Strontium hexaferrite nanomagnets suspended in a cosmetic preparation: a convenient tool to evaluate the biological effects of surface magnetism on human skin.

BACKGROUND/PURPOSE: Magnetic therapy has been popular for ages, but its therapeutic abilities remain to be demonstrated. We aimed to develop a homogeneous, stable dispersion of magnetic nanoparticles in a skin-care preparation, as a tool to analyze the biological and physiological effects of superficial magnetism in skin. METHODS: SrFe(12)O(19) nanoparticles were generated by ultrasound, dispersed in glycerol, stabilized in Dermud cream and permanently magnetized. The magnetic cream was applied on the epidermis of human skin organ cultures. The effects on UV-induced cell toxicity, apoptosis and inflammatory cytokine expression were analyzed. A clinical test was performed to check skin moisturization. RESULTS: Nanomagnets were found to be homogenously and stably dispersed. After magnetization, the preparation generated a magnetic field of 1-2 G. Upon cream application, no cytotoxicity and no impairment of cellular vitality were found after 24 and 48 h, respectively. The anti-apoptotic and anti-inflammatory properties of Dermud were not modified, but its long-term effect on moisturization in vivo was slightly increased. CONCLUSION: Nanomagnetic Dermud cream can be used as a tool to analyze the biological effects of nanomagnets dispersed on the skin surface at the cellular and molecular levels, thus allowing to explore the possible therapeutic uses of superficial magnetism for skin care.

The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

Inhibition of cholesterol transport into skin cells in cultures by phytosterol-loaded microemulsion.

Cholesterol and plant phytosterols are lipophilic compounds solubilized by intestinal micelles in a competitive manner. In this work, we used radioactive cholesterol- and phytosterol-loaded oil-in-water microemulsions to follow their incorporation and mutual competition in HaCaT keratinocytes, SZ95 sebocytes, and skin pieces in cultures. Dynamic light scattering showed homogenous nanostructures of 10.5+/-1.5 nm diameter and cryo-transmission electron microscopy confirmed the presence of uniform spherical droplets of 7.0+/-1.0 nm diameter. Up to 320 nmol/ml of cholesterol can be solubilized and transported into cells with minimal toxic effect by 0.5 wt% nanodroplets in a cell medium. Phytosterols inhibit incorporation of cholesterol into cells, in vitro, at molar ratios (phytosterols/cholesterol) of 4 and above. The loaded nanodroplets accumulate in intracellular vesicles (presumably endosomes). No metabolic conversion of cholesterol or phytosterols was found in these cells, in vitro, after 24 h, at 37 degrees C.

Characterization of PKC2, a gene encoding a second protein kinase C isotype of Saccharomyces cerevisiae.

BACKGROUND: Protein kinase C (PKC) has attracted considerable attention over the past decade, primarily because of its presumed role in cellular growth control and tumourigenesis. Mammalian cells express at least 10 different isozymes of PKC; it is this complexity that has made elucidating the precise functions of PKC: so difficult. The identification of PKC homologues in organisms such as Drosophila, Xenopus, Dictyostelium, Aplysia and Caenorhabditis indicates that the enzyme is evolutionarily conserved, and this has stimulated our search for counterparts in the yeast Saccharomyces cerevisiae, in which powerful genetic analyses can be used. To date, only one PKC homologue, PKC1, has been identified in yeast and no biochemical activity has been definitively ascribed to the encoded protein. This, and the inability to identify other PKC homologues in yeast by DNA hybridization, has led to doubts about the existence of PKC isozymes in yeast. We have taken the approach of screening yeast expression libraries with anti-PKC antibodies in an attempt to identify further homologues. RESULTS: We have identified a novel PKC isozyme, Pkc2p, encoded by the gene PKC2. We report here the sequence of PKC2 and a comparison showing its similarity to other PKCs. Phylogenetic analysis suggests that all known PKC genes, including PKC2, originated from a common ancestor. Disruption of the PKC2 protein-coding region, deleting the entire catalytic domain of the encoded enzyme, is not lethal to yeast growing on rich media. However, the pkc2 mutant, unlike wild-type strains, fails to grow on minimal media containing limited concentrations of amino acids. This implicates Pkc2p in the response of yeast cells to amino-acid starvation. CONCLUSION: We have shown that yeast cells do express more than one PKC isozyme, by identifying and characterizing a novel PKC gene PKC2, the product of which may be involved in the cellular response to amino-acid starvation.

