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Biomedical subjects

F M Watt

Publications and source records attributed to F M Watt.

141 records · Page 8Linked to original sources
PubMed1987

Influence of cell shape and adhesiveness on stratification and terminal differentiation of human keratinocytes in culture.

Human epidermal keratinocytes can be grown in culture under conditions in which they assemble a tissue with the same basic organization as normal epidermis. The cells stratify, mitosis is restricted to the basal layer and terminal differentiation occurs as the cells move through the suprabasal layers. Keratinocytes do not have to leave the basal layer in order to undergo terminal differentiation, but the two processes are normally linked, because during terminal differentiation the adhesive affinity of keratinocytes for the culture substratum and for other keratinocytes is reduced. Down-regulation of synthesis of basement membrane components and their receptors may provide the molecular basis for the reduction in cell-substratum adhesiveness. However, the molecules that mediate changes in cohesiveness have not yet been identified. Restriction of substratum contact, so that cells are prevented from spreading, appears to be one signal that induces keratinocytes to stop dividing and undergo terminal differentiation.

Cell Adhesion↗
PubMed1986

Immunolocalization of keratan sulphate in the human embryonic cornea and other human foetal organs.

An antibody raised against keratan sulphate was used to localize keratan sulphate in the human embryonic cornea and other human foetal organs. It was found that keratan sulphate was present in the corneal endothelium, as well as in the corneal stroma. In contrast, no keratan sulphate could be demonstrated in the corneal epithelium or in any posterior parts of the human foetal eye. Human corneal stromal cells grown in monolayers in tissue culture lost their capacity to bind this antibody. This suggests that explantation in vitro decreases or alters the expression of keratan sulphate in this cell type. Keratan sulphate was found to be present in abundant quantities in human embryonic cartilage and to a lesser extent in placenta. In contrast, keratan sulphate could not be detected in the foetal intestine, liver, suprarenal glands, the umbilical cord or in the foetal yolk sack.

Antibodies, Monoclonal↗
PubMed1985

Biosynthesis of EGF receptor, transferrin receptor and colligin by cultured human keratinocytes and the effect of retinoic acid.

The biosynthesis of EGF and transferrin receptor by human keratinocytes in culture has been followed using specific monoclonal antibodies. In addition, keratinocytes are shown to synthesise a Mr 47 000 protein that binds to gelatin-Sepharose. Peptide mapping confirms the identity of this protein with colligin, a newly described cell surface-associated glycoprotein that also binds to native collagens (Kurkinen et al., J biol chem 259 (1984) 5915) [9]. Vitamin A and its analogues have profound effects on the differentiation, morphology and motility of human keratinocytes in culture. We show here that retinoic acid (RA) has no effect on the growth rate of the cells or the synthesis of EGF receptor and colligin, but stimulates the synthesis of transferrin receptor.

Antibodies, Monoclonal↗
PubMed1985

Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate.

We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan sulfate. Keratan sulfate-negative chondrocytes were shown to come from the upper cartilage layers. There was therefore a direct correlation between biochemical heterogeneity of cartilage matrix and heterogeneity within the chondrocyte population. During growth in monolayer culture, superficial chondrocytes began to synthesize keratan sulfate, but the cells could still be distinguished from cultures of deep or unfractionated chondrocytes by their reduced substrate adhesiveness and tendency to remain rounded.

Animals↗
PubMed1985

Biochemical specificity of Xenopus notochord.

The biochemical composition and biosynthetic activity of Xenopus notochord were examined and compared with those of chick and mouse notochord. The notochords of all three species contain type-II collagen, and the notochords of Xenopus and chick synthesize a soluble glycoprotein with a molecular mass of 86 kilodaltons (kd). Mouse embryos were not tested for this molecule, because their notochords are too small to be dissected out. Most interestingly, Xenopus and chick notochords share a keratan-sulphate-containing proteoglycan which appears to be absent from mouse notochord. The presence or absence of keratan sulphate in the notochords of the different species reflects its presence or absence in cartilage. Since one role of the notochord in vivo is to stimulate chondrogenesis in the sclerotomes of the somites, this result provides support for the view that cells responding to the extracellular matrix produced by one tissue do so by increasing their production of the same matrix components.

