RESUMEN
The Blomia tropicalis (Blo t) mite species is considered a storage mite in temperate climate zones and an important source of indoor allergens causing allergic asthma and rhinitis in tropical and subtropical regions. Here, we report the crystal structure of one of the allergens from Blo t, recombinant proBlo t 1 (rproBlo t 1), determined at 2.1 Å resolution. Overall, the fold of rproBlo t 1 is characteristic for the pro-form of cysteine proteases from the C1A class. Structural comparison of experimentally mapped Der f 1/Der p1 IgG epitopes to the same surface patch on Blo t 1, as well as of sequence identity of surface-exposed residues, suggests limited cross-reactivity between these allergens and Blo t 1. This is in agreement with ELISA inhibition results showing that, although cross-reactive human IgE epitopes exist, there are unique IgE epitopes for both Blo t 1 and Der p 1.
Asunto(s)
Alérgenos/química , Alérgenos/inmunología , Antígenos Dermatofagoides/química , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/química , Proteínas de Artrópodos/inmunología , Reacciones Cruzadas/inmunología , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/inmunología , Inmunoglobulina E/inmunología , Conformación Proteica , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Modelos Moleculares , Péptidos/química , Péptidos/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Relación Estructura-ActividadRESUMEN
BACKGROUND: Neonates with a family history of atopy are at higher risk for developing wheezing in early life. OBJECTIVE: From a birth cohort of at risk infants (first-degree family with atopic disease), we evaluated the influence of distinct intrinsic immunologic risk factors on wheezing disorders in the first 2 years of life. METHODS: Cord blood samples were collected from 195 eligible subjects of a birth cohort of 253 subjects. The subjects studied were those who developed wheezing (n=34) or eczema (n=29) in the first 2 years of life, and 65 healthy control infants. At the time of thawing the viability of the cells were median 70% (range 67.5%-72.5%). Cytokines from lipopolysaccharide (LPS)-stimulated mononuclear cells were analysed using fluorescent-activated cell sorting-array and their profiles were evaluated using factor analysis. RESULTS: Infants with wheeze were significantly associated with enhanced combined LPS stimulated IL-1ß, IL-6, and IL-12/IL-23p40 compared with healthy controls (P=0.003). This profile was also associated with the increased risk for wheeze at 2 years of age (OR=2.45; 95% CI=1.50-3.93, P=0.001). LPS-stimulated cytokine IL-8 was also significantly higher in the wheeze group compared with healthy controls and eczema (P=0.003). Intracellular staining showed that monocytes are main producers of IL-6 and IL-8 from cord blood mononuclear cells. Most of the subjects were non-atopic with 3/34 (9%) wheeze and 9/29 (31%) eczema subjects sensitized to the common dietary or inhalant allergens. CONCLUSION AND CLINICAL RELEVANCE: In infants at genetic risk of atopy, wheeze but not eczema in the first 2 years of life is associated with intrinsic hyperresponsive innate cytokine responses which might predispose infants to wheeze development. Distinct pre-symptomatic hyperresponsive innate immune responses risk factors were found to be associated with early onset wheeze disorders, but not eczema.
