RESUMEN

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts

Asunto(s)

Bacteriología , Patología

Asunto(s)

Herpesvirus Humano 8/aislamiento & purificación , Sarcoma de Kaposi/diagnóstico , Anciano , ADN Viral/análisis , Herpesvirus Humano 8/genética , Humanos , Japón , Masculino , Reacción en Cadena de la Polimerasa , Recurrencia , Sarcoma de Kaposi/virología , Coloración y Etiquetado

RESUMEN

Multiple factors affect skin pigmentation, including those that regulate melanocyte and/or keratinocyte function. Such factors, particularly those that operate at the level of the melanosome, are relatively well characterized in mice, but the expression and function of structural and enzymatic proteins in melanocytes in human skin are not as well known. Some years ago, we generated peptide-specific antibodies to murine melanosomal proteins that proved to be instrumental in elucidating melanocyte development and differentiation in mice, but cross-reactivity of those antibodies with the corresponding human proteins often was weak or absent. In an effort to characterize the roles of melanosomal proteins in human skin pigmentation, and to understand the underlying mechanism(s) of abnormal skin pigmentation, we have now generated polyclonal antibodies against the human melanocyte-specific markers, tyrosinase, tyrosinase-related protein (TYRP1), Dopachrome tautomerase (DCT) and Pmel17 (SILV, also known as GP100). We used these antibodies to determine the distribution and function of melanosomal proteins in normal human skin (adult and newborn) and in various cutaneous pigmented lesions, such as intradermal nevi, lentigo simplex, solar lentigines and malignant melanomas. We also examined cytokeratin expression in these same samples to assess keratinocyte distribution and function. Immunohistochemical staining reveals distinct patterns of melanocyte distribution and function in normal skin and in various types of cutaneous pigmented lesions. Those differences in the expression patterns of melanocyte markers provide important clues to the roles of melanocytes in normal and in disrupted skin pigmentation.

Asunto(s)

Especificidad de Anticuerpos , Lentigo/patología , Melanocitos/química , Melanocitos/inmunología , Glicoproteínas de Membrana , Oxidorreductasas , Piel/química , Adulto , Secuencia de Aminoácidos , Animales , Células Cultivadas , Secciones por Congelación , Humanos , Inmunohistoquímica , Recién Nacido , Oxidorreductasas Intramoleculares/análisis , Oxidorreductasas Intramoleculares/inmunología , Queratinocitos/química , Queratinocitos/enzimología , Queratinocitos/inmunología , Melanocitos/enzimología , Melanoma/patología , Melanosomas/química , Melanosomas/enzimología , Melanosomas/inmunología , Datos de Secuencia Molecular , Monofenol Monooxigenasa/análisis , Monofenol Monooxigenasa/inmunología , Nevo Intradérmico/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas/análisis , Proteínas/inmunología , Conejos , Piel/citología , Piel/enzimología , Neoplasias Cutáneas/patología , Pigmentación de la Piel , Antígeno gp100 del Melanoma

RESUMEN

There is an urgent need for development of new serodiagnostic strategies for leptospirosis, an emerging zoonosis with worldwide distribution. We have evaluated the diagnostic utility of five recombinant antigens in enzyme-linked immunosorbent assays (ELISAs) for serodiagnosis of leptospirosis. Sera from 50 healthy residents of a high-incidence region were used to determine cutoff values for 96% specificity. In paired sera from 50 cases of leptospirosis confirmed by the microscopic agglutination test, immunoglobulin G (IgG) but not IgM reacted with the recombinant leptospiral proteins. The recombinant LipL32 IgG ELISA had the highest sensitivities in the acute (56%) and convalescent (94%) phases of leptospirosis. ELISAs based on recombinant OmpL1, LipL41, and Hsp58 had sensitivities of 16, 24, and 18% during the acute phase and 72, 44, and 32% during convalescence, respectively. Compared to sera from healthy individuals, patient sera did not react significantly with recombinant LipL36 (P > 0.05). Recombinant LipL32 IgG ELISA demonstrated 95% specificity among 100 healthy individuals, and specificities ranging from 90 to 97% among 30 dengue patients, 30 hepatitis patients, and 16 patients with diseases initially thought to be leptospirosis. Among 39 Venereal Disease Research Laboratory test-positive individuals and 30 Lyme disease patients, 13 and 23% of sera, respectively, reacted positively with the rLipL32 antigen. These findings indicate that rLipL32 may be an useful antigen for the serodiagnosis of leptospirosis.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Leptospira/inmunología , Leptospirosis/diagnóstico , Antígenos Bacterianos/genética , Ensayo de Inmunoadsorción Enzimática/normas , Humanos , Inmunoglobulina G/sangre , Lipoproteínas/genética , Lipoproteínas/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad

RESUMEN

Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.

Asunto(s)

Melanosomas/química , Proteínas de Neoplasias/análisis , Oxidorreductasas , Electroforesis/métodos , Humanos , Oxidorreductasas Intramoleculares/análisis , Melanosomas/ultraestructura , Glicoproteínas de Membrana/análisis , Microscopía Confocal/métodos , Microscopía Inmunoelectrónica/métodos , Monofenol Monooxigenasa/análisis , Proteínas/análisis , Células Tumorales Cultivadas , Antígeno gp100 del Melanoma

RESUMEN

Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine design.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/inmunología , Leptospirosis/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos , Fraccionamiento Celular , Electroforesis en Gel Bidimensional/métodos , Humanos , Leptospira/inmunología , Leptospirosis/sangre , Conejos

RESUMEN

In response to agouti signal protein, melanocytes switch from producing eumelanin to pheomelanin concomitant with the down-regulation of melanogenic gene transcription. We previously reported that a ubiquitous basic helix-loop-helix transcription factor, known as ITF2, is up-regulated during this switch, and we now report that treatment of melanocytes with melanocyte-stimulating hormone down-regulates expression of ITF2. To more fully characterize the involvement of ITF2 in regulating melanogenic gene transcription, ITF2 sense or antisense constructs were introduced into melan-a melanocytes. Gene and protein expression analyses and luciferase reporter assays using promoters from melanogenic genes showed that up-regulation of ITF2 suppressed melanogenic gene expression as well as the expression of Mitf, a melanocyte-specific transcription factor. In addition, stable ITF2 sense transfectants had significant reductions in pigmentation and a less dendritic phenotype compared with mock transfectants. In contrast, ITF2 antisense-transfected melanocytes were more pigmented and more dendritic. These results demonstrate that up-regulation of ITF2 during the pheomelanin switch is functionally significant and reveal that differential expression of a ubiquitous basic helix-loop-helix transcription factor can modulate expression of melanogenic genes and the differentiation of melanocytes.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Melanocitos/metabolismo , Proteínas del Tejido Nervioso , Transactivadores/fisiología , Transcripción Genética , Animales , Antígenos de Neoplasias , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Northern Blotting , Diferenciación Celular , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/química , Células Dendríticas/metabolismo , Regulación hacia Abajo , Genes Reporteros , Secuencias Hélice-Asa-Hélice , Luciferasas/metabolismo , Antígeno MART-1 , Melaninas/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Microscopía Electrónica , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Oligonucleótidos Antisentido/metabolismo , Fenotipo , Plásmidos/metabolismo , Pruebas de Precipitina , Regiones Promotoras Genéticas , Isoformas de Proteínas , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Factores de Transcripción TCF , Transactivadores/química , Factor de Transcripción 4 , Factores de Transcripción/metabolismo , Transfección , Regulación hacia Arriba

