the reduced level (50%) of DNA repair activity in AK cells is sufficiently different from that in normal cells to significantly affect cellular activity.
DNA Repair↗