Asunto(s)
Citocinas/metabolismo , Sangre Fetal/inmunología , Leucocitos Mononucleares/inmunología , Ruidos Respiratorios/etiología , Ruidos Respiratorios/inmunología , Edad de Inicio , Preescolar , Eccema/inmunología , Femenino , Sangre Fetal/citología , Humanos , Lactante , Recién Nacido , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/inmunología , Masculino , Factores de RiesgoRESUMEN
BACKGROUND: Eczema is a common chronic inflammatory skin disorder which shows strong genetic predisposition. To identify new potential molecular determinants of the disease pathogenesis, we performed a gene expression study in an eczema mouse model. This analysis identified a marked down regulation of the cornulin gene (CRNN), a member of the epidermal differentiation complex, in the eczema-like skin. We then investigated CRNN as an eczema candidate gene and studied its polymorphism and the expression in the skin of eczema patients. METHODS: An eczema-like phenotype was induced in mice by allergen (Der p2) patching. Gene expression analysis was performed with the subtractive suppression hybridization method and validated by real time PCR and the transmission disequilibrium test was used to test for genetic associations in 406 multiplex eczema families. RESULTS: Der p 2 patched mice developed a localized eczema and a Th 2 skewed systemic response. Real time PCR analysis confirmed a down regulation of CRNN mRNA in eczema-like skin in the mouse model and in human eczema. The CRNN polymorphism rs941934 was significantly associated with atopic eczema in the genetic analysis (P = 0.006), though only as part of an extended haplotype including a known associated variant (2282del4) in the filaggrin gene. CONCLUSIONS: CRNN mRNA expression is decreased in eczematous skin. Further studies are needed to verify whether the associated cornulin polymorphism contribute to the genetic susceptibility in eczema.
Asunto(s)
Dermatitis Atópica/genética , Regulación hacia Abajo/genética , Epidermis/inmunología , Predisposición Genética a la Enfermedad , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Adulto , Alérgenos/inmunología , Animales , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos , Citocinas/biosíntesis , Citocinas/inmunología , Dermatitis Atópica/inmunología , Epidermis/efectos de los fármacos , Epidermis/patología , Femenino , Proteínas Filagrina , Expresión Génica , Regulación de la Expresión Génica , Marcadores Genéticos , Genotipo , Haplotipos , Humanos , Inmunoglobulina E/sangre , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Psoriasis/diagnóstico , Psoriasis/genética , Psoriasis/inmunologíaRESUMEN
Except in animal models of cataractogenesis, the literature on the effects of tea infusion on ocular tissue is scant. In our patient, prolonged exposure to tea infusion may have led to a hypesthetic cornea with secondary limbal stem cell loss. In turn, the eye developed keratinization and corneal neovascularization.
Asunto(s)
Enfermedades de la Córnea/inducido químicamente , Enfermedades de la Córnea/patología , Té/efectos adversos , Anciano , Lentes de Contacto Hidrofílicos , Córnea/irrigación sanguínea , Enfermedades de la Córnea/terapia , Humanos , Queratinas/metabolismo , Masculino , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/patología , Flujo Sanguíneo Regional/fisiología , Células Madre/fisiología , Trastornos de la Visión/inducido químicamente , Trastornos de la Visión/patologíaRESUMEN
A troponin C-like phosphodiesterase activator from bovine thyroid has been purified to homogeneity. The overall purification was about 9,800-fold with a yield of 8%. Bovine thyroid activator protein is identical in biologic properties to that isolated from bovine brain. They have the same specific activity regarding stimulation of bovine brain cyclic nucleotide phosphodiesterase. Both proteins form a Ca2+-dependent complex with heart muscle troponin I which is stable in 6M urea-polyacrylamide gel and which is similar, but not identical, to the troponin C-troponin I complex. The physiochemical properties of bovine thyroid activator protein are identical with those of bovine brain and other phosphodiesterase activator proteins and are similar to heart muscle and skeletal muscle troponin C as follows: (A) they bind 3-4 exchangeable calcium ions/mol with dissociation constants between 10(-5) and 10(-6) M, (B) they are highly acidic with a high content of aspartic and glutamic acids and isoelectric points of approximately 4.1, (C) these proteins have an unusual ultraviolet absorption spectrum with six discrete maxima between 250 and 284 nm which are characteristic of phenylalanine and tyrosine, and (D) these proteins have a low content of cysteine, histidine, tyrosine and proline. The tryptic peptide maps of bovine thyroid and brain activator protein are very similar. However, despite a very similar amino acid composition, the peptide map of bovine heart muscle troponin C is significantly different from that of the other two proteins. The molecular weight of thyroid and brain activator protein is 16,500, while that of heart troponin C is 18,500. Thyroid and brain activator protein, as well as heart troponin C, appear to undergo significant Ca2+-dependent conformational changes, as measured by the difference in the circular dichroism spectrum and electrophoretic mobility observed in the presence and absence of calcium ion.
