RESUMO
A novel homolog of insect defensin, designated lucifensin II (Lucilia cuprina Wiedemann [Diptera: Calliphoridae] defensin), was purified from hemolymph extract from larvae of the blowfly L. cuprina. The full-length primary sequence of this peptide of 40 amino acid residues and three intramolecular disulfide bridges was determined by electrospray ionization-orbitrap mass spectrometry and Edman degradation and is almost identical to the previously identified sequence of lucifensin (Lucilia sericata Meigen defensin). Lucifensin II sequence differs from that of lucifensin by only one amino acid residue, that is, by isoleucine instead of valine at position 11. The presence of lucifensin II also was detected in the extracts of other larval tissues, such as gut, salivary glands, fat body, and whole body extract.
Assuntos
Defensinas/metabolismo , Dípteros/metabolismo , Proteínas de Insetos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Dípteros/crescimento & desenvolvimento , Larva/metabolismo , Especificidade de Órgãos , Compostos Organofosforados/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
PROBLEM: Immunosuppressive fraction of boar seminal vesicle fluid (ISF) was tested to muffle primary and secondary antibody responses to xenotranfusions. Contemporaneously, heparin non-binding fraction of seminal plasma (H- fraction), presumed to be identical to ISF, was used to support the results. METHOD: To study their similarity, ISF and H- fraction were analyzed by high-performance liquid chromatography and the separated proteins by N-terminal sequencing. In sera of mice treated with ISF or H- fraction, the productions of antibodies against rat erythrocytes and blood serum were evaluated by enzyme-linked immunosorbent assay (ELISA). The productions of IgM, IgA, and IgG subclasses were followed by sandwich ELISA. RESULTS: ISF and H- fraction were proved to be equal complexes of porcine seminal plasma (PSP) proteins PSP I and PSP II. Both inhibited antibody responses to rat erythrocytes and serum and the concentrations of IgM, IgG, IgG1 and IgG2 after the first transfusion with a long-lasting effect. Both suppressed the secondary antibody production if applied before the second transfusion. IgA and IgG3 stayed uninfluenced. ISF and H- fraction had an equal immunosuppressive effect. CONCLUSIONS: ISF was characterized biochemically, found to be identical to H- fraction, and determined to be powerful in overcoming unwanted exaggerated antibody responses to xenotransfusion.
Assuntos
Anticorpos Heterófilos/sangue , Antígenos Heterófilos/imunologia , Transfusão de Sangue , Sangue/imunologia , Proteínas/farmacologia , Sêmen/imunologia , Animais , Feminino , Imunização , Isotipos de Imunoglobulinas/sangue , Imunossupressores/química , Imunossupressores/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas/química , Ratos , Ratos Wistar , Proteínas de Plasma Seminal , SuínosRESUMO
Boar seminal plasma proteins were separated by affinity chromatography on immobilized heparin into two portions: heparin-binding (H+) and non-heparin-binding (H-) proteins. Gel chromatography of the H+ portion yielded four main protein fractions of >150, 45, 30 and 20 kDa, while that of the H- portion resulted in the separation into three main protein fractions of >150, 30 and 20 kDa. HPLC analysis and N-terminal sequencing used to characterize the composition of the protein fractions obtained by gel chromatography revealed that all consisted of low (12-16 kDa) molecular weight components: the H+ fraction consisted of DQH sperm protein, AQN and AWN spermadhesins whereas the H- fraction consisted of PSPI and PSPII spermadhesins. The high molecular weight values of fractions obtained by gel chromatography thus suggest that the proteins are present in boar seminal plasma in the form of aggregates. Interactions of individual boar seminal plasma proteins and their aggregates present in the H+ and H- fractions with acid polysaccharides were estimated.
Assuntos
Heparina/metabolismo , Proteínas/metabolismo , Sêmen/química , Suínos/metabolismo , Animais , Líquidos Corporais/química , Sulfatos de Condroitina/metabolismo , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Sulfato de Dextrana/metabolismo , Ácido Hialurônico/metabolismo , Substâncias Macromoleculares , Masculino , Peso Molecular , Polissacarídeos/metabolismo , Ligação Proteica , Proteínas/isolamento & purificação , Análise de Sequência de Proteína , Capacitação Espermática , Zona Pelúcida/metabolismoRESUMO
Cathepsin D, a lysosomal aspartic proteinase, is secreted in the form of enzymatically inactive proenzyme by many types of human breast cancer tissue and exerts mitogenic activity toward these tissues. Flow cytometry was used to test the binding of procathepsin D purified from the secretion of the breast cancer cell line ZR-75-1 to human breast cancer cells. No previously known surface antigens or soluble M6P-R or anti-M6P-R antibodies were found to inhibit the specific binding of procathepsin D-FITC. Similarly, none of these potential inhibitors was found to inhibit growth factor activity of procathepsin D. Our results indicate that procathepsin D growth factor activity is mediated by a new, previously unknown receptor moiety and that the binding activity can be localized in position 27-44 of the activation peptide of procathepsin D. Furthermore, in vivo experiments indicate that treatment with anti-procathepsin D antibodies can reverse the growth of human breast tumors in athymic nude mice.
