Cosmeceuticals.

The author uses kinetin, a plant-derived nucleotide, as an example to summarize the approach to advising a patient on a new product. (1) Does it penetrate the stratum corneum? Topically applied nucleotides can penetrate human skin, and one of the most active and useful of these for the treatment of actinic keratoses, 5-fluorouracil (5-FU), is used widely in dermatology. 5-FU derives some of its benefit from its uptake, specifically in actinic keratoses, which do not have a complete epidermal barrier. The molecule is able to penetrate the stratum corneum. It is not clear that topically applied nucleotides have the same degree of penetration on photodamaged skin without actinic keratoses. (2) Is there a plausible biochemical mechanism of action? 5-FU has a well-known mechanism of action (i.e., the inhibition of DNA/RNA synthesis by incorporation of a false pyrimidine analog). The mechanism of action for furfuryladenine in human skin remains unknown and needs to be shown. If furfuryladenine functions as an antioxidant, it may be useful as a photoprotectant. This function does not account for a mechanism of action for the reversal of photoaging, however. (3) Are there published peer-reviewed, double-blinded, placebo-controlled, statistically significant clinical trials to substantiate the efficacy claim? Peer-reviewed, double-blinded, statistically significant clinical trials on furfuryladenine have not been published to date. The field of cosmeceuticals presents a quandary. The list of cosmeceuticals for the dermatologist to assess grows longer each year. It is possible that some of the active ingredients are beneficial physiologically in human skin and that they can offer specific benefits, such as photoprotection. Further research needs to be conducted to validate these claims. It also is likely that a wide variety of molecules purported to be active in human skin do not have any physiologic benefit in human skin. Only further research can answer these difficult questions.

Neutrophilic dermatosis of the dorsal hands: pustular vasculitis revisited.

An entity termed "pustular vasculitis of the hands" was recently described. Patients with this condition presented with low-grade fevers and erythematous plaques, pustules, and bullae limited to the dorsal hands and fingers, which were characterized histologically by a dense neutrophilic infiltrate and leukocytoclastic vasculitis. We describe patients with a similar clinical presentation, but who lacked vasculitis on biopsy findings. We describe 3 otherwise asymptomatic patients with hemorrhagic bullae, plaques, and pustules solely on the dorsal hands. Biopsy specimens showed a neutrophilic infiltrate and leukocytoclasis, but no necrotizing vasculitis, and were reminiscent of Sweet's neutrophilic dermatoses. In our patients, corticosteroids or dapsone led to clearing of the lesions, and small maintenance doses of dapsone prevented their recurrence. Our 3 patients had clinical lesions similar to those termed pustular vasculitis of the hands, but which lacked leukocytoclastic vasculitis on biopsy findings. Because of histologic findings and a therapeutic response more characteristic of Sweet's syndrome, we propose the term neutrophilic dermatosis of the dorsal hands. In addition, low-dose dapsone is proposed as a possible first-line therapy in this condition, especially in those with recurrent disease.

Salicylic acid peels for the treatment of photoaging.

Several chemical agents are currently used to perform superficial chemical peels of the face. These include trichloracetic acid (15-30%), alpha-hydroxy acids (e.g., glycolic acid, 40-70%), and Jessner's solution (14% lactic acid, 14% resorcinol, and 14% salicylic acid). We have developed salicylic acid, a beta-hydroxy acid, at a higher strength (30% in a hydro-ethanolic vehicle) as an alternative peel. This peel has distinct advantages for resurfacing moderately photodamaged facial skin. We have peeled patients singly and multiply at 4-week intervals. The benefits are fading of pigment spots, decreased surface roughness, and reduction of fine lines.

Molecular analysis of microtubule-associated protein-2 kinase cDNA from mouse and rat brain.

