RESUMEN
BACKGROUND: Uterovaginal brachytherapy planning is conventionally based on the use of two orthogonal Xray projections. Currently, there is a large development of 3D brachytherapy planning based on the fusion of CT and MRI, which takes into account the extent of the tumor and the location of organs at risk. In this work, we evaluated the dosimetric data and first clinical results in patients with inoperable cervical cancer using MRI/âCT compatible applicator enabling 3D planning. PATIENTS AND METHODS: Between June 2012 and March 2013, we performed 52 uterovaginal applications in 13 patients with inoperable cervical cancer using Vienna Ring MRâCT applicator. Planning was carried out by the fusion of MRI and CT. Target volumes and organs at risk delineation were carried out on the basis of GECâESTRO and ABS recommendations as well as doses report-ing. RESULTS: Overall radiotherapy duration was 37-â52 days with median of 45 days. The median total dose delivered to the HR CTV was 88 Gy (70.7-â97.9) EQD2. The median single dose in brachytherapeutic applications was D90 = 6.45 Gy (3.2-â9.82). The median total doses delivered to the rectum, sigmoid colon and bladder were D2ccrectum = 64.2 Gy (54.3-â74.1), D2ccsigmoid = 68.6 Gy (57-â74.7) a D2ccbladder = 73.9 Gy (58.3-â92.6). In 11 patients (84.6%), complete locoregional remission was achieved, in the remaining two patients (15.4%), partial locoregional remission was achieved. Twelve patients (92.3%) had complete regression of the tumor in the cervix, one patient (7.7%) developed metastatic spread to the liver. Yet we did not observe manifestations of a higher degree of toxicity than the first grade, both GI and GU. Late GI toxicity was manifested in two patients (15.4%) and late GU toxicity was manifested in five patients (38.5%). CONCLUSION: 3D brachytherapy planning of inoperable cervical cancer using the fusion of MRI and CT conclusively raises the possibility of the dose escalation to the tumor and significantly spares the surrounding organs at risk. Subsequently, this way of planning leads to better local control of the disease and to lower radiation morbidity.
Asunto(s)
Braquiterapia/métodos , Imagen por Resonancia Magnética/métodos , Planificación de la Radioterapia Asistida por Computador , Neoplasias del Cuello Uterino/diagnóstico por imagen , Neoplasias del Cuello Uterino/radioterapia , Adulto , Anciano , Terapia Combinada , Femenino , Humanos , Imagenología Tridimensional , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Radiografía Intervencional , Dosificación Radioterapéutica , Neoplasias del Cuello Uterino/patologíaRESUMEN
Intratracheal immunization of mice with inactivated influenza B virus and delipidated Bacillus firmus as adjuvant increases protection of mice against infection with the homologous virus strain and induces cross-protection: mice immunized by influenza virus B/Yamanashi 166/98 were protected even against phylogenetically distant virus drift variant B/Lee/40 lethal for mice.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Inmunización/métodos , Virus de la Influenza B/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Administración por Inhalación , Animales , Bacillus/inmunología , Protección Cruzada , Femenino , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Análisis de Supervivencia , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Inactivated Bacillus firmus (BF), G+ nonpathogenic bacterium of the external environment, was coupled to ovalbumin (OVA) and used in immunization experiments as antigen carrier. Balb/c mice were immunized thrice intra-tracheally and intra-nasally with conjugates of OVA and BF. Surprisingly, administration of OVA-BF conjugates inhibited anti-OVA IgG response in both sera and mucosal secretions if compared to an exposure to OVA alone. The suppression of antigen-specific antibody production was accompanied by promotion of TH1 phenotype.
Asunto(s)
Antígenos/administración & dosificación , Bacillus/inmunología , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Administración Intranasal , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/sangre , Portadores de Fármacos , Femenino , Inmunidad Mucosa , Inmunización , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Técnicas In Vitro , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Células TH1/inmunología , TráqueaRESUMEN
Satisfactory mucosal immunity in the respiratory tract is very important for protection against influenza. It can be achieved only by mucosal immunization. Mucosal vaccination with inactivated influenza virus may not be sufficiently effective and suitable adjuvants are therefore sought. We tested intratracheal immunization of mice with inactivate B type influenza virus in a mixture with formolized G+ bacterium Bacillus firmus, whose adjuvant effects have previously been documented in another system. The treatment resulted in a marked increase of both systemic and mucosal antibody response in IgG and IgA classes. Stimulation of T lymphocytes after adjuvant immunization was very mild, no proliferation taking place after specific stimulation with antigen in vitro. However, slightly increased systemic (spleen) and local (lungs) production of cytokines without perceptible Th1/Th2 polarization was determined. B. firmus is an efficient adjuvant in respiratory tract immunization while with subcutaneous immunization it lowers the antibody response.