RESUMEN

BACKGROUND/PURPOSE: Twenty-two institutes have organized Keio University Prostate Cancer Study Group to study clinical efficacy and safety of Leuprolide acetate (Leuplin) for the treatment of advanced prostate cancer (clinical stage D1 and D2). Cotreatment of Leuplin and Estramustine phosphate disodium (Estracyt) has been performed to investigate its clinical efficacy. MATERIALS AND METHODS: One hundred and two cases of advanced prostate cancer were treated either with Leuplin alone (group I), Leuplin and Estracyt (group II) or Estracyt alone (group III). After 12 weeks treatment, clinical effects against subjective symptoms (pain, voiding difficulty, performance status and body weight), serum testosterone level, tumor size and serum PSA level were examined to investigate short-term effect of each treatment. The treatment had been continued for 24 months and the treatment effects including progression free survival and overall survival were analyzed. RESULTS: Clinical efficacy after 12 weeks treatment were examined among 97 cases (group I; 35 cases, group II; 36 cases, group III; 26 cases). The background of those patients in each group was statistically equal. Treatment effects against subjective symptoms and serum testosterone level statistically revealed no significant difference among 3 groups. Treatment effects against primary tumor, bone metastatic lesion, lymphnode metastatic lesion and serum PSA level were investigated and anti-tumor effect was characterized by total efficacy rate (complete remission rate plus partial remission rate) of each treatment group. Treatment efficacy rates for each lesion and PSA demonstrated no statistical difference among 3 treatment groups. Total efficacy rate of group I, II and III were 88.2%, 84.0% and 78.3%, respectively, which statistically revealed no significant difference. Total efficacy rate of each group after completing 24 months treatment was; group I 80.0%, group II 55.6% and group III 83.3%, which statistically showed no significant difference among 3 treatment groups. The median day for progression free survival of group I, II and III were 661, 731 and 517, respectively. The overall survival rate of group I, II and III after completing 24 months treatment were 77.5%, 83.0% and 72.4%, respectively. Both progression free survival rates and overall survival rates revealed no significant difference among 3 groups. Side effects during 24 months treatment were seen in 8.6% of group I, 47.2% of group II and 26.9% of group III, and these occurrence rates were significantly different among the groups (p = 0.0013). CONCLUSION: Although number of the cases had not been able to continue the treatment for their side effects, the statistical characterization demonstrated that cotreatment of Leuplin and Estracyt had no greater treatment effect than monotreatment of each drug.

Asunto(s)

Antineoplásicos Hormonales/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leuprolida/administración & dosificación , Neoplasias de la Próstata/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Esquema de Medicación , Estramustina/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/mortalidad , Tasa de Supervivencia

RESUMEN

The molecular bases of various types of congenital hypopigmentary disorders have been clarified in the past 10 years. Homozygous gene mutations of enzymes functional in melanogenesis such as tyrosinase, P protein and DHICA oxidase, result in oculocutaneous albinism (OCA) 1, OCA 2, and OCA 3, respectively. The genes responsible for Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS) have also recently been isolated and cloned. The transcription factor paired box 3 (PAX3) works at the promoter region of the microphthalmia-associated transcription factor (MITF) gene, and the MITF transcription factor orders the expression of c-kit, which encodes the receptor for stem-cell factor, which in turn stimulates melanoblast migration from the neural tube to the skin in the embryo. Heterozygous mutations of PAX3, MITF, or c-kit genes induce Waardenburg syndrome (WS) 1/3, WS 2 or Piebaldism, respectively. A defect of endothelin-3 or the endothelin-B receptor produces WS 4. In our examination of 26 OCA 1 patients in Japan, all were found to have homozygous or heterozygous tyrosinase gene mutations at codons 77 or 310. Therefore, mutations at codons 77 and 310 are the major ones in Japanese patients with OCA 1. An autosomal dominant pigmentary disease of dyschromatosis symmetrica hereditaria (DSH) is well known in Japan, and is characterized by a mixture of hypo- and hyper-pigmented macules of various sizes on the backs of the hands and feet. The disease gene and its chromosomal localization have not been identified yet. Our trial of linkage analysis and positional cloning to determine the disease gene is presented.