Asunto(s)
Calmodulina/aislamiento & purificación , Proteínas Portadoras/aislamiento & purificación , Proteínas Musculares , Glándula Tiroides/análisis , Troponina , Aminoácidos/análisis , Animales , Química Encefálica , Calcio/farmacología , Calmodulina/farmacología , Bovinos , Activación Enzimática , Peso Molecular , Miocardio/análisis , Proteínas del Tejido Nervioso/aislamiento & purificación , Especificidad de ÓrganosRESUMEN
PURPOSE: To report the ophthalmic manifestations of neonatal onset multisystem inflammatory disease, a recently recognized, rare systemic disorder characterized by the triad of arthropathy, rash, and abnormal central nervous system development. METHOD: Case report. RESULTS: A 2-year-old female with neonatal onset multisystem inflammatory disease presented with visual acuity of fix and follow with each eye, bilateral optic nerve head pallor and gliosis, as well as marked sheathing of the peripapillary vessels. No vitreous inflammation or macular edema was found. Visual acuity was stable from the neonatal period through the 3 months of follow-up after the changes involving the optic nerve heads and peripapillary vessels were observed (a total of 33 months). CONCLUSIONS: This report provides a description of the ocular manifestations of neonatal onset multisystem inflammatory disease and supports earlier suggestions that swelling of the optic nerve heads can occur. In this case, optic nerve head pallor may have been a sequela of such swelling. The pathogenesis of neonatal onset multisystem inflammatory disease is unknown.
Asunto(s)
Exantema/diagnóstico , Hidradenitis/diagnóstico , Artropatías/diagnóstico , Malformaciones del Sistema Nervioso/diagnóstico , Disco Óptico/patología , Enfermedades del Nervio Óptico/diagnóstico , Preescolar , Femenino , Humanos , Disco Óptico/irrigación sanguínea , Vasos Retinianos/patología , Tomografía Computarizada por Rayos X , Agudeza VisualRESUMEN
PURPOSE: To describe dense vitritis as the primary manifestation of ocular syphilis in three human immunodeficiency virus (HIV)-positive patients and to determine the response of these patients to the established regimen for neurosyphilis. METHODS: Anti-Toxoplasma gondii IgM and IgG antibody titers, tuberculin skin test, chest radiograph, and serum angiotensin-converting enzyme level were obtained because tuberculosis, sarcoidosis, and toxoplasmosis were in the differential diagnosis. Two of the three patients were not known to have HIV infection at the time of initial examination and consented to HIV testing. Treponemal and nontreponemal tests were performed on serum and cerebrospinal fluid to establish a definitive diagnosis. Treatment for neurosyphilis was initiated, and daily ophthalmic examinations were performed, with careful attention to signs commonly associated with syphilitic eye disease. RESULTS: All three patients exhibited improvement in visual acuity and resolution of vitreous haze. There was no evidence of other signs of posterior uveitis. The one patient for whom there has been a 6-month follow-up showed no sequelae of his eye disease. CONCLUSIONS: Human immunodeficiency virus-positive patients with syphilis may present atypically dense vitritis. In these patients, vitritis may be the first manifestation of syphilis. The regimen for neurosyphilis provides effective therapy. Moreover, in some patients, syphilitic vitritis may be the initial manifestation of HIV disease.
