RESUMEN
BACKGROUND: House dust mite (HDM) is the major indoor allergen for allergic diseases such as allergic rhinitis (AR) and asthma. Although sublingual immunotherapy is a curative treatment for HDM-induced AR, data from large-scale studies are limited. We evaluated the efficacy and safety of HDM tablets in adolescent and adult patients (aged 12-64 years) with HDM-induced AR with or without intermittent asthma. METHODS: In a double-blind trial in Japan, 968 subjects were randomized 1 : 1 : 1 to 300 index of reactivity (IR), 500 IR, or placebo groups. The primary endpoint was the Average Adjusted Symptom Score (AASS) in the last eight weeks of the 52-week treatment. Secondary endpoints included individual nasal and ocular symptom scores, rescue medication use, and the Japanese Rhinoconjunctivitis Quality of Life Questionnaire (JRQLQ) scores. RESULTS: The AASS in the last eight weeks of treatment significantly improved in both the 300 IR and the 500 IR groups compared to that in the placebo group (P < 0.001). In the 300 IR group, the onset of action occurred at week 8-10. All four nasal symptoms significantly improved in both active treatment groups; rescue medication use and JRQLQ outcome improved in the 300 IR group. Most adverse events (AEs) were mild, and 16 serious AEs (SAEs) were reported; however, none of them were drug-related. CONCLUSIONS: One-year treatment with 300 IR and 500 IR HDM tablets was effective without major safety concerns. The recommended therapeutic dose for AR is 300 IR.
Asunto(s)
Alérgenos/inmunología , Pyroglyphidae/inmunología , Rinitis Alérgica/inmunología , Rinitis Alérgica/terapia , Inmunoterapia Sublingual , Adolescente , Adulto , Alérgenos/administración & dosificación , Animales , Niño , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Japón , Masculino , Persona de Mediana Edad , Rinitis Alérgica/diagnóstico , Inmunoterapia Sublingual/efectos adversos , Inmunoterapia Sublingual/métodos , Comprimidos , Resultado del Tratamiento , Adulto JovenRESUMEN
Prostaglandin (PG) D2, the major cyclooxygenase metabolite generated from immunologically stimulated mast cells, is thought to contribute to the pathogenesis of allergic diseases due to its various inflammatory effects. However, since no DP receptor antagonist has been developed as an antiallergic drug, the role of PGD2 in the pathogenesis of allergic diseases remains uncertain. Here, we report the in vivo efficacy of our newly established DP receptor antagonist, S-5751 [((Z)-7-[(1R,2R,3S,5S)-2-(5-hydroxy benzo[b]thiophen-3-ylcarbonylamino)-10-norpinan-3-yl]hept-5- enoic acid)], using various allergic inflammation guinea pig models. In allergic rhinitis models, oral administration of S-5751 dramatically inhibited not only early nasal responses, as assessed by sneezing, mucosal plasma exudation, and nasal blockage, but also late responses such as mucosal plasma exudation and eosinophil infiltration. Even when S-5751 was administered after recovery from the early responses, these late phase responses were almost completely suppressed. In addition, S-5751 alleviated allergen-induced plasma exudation in the conjunctiva in an allergic conjunctivitis model and antigen-induced eosinophil infiltration into the lung in an asthma model. These findings provide evidence for the crucial role of PGD2 as a mediator of allergic inflammation in guinea pigs and suggest that DP receptor antagonists may be useful in the treatment of allergic diseases triggered by mast cell activation.