Assuntos
Neoplasias da Mama/metabolismo , Catepsina D/metabolismo , Precursores Enzimáticos/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Animais , Anticorpos/farmacologia , Neoplasias da Mama/química , Catepsina D/análise , Catepsina D/antagonistas & inibidores , Linhagem Celular , Ativação Enzimática , Precursores Enzimáticos/análise , Precursores Enzimáticos/antagonistas & inibidores , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de NeoplasiasRESUMO
Cells of Bacillus megaterium 27 were challenged by a 30-min heat shock at 45 degrees C during various sporulation stages and then shifted back to a temperature permissive for sporulation (27 degrees C), at which they developed spores. Heat shock applied at 120 min after the end of the exponential phase induced synthesis of heat shock proteins (HSPs) in the sporangia and delayed the inactivation of spores at 85 degrees C. Several HSPs, mainly HSP 70, could be detected in the cytoplasm of these spores. An analogous HSP, the main HSP induced by increased temperature during growth, belongs to the GroEL group according to its N-terminal sequence. The identity of this protein was confirmed by Western blot (immunoblot) analysis with polyclonal antibodies against B. subtilis GroEL. Sporangia treated by heat shock immediately or 240 min after exponential phase also synthesized HSPs, but none of them could be detected in the spores in an appreciable amount. These spores showed only a slightly increased heat resistance.
Assuntos
Bacillus megaterium/fisiologia , Proteínas de Choque Térmico/biossíntese , Sequência de Aminoácidos , Autorradiografia , Bacillus megaterium/crescimento & desenvolvimento , Bacillus megaterium/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Chaperonina 60 , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico/isolamento & purificação , Temperatura Alta , Cinética , Dados de Sequência Molecular , Peso Molecular , Esporos Bacterianos/fisiologia , Sulfatos/metabolismo , Radioisótopos de Enxofre , Fatores de TempoRESUMO
Proteinase-inhibiting components of the coelomic fluid of the earthworm Lumbricus terrestris were examined. Inhibition of proteinases of serine, aspartate and thiol families was tested. Very strong inhibition was observed only in the case of trypsin. Additional data suggest that the inhibition is related to proteins of molar mass of 42 kDa and 20 kDa, respectively. These two proteins are present in the coelomic fluid in several forms which differ in their isoelectric points.
Assuntos
Oligoquetos/química , Inibidores da Tripsina/isolamento & purificação , Animais , Ácido Aspártico Endopeptidases/metabolismo , Western Blotting , Fracionamento Químico , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cisteína Endopeptidases/metabolismo , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Serina Endopeptidases/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
The aminolysis of products of sequential degradation of proteins and peptides by methylamine is an alternative method of conversion of the unstable 5-alkyl-2-anilino-4-thiazolinones into the stable methyl amides of N alpha-phenylthiocarbamoyl amino acids. The volatility of methylamine permits use in the gas phase during both manual and automatic sequential degradation. Two procedures were studied: (mode A) aminolysis by methylamine in the sequencer reaction chamber after liberation of the thiazolinones by trifluoroacetic acid and (mode B) aminolysis by methylamine vapors passed through a 1-chlorobutane solution of thiazolinones in the conversion flask of the sequencer. The sequencing program was modified for both procedures by making use of the standard sequencer functions. The yields of aminolysis in the conversion flask (mode B) are comparable to those obtained by standard conversion in 25% trifluoracetic acid and the procedure does not affect the repetitive yield. Aminolysis on the glass filter (mode A) requires a major modification of the degradation process, yet gives higher yields of the degraded amino acid derivatives. A disadvantage of both procedures, especially of mode A, is the presence of N-methyl-N'-phenylthiourea in the methyl amide samples. We have not been able to achieve the expected improvement of the yields of degraded hydroxy amino acids. Therefore the replacement of acid conversion of anilinothiazolinones to phenylthiohydantiones by aminolysis for routine degradation cannot be recommended. High yields of methyl amides make aminolysis a promising candidate for the incorporation of fluorescent or other labels in the products of sequencing degradation.
Assuntos
Compostos de Anilina/química , Feniltioureia/química , Tiazóis/química , Amidas/química , Sequência de Aminoácidos , Aminoácidos/análise , Aminoácidos/metabolismo , Cromatografia Líquida de Alta Pressão , Métodos , Dados de Sequência Molecular , Estrutura MolecularRESUMO
Two propart peptides of aspartic proteinases, the propart peptide of chicken pepsin and human cathepsin D, respectively, were investigated from the point of view of their inhibitory activity for a set of aspartic proteinases. These peptides display a very broad inhibitory spectrum. The strongest inhibition was observed for pepsin A-like proteinases where propart peptides can be used as titrants of active enzymes.