Mouse epidermal cell renewal after topical treatment with different concentrations of the epidermal peptide pyroGlu-Glu-Asp-Ser-GlyOH

Earlier work has shown that epidermal cells contain a peptide, pyroGlu-Glu-Asp-Ser-GlyOH, that induces a moderate but long-lasting inhibition of epidermal cell proliferation when given at low (picomol) doses ip in vivo and in vitro. In the present study, the epidermal pentapeptide was applied topically to the back skin of hairless mice at different concentrations and in a water-miscible cream. A single topical application of either high (0.25% wt/wt) or low (0.004% or 0.02% wt/wt) doses of the pentapeptide was followed by oscillations in epidermal DNA synthesis and G2-M cell flux (mitotic rate). In general, epidermal cell proliferation was inhibited during the first 10-day period after treatment with the two lower doses, while the highest concentration of pentapeptide (0.25%) stimulated epidermal cell proliferation. In spite of the effects on epidermal cell proliferation the size of the epidermal cell population in the treated area (number of nucleated cells and epidermal thickness) showed no corresponding alterations. The results could imply that the epidermal pentapeptide modifies epidermal cell proliferation and terminal differentiation in such a way that the two are balance with each other.     

Control of epidermal stem cell clusters by Notch-mediated lateral induction

Stem cells in the basal layer of human interfollicular epidermis form clusters that can be reconstituted in vitro. In order to supply the interfollicular epidermis with differentiated cells, the size of these clusters must be controlled. Evidence suggests that control is regulated via differentiation of stem cells on the periphery of the clusters. Moreover, there is growing evidence that this regulation is mediated by the Notch signalling pathway. In this paper, we develop theoretical arguments, in conjunction with computer simulations of a model of the basal layer, to show that regulation of differentiation is the most likely mechanism for cluster control. In addition, we show that stem cells must adhere more strongly to each other than they do to differentiated cells. Developing our model further we show that lateral-induction, mediated by the Notch signalling pathway, is a natural mechanism for cluster control. It can not only indicate to cells the size of the cluster they are in and their position within it, but it can also control the cluster size. This can only be achieved by postulating a secondary, cluster wide, differentiation signal, and cells with high Delta expression being deaf to this signal.     

Spectrophotometric determination of peptide transport with chromogenic peptide mimetics

A spectrophotometric assay to determine peptide transport has been developed. Using two chromogenic peptide mimetics, L-phenylalanyl-L-2-sulfanilylglycine (PSG) and L-phenylalanyl-L-3-thiaphenylalanine (PSP), the peptide transport patterns in individual cell species can be evaluated effectively. After the addition of PSG to a HeLa cell suspension, sulfanilic acid accumulated progressively inside, but not outside, the cells, demonstrating that PSG was transported wholly intact. The addition of PSP to the same cell suspension was followed immediately by extracellular thiophenol production. Measurement of the rate of thiophenol release thereby provided direct determination of PSP transport. The thiophenol release was consistent with Michaelis-Menten kinetics, with a K(m) of 0.016 mM and a V(max) of 5.07 nmol/min (1 x 10(6) cells/ml, pH 7.4, 37 degrees C). The resulting kinetic constants estimated were in agreement with values determined by single-substrate enzyme kinetics. Using PSP, transport kinetics of various dipeptides was examined by competitive spectrophotometry. As a result, dipeptides tested could be ranked in order of kinetic power for their transport.     

The different structures containing tight junction proteins in epidermal and other stratified epithelial cells, including squamous cell metaplasia

In stratified squamous epithelia constituent proteins of tight junctions (TJs) are not restricted to the zonula occludens-related structures of the uppermost living cell layer such as the stratum granulosum of the epidermis but TJ membrane proteins such as occludin and certain members of the claudin family as well as TJ plaque proteins, notably cingulin and protein ZO-1, have also been identified by immunofluorescence and immunoelectron microscopy in more basal layers where they form special cell-cell-connecting structures such as the "lamellated" and the "sandwich" junctions. In the present study, we describe another TJ protein-containing structure, the very small puncta occludentia ("stud junctions"), as the smallest identifiable TJ-like unit that occurs in most, perhaps all strata. We have also determined the specific distributions of TJ proteins in the cell layers of squamous cell metaplasias of the human bronchial tract. Moreover, we show that the occludin-related tetraspanin protein tricellulin-alpha connects and seals the membranes of adjacent "three corner" cell structures of the uppermost layer in keratinocytes growing in culture. We hypothesize the possible occurrence of tricellulin-beta in more basal cell layers of keratinocyte cultures and the general occurrence of different tricellulin splice forms in stratified epithelia in situ, and discuss the possible functions of TJ proteins in stratified epithelia and tumors derived therefrom.     