Asunto(s)

Albinismo Oculocutáneo/genética , Hipopigmentación/genética , Proteínas de Transporte de Membrana , Proteínas Portadoras/genética , Síndrome de Hermanski-Pudlak/genética , Humanos , Japón , Proteínas de la Membrana/genética , Monofenol Monooxigenasa/genética , Piebaldismo/genética , Proteínas Proto-Oncogénicas c-kit/genética , Síndrome de Waardenburg/genética

RESUMEN

Oculocutaneous albinism (OCA) is an autosomal recessive disorder in which the biosynthesis of melanin is reduced or absent in skin, hair and eyes. Tyrosinase-related OCA (OCA1) is caused by mutations in the tyrosinase gene. Tyrosinase-negative OCA (OCA1A) is the most severe phenotype in which tyrosinase catalytic activity is completely lost, resulting in no mature melanin pigment. Yellow OCA (OCA1B) varies from very little pigment associated with whitish-blond hair to nearly normal pigment with dark-blond hair and skin. We determined the tyrosinase activity in melanocytes by the electron microscopic dihydroxyphenylalanine (EM-DOPA) reaction test using skin samples and analyzed tyrosinase gene mutations in nine Japanese patients with OCA. In 18 alleles of nine patients, the OCA1A-associated mutations, P310insC, R77Q and R278X, were found in seven, three and one alleles, respectively. Five patients who had these mutations in both alleles showed white hair, blue eyes and white skin and demonstrated no tyrosinase activity by the EM-DOPA reaction test. Three patients who had no tyrosinase gene mutation showed tyrosinase activity and heterogeneous clinical features. One patient in whom only an R77Q OCA1A mutation was found in one allele demonstrated a reduced tyrosinase activity, indicating OCA1B. This patient had white hair at birth, but it had turned blond by the age of 1 year. These results indicate that the EM-DOPA reaction test provides clear information on the status of tyrosinase activity which is essential for the identification of the disease subtype which in turn is important for the prognosis of patients with OCA.

Asunto(s)

Albinismo Oculocutáneo/genética , Melanocitos/enzimología , Monofenol Monooxigenasa/genética , Adulto , Anciano , Albinismo Oculocutáneo/diagnóstico , Albinismo Oculocutáneo/enzimología , Alelos , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Humanos , Lactante , Masculino , Melanocitos/ultraestructura , Melanosomas/enzimología , Melanosomas/ultraestructura , Microscopía Electrónica , Monofenol Monooxigenasa/análisis , Mutación , Linaje , Pruebas Cutáneas

RESUMEN

We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing [(3)H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl(2) (20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity with L. kirschneri infecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.

Asunto(s)

Leptospira/genética , Leptospira/inmunología , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Acilación , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Secuencia de Bases , Southern Blotting , Clonación Molecular , Cricetinae , Detergentes/farmacología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Ácidos Grasos/metabolismo , Immunoblotting , Infecciones , Riñón/microbiología , Riñón/patología , Leptospirosis/sangre , Lipoproteínas/genética , Mesocricetus , Datos de Secuencia Molecular , Octoxinol , Filogenia , Polietilenglicoles/farmacología , Pruebas de Precipitina , Factores de Tiempo

RESUMEN

Switching between production of eumelanin or pheomelanin in follicular melanocytes is responsible for hair color in mammals; in mice, this switch is controlled by the agouti locus, which encodes agouti signal protein (ASP) through the action of melanocortin receptor 1. To study expression and processing patterns of ASP in the skin and its regulation of pigment production in hair follicles, we have generated a rabbit antibody (termed alphaPEP16) against a synthetic peptide that corresponds to the carboxyl terminus of ASP. The specificity of that antibody was measured by ELISA and was confirmed by Western blot analysis. Using immunohistochemistry, we characterized the expression of ASP in the skin of newborn mice at 3, 6, and 9 days postnatally. Expression in nonagouti (a/a) black mouse skin was negative at all times examined, as expected, and high expression of ASP was observed in 6 day newborn agouti (A/+) and in 6 and 9 day newborn lethal yellow (A(y)/a) mouse skin. In lethal yellow (pheomelanogenic) mice, ASP expression increased day by day as the hair color became more yellow. These expression patterns suggest that ASP is delivered quickly and efficiently to melanocytes and to hair matrix cells in the hair bulbs where it regulates melanin production.