Be13, a human T-leukemia cell line highly sensitive to dexamethasone-induced cytolysis.

A unique human T-leukemia cell line highly sensitive to dexamethasone-induced lysis is described. The cell line designated Be13 is killed readily within 24 hr by 10(-9) M dexamethasone. No lysis is induced by nonglucocorticoid steroids. The lysis is mediated via specific cytoplasmic receptors and is efficiently blocked by the antagonist cortexolone. The inhibiting effect of actinomycin D and cycloheximide on the lytic process suggests the involvement of gene activation and destruction of the cells by an "autolytic protein." Kinetic studies imply that the lytic process is induced during a distinct phase of the cell cycle. Dexamethasone, however, does not cause an arrest in a distinct phase of the cell cycle. The Be13 cell is a unique human cell line killed directly by glucocorticoids, and it may serve as a suitable in vitro model for studying the lytic effect of glucocorticoids on the proliferating compartment of human leukemias.

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythrocyte protein kinase that phosphorylates spectrin.

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin) kinase according to physical and site-specificity criteria. The soluble enzyme shows an Mr of about 30000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that had been phosphorylated with [gamma-32P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with 32Pi. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.

A possible involvement of virus-associated protease in the fusion of Sendai virus envelopes with human erythrocytes.

A proteolytic activity is shown to be associated with relatively purified preparations of intact Sendai virus particles or with their reconstituted envelopes which are vesicles containing mainly the viral glycoproteins. Intact Sendai virus as well as reconstituted Sendai virus envelopes have been shown to be able to hydrolyze various protein molecules such as the human erythrocyte membrane polypeptide designated as band 3 and soluble polypeptides such as histone and insulin B-chain. The results of the present work raise the possibility that a direct correlation exists between the virus-associated proteolytic activity and the ability of the virions to lyse cells, to fuse with their membranes, and to promote cell-cell fusion. Inhibitors of proteolytic enzymes such as phenylmethylsulfonyl fluoride, tosyllysinechloromethylketone and tosylamidephenylethylchloromethylketone, or combinations thereof, inhibit the virus-associated proteolytic activity concomitantly with inhibition of its hemolytic and fusogenic activities. Electron microscopic studies showed that the various inhibitors did not affect the binding ability of the virus preparations. The possible involvement of a protease in the process of virus-membrane fusion is discussed.

Modulation of the binding and endocytosis of concanavalin A by guinea pig keratinocytes: reversible antagonistic effects of cholesterol and phospholipid-liposomes.

Alteration of guinea pig keratinocyte membrane microviscosities (eta) by liposomes of varying composition was determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Measurements performed either with whole cell suspensions or Percoll-separated cell subpopulations, indicate a similar membrane microviscosity (eta = 3.37 poise +/- 10%) compared to those microviscosities reported for other cell types. Our findings show that treatment of guinea pig keratinocytes with liposomes composed of phospholipids results in a decreased membrane microviscosity (1.95 poise), whereas treatment of the cells with an emulsion of cholesterol hemisuccinate, or liposomes composed of cerebrosides, causes an increase in membrane microviscosity (3.85 poise and 5.55 poise +/- 10%, respectively). Changes in membrane fluidity had no significant effect on cell viability. A reduced membrane microviscosity resulted in a decrease in the binding of Concanavalin A to keratinocytes, whereas an increased microviscosity resulted in an increased binding of Concanavalin A. Furthermore, endocytosis of Concanavalin A bound to keratinocytes plasma membranes was not significantly affected by a reduced membrane microviscosity, whereas an increased membrane microviscosity completely blocked the endocytosis of Concanavalin A. Another novel observation was that membranes "fluidified" by phospholipid liposomes could be "rigidified" by treatment with cholesterol hemisuccinate and vice versa. Moreover, these alternate changes in membrane microviscosity resulted in simultaneous alternate changes in the binding of Concanavalin A to the keratinocyte surface.