Buckling of inner cell wall layers after manipulations to reduce tensile stress: observations and interpretations for stress transmission

The inner layer of the cell wall in tissues that are under tensile stress in situ, e.g. epidermis and collenchyma of etiolated sunflower hypocotyls, shows a pattern of transverse folds when the tissues are detached and plasmolysed. This can be observed by Nomarski imaging of inner surfaces of the outer cell walls and electron microscopy of longitudinal sections after peeling the epidermis and bathing it in plasmolysing solutions. The folds are apparently caused by buckling of the inner layer due to the longitudinal compressive force exerted on this layer by the outer wall layer, when it shrinks after the removal of the longitudinal tensile stresses. In these stresses, two components can be distinguished: the tissue stress, disappearing on peeling, and that caused directly by turgor pressure, disappearing in hyperosmotic solution. Investigation of the buckling indicates that the outer layer of the cell wall transmits in situ most of the longitudinal tensile stress in the wall. The common concept that the inner layer of the wall is the region bearing most stress and therefore regulating growth can still be valid with respect to the transverse stress component.     

Consensus sequence for processing of peptide precursors at monobasic sites

Many regulatory peptide precursors undergo post-translational processing at mono- and/or dibasic residues. Comparison of amino acids around the monobasic cleavage sites suggests that these cleavages follow certain sequence motifs and can be described as the rules that govern monobasic cleavages: (i) a basic amino acid it present at either 3, 5, or 7 amino acids N-terminal to the cleavage site, (ii) hydrophobic aliphatic amino acids (leucine, isoleucine, valine, or methionine) are never present in the position C-terminal to the monobasic amino acid at the cleavage site, (iii) a cysteine is never present in the vicinity of the cleavage site, and (iv) an aromatic amino acid is never present at the position N-terminal to the monobasic amino acid at the cleavage site. In addition to these rules, the monobasic cleavages follow certain tendencies: (i) the amino acid at the cleavage site tends to be predominantly arginine, (ii) the amino acid at the position C-terminal to the cleavage site tends to be serine, alanine or glycine in more than 60% of the cases, (iii) the amino acid at either 3, 5, or 7 position N-terminal to the cleavage site tends to be arginine, (iv) aromatic amino acids are rare at the position C-terminal to the monobasic amino acid at the cleavage site, and (v) aliphatic amino acids tend to be in the two positions N-terminal to and the two positions C-terminal to the cleavage site, except as noted above. When compared with a large number of sequence containing single basic amino acids, these rules and tendencies are capable of not only correctly predicting the processing sites, but also are capable of excluding most of the single basic sequences that are known to be uncleaved. Many or these rules can also be applied to correctly predict the dibasic and multibasic cleavage sites suggesting that the rules and tendencies could govern endoproteolytic processing at the monobasic, dibasic and multibasic sites.     

Total epidermal cell walls of pea stems respond differently to auxin than does the outer epidermal wall alone

The effect of auxin on cell wall mass in the epidermis of third internodes of Pisum sativum L. cv. Alaska grown in dim red light was investigated using epidermal peels, to determine whether epidermal peels reflect the behavior of the outer epidermal cell wall. In contrast to the outer epidermal wall itself, where auxin caused thinning in proportion to growth (M.S. Bret-Harte et al, 1991, Planta 185, 462–471), auxin promoted an increase in wall mass in epidermal peels from treated internode segments in the absence of exogenously supplied sugar. The percentage gain in mass was smaller than the percentage elongation, however, so mass per unit length decreased in peels from auxin-treated segments. Epidermal peels from auxin-treated segments gained more wall mass than control peels even when adhering internal tissue at the basal end of the peel was removed. Epidermal peels also had a gross composition different from that of the outer wall alone (M.S. Bret-Harte and L.D. Talbott, 1993, Planta 190, 369–378). These discrepancies can be explained by the observation that the outer wall makes up only 30% of the mass of the epidermal peel. It appears that the inner walls of the epidermis, and walls of the outer layer of cortical cells that remain attached to the epidermis during peeling, nearly maintain their thickness by biosynthesis while the outer wall loses mass as previously described (Bret-Harte et al. 1991). These results indicate that epidermal peels may not be a good system for examining the biochemical and physiological properties of the outer epidermal cell wall.I would like to thank Dr. Peter M. Ray, of Stanford University, for the use of experimental facilities, helpful discussions, and technical and editorial assistance, Dr. Winslow R. Briggs, of the Carnegie Institute of Washington, for helpful discussions and for the use of experimental facilities, Dr. Paul B. Green, of Stanford University, for financial support, and Dr. Wendy K. Silk, of the Department of Land, Air, and Water Resources, University of California, Davis, for financial support. This work was supported by a National Science Foundation Graduate Fellowship, National Science Foundation grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk in the final writing.     