Asunto(s)
Infecciones Bacterianas del Ojo/complicaciones , Infecciones por VIH/complicaciones , Sífilis/complicaciones , Uveítis Posterior/etiología , Cuerpo Vítreo/patología , Adulto , Oftalmopatías/tratamiento farmacológico , Oftalmopatías/etiología , Infecciones Bacterianas del Ojo/tratamiento farmacológico , Fondo de Ojo , Seropositividad para VIH/complicaciones , Humanos , Inyecciones Intravenosas , Masculino , Penicilina G/uso terapéutico , Penicilina G Benzatina/uso terapéutico , Penicilinas/uso terapéutico , Sífilis/tratamiento farmacológico , Serodiagnóstico de la Sífilis , Uveítis Posterior/tratamiento farmacológico , Agudeza Visual , Cuerpo Vítreo/efectos de los fármacosRESUMEN
PURPOSE: To report a case of corneal haze after attempts to eliminate post-laser in situ keratomileusis (LASIK) lamellar striae. METHODS: Case report. RESULTS: A 24-year-old woman with visually significant flap striae 2 months after LASIK underwent manual epithelial debridement and flap hydration, refloating, and stretching to eliminate the striae. Three weeks after this intervention, she developed visually significant haze that was confined to the stroma of the flap. The haze slowly improved with use of a topical steroid. CONCLUSION: Stromal haze can develop after treatment of flap striae with epithelial debridement and hypo-osmolar irrigation. We speculate that these maneuvers may have induced cell death of anterior keratocytes and led to haze formation, as can occur after simple epithelial debridement and epithelial scrape-photorefractive keratectomy.
Asunto(s)
Opacidad de la Córnea/etiología , Sustancia Propia/patología , Desbridamiento/efectos adversos , Epitelio Corneal/cirugía , Queratomileusis por Láser In Situ/efectos adversos , Colgajos Quirúrgicos , Adulto , Agua Corporal/metabolismo , Opacidad de la Córnea/metabolismo , Opacidad de la Córnea/patología , Sustancia Propia/metabolismo , Células Epiteliales/patología , Epitelio Corneal/metabolismo , Femenino , Humanos , Irrigación Terapéutica , Agudeza VisualRESUMEN
PURPOSE: To report a case of Aspergillus fumigatus keratitis after a laser in situ keratomileusis (LASIK) enhancement procedure. METHOD: Case report. RESULTS: A 56-year-old woman developed an ulcer in the flap 13 days after LASIK enhancement. A 4-week course of fortified antibiotics for a presumed bacterial infection followed. The ulcer progressed, causing 60% thinning of the corneal stroma. A biopsy was performed 5 weeks after onset of symptoms, and antifungal agents were initiated. Cultures showed A. fumigatus. Her cornea perforated after the biopsy, requiring cyanoacrylate and lamellar overlay sutures, but the infiltrate resolved on antifungal agents. CONCLUSION: This report is the first description of Aspergillus keratitis after LASIK. We hypothesize that the infection became established on the stromal bed during surgery and led to melting, anteriorly through the flap and posteriorly through the stroma. Diagnosis was made by a corneal biopsy and inoculation of a wide array of media. This case demonstrates the need to consider atypical organisms, including fungi, in the differential diagnosis of post-LASIK infections when there is no response to therapy and highlights the role of corneal biopsy and flap lifting in the diagnosis of this condition.