The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae.

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.

Alterations in membrane protein and phosphorylation pattern in beta-thalassemic red blood cells.

The architecture and phosphorylation pattern of RBC membranes were studied in intact RBC and ghosts of patients with beta-thalassemia intermedia. Electron microscopic studies showed severe morphological alterations in ghosts from spx thalassemic patients. The polypeptide pattern obtained on SDS-PAGE revealed a fourfold increase in globin content of ghosts from spx patients. In addition, multiple protein bands were detected migrating below band 4.2, accompanied by alterations in the band 3 zone. When membrane protein phosphorylation was examined by SDS-PAGE and auto-radiography following incubation of intact RBC with [32P]Pi, a reduced labeling of the normally phosphorylated polypeptides was found in the thalassemic RBC. In addition, new phosphorylated peptides appeared in the region of band 3 and below band 4. On the other hand, phosphorylation of isolated membranes with [gamma-32P]ATP showed no major differences in the labeling of the major phosphorylated proteins. An analysis of the initial rate of spectrin-band 2.1 phosphorylation obtained by counting the excised bands from the SDS gels showed that there was a twofold increase in spectrin-band 2.1-phosphorylation rate catalyzed by cAMP-dependent protein-kinase in the thalassemic ghosts, although no differences were found in the extent of spectrin phosphorability. The results are consistent with major changes in membrane protein disposition in thalassemic RBC, most probably caused by the precipitation of excess globin chains.

Increase of oxidatively modified protein is associated with a decrease of proteasome activity and content in aging epidermal cells.

For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker 1-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins.

Are desmoglein autoantibodies essential for the immunopathogenesis of pemphigus vulgaris, or just "witnesses of disease"?

Pemphigus vulgaris (PV) is fascinating to dermatologists, epithelial biologists and immunologists alike, as its pathogenesis has been clarified to a much greater extent than that of most other organ-specific autoimmune diseases, and as it has provided abundant novel insights into desmoglein biology and pathology along the way. Historically, the most influential PV pathogenesis concept is that of Stanley and Amagai. This concept holds that autoantibodies against desmogleins are both essential and sufficient for epidermal blister formation (acantholysis) by impeding the normal functioning of these major adhesion proteins. However, as with most good theories, this landmark concept has left a number of intriguing and important questions open (or at least has not managed to answer these to everyone's satisfaction). Moreover, selected dissenting voices in the literature have increasingly called attention to what may or may not be construed as inconsistencies in this dominant PV pathogenesis paradigm of the recent past. The present debate feature therefore bravely rises to the challenge of re-examining the entire currently available evidence, as rationally and as undogmatically as possible, by provocatively asking a carefully selected congregation of experts (who have never before jointly published on this controversial topic!) to discuss how essential anti-desmoglein autoantibodies really are in the immunopathogenesis of PV. Not surprisingly, some of our expert "witnesses" in this animated debate propose diametrically opposed answers to this question. While doing so, incisive additional questions are raised that relate to the central one posed, and our attention is called to facts that may deserve more careful consideration than they have received so far. Together with the intriguing (often still very speculative) complementary or alternative pathogenesis scenarios proposed in the following pages, this offers welcome "food for thought" as well as very specific suggestions for important future research directions--within and beyond the camp of PV aficionados. The editors trust that this attempt at a rational public debate of the full evidence that is currently at hand will constructively contribute to further dissecting the exciting--and clinically very relevant!--immunopathogenesis of PV in all its complexity.

