Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [³²P]phosphate labeling in vivo.It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.     

Calcium and gibberellin-induced elongation of lettuce hypocotyl sections

The relationship between calcium ions and gibberellic acid (GA₃)-induced growth in the excised hypocotyl of lettuce (Lactuca sativa L.) was investigated. The short-term kinetics of growth responses were measured using a linear displacement transducer. Test solutions were added either as drops to the filter paper on which the hypocotyl stood (non-flow-past) or by switching solution flowing past the base of hypocotyl (flow-past), resulting in differences in growth behavior. Drops of CaCl₂ added at a high concentration (10 mM) inhibited growth within a few minutes. This inhibition was reversed by ethylenediaminetetraacetic acid (EDTA). Drops of EDTA or ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid caused a rapid increase in growth rate. Growth induced by EDTA was not further promoted by GA₃. A continuous H₂O flow resulted in growth rates comparable to those in response to GA₃. Addition of CaCl₂ to the flow-past medium inhibited growth and this inhibition was reversed by a decrease in CaCl₂ concentration. The growth rate was found to be a function of CaCl₂ concentration. When a constant CaCl₂ concentration was maintained by the flow-past medium, a shift in pH from 5.5 to 4.25 had no obvious effect on hypocotyl elongation. Gibberellic acid was found to reverse the inhibitory effect of CaCl₂, causing an increase in growth rate similar to that found previously when GA₃ was added to hypocotyls grown in H₂O under non-flow-past conditions. We propose that gibberellin controls extension growth in lettuce hypocotyl sections by regulating the uptake of Ca²⁺ by the hypocotyl cells.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid - GA gibberellin - GA₃ gibberellic acid - IAA indole-3-acetic acid     

Experimental Evaluation of Relationships among Populations Resulting from Intergradation among Cultivars of ZEA MAYS L

Populations were developed as intergradations among three maize cultivars, two adapted to North Carolina and one to Mexico, with the objective of testing whether diallel information could be used to identify the relationships among populations. The data substantiated theoretical observations that the technique would identify relationships among populations resulting from recent intergradations.     

Regeneration of the supraoptico-hypophyseal and paraventriculo-hypophyseal tracts in the hypophysectomized rat

Summary The effect of hypophysectomy on the nerve fiber pattern in the median eminence and infundibular stem of the rat has been investigated by a slightly modified Bodian technique. Postoperative changes in the distribution of neurosecretory material and connective tissue and changes in vascularity have also been studied.Extensive regeneration of the fibers of the supraoptico-hypophyseal and paraventriculo-hypophyseal tract could be demonstrated. It is most pronounced at the distal extremity of the infundibular stem but occurs also in the rostral part of the infundibular stem and in the median eminence. Regeneration starts in the second postoperative week and is completed about four weeks later. The nervous regeneration observed in the pituitary area after hypophysectomy is more extensive than is usually encountered after lesions elsewhere in the central nervous system.It could be demonstrated moreover that neurosecretory material accumulates at the same sites in which the terminals of the regenerated nerve fibers can be found. Hypophysectomy also causes an increase in capillary density and connective tissue content of the infundibular stem. Accumulations of neurosecretory material are always found in areas showing a high capillary density and a considerable amount of connective tissue.Factors which might be responsible for the extensive nervous regeneration in the pituitary area are discussed as are the factors determining the pattern of outgrowth of the regenerating nerve fibers. Morphological aspects of storage of posterior pituitary hormones are considered in the light of data in the literature and the results of the present work.Partly supported by a U.S. Public Health grant to Dr. E. Scharrer and a travel grant to the author from the Netherlands Organization for pure scientific Research (ZWO).     

Immunoregulation by mouse T-cell clones. I. Suppression and amplification of cytotoxic responses by cloned H-Y-specific cytolytic T lymphocytes

H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.     

Patterns of cytokeratin and vimentin expression in the human eye

We studied the expression of the various cytokeratin (CK) polypeptides and vimentin in tissues of the human eye by applying immunocytochemical procedures using a panel of monoclonal antibodies as well as by performing biochemical analyses of microdissected tissues. Adult corneal epithelium was found to contain significant amounts of the cornea-specific CKs nos. 3 and 12 as well as CK no. 5, and several additional minor CK components. Among these last CKs, no. 19 was found to exhibit an irregular mosaic-like staining pattern in the peripheral zone of the corneal epithelium, while having a predominantly basal distribution in the limbal epithelium. Both the fetal corneal epithelium and the conjunctival epithelium were uniformly positive for CK no. 19. In the ciliary epithelium, co-expression of CKs nos. 8 and 18 and vimentin was detected, whereas in the retinal pigment epithelium, CKs nos. 8 and 18 were dominant. The present data illustrate the remarkable diversity and complexity of CK-polypeptide expression in the human eye, whose significance with respect to histogenetic and functional aspects is, as yet, only partially clear. The unusual distribution of CK no. 19 in different zones of the corneal epithelium may be related to the specific topography of corneal stem cells. The occurrence of the expression of simple-epithelium CKs in the ciliary and pigment epithelium demonstrates that, despite their neuroectodermal derivation, these are true epithelia.     