Animals↗
PubMed1984

Selective migration of terminally differentiating cells from the basal layer of cultured human epidermis.

How terminally differentiating cells are selectively expelled from the basal layer of epidermis has been a source of interest and speculation for many years. The problem can now be studied in culture, using involucrin synthesis as an early marker of terminal differentiation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the calcium ion concentration of the culture medium, they still begin to synthesize involucrin. Raising the level of calcium ions induces stratification, and cells that are synthesizing involucrin are selectively expelled from the basal layer. I have found that during calcium-induced stratification no new proteins or glycoproteins are synthesized, and the rate of cell division does not change. Movement of involucrin-positive cells out of the basal layer was found to be unaffected by cycloheximide, tunicamycin, or cytosine arabinoside. These results suggest that keratinocytes growing as a monolayer already have the necessary properties to determine their position when stratification is induced. Addition of calcium simply allows formation of desmosomes and other intimate cell contacts required for stratification. The properties of involucrin-positive cells that determine their suprabasal position include a reduced affinity for the culture substrate and preferential adhesion to other cells at the same stage of terminal differentiation. The molecular basis of these adhesive changes is discussed.

Calcium↗
PubMed1984

Calcium-induced reorganization of desmosomal components in cultured human keratinocytes.

We used antibodies raised against individual desmosomal components to study calcium-induced desmosome formation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the level of calcium ions in the culture medium, there is little contact between adjacent cells. Raising the level of calcium ions rapidly induces desmosome formation, and stratification occurs within 24 h. We found that before addition of calcium the 115,000- and 100,000-mol-wt core glycoproteins were distributed over the entire cell surface, whereas the plaque proteins (205,000 and 230,000 mol wt), the 82,000- and 86,000-mol-wt proteins, and the 150,000-mol-wt glycoprotein were located throughout the cytoplasm. 15 min after increasing the calcium ion concentration, all of these molecules appeared at the cell margins. The intensity of peripheral staining increased over the next 2 h and during this time the distribution of keratin filaments changed from predominantly perinuclear to extend throughout the cytoplasm. Keratinocytes could be dissociated with EDTA for up to 2 h after exposure to calcium. After 3 h of exposure to calcium the cells were no longer susceptible to EDTA dissociation and staining for desmosomal plaque antigens persisted in regions of intercellular contact. Desmosomal staining in stratified cultures became greatly reduced within 24 h of lowering the calcium ion concentration again. We have preliminary evidence that stratification occurs by breakdown of desmosomes at lateral surfaces and reformation at surfaces of contact between basal and suprabasal cells, rather than by rearrangement of existing desmosomes. Involucrin-positive cells in the monolayer appeared to contain more 205,000- and 230,000-mol-wt proteins free in the cytoplasm than involucrin-negative cells.

Antigens↗
PubMed1983

Involucrin and other markers of keratinocyte terminal differentiation.

Human epidermal keratinocytes in culture form stratified colonies that retain many of the properties of intact epidermis. Mitosis is restricted to the basal layer, and cells that leave it undergo terminal differentiation, increasing in size and synthesizing a range of specialized molecules as they pass through the different cell layers. One useful marker for an early stage in the pathway of terminal differentiation is involucrin, a soluble protein precursor of the cross-linked envelope. Involucrin is synthesized after keratinocytes have left the basal layer and begun to enlarge, but some time before the onset of envelope cross-linking, which occurs only in the outermost cell layers. Small, involucrin-negative cells can be isolated and cultured in medium containing a low concentration of calcium ions, to prevent stratification, but not cell division and enlargement. Under these conditions, involucrin synthesis is initiated by some cells in the monolayer, indicating that attainment of a suprabasal position is not required for this step in terminal differentiation. Furthermore, if the level of calcium is raised to induce stratification, involucrin-positive cells are selectively expelled from the basal layer. This suggests that migration from the basal layer may be a consequence, not a cause, of terminal differentiation. Changes in cell-surface properties during terminal differentiation might explain the observed sorting out of involucrin-positive and -negative cells. We have therefore investigated lectin binding to different cell layers in natural and cultured human epidermis. Some lectins bind only to nonbasal keratinocytes, and the binding of peanut agglutinin, in particular, shows interesting correlations with involucrin expression, both in stratified colonies and in monolayers.