Protective effect of intravenous immunoglobulin (IVIG) in an experimental model of pemphigus vulgaris.

Uncontrolled studies have found intravenous immunoglobulin (IVIG) to be effective in the treatment of pemphigus vulgaris (PV). The aim of this study was to evaluate the role of IVIG in preventing IgG autoantibodies binding to desmoglein-3 and blister formation using a controlled experimental design. The ability of IVIG to affect the binding of IgG affinity purified from two patients with PV to desmoglein-3 in comparison to IgG from one donor, was conducted by enzyme-linked immunosorbent assay (ELISA). The specificity was confirmed by competition assay. We assessed the effect of IVIG on the induction of experimental-PV in CD1 newborn mice by subcutaneous subjection of IgG affinity purified from two patients with PV. The treatment was conducted by subcutaneous administration of IVIG together with IgG from the pemphigus patients or appropriate control. The skin of the newborns was examined 24-48 h later for blisters, and samples of the affected areas were analysed by immunohistochemistry. IVIG as a whole molecule and its F(ab)(2) portion inhibited the binding of anti-desmoglein-3 antibody to recombinant desmoglein-3 in a dose-dependent manner. The specificity was confirmed by competition assays. In-vivo, IVIG and its F(ab)(2) portion prevented blister formation in the newborn mice. Cutaneous lesions were noted only in the groups of newborn mice who were injected with IgG fractions from the PV patients. Immunopathological evaluation revealed that IVIG prevented the formation of acanthylosis with IgG deposition in the intercellular spaces. These results point to the efficacy of IVIG in the prevention of blister formation in an experimental PV model.

Steady state and exchange kinetics of pyruvate, phosphate dikinase from Propionibacterium shermanii.

Evidence is presented based on requirements for exchange in the partial reactions, initial velocity and exchange kinetics and product inhibition, that the pyruvate, phosphate dikinase reaction of propionibacteria occurs by a nonclassical Tri Uni Uni Ping Pong mechanism. The mechanism involves a pyrophosphoryl enzyme, a phosphoryl enzyme, and the free enzyme, and three functionally distinct and independent substrate sites. On the first site, there is pyrophosphorylation of the enzyme by ATP with subsequent release of AMP. The pyrophosphoryl moiety then reacts at the second site with Pi yielding the product PPi and the phosphoryl from of the enzyme. At the third site pyruvate is phosphorylated yielding P-enolpyruvate and the free enzyme. The three catalytic sites are proposed to be linked by a histidyl residue which functions as a pyrophosphoyrl- and phosphoryl-carrier between the three sites. This proposal is based on the following observations. (A) The patterns of the double reciprocal plots of the initial velocities were all parallel; (b) product inhibition between each pair of substrates and products of the three partial reactions were competitive, i.e. ATP against AMP, Pi against PPi, and pyruvate against P-enolpyruvate; (c) the other product inhibitions, with one exception, were noncompetitive as required by the nonclassical ping-pong mechanism; (d) ATP or P-enolpyruvate was required for the Pi in equilibrium PPi exchange reaction which is in accord with the participation of a pyrosphosphoryl or phosphoryl form of the enzyme in this exchange; (e) the ATP in equilibrium AMP exchange and pyruvate in equilibrium P-enolpyruvate exchange did not require additional substrates. In addition, the inhibition and participation in the exchange reactions of the alpha,beta and beta,gamma-methylene analogues of ATP and of the methylene analogue of inorganic pyrophosphate were investigated and the results were in accord with the proposed mechanism. The combined evidence provides a well documented example of a three site nonclassical Tri Uni Uni Ping Pong mechanism.

Isolation of a pyrophosphoryl form of pyruvate, phosphate dikinase from Propionibacteria.