Asunto(s)
Aspergilosis/microbiología , Aspergillus fumigatus/aislamiento & purificación , Úlcera de la Córnea/microbiología , Infecciones Fúngicas del Ojo/microbiología , Queratomileusis por Láser In Situ/efectos adversos , Antifúngicos/uso terapéutico , Aspergilosis/diagnóstico , Aspergilosis/tratamiento farmacológico , Úlcera de la Córnea/diagnóstico , Úlcera de la Córnea/tratamiento farmacológico , Infecciones Fúngicas del Ojo/diagnóstico , Infecciones Fúngicas del Ojo/tratamiento farmacológico , Femenino , Humanos , Pruebas de Sensibilidad Microbiana , Persona de Mediana EdadRESUMEN
OBJECTIVE: To determine whether zinc, which blocks apoptosis in many systems, including in leukemic cells and possibly in retinal dystrophies, can prevent the unwanted loss of anterior stromal keratocytes after superficial keratectomy. METHODS: After mechanical central corneal epithelial debridement, the left eyes of nine New Zealand white rabbits were treated with 25 mM zinc chloride (ZnCl2) in Earle's salts minimal essential medium (MEM) either every 30 min (n = 3), every 2 h (n = 3), or every 4 h (n = 3). The left eyes of nine additional animals, divided into three equal groups, were deepithelialized, and each received pure culture medium at one of the same three frequencies. One eye of each of another six rabbits was deepithelialized but received no drops. After 24 h, all 24 animals were sacrificed and the globes were enucleated. The corneas were processed and sections were stained with hematoxylin and eosin. RESULTS: The cell count of MEM-treated corneas exceeded that of untreated corneas (p = 0.03, analysis of variance [ANOVA]), but there was no difference among eyes that received the different frequencies of MEM application (p = 0.36, ANOVA). Cell counts increased with frequency of zinc application, but the differences were not statistically significant (p = 0.09, ANOVA). Only in the group receiving the most frequent zinc applications were superficial keratocytes retained. This group also possessed a greater number of stromal keratocytes than untreated controls and MEM-treated corneas (p = 0.01). CONCLUSIONS: At a significantly high dosage, zinc can prevent loss of superficial keratocytes to a greater extent than culture medium alone can. Moreover, zinc has the advantage of preserving the most anterior layer of keratocytes. Retention of these cells may prevent the reactive overproliferation that constitutes haze after photorefractive surgery.
Asunto(s)
Apoptosis/efectos de los fármacos , Cloruros/farmacología , Sustancia Propia/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Complicaciones Posoperatorias/prevención & control , Compuestos de Zinc/farmacología , Administración Tópica , Animales , Recuento de Células , Cloruros/administración & dosificación , Sustancia Propia/patología , Sustancia Propia/cirugía , Desbridamiento , Fibroblastos/patología , Soluciones Oftálmicas , Conejos , Cicatrización de Heridas/efectos de los fármacos , Compuestos de Zinc/administración & dosificaciónRESUMEN
Neovascularization is an infrequent but serious complication of uveitis. The retina and optic disk appear to be affected most often, although new blood vessels may arise from the iris, ciliary body, and choroid as well. Although neovascularization can usually be identified on careful clinical examination, some patients may require fluorescein angiography or UBM. Numerous neovascular growth and inhibitory factors have been identified experimentally. Clinically, however, uveitic neovascularization appears to be determined most directly by the severity of the inflammation and the presence of retinal nonperfusion. Virtually all patients with uveitic neovascularization deserve a trial of local or systemic corticosteroids. Laser photocoagulation can be considered in those patients who fail to respond to corticosteroid therapy, but only when retinal nonperfusion has been demonstrated on fluorescein angiography. Surgical excision of newly formed vessels is reserved for selected patients with CNV and uveitis, but should only be considered when corticosteroids and focal photocoagulation are ineffective or are otherwise contraindicated.
Asunto(s)
Neovascularización Coroidal/etiología , Neovascularización Retiniana/etiología , Úvea/irrigación sanguínea , Uveítis/complicaciones , Neovascularización Coroidal/diagnóstico , Neovascularización Coroidal/terapia , Diagnóstico Diferencial , Angiografía con Fluoresceína , Fondo de Ojo , Humanos , Neovascularización Retiniana/diagnóstico , Neovascularización Retiniana/terapia , Uveítis/patologíaRESUMEN
Blomia tropicalis (Bt) and Dermatophagoides pteronyssinus (Dp) are the predominant domestic mites species in Singapore and Taiwan. This study aims to characterize and compare the mite sensitization profiles in both countries. Skin prick tests were performed on 203 Singaporeans with Dp and Bt crude extracts. In vitro IgE and IgG4 reactivity to extracts and specific allergens (Der p 1, Der p 2 Der p 5 and Blo t 5) were determined by immunoassays. Approximately 91% of the tested Singaporeans were skin test positive for both Bt and Dp. Both populations share similar frequencies of in vitro IgE reactivity to all the allergens tested, but they differ in the pattern and magnitude of allergen sensitization. Although Der p 1, Der p 2 and Blo t5 are major sensitizing allergens in both countries, Blo t 5 is a more potent one in Singapore, probably reflecting the high level of exposure to Bt. The unique major Bt and Dp allergens should be included for precise diagnosis and effective immuno-therapeutic treatment of mite allergy in both countries.