Asunto(s)
Hipersensibilidad/prevención & control , Inflamación/prevención & control , Antagonistas de Prostaglandina/farmacología , Receptores de Prostaglandina/efectos de los fármacos , Tiofenos/farmacología , Animales , Plaquetas/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Conjuntivitis/tratamiento farmacológico , Conjuntivitis/patología , AMP Cíclico/sangre , Cobayas , Células HL-60 , Humanos , Hipersensibilidad/complicaciones , Técnicas In Vitro , Inflamación/etiología , Ligandos , Masculino , Enfermedades Respiratorias/tratamiento farmacológico , Enfermedades Respiratorias/patología , Rinitis Alérgica Estacional/tratamiento farmacológico , Rinitis Alérgica Estacional/patologíaRESUMEN
Various 1,2-isothiazolidine-1,1-dioxide (gamma-sultam) derivatives containing an antioxidant moiety, 2,6-di-tert-butylphenol substituent, were prepared. Some compounds, which have a lower alkyl group at the 2-position of the gamma-sultam skeleton, showed potent inhibitory effects on both cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LO), as well as production of interleukin (IL)-1 in in vitro assays. They also proved to be effective in several animal arthritic models without any ulcerogenic activities. Among these compounds, (E)-(5)-(3,5-di-tert-butyl-4-hydroxybenzylidene)-2-ethyl-1, 2-isothiazolidine-1,1-dioxide (S-2474) was selected as an antiarthritic drug candidate and is now under clinical trials. The structure-activity relationships (SAR) examined and some pharmacological evaluations are described.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Artritis/tratamiento farmacológico , Óxidos S-Cíclicos/síntesis química , Isoenzimas/farmacología , Inhibidores de la Lipooxigenasa , Prostaglandina-Endoperóxido Sintasas/farmacología , Tiazoles/síntesis química , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Óxidos S-Cíclicos/efectos adversos , Óxidos S-Cíclicos/uso terapéutico , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/síntesis química , Inhibidores de la Ciclooxigenasa/uso terapéutico , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Edema/inducido químicamente , Edema/tratamiento farmacológico , Femenino , Mucosa Gástrica/efectos de los fármacos , Humanos , Interleucina-1/antagonistas & inhibidores , Interleucina-1/farmacología , Leucotrieno B4/antagonistas & inhibidores , Leucotrieno B4/biosíntesis , Inhibidores de la Lipooxigenasa/síntesis química , Inhibidores de la Lipooxigenasa/uso terapéutico , Masculino , Proteínas de la Membrana , Ratas , Ratas Endogámicas Lew , Relación Estructura-Actividad , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Tiazoles/efectos adversos , Tiazoles/uso terapéuticoRESUMEN
Bone is continuously being formed and resorbed. This process is accomplished by the precise coordination of two cell types: osteoblasts and osteoclasts. Osteoclasts are large, multinucleated cells that are derived from the same hematopoietic precursors as macrophages. However, these bone-resorbing cells are difficult to study directly because of their relative inaccessibility. The purification of primary osteoclasts from rabbit bones by their adherent nature provides an opportunity for investigating the molecules in osteoclasts. We have examined the expression of receptor tyrosine kinase by polymerase chain reaction (PCR) and found that Tyro 3 was frequently identified from primary osteoclasts in PCR cloning. Immunohistochemistry revealed that Tyro 3 was expressed on the multinucleated osteoclasts which were positive for tartrate-resistant acid phosphatase (TRAP), but not on mononuclear TRAP-positive cells. The Tyro 3 ligand, Gas6, induced the phosphorylation of Tyro 3 receptors in osteoclasts in two to five min. Gas6 and protein S directly enhanced the bone resorbing activity of mature osteoclasts. This effect of Gas6 was inhibited by the addition of a tyrosine kinase inhibitor, herbimycin A. However, Gas6 did not affect the differentiation of osteoclasts from bone marrow cells. Gas6 and protein S are dependent on vitamin K, a cofactor for the enzyme responsible for carboxylation of glutamic acid residues. The findings in this study are the first to indicate a new biological activity of Gas6 and protein S as a direct regulator of osteoclastic function; they give an insight into the role of these vitamin K-dependent ligands in bone resorption in vivo.
