Lucifensin II, a defensin of medicinal maggots of the blowfly Lucilia cuprina (Diptera: Calliphoridae).

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.

Inhibition of the antibody responses to rat blood transfusion antigens in mice by boar seminal immunosuppressive fraction.

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.

Aggregated forms of heparin-binding and non-heparin-binding proteins of boar seminal plasma and their binding properties.

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.

Analysis of the interaction of procathepsin D activation peptide with breast cancer cells.

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.

Heat shock applied early in sporulation affects heat resistance of Bacillus megaterium spores.

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.

New trypsin inhibitors are present in the coelomic fluid of the earthworm Lumbricus terrestris.

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.

Evaluation of aminolysis of anilinothiazolinones to phenylthiocarbamoyl amino acid methyl amides as an alternative conversion method in protein sequencing.

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.

Inhibition of aspartic proteinases by propart peptides of human procathepsin D and chicken pepsinogen.

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.