Asunto(s)
Adyuvantes Inmunológicos , Betainfluenzavirus/inmunología , Inmunidad Mucosa/inmunología , Vacunas contra la Influenza/inmunología , Vacunas de Productos Inactivados/inmunología , Inactivación de Virus , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/inmunología , Línea Celular , Citocinas/biosíntesis , Citocinas/genética , Citocinas/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática , Femenino , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Activación de Linfocitos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Pruebas de Neutralización , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T/inmunologíaRESUMEN
Functions of T cells were determined after intranasal and intratracheal immunization of mice with ovalbumin (Ova) and Bacillus firmus (Bf), a Gram-positive nonpathogenic bacterium of the external environment, or delipidated Bf (dBf) as adjuvants, with the aim to elucidate the mechanism of support of Ova-specific antibody production caused by Bf that had been observed in an identical experiment. Neither Bf nor dBf in a mixture with Ova stimulated Ova-specific T-cell response tested as antigen-specific blast transformation. By contrast, a mild polyclonal stimulation was observed in splenocytes from mice given dBf. In vitro incubation of splenocytes with 100 micrograms (but not 10 micrograms) of Bf or dBf led to a highly significant inhibition of proliferation below the control level in all groups of animals. Supernatants of splenocyte cultures were further tested for cytokine production. IL-10 and IFN-gamma were released after in vitro challenge with dBf and in some cases also with Bf. Analysis of sera demonstrated that administration of Ova + adjuvant brought about an increase in anti-Ova IgG1, IgG2a and IgG2b whereas treatment with Ova alone caused a rise in IgG1 only. The role of Bf or dBf in the enhancement of antigen-specific antibody production could be in influencing macrophages and inducing cytokine milieu composed of IL-10, IFN-gamma and other factors that leads to a bystander stimulation of specifically activated Ova-B cell receptor (Ova-BCR)-bearing cells.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Bacillus/inmunología , Ovalbúmina/inmunología , Linfocitos T/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , Animales , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Inmunización/métodos , Inmunización/normas , Inmunoglobulina G/sangre , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/administración & dosificaciónRESUMEN
Bacillus firmus (a Gram-positive nonpathogenic and harmless bacterium), was shown to be a strong polyclonal activator of mouse B lymphocytes as estimated by ELISA testing of Ig concentrations in culture supernatants after incubation of BALB/c mouse splenocytes with inactivated bacillus. Synthesis of all main Ig classes and all IgG subclasses was stimulated in vitro, the considerable effect on IgA formation being the most interesting feature. B cell stimulation was T cell dependent, as was demonstrated by the effect of B. firmus on all Ig isotypes and by comparison of lymphocyte response of nu/nu mice and heterozygous nu/+ mice. The effect of B. firmus on splenocyte proliferation was stimulatory or suppressive depending on the dose of the bacterium. Increased synthesis of IFN-gamma and IL-10 (detected by ELISA in splenocyte culture supernatants) showed probable stimulation of Th1 and Th2 subpopulations. Considering the stimulatory effect on IgA formation and macrophage stimulation, B. firmus seems to be a prospective mucosal adjuvant and/or probiotic.
Asunto(s)
Linfocitos B/inmunología , Bacillus/inmunología , Inmunoglobulina G/biosíntesis , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Linfocitos B/efectos de los fármacos , Bacillus/química , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB CRESUMEN
A nonpathogenic bacterium of external environment possessing remarkable immunomodulatory activity, Bacillus firmus (BF) inactivated with formaldehyde, was given intragastrically to two genetically different mouse strains BALB/c (H-2d) and B10.BR/SnPh (B10.BR, H-2k) reared in conventional (CV) and B10.BR strain also in germ-free (GF) conditions. Repeated intragastric administration of BF (500 micrograms every other day over two weeks, starting at the age of 3 months) significantly enhanced intestinal IgA levels in CV BALB/c mice but did not affect intestinal IgA in CV B10.BR mice. In GF B10.BR mice, IgG levels in sera and intestinal washings increased after BF administration compared to CV B10.BR mice. In CV BALB/c mice, specific activity of enterocyte brush-border enzymes (lactase, gamma-glutamyltransferase, alkaline phosphatase) decreased after BF treatment; sucrase (sucrose alpha-glucosidase) activity was not affected. On the other hand, in B10.BR mice, specific activity of gamma-glutamyltransferase and dipeptidyl peptidase IV were higher after administration of BF in both CV and GF groups relative to untreated controls. The activities of lactase and glucoamylase (glucan 1,4-alpha-glucosidase) were significantly stimulated only in the group of GF B10.BR mice treated with formolized BF. The stimulation of immunoglobulin production after BF treatment was accompanied by changes in the levels of enterocyte brush-border enzymes; this responsiveness to BF treatment was genetically regulated.