Pyruvate, phosphate dikinase from Propionibacterium shermanii catalyzes the formation of P-enolpyruvate, AMP, and inorganic pyrophosphate from pyruvate, ATP, and orthophosphate; the mechanism involves three partial reactions and three forms of the enzyme: pyrophosphoryl-enzyme, phosphoryl-enzyme, and free enzyme. The phosphoryl-enzyme was prepared by incubation with P-enolpyruvate and isolated by gelchromatography. The phosphoryl-enzyme was converted to (32)P(31)P-enzyme and [(32)P]Pi by incubation with [(32)P]PPi; 1 mol of pyrophosphoryl-enzyme was formed per mol of enzyme of molecular weight 150,000. The labeled enzyme released its radioactivity upon incubation with Pi or AMP to produce the expected [(33)P]PPi or [gamma-(32)P]ATP, respectively. Hydrolysis of the pyrophosphoryl-enzyme with dilute acid yielded PPi. The beta,gamma-methylene analogue of ATP was reactive in exchange reactions with [(14)C]AMP. To our knowledge, this is the first proven example of a pyrophosphoryl-enzyme.

Vitamin A inhibits cytokines produced by type 1 lymphocytes in vitro.

The effect of vitamin A (retinol) on cell-mediated immune responses was studied. As an experimental model, Leishmania major infection in mice was used. In this model, resistant mouse strains develop a type 1 response, while susceptible strains develop a type 2 response. Using lymph node cells and T-cell lines developed from infected susceptible and resistant mice, it was found that vitamin A inhibited lymphocyte proliferation in a dose-dependent manner. By separately incubating antigen-presenting cells and T cells with vitamin A, it was shown that the inhibitory effect was on the T cells. Type 1 cytokine (IFN-gamma, GM-CSF, IL-2) secretion in vitro in response to stimulation with specific antigen was also inhibited in a dose-dependent manner, whereas secretion of type 2 cytokines (IL-4 and IL-10) was not affected by vitamin A. The inhibitory effect was also observed in PMA-stimulated (but not Con A-stimulated) lymphocytes and was noticeable even if the vitamin was added as late as 24 h after initiation of the incubation period. Since PMA does not operate via a receptor-coupled signaling pathway but rather directly affects the protein kinase C (PKC) pathway, we have measured the effect of vitamin A on PKC in situ activation. Incubation of lymphocytes and antigen in the presence of vitamin A caused inhibition of PKC isoenzymes translocation to the particulate cell fraction, as measured by immunoblotting. The results presented indicate that, when added to cell cultures in vitro, vitamin A inhibits only secretion of type 1 but not type 2 cytokines, possibly through an inhibitory effect on protein kinase C activity.

Effect of androgenic gland ablation on morphotypic differentiation and sexual characteristics of male freshwater prawns, Macrobrachium rosenbergii.

Mature males of the freshwater prawn, Macrobrachium rosenbergii (de Man), may change from one to another morphotype, according to a set sequence. Small males may develop into orange-claw males and orange-claw males into dominant blue-claw males. Each of the three morphotypes demonstrates distinctive reproductive behavior and secondary sexual characteristics. The role of the androgenic gland in this morphotypic transformation was examined experimentally by bilateral androgenic gland ablation (andrectomy) of small males and orange-claw males. For andrectomy initiated in the small male morphotype, transformation to the next morphotype was permitted (orange-claw), but subsequent transformation to the blue-claw morphotype was blocked. Andrectomy of orange-claw males did not prevent transformation into the blue-claw. Andrectomy on both small and orange-claw males caused disappearance of the genital papillae and atrophy of the sperm ducts and testes. The growth rates of the andrectomized small and orange-claw males were significantly lower than those of the unoperated and sham-operated controls. We conclude that androgenic gland factors control not only the differentiation of male secondary sexual characteristics but also morphotypic differentiation. Bioassays based on the results of this study will be instrumental in the characterization of such a factor(s).