Asunto(s)
Alérgenos/inmunología , Glicoproteínas/inmunología , Hipersensibilidad/inmunología , Ácaros/inmunología , Adolescente , Adulto , Animales , Antígenos Dermatofagoides , Antígenos de Plantas , Niño , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoensayo , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Singapur , Pruebas Cutáneas , TaiwánRESUMEN
BACKGROUND: Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems. OBJECTIVE: To assess the allergenicity of native and recombinant mite glutathione S-transferase (GST) (Der p 8) and study the IgE cross-reactivity between Der p 8 and cockroach GST. METHODS: Der p 8 cDNA encoding a new isoform was isolated and expressed in yeast. Native Der p 8 was affinity purified from mite extract. IgE reactivity to native and recombinant Der p 8 was assessed by ELISA using sera from allergic subjects from Taiwan, Singapore and Malaysia. IgE cross-reactivity between Der p 8 and cockroach GST was examined by IgE inhibition assays. RESULTS: Our Der p 8 cDNA encoded a basic isoform (pI=8.5) containing six polymorphic residues located at positions 46, 106, 149, 160, 167 and 184. At least 8 isoforms of native Der p 8 were detected by two-dimensionalgel and immunoblot analyses. Sera from Taiwanese asthmatics showed 96% and 84% IgE reactivity to native Der p 8 and recombinant Der p 8, respectively. Native Der p 8 showed 75% and 65% IgE reactivity with sera from Malaysia and Singapore, respectively. CONCLUSIONS: A high frequency of sensitization to mite GST among allergic subjects was observed but the titres of IgE reactivity were low. The IgE cross-reactivity between mite and cockroach GST suggests that GST is a panallergen.
Asunto(s)
Alérgenos/inmunología , Cucarachas/inmunología , Dermatophagoides pteronyssinus/inmunología , Glutatión Transferasa/inmunología , Inmunoglobulina E/inmunología , Adolescente , Adulto , Alérgenos/genética , Animales , Reacciones Antígeno-Anticuerpo/inmunología , Antígenos Dermatofagoides , Antígenos de Plantas , Proteínas de Artrópodos , Asma/inmunología , Secuencia de Bases , Niño , Preescolar , Reacciones Cruzadas/inmunología , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Hipersensibilidad/inmunología , Lactante , Isoformas de Proteínas/inmunología , Proteínas Recombinantes/inmunologíaRESUMEN
A low molecular weight protein found in the soluble extract of bovine adrenal medulla, and having a high affinity for calcium ions has been purified to apparent homogeneity. The purification requires three steps, including ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. The protein was homogeneous by the criteria of both standard and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sedimentation velocity analysis, and NH2-terminal analysis. The calcium-binding protein is very acidic and has an isoelectric point of 4.27. Aspartic and glutamic acid together account for 30% of the total amino acid composition. The ultraviolet absorption spectrum reveals a A280/A260 ratio of 0.83 and shows discrete maxima at 258, 264, 269, 278, and 284 nm. Two moles of calcium are bound per mole of protein. The apparent Kp is approximately 20 muM. The molecular weight was found to be 16,000 +/- 1,000 by both sodium dodecyl sulfate gel electrophoresis and sedimentation equilibrium ultracentrifugation. The protein was found to consist of a single polypeptide chain by the criteria of tryptic peptide mapping and NH2-terminal analysis. Amino acid analysis revealed the absence of tryptophan, single residues of cysteine and histidine, and 2 residues of tyrosine. The protein was void of carbohydrate and phosphate. The structural similarities and possible functional correlation between adrenal medulla calcium-binding protein and troponin-C from muscle tissue are discussed.