Asunto(s)
Bacillus/inmunología , Intestinos/inmunología , Intestinos/microbiología , Fosfatasa Alcalina/metabolismo , Animales , Dipeptidil Peptidasa 4/metabolismo , Enterocitos/enzimología , Enterocitos/microbiología , Femenino , Predisposición Genética a la Enfermedad , Vida Libre de Gérmenes , Glucano 1,4-alfa-Glucosidasa/metabolismo , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/sangre , Inmunohistoquímica , Mucosa Intestinal/enzimología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Intestinos/enzimología , Lactasa , Ratones , Ratones Endogámicos BALB C , Microvellosidades/enzimología , Sacarasa/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Bacillus firmus, a non-pathogenic Gram positive (G+) bacterium of the external environment was investigated for immunomodulatory properties. It stimulated an increase in anti-ovalbumin IgG in sera, bronchoalveolar lavages and intestinal washings after both intranasal (i.n.) and intratracheal (i.t.) immunisation, and enhanced anti-ovalbumin IgA in intestinal secretions and in bronchoalveolar lavage fluid after i.n. or i.t. immunisation, respectively. The immunomodulatory effect of B. firmus on antibody formation was antigen specific.
Asunto(s)
Adyuvantes Inmunológicos , Anticuerpos/inmunología , Bacillus/inmunología , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Mucosa Respiratoria/inmunología , Administración Intranasal , Animales , Anticuerpos Antibacterianos/sangre , Formación de Anticuerpos , Antígenos/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Femenino , Inmunidad Mucosa/inmunología , Inyecciones , Intestinos/inmunología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , TráqueaRESUMEN
PROBLEM: The role of Ala-Trp-Asn (AWN) and Ala-Gln-Asn (AQN) families of spermadhesive sperm proteins in fertilization. METHOD OF STUDY: The preparation and characterization of polyclonal antibodies against AWN and AQN spermadhesins and one monoclonal antibody (MAb), designated Bo.5, against AWN spermadhesin. The use of biochemical and immunocytochemical methods for characterization of spermadhesins on the sperm membrane of boar spermatozoa and in the cryostat sections of boar reproductive organs. RESULTS: Polyclonal anti-AWN and anti-AQN antibodies specifically reacted with AWN and AQN proteins, respectively. MAb Bo.5 detected the 17-, 16-, and 14-kDa protein members of AWN subfamily. The monoclonal, as well as the polyclonal, AWN antibodies remarkably decreased the sperm binding to the egg surface in an in vitro sperm zona pellucida binding assay. CONCLUSIONS: Presented results demonstrate that polyclonal antibodies and MAb Bo.5 against spermadhesins specifically recognize the membrane-associated antigens and inhibit the binding of sperm to oocytes. Reduced binding of sperm to oocytes, due to the antibodies, indicates the role of these spermadhesins in sperm-egg primary binding.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Moléculas de Adhesión Celular/inmunología , Glicoproteínas/inmunología , Oocitos/metabolismo , Interacciones Espermatozoide-Óvulo/inmunología , Espermatozoides/fisiología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/fisiología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Glicoproteínas/aislamiento & purificación , Glicoproteínas/fisiología , Humanos , Masculino , Oocitos/inmunología , Oocitos/fisiología , Conejos , Receptores de Superficie Celular , Espermatozoides/inmunología , Espermatozoides/metabolismo , Porcinos , Zona Pelúcida/metabolismo , Zona Pelúcida/fisiologíaRESUMEN
Heparin-binding proteins (designated BHB-2-BHB-9) were isolated from boar seminal plasma by affinity chromatography on heparin immobilized on polyacrylamide gel, followed by reverse phase HPLC. According to their N-terminal amino acid sequences, BHB-3-BHB-5 belong to the AQN family of spermadhesins and BHB-7-BHB-9 to the AWN family. BHB-6 is composed of two different proteins. The dominant protein (14 kDa) has the N-terminal amino acid sequence HNKQEGRDHD that is identical to the sequence of human semenogelin at positions 85-94. The minor proteins (16 and 17 kDa) belong to the AWN family of spermadhesins. The 14 kDa HNK protein does not crossreact with antibodies against AQN or AWN spermadhesins. BHB-2 also binds to the acrosome of boar epididymal spermatozoa but has the N-terminal sequence DQH. Therefore, basic protein BHB-2 belongs to a new family of DQH sperm surface proteins that are homologous to the acidic proteins from bull and stallion seminal plasma, to the collagen binding domain II in fibronectin and to the leucocyte cell-cell adhesion regulator, but are not homologous to AQN or AWN spermadhesins. Nevertheless, anti-AQN-1 spermadhesin antibodies crossreact strongly with DQH protein. All boar heparin-binding proteins bind concanavalin A indicating their glycoprotein nature, which was proved by the detection of glucosamine and galactosamine residues in their molecules. Furthermore, spermadhesins interact with zona pellucida, protease inhibitors and a polyacrylamide derivative of heparin. Affinity chromatography experiments showed that the DQH protein bound to gelatin-agarose together with the AWN proteins and that the DQH protein and AQN-1 spermadhesin belong to the phosphoryl choline binding proteins.