Asunto(s)

Técnica del Anticuerpo Fluorescente , Color del Cabello/fisiología , Folículo Piloso/ultraestructura , Péptidos y Proteínas de Señalización Intercelular , Melanocitos/ultraestructura , Proteínas/aislamiento & purificación , Proteína de Señalización Agouti , Animales , Especificidad de Anticuerpos , Melaninas/biosíntesis , Hormonas Estimuladoras de los Melanocitos/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Monofenol Monooxigenasa/aislamiento & purificación , Proteínas/inmunología , Receptores de Corticotropina , Receptores de Melanocortina

RESUMEN

Dyschromatosis symmetrica hereditaria (DSH) is an autosomal dominant pigmentary disorder, first reported by Toyama in 1910. It is characterized by a mixture of hypopigmented and hyperpigmented macules of various sizes on the backs of the hands and feet. The disease gene of DSH and its chromosomal localization have not yet been identified. A family with DSH and idiopathic torsion dystonia (ITD), a rare neurological disease, was recently reported. Therefore, we speculated that there was a linkage between the DSH gene and the ITD gene, named DYT1 and localized on chromosome 9, and performed linkage analysis between DSH and microsatellite markers on chromosome 9 in three Japanese DSH families (36 patients in total). We obtained a LOD score of < -2 over the whole region of chromosome 9 encompassing DYT1. Thus, we conclude that there is no linkage between DSH and DYT1 as well as any region of chromosome 9.

Asunto(s)

Cromosomas Humanos Par 9 , Ligamiento Genético , Trastornos de la Pigmentación/genética , Femenino , Humanos , Masculino , Linaje , Enfermedades de la Piel/genética

RESUMEN

New vaccine strategies are needed for prevention of leptospirosis, a widespread human and veterinary disease caused by invasive spirochetes belonging to the genus Leptospira. We have examined the immunoprotective capacity of the leptospiral porin OmpL1 and the leptospiral outer membrane lipoprotein LipL41 in the Golden Syrian hamster model of leptospirosis. Specialized expression plasmids were developed to facilitate expression of leptospiral proteins in Escherichia coli as the membrane-associated proteins OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic in E. coli, this was accomplished by using plasmid pMMB66-OmpL1, which has undetectable background expression without induction. LipL41-M expression and processing were enhanced by altering its lipoprotein signal peptidase cleavage site to mimic that of the murein lipoprotein. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with Leptospira kirschneri serovar grippotyphosa. At 28 days after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% confidence interval [CI], 53 to 89%), compared with only 25% (95% CI, 8 to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. The protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with either OmpL1-M or LipL41-M alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with His(6)-OmpL1 and His(6)-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 associates with membranes is an important determinant of immunoprotection.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/inmunología , Inmunización , Leptospira/inmunología , Leptospirosis/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/genética , Cricetinae , Modelos Animales de Enfermedad , Leptospira/genética , Leptospira/metabolismo , Leptospirosis/inmunología , Leptospirosis/mortalidad , Dosificación Letal Mediana , Mesocricetus , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes/inmunología