Asunto(s)
Médula Suprarrenal/análisis , Proteínas Musculares , Troponina/aislamiento & purificación , Aminoácidos/análisis , Animales , Sitios de Unión , Bovinos , Ácido Edético , Peso Molecular , Proteínas Musculares/aislamiento & purificación , Fragmentos de Péptidos/análisis , Unión Proteica , Espectrofotometría Ultravioleta , TripsinaRESUMEN
A high affinity calcium-binding protein has recently been purified from the adrenal medulla (Kuo, I.C.Y., and Coffee, C.J. (1976) J. Biol. Chem. 251, 1603-1609). This protein is closely related in its chemical and physical properties to troponin-C (TN-C) of muscle tissue. Further examination of the adrenal medulla protein indicates that the removal of calcium is accompanied by a marked change in the conformation. This change in structure is similar, if not identical, to the calcium-dependent conformational change which has been described for skeletal muscle TN-C (Murray, A.C., and Kay, C.M. (1972) Biochemistry 11, 2622). The far ultraviolet circular dichroism spectrum of native adrenal medulla calcium-binding protein (AM-CBP) shows characteristic helical ellipticity bands at 222 and 207 nm. The helical content, as estimated from these data, is between 40 and 45%. Removal of calcium is accompanied by a change in ellipticity which corresponds to a decrease from 40 to 20% in the helical content. The near-ultraviolet circular dichroism spectrum shows negative dichroic bands at 262 and 268 nm which are characteristic of phenylalanine. These bands are relatively insensitive to changes in the calcium ion concentration. Sedimentation velocity studies likewise are indicative of a calcium-dependent structural alteration. The sedimentation coefficient of the native protein was observed to be 1.89 S. Similar measurements performed in the presence of 3 mM ethylene glycol bis(beta-aminoethyl ether) N,N, N', N'-tetraacetic acid (EGTA) gave a sedimentation coefficient of 1.50 S. The molecular weight, as determined by sedimentation equilibrium studies, was 16,000 regardless of whether the measurements were made in the presence of CaCl2 or EGTA. From the elution properties of AM-CBP on Sephadex G-100, the Stokes radius was observed to be 19.8 A in the presence of calcium and 21.9 A in the presence of EGTA. All of these changes which were induced by the addition of EGTA were completely reversible by the readdition of excess CaCl2. These data suggest that the removal of calcium from AM-CBP is accompanied by a pronounced conformational change which occurs without a molecular weight change. The decreased sedimentation coefficient, the increased Stokes radius, and the reduced helical content, which are observed for the apoprotein, indicate that removal of calcium results in a transformation from a compact symmetrical structure to one that is less ordered and more asymmetrical.
Asunto(s)
Médula Suprarrenal/metabolismo , Calcio , Proteínas Musculares , Receptores de Droga , Troponina , Animales , Sitios de Unión , Proteínas Portadoras/aislamiento & purificación , Bovinos , Dicroismo Circular , Peso Molecular , Proteínas Musculares/metabolismo , Unión Proteica , Conformación Proteica , Receptores de Droga/metabolismo , Espectrofotometría Ultravioleta , Troponina/metabolismoRESUMEN
A simple method to examine the optical parallelism between planar end faces of a ruby rod is suggested. When the ruby rod is illuminated by an expanded He-Ne laser beam with linear polarization, the interference pattern due to the recombination of the E and O rays at a Polaroid analyzer shows its optical parallelism. This optical parallelism, which depends not entirely on the mechanical wedge angle but also on the equivalent wedge angle produced by lineage, can be employed as a criterion to show whether or not the ruby rod is suitable in an E-ray oscillation laser system.