Asunto(s)
Semen/química , Porcinos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Proteínas Portadoras/metabolismo , Femenino , Immunoblotting , Masculino , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Unión Proteica , Espermatozoides/metabolismo , Zona Pelúcida/metabolismoRESUMEN
Heparin-binding proteins BHB 2-BHB 5 were purified from boar seminal plasma by affinity chromatography on a heparin-polyacrylamide column and reversed phase HPLC. Three of the proteins, BHB 3-BHB 5, were found to be identical to spermadhesins AQN 1-AQN 3 isolated from boar spermatozoa. The lectin-like properties of the isolated proteins BHB 2-BHB 5 were studied using double-diffusion in agarose gel, enzyme-linked binding assay, and inhibition assays of erythroagglutinating activity. It was found that proteins BHB 3-BHB 5 (spermadhesins AQN 1-AQN 3) interacted with glycoproteins containing O-glycosidically bound oligosaccharide chains, but not with those containing only N-linked carbohydrate chains. The strongest interaction was observed between BHB 3 (AQN 1) and desialyzed bovine submaxillary gland mucin, the glycoprotein containing only O-glycosidically linked saccharides. No interaction of BHB 3-BHB 5 proteins with simple saccharides, their derivatives or acidic polysaccharides was observed. Both the hemagglutinating activity and saccharide-binding properties of BHB 2 protein were quite different. Agglutinating activity of human erythrocytes by BHB 2 protein was significantly higher than that by BHB 3-BHB 5 proteins (AQN spermadhesins). In contrast to AQN spermadhesins, BHB 2 protein (DQH sperm surface protein) interacted strongly with acidic polysaccharides and sialyzed glycoproteins, but no binding of desialyzed glycoproteins as well as N-acetyl-alpha-D-galactosaminyl-O-serine,simple monosaccharides and amino sugars was observed.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Plasma Seminal , Espermatozoides/metabolismo , Animales , Proteínas Portadoras/aislamiento & purificación , Bovinos , Moléculas de Adhesión Celular/aislamiento & purificación , Femenino , Hemaglutinación , Humanos , Técnicas In Vitro , Masculino , Glicoproteínas de Membrana/aislamiento & purificación , Unión Proteica , Semen/metabolismo , Interacciones Espermatozoide-Óvulo , Porcinos , Zona Pelúcida/metabolismoRESUMEN
A mouse monoclonal antibody against boar acrosin and antiserum prepared to highly purified acrosin in female rabbits were used to detect the antigen in various fluids and tissues of boars using an indirect immunofluorescence technique. A strong reaction was found in fluid and epithelial tissue of the seminal vesicles as well as in the germinal cells in the testis. No immunoreactivity was detected in tissues of the epididymides and other organs of the boar. The antigens present in seminal vesicle fluid of boars were partially purified by column chromatography. It was demonstrated that two antigens differing in molecular mass were present and both possessed protease and amidase activity. The higher molecular mass antigen eluted from a gel filtration column in a volume identical to that of proacrosin. The same result was obtained in polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). The low molecular mass antigen was eluted from Sephadex G-75 column together with natural protease inhibitors corresponding in molecular mass to less than 20 kDa. The mobility of the antigen in SDS-PAGE was greater than that of chymotrypsin. It is assumed that the protease from seminal vesicle epithelial resembled acrosin in structure and function. Acrosin may therefore not be specific for spermatozoa.