We have isolated and characterized brain cDNA clones encoding microtubule-associated protein-2 (MAP-2) kinase for rat (rMNK1) and mouse (mMNK1). The nucleotide sequences diverged by only 5% whereas the amino acid sequences were identical except for one conservative residue change. Conservation of the expressed sequence extended into other mammalian species. These findings constitute the first demonstration of a strict evolutionary conservation of MAP-2 kinase. Genomic restriction patterns revealed a single MAP-2 kinase gene that shares homology with other genomic sequences. The 3' terminal half of the gene appears to be encoded by four exons. rMNK1 and mMNK1 differed from a recently reported MAP-2 kinase cDNA, termed ERK1, because of a nonconservative change in position 82, from Gly in ERK1 to Arg in rMNK1. The rMNK1 gene was found to be expressed mainly as a 1.8-kb transcript that was highest in brain and in lung. In contrast to ERK1, rMNK1 showed two equally prominent mRNA species in liver, at 1.8 kb and 5 kb, which imply differential processing of the primary transcript. Results derived from the immunological screening of an expression library showed that MAP-2 kinase might share epitopes with two prominent protein kinase C substrates, MARCKS (an 80-kD protein kinase C substrate) and GAP-43, suggesting the possibility that MAP-2 kinase could interact with kinase C.

Differential expression of an 80-kDa protein kinase C substrate in preneoplastic and neoplastic mouse JB6 cells.

An 80-kDa protein (p80), previously reported to be a major protein kinase C substrate in preneoplastic JB6 mouse epidermal cells, has been shown to be transiently phosphorylated by phorbol 12-O-tetradecanoate 13-acetate. Phosphorylation was maximal at 2 hr of phorbol 12-O-tetradecanoate 13-acetate treatment and returned to basal levels by 24 hr. In contrast, using a p80-specific antibody, we found that phorbol 12-O-tetradecanoate 13-acetate treatment produced no increase in p80 concentration. p80 showed a progressive decrease in JB6 cells during progression from a preneoplastic to neoplastic phenotype. The lack of p80 expression in neoplastic cells was not attributable to lack of protein kinase C; the protein kinase activity and protein concentration were similar in cells of all three phenotypes. When p80 mRNA was analyzed by hybridization to a putative p80 cDNA clone, its relative concentration paralleled that of p80 protein, with high levels present in preneoplastic JB6 cells, and little or no evidence for p80-hybridizing RNA in transformed cells. Thus, p80 appears to be regulated pretranslationally at the level of mRNA concentration during preneoplastic progression in mouse epidermal JB6 cells.

A major myristylated substrate of protein kinase C and protein kinase C itself are differentially regulated during murine B- and T-lymphocyte development and activation.

The regulation and expression of protein kinase C (PKC) and phosphomyristin C (PMC) (a principal substrate of PKC which is the major myristylated protein in lymphocyte and glioma lines that express it) in murine B and T lymphocytes were investigated. Both PMC and PKC are differentially regulated during T-cell development. The level of PMC expression is highest in CD4-8-, intermediate in CD4+8+, and lowest in J11d-, CD4, or CD8 single-positive thymocytes. PKC is equally expressed by all three thymic populations. In striking contrast to thymocytes, resting peripheral lymph node T cells and T-cell clones express little if any PMC and reduced levels of PKC. Neither PKC nor PMC is significantly induced upon the activation of lymph node T cells: treatment with anti-CD3 antibodies or anti-CD3 and interleukin-2 fails to induce PKC, whereas PMC is not induced by anti-CD3 alone and is only slightly induced by anti-CD3 and interleukin-2. In contrast to the situation with T cells, PMC and PKC are constitutively expressed at moderate levels in mature B cells. PMC is greatly increased in B-cell blasts generated by cross-linking the antigen receptor with anti-immunoglobulin. These results demonstrate that PMC and PKC are differentially regulated during the development and activation of B and T cells, suggesting that cellular events that rely upon PKC and PMC may differ during ontogeny and activation of different lymphocyte subsets.

Protein kinase C substrates from bovine brain. Purification and characterization of neuromodulin, a neuron-specific calmodulin-binding protein.