RESUMEN
Crystals of the 14-kDa group 5 allergen from Dermatophagoides pteronyssinus (Der p 5) have been obtained at low pH and diffract to 3-A resolution using a conventional x-ray source. The crystals belong to tetragonal space group P41212 or P43212, with unit cell parameters a = b = 114 A and c = 234 A. A self-rotation search revealed a 432 point symmetry and thus suggested 96 molecules in one unit cell, hence 12 monomers in each asymmetric unit.
Asunto(s)
Glicoproteínas/química , Ácaros/inmunología , Animales , Antígenos Dermatofagoides , Cristalización , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Proteínas de Insectos/química , Proteínas Recombinantes/químicaRESUMEN
BACKGROUND: A 98-kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned. This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11. METHODS: The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages. The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E. coli. These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays. RESULTS: Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28-residue repeats and lacks the N-terminus and the C-terminus. The sequence identity of Der f 11 with other known paramyosins is 34-60%. The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2. These peptides are more reactive than whole rDf642. CONCLUSIONS: Mite paramyosin is very similar to other known paramyosins. The human IgE and IgG epitopes are scattered throughout the entire molecule. Data also indicate the presence of unique IgE and IgG epitopes in Der f 11.
Asunto(s)
Alérgenos/genética , Epítopos/genética , Glicoproteínas/inmunología , Inmunoglobulina E/genética , Inmunoglobulina G/genética , Ácaros , Tropomiosina/genética , Alérgenos/inmunología , Animales , Afinidad de Anticuerpos , Antígenos Dermatofagoides , Secuencia de Bases , Epítopos/inmunología , Glicoproteínas/genética , Humanos , Immunoblotting , Inmunoglobulina G/inmunología , Ácaros/genética , Ácaros/inmunología , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Tropomiosina/inmunologíaRESUMEN
OBJECTIVE: To describe subretinal fibrosis as a long-term complication of Vogt-Koyanagi-Harada (VKH) disease. DESIGN: Retrospective, clinic-based, cross-sectional study and clinical correlation. PARTICIPANTS: Ten patients with VKH disease and subretinal fibrosis were seen at two uveitis referral centers between 1977 and 1997. INTERVENTION: A review of the historical, clinical, and fluorescein angiographic features was performed. MAIN OUTCOME MEASURES: The prevalence, demographic and clinical features, and time to development of subretinal fibrosis were summarized. RESULTS: Subretinal fibrosis occurred in 20 eyes of 10 patients with VKH disease, an overall prevalence of 8% between the two institutions. Patient age ranged from 16 years to 48 years, with a median of 34.5 years. Five patients were Hispanic, one was mixed Hispanic and American Indian, three were Asian or mixed Asian and Caucasian, and one was African-American. Eight of the 10 patients were men. All patients were in the chronic, recurrent phase of their disease when they had subretinal fibrosis develop, and all patients had recurrent episodes of posterior uveitis. Presenting vision ranged from 20/20 to light perception, with a median acuity of 20/200. All patients were initially treated with oral and topical corticosteroids. Four patients required additional noncorticosteroid immunosuppressive therapy. Time from diagnosis of VKH disease to development of subretinal fibrosis ranged from zero (fibrosis present at time of diagnosis) to 27 years, with a median time of 10 months. The median time from diagnosis of VKH to development of subretinal fibrosis in Hispanic patients was 6.5 months, whereas in non-Hispanic patients it was 6.5 years. Final vision ranged from 20/25 to light perception, with a median acuity of 20/60. Seven of 20 eyes had a final visual acuity of 20/40 or better, and seven eyes saw 20/200 or worse. Five of the eyes with 20/200 or worse vision had fibrosis involving the fovea. CONCLUSIONS: Subretinal fibrosis occurs in a sizeable proportion of patients with VKH disease and may contribute to permanent loss of vision.