Asunto(s)
Acrosina/metabolismo , Vesículas Seminales/enzimología , Serina Endopeptidasas/metabolismo , Porcinos/metabolismo , Acrosina/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Masculino , Serina Endopeptidasas/análisis , Serina Endopeptidasas/inmunología , Espermatozoides/enzimologíaRESUMEN
A highly purified 15 kDa glycoprotein isolated from ejaculated spermatozoa was used to raise antisera in female rabbits. An indirect immunofluorescence technique was used to detect the antigen in the seminal vesicle tissue and on the acrosomes of ejaculated, native and capacitated, boar spermatozoa. No immunoreactivity was detected on cells of the seminiferous tubules (spermatogonia, spermatocytes, and spermatids), on spermatozoa in the ductus epididymis and in cells of the epididymal and testicular tissues. These observations support the view that the 15 kDa protein is produced in the seminal vesicle secretory epithelium, and is attached to the sperm plasma membrane during the exposure of spermatozoa to seminal vesicle compounds. The observations that the antigen remained on the acrosome of ejaculated spermatozoa after capacitation and blocked sperm-oocyte binding in vitro suggest that the antigen plays a role in sperm-egg interactions. The strong immunoreactivity exhibited by cumulus cells after incubation of antisera with the porcine egg surrounded by cumulus cells shows the possible importance of the 15 kDa glycoprotein for contact of spermatozoa with cells of the cumulus oophorus surrounding the egg.
Asunto(s)
Glicoproteínas/metabolismo , Folículo Ovárico/citología , Interacciones Espermatozoide-Óvulo/inmunología , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Animales , Bovinos , Ensayo de Inmunoadsorción Enzimática , Epidídimo/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Glicoproteínas/inmunología , Inmunoelectroforesis , Masculino , Folículo Ovárico/metabolismo , Próstata/inmunología , Conejos , Vesículas Seminales/metabolismo , Aglutinación Espermática , Capacitación Espermática , Espermatozoides/inmunología , Porcinos , Testículo/inmunologíaRESUMEN
The serine proteinase acrosin plays an important role in sperm penetration of the zona pellucida. In the present study we investigated the effect of the enzyme on various matrix proteins. Acrosin degraded proteolytically fibronectin, type IV collagen and heat denatured type I collagen, whereas neither native type I collagen nor laminin were cleaved by the enzyme. The specific activity of acrosin with type IV collagen as substrate (66.6 g/h/g) was 125-fold higher than that of known type IV collagenase or stromelysin. These results suggest that acrosin may act as a matrix-degrading proteinase.
Asunto(s)
Acrosina/metabolismo , Colágeno/metabolismo , Fibronectinas/metabolismo , Espermatozoides/enzimología , Acrosina/antagonistas & inhibidores , Acrosina/farmacología , Animales , Calor , Humanos , Laminina/metabolismo , Masculino , Metaloproteinasa 3 de la Matriz , Metaloendopeptidasas/metabolismo , Colagenasa Microbiana/metabolismo , Desnaturalización Proteica , Ratas , Especificidad por SustratoRESUMEN
Human cumuli-oophori were cultured in vitro in the presence of radioactive protein and polysaccharide precursors. The time course of the cumulus cell secretion was traced by histoautoradiography. Matrix solubilization, and sodium dodecyl sulphate polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that proteoglycan (Mr greater than 1,700,000) was the main cumulus cell product that was prevailingly deposited in the cumulus intercellular matrix and partly released into the culture medium. It was capable of accelerating the conversion of proacrosin to acrosin and this activity was abolished by enzymatic removal of chondroitin sulphate, the predominant glycosaminoglycan of this proteoglycan fraction. None of the other fractions, including a proteoglycan of Mr 80,000-90,000, containing heparan sulphate, accelerated the conversion of proacrosin to acrosin under the conditions used. The results suggest that chondroitin sulphate is the active component of the high-Mr proacrosin activator of the human cumulus-oophorus.