Asunto(s)
Células de la Médula Ósea/citología , Péptidos y Proteínas de Señalización Intercelular , Osteoclastos/fisiología , Proteínas/farmacología , Proteínas Tirosina Quinasas Receptoras/fisiología , Fosfatasa Ácida/biosíntesis , Animales , Biomarcadores , Resorción Ósea , Calcitriol/farmacología , Diferenciación Celular , Células Cultivadas , Clonación Molecular , Colágeno , Cartilla de ADN , Células Madre Hematopoyéticas/citología , Isoenzimas/biosíntesis , Ratones , Ratones Endogámicos C57BL , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Fosforilación , Reacción en Cadena de la Polimerasa , Conejos , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Recombinantes/biosíntesis , Fosfatasa Ácida TartratorresistenteRESUMEN
To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (< 1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (< 5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (> 8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.
Asunto(s)
Carcinógenos/toxicidad , Comunicación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/toxicidad , Uniones Comunicantes/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Acetato de Tetradecanoilforbol/toxicidad , Células 3T3/citología , Células 3T3/efectos de los fármacos , Animales , Northern Blotting , Western Blotting , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Humanos , Indoles/toxicidad , Maleimidas/toxicidad , Ratones , Osteoblastos/citología , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Recombinantes/toxicidadRESUMEN
Concanavalin A (Con A), a plant lectin that recognizes cell-surface glycoproteins, up-regulates osteoclastic bone-resorbing activity of cultured isolated rabbit pure osteoclasts as well as unfractionated bone cells. The effect of Con A was blocked by alpha-methyl mannopyranoside. Several Con A-binding proteins were detected in the plasma membranes from osteoclasts. Furthermore, Con A increased the levels of transcripts of cathepsin K/OC-2, one of the proteases responsible for osteoclastic bone-resorbing activity. These results indicate that Con A directly enhances the function of osteoclasts by the association with surface glycoproteins of osteoclasts.
Asunto(s)
Resorción Ósea , Catepsinas/genética , Concanavalina A/farmacología , Expresión Génica/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Animales , Catepsina K , Células Cultivadas , ConejosRESUMEN
We previously reported the isolation of the cartilage-derived growth promoting factors chondromodulin-I (ChM-I) and chondromodulin-II (ChM-II) from fetal bovine epiphyseal cartilage. Both of these factors stimulate the growth and matrix formation of chondrocytes in vitro. In the present study, we found that ChM-I and ChM-II stimulated the proliferation of clonal mouse osteoblastic MC3T3-E1 cells as well as primary mouse osteoblasts in culture. Unlike other known growth factors, these factors did not support the proliferation of fibroblasts. Concomitantly with growth stimulation of osteoblasts, there was a reduction of alkaline phosphatase (ALP) activity in the cells, the expression of the differentiated phenotype. These results suggest that epiphyseal cartilage may play a functional role in longitudinal bone growth by production of these unique growth-promoting factors.
Asunto(s)
Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana , Osteoblastos/citología , Proteínas/farmacología , Fosfatasa Alcalina/metabolismo , Animales , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , ADN/biosíntesis , Factor 2 de Crecimiento de Fibroblastos/farmacología , Ratones , Ratones Endogámicos ICR , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismoRESUMEN
Human calcitonin (hCT) has been reported to have a less hypocalcemizing effect on rats and to have a lower binding affinity for the receptor of mouse osteoclasts than salmon CT(sCT). In this study we comparatively examined the effect of hCT and sCT on osteoclastic bone-resorbing activity of unfractionated cells obtained from human giant cell tumor of bone and from rabbit and mouse long bones. We found that hCT had the same inhibitory effect as sCT on the bone-resorbing activity of human and rabbit osteoclastic cells, but a different one on that of mouse cells. These results indicate that the activity of drugs should be assayed using human cells if possible.