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues. I. Human and bovine hair follicles

The cytokeratin family of intermediate filament (IF) proteins can be grouped into the epithelial polypeptides ("soft alpha-keratins"), of which at least 19 exist in the various human epithelia, and the hair-type cytokeratins ("hard alpha-keratins"), which are typical of trichocytes, i.e., the living hair-forming cells. We have recently shown [34] that the hair follicles from diverse mammalian species contain a set of eight major cytokeratin polypeptides, four each of the acidic (type I) and the basic (type II) subfamily, which are different from all known epithelial cytokeratins. In addition, we have identified two new minor trichocytic cytokeratin polypeptides, designated Hax (type I) and Hbx (type II). Antibodies against trichocytic cytokeratins that do not crossreact with any of the epithelial cytokeratins have enabled us to study the expression of both kinds of cytokeratin in the various cell types of human and bovine hair follicles. Using immunofluorescence microscopy, we have observed intense reactions of trichocytic cytokeratins only in cells contributing to the forming hairs, i.e., hair shaft, medulla and cuticle, whereas immunostaining of the peribulbar matrix cells was weaker, if at all detectable. In contrast, epithelial cytokeratins were localized in both the inner and outer root sheath epithelia but, surprisingly, also in certain portions of the trichocyte column, notably cells of the cuticle, certain medullary cells, and trichocytes of the basalmost peripapillary cell layers. Cells coexpressing trichocytic and epithelial cytokeratins have been identified by double-label immunofluorescence microscopy. Epithelial cytokeratins of the inner and outer root sheath epithelia include, most remarkably, "simple-epithelium-type" cytokeratins 8, 18, and 19; these occur in certain peribulbar regions, in distinct patterns, but with variable frequencies. The occurrence of simple epithelial cytokeratins in hair follicles has also been confirmed by high-sensitivity immunoblotting of follicular polypeptides separated by gel electrophoresis. Vimentin-positive cells were abundantly interspersed (in some follicles, but not in all) between the trichocytes of the peripapillary cone, most of them probably being melanocytes. The cell-type complexity of the hair follicle and the different patterns of cytoskeletal protein expression in the various hair follicle cells are discussed in relation to the development and growth of this organ.     

Identifying pedigrees segregating at a major locus for a quantitative trait: an efficient strategy for linkage analysis

Having found evidence for segregation at a major locus for a quantitative trait, a logical next step is to identify those pedigrees in which major-locus segregation is occurring. If the quantitative trait is a risk factor for an associated disease, identifying such segregating pedigrees can be important in classifying families by etiology, in risk assessment, and in suggesting treatment modalities. Identifying segregating pedigrees can also be helpful in selecting pedigrees to include in a subsequent linkage study to map the major locus. Here, we describe a strategy to identify pedigrees segregating at a major locus for a quantitative trait. We apply this pedigree selection strategy to simulated data generated under a major-locus or mixed model with a rare dominant allele and sampled according to one of several fixed-structure or sequential sampling designs. We demonstrate that for the situations considered, the pedigree selection strategy is sensitive and specific and that a linkage study based only on the pedigrees classified as segregating extracts essentially all the linkage information in the entire sample of pedigrees. Our results suggest that for large-scale linkage studies involving many genetic markers, the savings from this strategy can be substantial and that, compared with fixed-structure sampling, sequential sampling of pedigrees can greatly improve the efficiency for linkage analysis of a quantitative trait.     

Control of Adipose Tissue Expandability in Response to High Fat Diet by the Insulin-like Growth Factor-binding Protein-4

Adipose tissue expansion requires growth and proliferation of adipocytes and the concomitant expansion of their stromovascular network. We have used an ex vivo angiogenesis assay to study the mechanisms involved in adipose tissue expansion. In this assay, adipose tissue fragments placed under pro-angiogenic conditions form sprouts composed of endothelial, perivascular, and other proliferative cells. We find that sprouting was directly stimulated by insulin and was enhanced by prior treatment of mice with the insulin sensitizer rosiglitazone. Moreover, basal and insulin-stimulated sprouting increased progressively over 30 weeks of high fat diet feeding, correlating with tissue expansion during this period. cDNA microarrays analyzed to identify genes correlating with insulin-stimulated sprouting surprisingly revealed only four positively correlating (Fads3, Tmsb10, Depdc6, and Rasl12) and four negatively correlating (Asph, IGFbp4, Ppm1b, and Adcyap1r1) genes. Among the proteins encoded by these genes, IGFbp4, which suppresses IGF-1 signaling, has been previously implicated in angiogenesis, suggesting a role for IGF-1 in adipose tissue expandability. Indeed, IGF-1 potently stimulated sprouting, and the presence of activated IGF-1 receptors in the vasculature was revealed by immunostaining. Recombinant IGFbp4 blocked the effects of insulin and IGF-1 on mouse adipose tissue sprouting and also suppressed sprouting from human subcutaneous adipose tissue. These results reveal an important role of IGF-1/IGFbp4 signaling in post-developmental adipose tissue expansion.