Asunto(s)
Acrosina/biosíntesis , Acrosina/metabolismo , Sulfatos de Condroitina/metabolismo , Precursores Enzimáticos/metabolismo , Matriz Extracelular/metabolismo , Folículo Ovárico/metabolismo , Proteoglicanos/biosíntesis , Autorradiografía , Células Cultivadas , Cromatografía Líquida de Alta Presión , Activación Enzimática/fisiología , Matriz Extracelular/química , Femenino , Humanos , Microscopía Electrónica de Rastreo , Folículo Ovárico/química , Folículo Ovárico/citología , Proteoglicanos/análisisRESUMEN
Boar seminal vesicle fluid inhibits blast transformation of porcine lymphocytes. Boar seminal vesicle fluid was precipitated in 8% ethanol and the dissolved precipitate separated into two peaks upon chromatography on a Sephacryl S-200 column. The inhibitory activity was found predominantly in the second peak. This peak was rechromatographed on the Sephacryl S-200 column, and the fractions with inhibitory activity were pooled and passed down a Sephadex G-75 column. This chromatography run separated the accompanying protease inhibitor from the immunosuppressive fraction. The molecular mass, estimated by chromatography on the Sephadex G-75 column, was 25,000-27,000 for the immunosuppressive factor and 7,500 for the protease inhibitor. Activity for the immunosuppressive fraction was also demonstrated in vivo.
Asunto(s)
Tolerancia Inmunológica/inmunología , Semen/inmunología , Animales , Cromatografía en Gel/métodos , Masculino , PorcinosRESUMEN
Lectin-like molecules on the sperm surface are implicated in the process of gamete recognition and adhesion. We have isolated and biochemically characterized a 15 kDa glycoprotein from ejaculated boar sperm which possess zona pellucida-binding- and haemagglutinating-activity. The zona/15 kDa protein interaction is inhibited by fucoidan, suggesting that the glycoprotein is one of the sperm components which participate in the initial gamete interaction. N-Terminal sequence analysis of the isolated 15 kDa glycoprotein showed that it may belong to the same sperm/egg recognition-mediating protein family as the sea urchin sperm protein binding.
Asunto(s)
Glicoproteínas/química , Receptores de Superficie Celular/química , Espermatozoides/química , Zona Pelúcida/química , Secuencia de Aminoácidos , Animales , Evolución Biológica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Masculino , Datos de Secuencia Molecular , Peso Molecular , Polisacáridos/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Erizos de Mar , Espermatozoides/efectos de los fármacos , Porcinos , Zona Pelúcida/efectos de los fármacosRESUMEN
A new acrosin inhibitor with a relative molecular mass of about 8000 was isolated to apparent homogeneity from ejaculated boar spermatozoa. The inhibitor is effective against boar acrosin and bovine trypsin. It interacts with polyvalent antibodies against the acrosin inhibitor from boar seminal plasma, but differs from all known acrosin inhibitors in its amino acid composition and N-terminal sequence.
Asunto(s)
Acrosina/antagonistas & inhibidores , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Masculino , Datos de Secuencia Molecular , Peso Molecular , Porcinos , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificaciónRESUMEN
The effect of heat-solubilized zonae pellucidae (ZP) isolated from mature pig ovaries on the conversion of pig proacrosin to acrosin was examined and compared with the effects of different sulphated polysaccharides. At low concentrations, zona glycoproteins potently stimulated acrosin auto-activation. Up to 2.5 micrograms ml-1 ZP, the acceleration of acrosin activation was shown to be dependent on the ZP concentration. At higher concentrations zona glycoproteins exerted an inhibitory effect on acrosin amidase activity. A similar effect was demonstrated for fucoidan, heparin, and chondroitin sulphate. The results intimate that in vivo, the conversion of proacrosin to acrosin is regulated at the sperm-zona interface.
Asunto(s)
Acrosina/biosíntesis , Acrosina/metabolismo , Precursores Enzimáticos/metabolismo , Óvulo/fisiología , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/metabolismo , Zona Pelúcida/fisiología , Animales , Sulfatos de Condroitina/farmacología , Activación Enzimática/efectos de los fármacos , Heparina/farmacología , Mananos/farmacología , Polisacáridos/farmacología , Albúmina Sérica/farmacología , PorcinosRESUMEN
Low-molecular-mass zymogen was extracted from boar spermatozoa together with proacrosin using 10% acetic acid supplemented with 10% glycerol, and was purified by the sequential use of gel filtration on Sephadex G-75 and (FPLC) reversed-phase chromatography. LMM zymogen represented approximately 5% of the latent trypsin-like activity present in the sperm extract. SDS-PAGE indicated a molecular mass of 33 kDa. The zymogen reacted with both mouse monoclonal and rabbit polyclonal antibodies to boar acrosin. Determination of the N-terminal sequence of 34 amino-acid residues revealed its identity with the known N-terminal sequence of boar proacrosin.