Free Edges in Epithelial Cell Sheets Stimulate Epidermal Growth Factor Receptor Signaling

The ability of epithelia to migrate and cover wounds is essential to maintaining their functions as physical barriers. Wounding induces many cues that may affect the transition to motility, including the immediate mechanical perturbation, release of material from broken cells, new interactions with adjacent extracellular matrix, and breakdown of physical separation of ligands from their receptors. Depending on the exact nature of wounds, some cues may be present only transiently or insignificantly. In many epithelia, activation of the epidermal growth factor receptor (EGFR) is a central event in induction of motility, and we find that its continuous activation is required for progression of healing of wounds in sheets of corneal epithelial cells. Here, we examine the hypothesis that edges, which are universally and continuously present in wounds, are a cue. Using a novel culture model we find that their presence is sufficient to cause activation of the EGFR and increased motility of cells in the absence of other cues. Edges that are bordered by agarose do not induce activation of the EGFR, indicating that activation is not due to loss of any specific type of cell–cell interaction but rather due to loss of physical constraints.     

Human transferrin polymorphism

The application of isoelectric focussing (IEF) has revealed a large amount of heterogeneity in the human transferrin (TF) system and has enhanced its potential value in anthropological and genetic studies. The average heterozygosity has been elevated from 0.05, observed by conventional methods of electrophoresis, to 0.29 detected by IEF. So far approximately 30,000 individuals from 122 population groups have been analyzed for TF subtypes to evaluate the magnitude of genetic variation at the TF locus. Possible environmental and biological factors, which may be operating to maintain the TF polymorphism, are discussed.     

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.     

DNA “fingerprints” and paternity ascertainment in chimpanzees (Pan troglodytes)

Highly variable regions of DNA are found in a wide diversity of organisms and are typically composed of alleles consisting of a variable number of tandem repeats (VNTRs) of a short core sequence. DNA fingerprinting probes are VNTR probes that simultaneously detect a large number of similar VNTRs in the target DNA. The highly polymorphic pattern observed in a DNA fingerprint allows resolution of questions concerning individual identification. M13 phage was used to fingerprint captive chimpanzees for paternity ascertainment. Although the probability of band sharing among captive chimps appears to be higher than among some other reported captive and feral animal populations, the probe is highly useful and can be expected to become more widely used in the genetic management of captive populations.     

Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.

Intact human platelets, terminally differentiated cells with no growth potential, were found to possess unusually high levels of tyrosine-specific protein phosphorylation. The physiological platelet activator thrombin transiently elevated platelet phosphotyrosine content, apparently through stimulation of one or more tyrosine-specific protein kinases. Immunoblotting with antiphosphotyrosine antiserum showed that thrombin caused dramatic changes in the tyrosine phosphorylation of a number of individual protein bands and that these changes occurred in three distinct temporal waves. Most but not all of the protein bands phosphorylated at tyrosine in response to thrombin were also tyrosine phosphorylated in response to chilling or the combination of ionophore A23187 and tetradecanoylphorbol acetate. Thrombin stimulated the phosphorylation of the tyrosine kinase pp60c-src, primarily at Ser-12 and Tyr-527, although the effects of these phosphorylations on platelet pp60c-src function were not apparent. Together, these results suggest that tyrosine-specific protein kinases of uncertain identity are involved in signal transduction in platelets.     

Genetic studies of human apolipoproteins. V. A novel rapid procedure to screen apolipoprotein E polymorphism

A simple and new method has been developed to detect apolipoprotein E polymorphism directly from plasma or serum without prior ultracentrifugation and delipidation. The method combines the use of dialyzed plasma or serum samples with or without neuraminidase treatment followed by monodimensional isoelectric focusing in simple or 3 M urea gels at a constant low power and progressively increasing voltage over a 3-hr period, and finally protein blotting to a nitrocellulose membrane. Apolipoprotein E phenotypes are identified immunologically using a double antibody reaction, the primary antibody being a monospecific, polyclonal goat anti-apolipoprotein E, and the secondary antibody being a rabbit anti-goat IgG conjugated with alkaline phosphatase. The method was employed to screen apolipoprotein E polymorphism in two white populations in the United States. The frequency values are comparable to those reported previously by other investigators using conventional detection methods. The procedure is simple, accurate, suitable for large scale epidemiologic, clinical, and genetic studies.     

Comparison of five tandem repeat loci between humans and chimpanzees.

Five tandem repeat loci were studied in humans and chimpanzees using VNTR probes derived from human DNA. Shared alleles were found at three loci and were often the modal allele in one species but never in both. There was no difference in the mean number of alleles per locus. However, these species exhibited substantially different levels of gene diversity, with chimpanzees monomorphic at two loci. Evidence of reduced variability in chimpanzees corroborates earlier comparisons using isozymes and plasma proteins. Molecular mechanisms, population dynamics, or both may be responsible for these differences. Equal numbers of alleles per locus may reflect high mutation rates. By one test, chimpanzees were out of equilibrium at one locus, which may reflect a typing error or population substructure. The long divergence time, and the high probability of backward mutations, precludes accurate estimation of genetic distance between these species.