Although such solubility is uncommon among proteins generally, several bovine brain proteins were found to be soluble in 2.5% perchloric acid, and many of them were in vitro substrates for protein kinase C (Ca2+/phospholipid-dependent enzyme). Two of the perchloric acid-soluble brain proteins were purified, p43 and p17. P43 and p17 could be phosphorylated by protein kinase C only in the presence of Ca2+ and phospholipids and neither was a substrate for protein kinase II. P43 was subsequently identified as the neurospecific, calmodulin-binding protein, neuromodulin (also designated P-57, GAP43, B50, or F1) (Alexander, K. H., Wakim, B. T., Doyle, G. S., Walsh, K. A., and Storm, D. R. (1988) J. Biol. Chem. 263, 7544-7549). A rapid purification method for neuromodulin was developed taking advantage of its newly discovered property, solubility in 2.5% perchloric acid, and of its previously recognized calmodulin-binding property. Evidence was obtained that neuromodulin isolated from cytosolic extract exists as a mixture of molecular forms and that the Ca2+-binding S100 protein-beta discriminates among the different neuromodulin isoforms in forming covalent complexes via disulfide bridges; this discrimination may be explained by analogous differences observed between the NH2-terminal amino acid sequences of p57 and F1. Solubility in 2.5% perchloric acid was demonstrated for another rat brain protein kinase C substrate, p87. We suggest that perchloric acid solubility might be a common property of protein kinase C substrates.

Purification and characterization of an Mr 87,000 protein kinase C substrate from rat brain.

We (Kligman, D., and Patel, J. (1986) J. Neurochem. 47, 298-303) and others have previously identified a major protein kinase C substrate of apparent molecular weight 87,000 (Mr 87,000). To gain insight into the function of this potentially important phosphoprotein, we have undertaken its purification and characterization from rat brain. We now report a purification scheme involving heat treatment, ammonium sulfate precipitation, anion ion-exchange and reversed-phase chromatography. This procedure gave a Mr 87,000 that was homogeneous (based on silver staining), 1,600-fold enriched relative to heat-treated material and at a yield of approximately 58 micrograms/kg wet weight. We also report the amino acid composition to be high in acidic residues and in alanine and show the protein to be phosphorylated on serine residues with a stoichiometry of 2 mol of phosphate/mol of substrate. The subcellular distribution indicates Mr 87,000 is present in two forms, membrane-bound and soluble. The membrane-bound Mr 87,000 represents 45% of the total phosphoprotein content and is enriched in microsomal and synaptic membranes. Ontogenic study has revealed this protein to be developmentally regulated, with the highest concentrations of Mr 87,000 found in prenatal animals. The availability of a purification procedure should greatly facilitate further structural characterization and elucidation of the function of Mr 87,000.

Neurite extension factor induces rapid morphological differentiation of mouse neuroblastoma cells in defined medium.

The present study describes an in vitro bioassay for neurite-promoting factors using a mouse neuroblastoma cell line (Neuro-2A) in defined medium at low cell density. Neurite extension factor (NEF) is a disulfide-bonded dimer of a protein closely related to S100 beta, a calcium-binding protein made by glial cells. NEF induces process outgrowth from chick embryo cerebral cortical neurons. We now report that NEF induces rapid morphological differentiation of Neuro-2A cells. Within 4-6 h of addition of NEF, the cells elaborate multipolar neurites and the cell body becomes rounded. In the absence of NEF, the cells do not extend neurites and the cell body appears flattened. This response is dose-dependent, with half-maximal stimulation at a concentration of about 300 ng/ml (15 nM) of NEF. This completely defined system should be useful for characterizing NEF receptors and studying the mechanism of action of NEF.

A protein modulator stimulates C kinase-dependent phosphorylation of a 90K substrate in synaptic membranes.