RESUMEN

BACKGROUND: Yellow oculocutaneous albinism (OCA) that is caused by tyrosinase gene mutations shows two characteristics: extreme hypopigmentation at birth and the eventual development of yellow or blond hair. OBJECTIVE: We studied a Japanese girl who had brown hair, a lighter skin color than her unaffected family and brown eyes at 9 months of age. METHODS: We performed direct sequencing analyses of the tyrosinase gene in her genomic DNA. RESULTS: The patient was a compound heterozygote for the +DeltaC310 mutation (known to result in absent melanogenic activity) and a second t-->a transition at the 3' end of intron 2. CONCLUSION: The t-->a transition has previously been reported as a splicing mutation in other Caucasian patients with a typical yellow OCA phenotype. However, this patient showed much more pigmentation than that reported in Caucasians. Therefore, we estimate that the mild phenotype results from her genetic pigment background.

Asunto(s)

Albinismo Oculocutáneo/genética , Albinismo Oculocutáneo/patología , Empalme Alternativo/genética , Monofenol Monooxigenasa/genética , Pigmentación/genética , Análisis Mutacional de ADN , Femenino , Pruebas Genéticas , Genotipo , Heterocigoto , Humanos , Lactante , Japón , Melaninas/deficiencia , Mutación , Fenotipo , Eliminación de Secuencia

RESUMEN

The purpose of this study was to investigate the mechanism of fatty acid-induced regulation of melanogenesis. An apparent regulatory effect on melanogenesis was observed when cultured B16F10 melanoma cells were incubated with fatty acids, i.e., linoleic acid (unsaturated, C18:2) decreased melanin synthesis while palmitic acid (saturated, C16:0) increased it. However, mRNA levels of the melanogenic enzymes, tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2), were not altered. Regarding protein levels of these enzymes, the amount of tyrosinase was decreased by linoleic acid and increased by palmitic acid, whereas the amounts of TRP1 and TRP2 did not change after incubation with fatty acids. Pulse-chase assay by [35S]methionine metabolic labeling revealed that neither linoleic acid nor palmitic acid altered the synthesis of tyrosinase. Further, it was shown that linoleic acid accelerated, while palmitic acid decelerated, the proteolytic degradation of tyrosinase. These results suggest that modification of proteolytic degradation of tyrosinase is involved in regulatory effects of fatty acids on melanogenesis in cultured melanoma cells.

Asunto(s)

Ácidos Grasos/fisiología , Melaninas/biosíntesis , Glicoproteínas de Membrana , Monofenol Monooxigenasa/metabolismo , Animales , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Ácido Linoleico/farmacología , Ratones , Monofenol Monooxigenasa/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Ácido Palmítico/farmacología , Proteínas/genética , Proteínas/metabolismo , Células Tumorales Cultivadas

RESUMEN

Varied effects of chemical or biological compounds on mammalian pigmentation have been reported by many groups, but to date, no standardized method has established necessary and/or optimal parameters for testing such agents. A standardized method has been developed to screen compounds with potential effects on pigmentation. The protocol comprises basic parameters to analyze melanogenic effects and allows for further characterization of candidate compounds, providing important insights into their mechanism of action. In this protocol (termed STOPR, for standardized testing of pigmentation regulators), compounds are initially screened using purified tyrosinase and are then tested on melanocytes in culture. After treatment of melanocytes with potentially bioactive compounds, cell proliferation and viability, total melanin accumulated, and melanogenic potential are measured. This protocol is an important first step in characterizing chemical regulation of effects on melanogenesis. When bioactive candidate compounds are identified, testing may proceed for pharmacological or otherwise commercial applications in coculture and/or organ culture models followed by in vivo testing. As an application of this method, results for compounds known to stimulate and/or inhibit melanogenesis (including arbutin, hydroquinone, kojic acid, melanocyte-stimulating hormone, and thymidine dimers) as well as some commercial skin whiteners are reported.