Asunto(s)
Resorción Ósea/tratamiento farmacológico , Calcitonina/uso terapéutico , Células Gigantes/patología , Osteoclastos/efectos de los fármacos , Animales , Neoplasias Óseas/patología , Calcitonina/farmacología , Fraccionamiento Celular , Células Gigantes/citología , Células Gigantes/ultraestructura , Humanos , Ratones , Microscopía Electrónica , Osteoclastos/citología , Osteoclastos/ultraestructura , Conejos , Especificidad de la Especie , Células Tumorales CultivadasRESUMEN
Vitamin A metabolites such as all-trans-retinoic acid (all-trans-RA) affect several steps of metabolic processes in vertebrates. In the last few years, several studies have shown the effect of RA on bone formation and metabolism. However, mechanisms of its action still remain unclear, especially with respect to the regulation of bone cells. Therefore, this study was carried out to clarify how RA regulates the activity of osteoclasts. Using a pit assay involving unfractionated bone cells, including osteoclasts obtained from rabbits, we found that RA stimulated an increase in the bone-resorbing activity in a dose- and time-dependent manner. Furthermore, this effect occurred more rapidly than that of treatments with 1 alpha,25-dihydroxyvitamin D3. However, this effect of RA may be partly related to cross-talk between osteoclasts and other types of cells. Therefore we studied the effect of RA on isolated osteoclasts. We found that all-trans-RA regulates the gene expression of cathepsin K/OC-2, a dominant cysteine proteinase, at the transcriptional level in mature osteoclasts isolated from rabbits. Moreover, retinoic acid-receptor alpha mRNA and retinoid X-receptor beta mRNA were expressed in these mature osteoclasts. Our results indicate that osteoclasts are target cells for RA and that RA might regulate a part of bone formation and metabolism through osteoclasts.
Asunto(s)
Resorción Ósea , Catepsinas/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Tretinoina/farmacología , Animales , Catepsina K , Células Cultivadas , Conejos , Receptores de Ácido Retinoico/genética , Transcripción Genética/efectos de los fármacosRESUMEN
The inhibitory effect of murine interferon gamma (muIFN gamma) on humoral hypercalcemia in nude mice bearing lower-jaw cancer (LJC-1-JCK), in which parathyroid-hormone(PTH)-related protein is responsible for causing humoral hypercalcemia by activating bone resorption, was examined in comparison with that of a new bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). muIFN gamma was injected into tumor-bearing nude mice for 5 days before the establishment of hypercalcemia. The increase of plasma calcium concentration was delayed and this effect continued for more than 6 days even after the injection was stopped. Alendronate markedly suppressed hypercalcemia in tumor-bearing nude mice but this inhibitory effect continued for less than 6 days. Neither muIFN gamma nor alendronate affected the tumor volume or serum PTH-related protein concentration. Injection of muIFN gamma into mice for 3 days almost completely abolished the formation of multinucleated osteoclast-like cells from bone marrow cells in vitro, whereas injection of alendronate into mice had no effect. These findings suggested that muIFN gamma suppressed the formation of osteoclasts, resulting in the prolonged decrease of plasma calcium concentration in hypercalcemic tumor-bearing nude mice, whereas alendronate is cytotoxic to functionally mature osteoclasts and inhibited osteoclastic bone resorption, resulting in a marked decrease in the plasma calcium concentration in tumor-bearing hypercalcemic nude mice.
Asunto(s)
Difosfonatos/farmacología , Hipercalcemia/sangre , Hipercalcemia/tratamiento farmacológico , Interferón gamma/farmacología , Neoplasias Maxilomandibulares/sangre , Neoplasias Maxilomandibulares/tratamiento farmacológico , Adulto , Alendronato , Animales , Resorción Ósea/metabolismo , Calcio/sangre , Femenino , Factor Estimulante de Colonias de Granulocitos/sangre , Humanos , Hipercalcemia/complicaciones , Interleucina-1/sangre , Interleucina-6/sangre , Neoplasias Maxilomandibulares/complicaciones , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/sangre , Trasplante de Neoplasias , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A new type of nylon oligomer degradation enzyme (EIII) was purified from an Escherichia coli clone harboring the EIII gene (nylC). This enzyme hydrolyzed the linear trimer, tetramer, and pentamer of 6-aminohexanoate by an endo-type reaction, and this specificity is different from that of the EI (nylA gene product) and EII (nylB gene product). Amino acid sequencing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified EIII demonstrated that the enzyme is made of two polypeptide chains arising from an internal cleavage between amino acid residues 266 and 267.