We have identified and partially purified an acidic, heat-stable, noncalmodulin protein from bovine brain cytosol that stimulates Ca2+-dependent phosphorylation of an Mr 90K substrate in crude rat brain synaptic membranes. We show that this modulator of phosphorylation (MOP) enhances Ca2+- and phospholipid-dependent protein kinase (C kinase) phosphorylation of this 90K substrate. The 90K substrate is a higher Mr form of an 87K substrate that is a major C kinase substrate in rat brain. The Ca2+-dependent phosphorylation of both substrates is inhibited by the Ca2+-binding proteins S-100 and calmodulin. Both substrates yield phosphopeptide fragments of Mr 9K and 13K after limited proteolysis with V8 protease. Two-dimensional polyacrylamide gel electrophoresis reveals that they have similar acidic isoelectric points (pI 5.0). MOP enhances Ca2+-dependent phosphorylation of the 90K substrate whereas the phosphorylation of 87K is diminished. This reciprocal relationship suggests that the mobility of the 87K substrate in sodium dodecyl sulfate-polyacrylamide gels is decreased to 90K with increasing phosphorylation. MOP may be a novel protein modulator of C kinase-mediated phosphorylation in the nervous system.

Purification and characterization of a neurite extension factor from bovine brain.

The extension of neurites by chicken embryo cerebral cortical neurons can be measured quantitatively at low cell density in serum-free, defined medium. An acidic, heat-stable protein fraction from bovine brain has been shown to have neurite extension activity in this assay. We report the use of reversed-phase HPLC to purify a neurite extension factor from this fraction to apparent homogeneity. The protein was characterized by NaDodSO4/PAGE. In the presence of reducing agents, the protein migrated as a single band, with an apparent molecular weight of 6500. In the absence of reducing agents, the protein showed bands at apparent molecular weights of 6500, 21,000-22,000, 30,000, and 40,000. Reduction and S-carboxymethylation of the protein abolished all biological activity and resulted in a shift of the apparent molecular weight to 11,000. The amino acid composition of the purified neurite-extension factor was nearly identical to that of bovine brain S100 beta. The amino acid sequences of peptides derived from trypsin or cyanogen bromide digests of the protein were identical to those found in S100 beta and accounted for 71 of 91 amino acids in the protein. However, three peptides obtained from cyanogen bromide digestion of the nonreduced protein appeared to be disulfide-linked dimers. Our results indicate that a biological activity, neurite extension, which is critical for the development of the nervous system, is associated with a disulfide form of S100 beta.

Isolation of a protein from bovine brain which promotes neurite extension from chick embryo cerebral cortex neurons in defined medium.

Seven-day-old chick embryo cerebral cortex neurons cultured at low density (10,000 cells/16 mm well) in defined medium in polylysine-coated wells fail to extend neurites and assume a flattened phase-dark morphology. Addition of soluble bovine brain extract promotes neurite outgrowth and rounding of the cell body (which becomes phase-bright). A quantitative bioassay was utilized to purify this neurite extension factor (NEF), based on counting the number of phase-bright neurons with processes at least equal to one cell body diameter after 20 h in culture. Using a combination of heat treatment, DEAE-cellulose chromatography, and gel filtration, an acidic protein with a native Mr = 75,000 has been purified. Upon reduction it yields subunits of Mr = 37,000. Purified fractions are active at 100 ng/ml in inducing neurite outgrowth in this bioassay.

Neurite outgrowth from cerebral cortical neurons is promoted by medium conditioned over heart cells.

Seven day chick embryo cerebral cortical neurons cultured at low density (10,000 cells/16 mm well) in serum-free, defined medium in polylysine-coated wells fail to extend neurites and assume a flattened phase-dark morphology. Addition of serum-free, defined medium conditioned over chick embryo heart cells promotes neurite outgrowth and rounding of the cell body (which becomes phase bright). A sensitive bioassay, based on counting the number of phase-bright neurons with processes at least equal to one cell body diameter after 20 hours in culture, was used to titrate neurite-promoting activity in heart conditioned medium. This activity is completely destroyed by exposure to trypsin. As little as 2 micrograms/ml total conditioned medium protein elicits a half-maximal response in this bioassay.

Aggressive preschoolers: a pilot study.

The authors studied the relationship between the level of aggressivity in 12 aggressive preschoolers and five hypothesized pathogenic variables: constitutional, lack of maternal attunement to the child's needs, passive experience of aggression, parental overindulgence and the presence of a precipitant. The data tend to support a strong predictive association between the pathogenic variables as a group and the level of aggressivity, although the results must be regarded as preliminary because of the small number of cases.