Asunto(s)

Pigmentación de la Piel/efectos de los fármacos , Animales , Arbutina/farmacología , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/normas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hidroquinonas/farmacología , Melaninas/biosíntesis , Hormonas Estimuladoras de los Melanocitos/farmacología , Melanocitos/citología , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Ratones , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Niacinamida/farmacología , Dímeros de Pirimidina/farmacología , Pironas/farmacología

RESUMEN

Macrophage migration inhibitory factor (MIF) is a relatively small, 12.5-kDa protein that is structurally related to some isomerases and for which multiple immune and catalytic roles have been proposed. MIF is widely expressed in tissues with particularly high levels in neural tissues. Here we show that MIF is able to catalyze the conversion of 3,4-dihydroxyphenylaminechrome and norepinephrinechrome, toxic quinone products of the neurotransmitter catecholamines 3,4-dihydroxyphenylamine and norepinephrine, to indoledihydroxy derivatives that may serve as precursors to neuromelanin. This raises the possibility that MIF participates in a detoxification pathway for catecholamine products and could therefore have a protective role in neural tissues, which as in Parkinson's disease, may be subject to catecholamine-related cell death.

Asunto(s)

Catecolaminas/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Envejecimiento/metabolismo , Animales , Secuencia de Bases , Encéfalo/metabolismo , Cartilla de ADN , Humanos , Ratones , Oxidación-Reducción , Proteínas Recombinantes/metabolismo

RESUMEN

Macrophage migration inhibitory factor (MIF) was originally identified several decades ago as a lymphokine-derived protein that inhibited monocyte migration. Recently, it has been reported that MIF has D-dopachrome tautomerase, phenylpyruvate tautomerase and thiol protein oxidoreductase activities, although the physiological significance of those activities is not yet clear. Here we show that MIF is able to catalyze the conversion of dopaminechrome and norepinephrinechrome, toxic quinone products of the neurotransmitters dopamine and norepinephrine, respectively, to indole derivatives that may serve as precursors to neuromelanin. Since MIF is highly expressed in human brain, these observations raise the possibility that MIF participates in a detoxification pathway for catecholamine products and could therefore have an important role for neural tissues. The potential role of MIF in the formation of neuromelanin from catecholamines is also an extremely interesting possibility.

Asunto(s)

Catecolaminas/metabolismo , Factores Inhibidores de la Migración de Macrófagos/fisiología , Animales , Encéfalo/metabolismo , Catálisis , Dopamina/metabolismo , Indoles/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Melaninas/biosíntesis , Ratones , Proteínas del Tejido Nervioso/metabolismo , Norepinefrina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

The purpose of this study was to determine whether the hypothesis on the levels of psychological processes (Hatta, 1977, 1978) accounts for lateral differences in arm positioning movement (a spatial localization task). Fifteen right-handed male subjects were asked to perform a constrained criterion movement, 12 cm in length, with the left or right arm. Then, after a 10-sec retention interval, they were asked to perform the movement with the same arm, estimating lengths of 6, 12 or 24 cm. Different levels of psychological processes were assumed to be involved in estimating these various movement lengths--half, the same, or double that of the original. All possible combinations of the arm (left/right) and three movement length were tested. The CE scores were lower (more accurate) for the left arm (half; 1.5 +/- 8.1 mm, same; 4.3 +/- 6.2mm, double; 5.9 +/- 20.3 mm) than those for the right arm (half; 5.9 +/- 7.6 mm, same; 10.6 +/- 10.6 mm, double; 11.8 +/- 23.6 mm) in all conditions, indicating a lateral difference (the right hemisphere dominance) in arm positioning tasks. This typical lateral difference, which displayed no significant difference among conditions, is supposed to be mediated by complex or high-level psychological processes. These psychological processes are required by the subjects in the estimation of the various movement lengths. This study suggests that the level of psychological processes is a crucial factor in the manifestation of lateral differences in the performance of arm positioning movements.

Asunto(s)

Brazo/fisiología , Lateralidad Funcional/fisiología , Propiocepción/fisiología , Percepción Espacial/fisiología , Adulto , Análisis de Varianza , Humanos , Masculino