Mouse liver regeneration after carbon tetrachloride injury as test system for hepatic growth regulators

A test system for growth regulators based on the time course of liver regeneration in male NMRI mice injected intraperitonelly (ip) with 50 nmol CC1₄ at 12 is described. Regenerative DNA synthesis (labelling index) peaked at 36 h after CC1₄ injury, and the Colcemid-assessed mitotic rate (MR) at 42 h, i.e., 6 h later. This response pattern was used to assess the effects of factors in liver extracts that regulate or modulate hepatocyte proliferation. The effect of one, two, four or eight ip injections of an aqueous mouse liver extract on MR was tested at 48 h. A 30–70% inhibition was seen only after single injections at 12 h, 29 h or 44 h after CCl₄ treatment. A 30–80% stimulation was observed after a single injection of the liver extract at 0, 5 or 24 h, and after two or four injections. The assay system could thus detect the presence of growth modulators in the extract. The experiments also showed that the timing was crucial. We recently isolated and characterized a growth inhibitory pentapeptide from mouse liver extracts. Using a synthetic pentapeptide with the same structure we reassessed the timing for growth inhibition seen with the liver extract. The following test system for growth inhibitors seemed most expedient: inhibitor administration at 29 h to affect G₁-S transition, measured as reduced DNA synthesis at 36 h, or inhibitor administration at 44 h to affect G₂-M transition, measured as reduced MR at 48 h.     

A study of cell interactions involved in Pleurodeles waltlii epidermal differentiation

Summary A polyclonal antibody (SP-2) has been produced, which recognizes antigens expressed in epidermal cells of Pleurodeles waltlii embryos. The antigens appear first at the end of gastrulation in the external surface of the embryo and are selectively expressed in ectodermally derived epidermal structures. Ectodermal commitment was investigated using cell cultures and blastocoel graft experiments. The four animal blastomeres of the 8-cell stage as well as the animal cap explants of the early gastrula stage cultured in vitro differentiate into epidermis, and SP-2 antigens are expressed. The expression of SP-2-defined antigens is inhibited both in vivo and in vitro by the inductive interaction of chordomesoderm. Once dissociated, ectodermal cells do not react with SP-2. Conversely, the aggregation of ectodermal cells may restore the expression of SP-2 antigens. Transplantation of animal cap explants or isolated ectodermal cells into the blastocoel of a host embryo at the early gastrula stage shows that only cells integrated into the epidermis express the marker antigens. When vegetal cells were dissociated from donor embryos before the mid-blastula stage and implanted into the blastocoel of host embryos at the early gastrula stage, their progeny were found in all germ layers, cells that were found in the host epidermis were stained with SP-2, whereas those contributing to mesoderm and endoderm were not. Thus the acquisition of cell polarity in epidermal differentiation and the organization of cells into epithelial structures are essential for SP-2-defined antigen expression.     