Cell Differentiation↗
PubMed1982

Regulation by vitamin A of envelope cross-linking in cultured keratinocytes derived from different human epithelia.

When keratinocytes derived from different squamous epithelia are cultured in the absence of vitamin A, they form cross-linked envelopes during the last stage of terminal differentiation. Addition of the vitamin inhibits envelope formation, but the degree of inhibition is not the same for different keratinocyte subtypes. In the presence of low concentrations of retinyl acetate, conjunctival keratinocytes form virtually no cross-linked envelopes; esophageal and vaginal keratinocytes are less sensitive to the vitamin, and epidermal keratinocytes are the least sensitive. The suppression of cross-linked envelope formation is not associated with a proportional decrease in the concentration of involucrin, a precursor of the envelope, but occurs at the level of cross-linking itself, a process dependent on an increase in the intracellular concentration of calcium ions. Keratinocytes in which spontaneous envelope cross-linking has been prevented by retinyl acetate promptly form cross-linked envelopes if Ca2+ is introduced into the cytoplasm.

Calcium↗
PubMed1981

Involucrin synthesis is correlated with cell size in human epidermal cultures.

Late in terminal differentiation, human epidermal keratinocytes form an insoluble protein envelope on the cytoplasmic side of the plasma membrane. Involucrin, a soluble protein precursor of the envelope, is synthesized at an earlier stage of differentiation, both in the natural epithelium and in cultured keratinocytes. Because keratinocytes are known to enlarge during differentiation, we looked for a correlation between involucrin synthesis and cell size, using antiserum raised against the purified protein. We found that virtually no cultured epidermal keratinocytes with a diameter less than or equal to 14 micrometer contained involucrin, but most cells greater than 17 micrometer did. Using density gradient centrifugation, we were able to isolate a population of small cells containing almost no involucrin, as judged by immunodiffusion, PAGE, and immunoprecipitation. Large cells possessed translatable mRNA for involucrin, whereas small cells did not. We conclude that when cultured keratinocytes reach a certain size (approximately 14 micrometer in diameter) the specific mRNA for involucrin begins to accumulate and synthesis of the protein begins.

Cell Separation↗
PubMed1980

Microtubule-organizing centres in mammalian cells in culture.

When microtubules in animal cells are dissociated and then allowed to recover, they re-grow from discrete points in the cytoplasm known as microtubule-organizing centres (MTOC). The microtubules may be dissociated by antimitotic drugs such as colcemid, vinblastine and griseofulvin, or by chilling the cells. Immunofluorescence studies show that the number of MTOC seen does not depend on the concentration of antimitotic drug used or the duration of exposure to the drug; the same results are obtained with vinblastine, griseofulvin and colcemid. However, after cold treatment, the proprotion of cells showing only a single MTOC is greater than that seen with the antimitotic drugs and may exceed 90%. Evidence is presented for the view that procedures involving the use of antimitotic drugs give artificially high values for the number of MTOC per cell.

Animals↗
PubMed1980

Microtubule-organizing centres in binucleate cells and homosynkaryons.

Immunofluorescence studies showed that most binucleate Vero cells formed by virus-induced fusion or by inhibition of cytokinesis had a single microtubule-organizing centre (MTOC) when examined during the reassembly of microtubules after chilling, but two or more organizing centres when examined after exposure to colcemid. These findings suggest that although binucleate cells initially contain more MTOC than mononucleate cells, the extra MTOC are normally aggregated, so that the number of MTOC in binucleate cells tends to be reduced very quickly to that in mononucleate cells.

Animals↗
PubMed1978

The distribution of actin cables and microtubules in hybrids between malignant and non-malignant cells, and in tumours derived from them.

Cell fusion provides a genetic test for linkage between cellular phenotypic markers and malignancy. We have examined hybrid cells in which malignancy is suppressed and malignant segregants derived from them, to test whether the reappearance of malignancy is regularly associated with loss or abnormality of actin cables or microtubules. Immunofluorescence studies did not reveal any difference between the malignant and non-malignant hybrids in the number and distribution of microtubules that they contained. All the hybrids, whether tumorigenic or not, contained elaborate patterns of cytoplasmic microtubules. Furthermore, no consistent relationship was observed between the absence of actin cables and malignancy.

Actins↗