RESUMEN
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.
Asunto(s)
Terapias Complementarias/instrumentación , Terapias Complementarias/métodos , Magnetismo/instrumentación , Magnetismo/métodos , Piel/efectos de la radiación , Cosméticos/farmacología , Capacidad Eléctrica , Campos Electromagnéticos , Emolientes/farmacología , Compuestos Férricos , Humanos , Nanopartículas del Metal , Modelos Teóricos , Técnicas de Cultivo de Órganos , EstroncioRESUMEN
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.
Asunto(s)
Colesterol/metabolismo , Queratinocitos/metabolismo , Fitosteroles/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colestadienoles/farmacología , Microscopía por Crioelectrón , Emulsiones/farmacología , Humanos , Queratinocitos/efectos de los fármacos , Microscopía Electrónica de Transmisión , Sitoesteroles/metabolismo , Sitoesteroles/farmacologíaRESUMEN
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.
Asunto(s)
Autoanticuerpos/inmunología , Desmogleína 1/inmunología , Desmogleína 3/inmunología , Pénfigo/inmunología , Animales , Autoanticuerpos/fisiología , Desmogleína 1/fisiología , Desmogleína 3/fisiología , Desmosomas/fisiología , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Queratinocitos/inmunología , Queratinocitos/patología , Ratones , Pénfigo/patología , Pénfigo/fisiopatología , Índice de Severidad de la EnfermedadRESUMEN
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.
Asunto(s)
Inmunoglobulinas Intravenosas/inmunología , Pénfigo/inmunología , Animales , Animales Recién Nacidos , Especificidad de Anticuerpos/inmunología , Autoanticuerpos/inmunología , Vesícula/inmunología , Vesícula/patología , Vesícula/prevención & control , Desmogleína 3/inmunología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta Inmunológica , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulinas Intravenosas/administración & dosificación , Inmunohistoquímica/métodos , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos , Pénfigo/patología , Proteínas Recombinantes/inmunología , Piel/inmunología , Piel/patologíaRESUMEN
Through a still unclear mechanism, pemphigus vulgaris autoantibodies (PV-IgG) induce intra-epidermal acantholytic lesions responsible for severe to fatal skin wounding. We present evidence that PV lesions contain apoptotic keratinocytes, and that cell death is induced in the lesional tissue apparently before cell separation. These data suggest that apoptosis could be the cause of the acantholytic phenomenon. We show that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) co-aggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation. Moreover, we have shown that in skin organ cultures and in keratinocyte cultures, PV-IgG can induce caspase activation and DNA fragmentation, and caspase inhibitors can prevent the formation of PV-IgG-induced epidermal lesions. Altogether, these results suggest that PV-IgG-induced acantholysis may proceed through the death-signaling pathway. They highlight new perspectives on mechanisms of tissue damage in autoimmune diseases.
Asunto(s)
Acantólisis/inmunología , Apoptosis/inmunología , Autoanticuerpos/inmunología , Epidermis/inmunología , Pénfigo/inmunología , Inhibidores de Caspasas , Caspasas/metabolismo , Técnicas de Cultivo de Célula , Humanos , Queratinocitos/enzimología , Queratinocitos/inmunología , Queratinocitos/metabolismo , Cinética , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND: Pemphigus vulgaris (PV) autoantibodies (PV-IgG) have been found in 40-70% of sera of first-degree relatives of pemphigus patients. OBJECTIVES: To determine the possible role of PV-IgG subclasses in the pathogenesis of the disease. PATIENTS AND METHODS: Study groups comprised 25 PV patients, 55 unaffected family members and 56 sera of healthy individuals. Indirect immunofluorescence (IIF) staining and Western immunoblotting (WB) techniques were used to determine total PV-IgG and PV-IgG subclasses and their reactivity to desmoglein (Dsg) 1 and 3. RESULTS: By IIF staining, circulating PV-IgG were found in 64% of the patients, in 15% of the relatives and in none of the controls (P < or = 0.001); by WB the results were 91%, 49% and 12%, respectively (P < or = 0.001). The distribution of PV-IgG subclasses 1-3 was similar among patients and their relatives. PV-IgG4 was found in 62% of the patients but in only one relative and was absent in the controls (P < or = 0.001). PV-IgG1, 2 and 4 were found to react mainly with Dsg3 and PV-IgG3 mainly with Dsg1 and 3. CONCLUSIONS: These results support the concept of a genetic predisposition in pemphigus. The non-complement-fixing PV-IgG4 and at least one complement-fixing PV-IgG subclass appear to be involved in the pathogenesis of the disease. The absence of PV-IgG4 among relatives who were PV-IgG carriers seems to be linked to the fact that they do not develop pemphigus. The exact nature of this linkage is still unclear.