Asunto(s)
Amidohidrolasas/genética , Flavobacterium/enzimología , Flavobacterium/genética , Genes Bacterianos , Amidohidrolasas/aislamiento & purificación , Aminocaproatos , Ácido Aminocaproico/metabolismo , Biodegradación Ambiental , Nylons/metabolismo , Especificidad por SustratoRESUMEN
Flavobacterium sp. strain KI725 harbors plasmid pOAD21, a derivative of nylon oligomer-degradative plasmid pOAD2, in which all of nylA (the gene for 6-aminohexanoate cyclic dimer hydrolase [EI]) was deleted but nylB (the gene for 6-aminohexanoate dimer hydrolase [EII]) was retained. KI725 showed no growth on unfractionated nylon oligomers (Nom1) obtained from a nylon factory as a sole carbon and nitrogen source (Nom1 minimum plate). Extracts of KI725 cells possessed hydrolytic activity for Nom1 (approximately 5% of the activity of KI72), but pOAD2-cured strains (KI722 and KI723) showed no activity. KI725R strains which grew on the Nom1 minimum plate were spontaneously isolated from KI725 at a frequency of 10(-7) per cell. Activity toward Nom1 was enhanced in KI725R strains (10 to 30% of the activity of KI72). This new Nom1 degrading enzyme (EIII, the nylC gene product) hydrolyzed not only Nom1 but also the N-carbobenzoxy-6-aminohexanoate trimer, a substrate which was not hydrolyzed by either EI or EII. Cloning and sequence analysis showed that the nylC gene is located close to nylB on pOAD21 and is a 1,065-bp open reading frame corresponding to 355 amino acid residues. The nucleotide sequence of the nylC gene and the deduced amino acid sequence of EIII had no detectable homology with the sequences of nylA (EI) and nylB (EII).
Asunto(s)
Amidohidrolasas/genética , Proteínas Bacterianas/genética , Flavobacterium/genética , Nylons/metabolismo , Plásmidos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Escherichia coli , Datos de Secuencia Molecular , Mapeo RestrictivoRESUMEN
A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.
Asunto(s)
Bacillus/enzimología , Bacillus/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas , Secuencia de Bases , Bovinos , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Quimotripsina/genética , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Oligopéptidos/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato , Tripsina/genéticaRESUMEN
In order to obtain a large quantity of glutamic-acid-specific endopeptidase of Staphylococcus aureus ATCC12600 (SPase) without cultivating its pathogenic host bacterium, expression plasmids enabling secretion of SPase from Bacillus subtilis were constructed by inserting the SPase gene into B. subtilis-Escherichia coli shuttle vectors. B. subtilis harbouring a simple recombinant plasmid containing the coding and the 5'-flanking regions of SPase in the shuttle vector pHY300PLK secreted 22 mg/l of SPase into the medium. As this level was lower than that of the natural strain (45 mg/l), we tried to increase the expression level by constructing a series of hybrid plasmids with the following features: (1) the terminator sequence of the alkaline protease gene from B. subtilis, (2) the promoter and the leader sequences of the alpha-amylase gene or of alkaline protease gene from B. amyloliquefaciens, (3) the vector pHY300PLK and the fused vector of pHY300PLK and pUB110. By using a variety of hybrid plasmids, the resulting transformants secreted SPase at levels of 33-120 mg/l. The recombinant SPase isolated from the medium was indistinguishable from the natural one with respect to its behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting as well as its enzyme activity.