In ovo peptide YY and epidermal growth factor administration and their effects on growth and yolk utilization in neonatal meat-type chickens (Gallus domesticus)

The effects of in ovo peptide YY (PYY) or epidermal growth factor (EGF) administration on chick growth, yolk absorption and yolk stalk function in posthatch (0–5 days) meat-type or broiler chicks were determined. At Day 18 of incubation, treated eggs were injected into the air cell with 100 μl of either PYY (Trial 1) or EGF (Trial 2) at a dosage of 600 μg/kg egg weight. Saline-treated control eggs were injected similarly with 0.9% saline. At hatch, 200 μl of ⁵¹Cr-labeled microspheres were injected into chick yolk sacs. Epidermal growth factor increased ileal wet weight adjusted for body weight as well as ileal serosal dry matter. Body weight, feed consumption and excreta weight per bird, and relative weights of the yolk sac, intestine and liver were significantly affected by age of the chick in both trials. Relative radioactivity of the yolk sac, yolk stalk, blood, liver, and kidneys were affected by bird age in Trial 2; however, there were no significant effects due to PYY or EGF treatments on relative radioactivity of the tissues and organs examined. These data suggest that PYY and EGF had no effect on yolk absorption or yolk stalk function through 5 days in the posthatch chick.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphophate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.     

The flexible termini of conantokin G define its interactions with NMDA receptors

Converging lines of evidence suggest that the N-methyl-D-aspartic acid (NMDA) antagonistproperties of conantokin G (ConG) are mediated through a novel polyamine-associated site.Moreover, structural modification of the heptadecapeptide yields peptides that can eithermimic the NMDA antagonist properties of the parent peptide or produce polyamine-likeactions at NMDA receptors. We synthesized a panel of ConG fragments and evaluated theireffects using a neurochemical assay that predicts pharmacological actions at NMDA receptors.While the C-terminal tetrapeptide elicited a polyamine-like activation of [3H]MK-801 bindingwith a potency comparable to spermine, the N-terminal pentapeptide produced a marginalinhibition of spermine-enhanced [3H]MK-801 binding. These observations suggest that theparent peptide interacts with two distinct sites on NMDA receptors. In contrast, amino acidreplacements in the middle region of ConG resulted in analogues that were of comparable orgreater potency than the parent peptide. The Ala7,Tyr10 derivative is of particular interestsince it is a potent inhibitor (IC50 80 nM) of spermine-enhanced [3H]MK-801 binding, andmay thus serve as a precursor for studies designed to 125I-label putative ConG binding sites.Our observations are also consistent with the hypothesis that the termini of ConG are essentialfor an interaction with NMDA receptors, while the middle region of this peptide serves as aspacer unit. This hypothesis is consonant with spectroscopic evidence that ConG possessesa central rigid helical backbone with flexible N- and C-terminal regions. Nonetheless, ConGvariants in which the termini were connected with conformationally stabilized- or 310-helical spacers grew progressively less potent as NMDA antagonists as the structural stabilityof these peptides increased. Thus, the middle region of ConG appears to possess functionsother than providing conformational stability. These newly synthesized ConG derivatives mayserve as a basis for the design of novel peptidic or peptidomimetic agents.     

The flexible termini of conantokin G define its interactions with NMDA receptors

Summary Converging lines of evidence suggest that theN-methyl-d-aspartic acid (NMDA) antagonist properties of conantokin G (ConG) are mediated through a novel polyamine-associated site. Moreover, structural modification of the heptadecapeptide yields peptides that can either mimic the NMDA antagonist properties of the parent peptide or produce polyamine-like actions at NMDA receptors. We synthesized a panel of ConG fragments and evaluated their effects using a neurochemical assay that predicts pharmacological actions at NMDA receptors. While the C-terminal tetrapeptide elicited a polyamine-like activation of [³H]MK-801 binding with a potency comparable to spermine, the N-terminal pentapeptide produced a marginal inhibition of spermine-enhanced [³H]MK-801 binding. These observations suggest that the parent peptide interacts with two distinct sites on NMDA receptors. In contrast, amino acid replacements in the middle region of ConG resulted in analogues that were of comparable or greater potency than the parent peptide. The Ala⁷, Tyr¹⁰ derivative is of particular interest since it is a potent inhibitor (IC₅₀≈80 nM) of spermine-enhanced [³H]MK-801 binding, and may thus serve as a precursor for studies designed to¹²⁵I-label putative ConG binding sites. Our observations are also consistent with the hypothesis that the termini of ConG are essential for an interaction with NMDA receptors, while the middle region of this peptide serves as a spacer unit. This hypothesis is consonant with spectroscopic evidence that ConG possesses a central rigid helical backbone with flexible N- and C-terminal regions. Nonetheless, ConG variants in which the termini were connected with conformationally stabilized α-or 3₁₀-helical spacers grew progressively less potent as NMDA antagonists as the structural stability of these peptides increased. Thus, the middle region of ConG appears to possess functions other than providing conformational stability. These newly synthesized ConG derivatives may serve as a basis for the design of novel peptide or peptidomimetic agents.     