Asunto(s)
Autoanticuerpos/sangre , Cadherinas/inmunología , Predisposición Genética a la Enfermedad , Inmunoglobulina G/sangre , Pénfigo/inmunología , Adulto , Anciano , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Western Blotting , Desmogleína 1 , Desmogleína 3 , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Pénfigo/genéticaRESUMEN
During ovarian maturation in the crayfish Cherax quadricarinatus, changes in ovarian protein kinase C (PKC) isoenzymes take place in parallel to yolk accumulation (as shown by immunoblot analysis). Significant changes were recorded in the amounts of specific isoenzymes and in their distribution between the cytosol and the membranes. Ovarian maturation was accompanied by the appearance of high- and low-molecular-weight immunoreactive PKC isoenzyme species. Among the isoenzymes tested, PKC alpha was the most clearly activated during ovarian maturation, as shown by significant translocation from the cytosol to the particulate fraction and the appearance of high-molecular-weight species. Moreover, a similar picture was obtained in the ovaries of intersex individuals upon induction of secondary vitellogenesis by androgenic gland ablation. Immunohistological staining showed PKC alpha to be localized mainly in the cytosol of premature oocytes, whereas in later maturation stages, it was concentrated around the nucleus in a vesicular structure and in the oocyte membrane. In secondary vitellogenic stages, PKC was localized in the plasma membrane and apparently in follicular cells. In addition, its activity was demonstrated by in vitro phosphorylation assays of a crayfish ovarian homogenate. Activation of total PKC phosphorylation of histone, an external substrate, was induced by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate (TPA) or methyl farnesoate. Both TPA and methyl farnesoate stimulated activation of PKC alpha in organ culture, causing its translocation from the cytosol to the membranes and inducing autophosphorylation of threonine residues. The changes in PKC isoenzymes during ovarian maturation in the crayfish suggest their involvement in this process as well as a possible regulatory role for methyl farnesoate through a direct effect on some PKC isoenzymes.
Asunto(s)
Astacoidea/fisiología , Ácidos Grasos Insaturados/farmacología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Vitelogénesis , Animales , Activación Enzimática/efectos de los fármacos , Femenino , Oocitos/enzimología , Técnicas de Cultivo de Órganos , Ovario/enzimología , Ovario/fisiología , Acetato de Tetradecanoilforbol/farmacología , Distribución TisularRESUMEN
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.
Asunto(s)
Envejecimiento/fisiología , Cisteína Endopeptidasas/metabolismo , Queratinocitos/enzimología , Complejos Multienzimáticos/metabolismo , Adolescente , Adulto , Anciano , Análisis de Varianza , Western Blotting , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epidermis/enzimología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Complejo de la Endopetidasa Proteasomal , Proteínas/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
In response to crowding, locusts develop characteristic black patterns that are well discernible in the gregarious phase at outbreaks. We report here a dark-color-inducing neuropeptide (dark-pigmentotropin) from the corpora cardiaca of two plague locusts, Schistocerca gregaria and Locusta migratoria. The chromatographic isolation of this neuropeptide was monitored by using a bioassay with an albino mutant of L. migratoria. The neurohormone, consisting of 11 amino acids, is identical to [His7] corazonin, previously isolated from corpora cardiaca of another acridid without known function. The present results show that even in isolated (solitary) nymphs, [His7] corazonin induces gregarious black patterns. Its primary structure shows some similarity with the vertebrate melanophore stimulating hormone.
Asunto(s)
Saltamontes/fisiología , Neuropéptidos/fisiología , Animales , Luz , Mutación , Pigmentos Biológicos/fisiología , Polimorfismo GenéticoRESUMEN
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.
Asunto(s)
Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Inmunosupresores/farmacología , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Vitamina A/farmacología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , División Celular/efectos de los fármacos , División Celular/inmunología , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta Inmunológica , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Femenino , Inhibidores de Crecimiento/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células TH1/inmunología , Factores de TiempoRESUMEN
A case of paraneoplastic pemphigus associated with pancreatic carcinoma is presented. The histopathological and immunological features of the case, which are consistent with and differ from the accepted diagnostic criteria, are discussed.
Asunto(s)
Neoplasias Pancreáticas/complicaciones , Síndromes Paraneoplásicos/diagnóstico , Pénfigo/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Pénfigo/diagnósticoRESUMEN
BACKGROUND: Patients with pemphigus vulgaris (PV) are genetically linked to two alleles of the HLA subgroup, and circulating antibodies were found in first-degree relatives of these patients, thus showing genetic predisposition. OBJECTIVE: Our purpose was to determine the occurrence of circulating true PV-IgG in patients' relatives. METHODS: Circulating PV-IgG was determined in 21 first-degree relatives of 12 patients with PV by indirect immunofluorescence on monkey esophagus, carcinoma A431 cultures, and Western immunoblotting. Direct immunofluorescence was performed on skin biopsy specimens of 20 relatives. RESULTS: Circulating PV-IgG was detected in 15 relatives (71%) by all methods tested. Good correlation was found between immunoblot reactivity and immunofluorescence. Of the 15 "positive" relatives, only five showed fixation of IgG to epidermal cells in vivo. CONCLUSION: The permeability of the epidermis or epidermal cell reactivity in vivo probably controls the expression of disease in patients' relatives.
Asunto(s)
Western Blotting , Técnica del Anticuerpo Fluorescente Directa , Técnica del Anticuerpo Fluorescente Indirecta , Inmunoglobulina G/sangre , Pénfigo/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Animales , Biopsia , Carcinoma/inmunología , Epidermis/inmunología , Epidermis/patología , Esófago/inmunología , Femenino , Técnica del Anticuerpo Fluorescente Directa/métodos , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Expresión Génica , Predisposición Genética a la Enfermedad , Antígenos HLA/genética , Haplorrinos , Humanos , Inmunoglobulina G/genética , Israel , Judíos/genética , Masculino , Persona de Mediana Edad , Pénfigo/genética , Permeabilidad , Piel/inmunología , Piel/patología , Células Tumorales CultivadasRESUMEN
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.
RESUMEN
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.
Asunto(s)
Catecoles/farmacología , División Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/fisiología , Queratinocitos/citología , Nitrilos/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Tirfostinos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Receptores ErbB/antagonistas & inhibidores , Cobayas , Humanos , Técnicas In Vitro , Peso Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Fosfotirosina , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Bacillus cereus/enzimología , Bacillus subtilis/enzimología , Tripsina/metabolismo , Arginina/análogos & derivados , Arginina/metabolismo , Bacillus cereus/efectos de los fármacos , Bacillus subtilis/efectos de los fármacos , Benzoilarginina-Nitroanilida , Compuestos Cromogénicos/metabolismo , Calor , Hidrólisis , Leupeptinas/farmacología , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/enzimología , Tosilarginina Metil Éster/farmacología , Clorometilcetona Tosilisina/farmacologíaRESUMEN
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.
Asunto(s)
Proteína Quinasa C/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Animales , Sitios de Unión , Western Blotting , Cromatografía/métodos , Cromatografía DEAE-Celulosa/métodos , Diglicéridos/farmacología , Durapatita , Activación Enzimática , Hidroxiapatitas , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Mamíferos , Peso Molecular , Fosforilación , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
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).
Asunto(s)
Palaemonidae/crecimiento & desarrollo , Diferenciación Sexual , Testículo/fisiología , Animales , Extremidades/crecimiento & desarrollo , Femenino , Masculino , Tamaño de los Órganos , Palaemonidae/fisiología , EspermatozoidesRESUMEN
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.