Chronic systemic treatment with epidermal growth factor in the rat increases the mucosal surface of the small intestine

We examined the effects of treatment with human recombinant epidermal growth factor (EGF) on the functioning small intestine in the rat. Male Wistar rats, 7–8 weeks old, were treated with EGF administered subcutaneously in doses of 0 (n = 7) or 150 μg/kg/day (n = 8) for 4 weeks. The histological composition and mucosal surface area of the perfusion-fixed small intestine was quantified with stereological principles. The length of the gut remained unchanged. The amount of tissue and surface area per length of gut (median (ranges)) were increased from 117 (101–131) mg/cm and 2.6 (2.1–3.5) cm²/cm in the controls to 146 (138–152) mg/cm and 3.5 (2.5–3.8) cm²/cm for the complete small intestine (both comparisons p < 0.02). The weight increase was due to mucosal growth in all parts of the intestine, whereas the surface area was only increased in proximal and middle parts. It is concluded that EGF treatment in rats increases the mucosal weight and surface area of the functioning small intestine.     

Evaluation on the effect of different in-gel peptide isoelectric focusing parameters in global proteomic profiling

Peptide isoelectric focusing (IEF) is a common technique used in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) proteomic workflow, in which the tryptic peptide is first pre-fractionated based on pI values before being subjected to reverse phase LC–MS analysis. Although this method has been widely used by many research groups, a systemic study on the optimal conditions and fundamental parameters influencing the experimental outcomes has been lacking, including the effect of peptide extraction methods, the extent of pre-fractionation, and the choice of pH range. In this study, we compared the effect of different parameters on the numbers of peptides and proteins identified using two complex mouse proteomes. The results indicated that extraction of peptides from immobilized pH gradient (IPG) strips by sequential elution of increasingly organic solvents provided the highest number of peptide identification. In addition, we showed that approximately 45 more unique proteins were identified for every additional fraction collected during peptide IEF. Although narrow pH ranges provided higher resolution in peptide separation as expected, different pH ranges yielded similar numbers of peptide and protein identification. Overall, we demonstrated that the extraction solvent influenced the numbers of peptide and protein identification and quantitatively demonstrated the advantage of extensive fractionation and the performance of different pH ranges in practice.     

Dendritic cell acquisition of epitope cargo mediated by simple cationic peptide structures

In this study we evaluate the uptake by murine dendritic cells (DCs) of different synthetic, branched cationic peptide structures with a view to facilitating peptide epitope delivery. The level of cell uptake by fluorescenated peptides was measured by flow cytometry following quenching of extracellular fluorescence with trypan blue. Branched peptides containing either N-terminal arginine or N-terminal lysine residues were able to mediate cell entry but the peptide containing four arginine residues in a branching configuration (R₄) was found to be superior not only to other branched peptides in translocating to the cell interior and also to a peptide containing four arginine residues arranged linearly. Fluorescenated R₄ was found to be localized within intracellular vesicle-like compartments as well as being distributed throughout the cell cytoplasm. Uptake of R₄ utilized an energy-dependent process that appeared to involve phosphatidylinositol-3-kinase and could induce intermediate levels of DC maturation. R₄ when conjugated to a T-helper cell and CTL epitope construct was able to induce antigen-specific CD8⁺ T-cell mediated immune responses in mice when administered in adjuvant as were DCs that were pulsed with this construct and then matured with LPS. Fluorescenated R₄ was also found to translocate into the interior of other cell types indicating that it may be useful for the delivery of peptide cargo into other specialized cells.     

A leucine zipper-based peptide hybrid delivers functional Nanog protein inside the cell nucleus

We synthesized a pair of compounds containing leucine zipper peptides to deliver protein cargo into cells. One is a cell-penetrating peptide (CPP) with Lz(E), a leucine zipper peptide containing negatively charged amino acids, and the other is a Nanog protein with Lz(K), a leucine zipper peptide containing positively charged amino acids. When cells were treated with these equimolar mixtures, Nanog-Lz(K) hybridized with Lz(E)-CPP was successfully delivered into the cells. Furthermore, Nanog-Lz(K) exerted its proper function after nuclear transport.