Proline-rich cell surface antigens of horseshoe crab hemocytes are substrates for protein cross-linking with a clotting protein coagulin

Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites.     

Parthenogenetic female populations in the brown alga Scytosiphon lomentaria (Scytosiphonaceae,Ectocarpales): decay of a sexual trait and acquisition of asexual traits

In isogamous brown algae, the sexuality of populations needs to be tested by laboratory crossing experiments, as the sexes of gametophytes are morphologically indistinguishable. In some cases, gamete fusion is not observed and the precise reproductive mode of the populations is unknown. In the isogamous brown alga Scytosiphon lomentaria in Japan, both asexual (gamete fusion is unobservable) and sexual populations (gamete fusion is observable) have been reported. In order to elucidate the reproductive mode of asexual populations in this species, we used PCR‐based sex markers to investigate the sex ratio of three asexual and two sexual field populations. The markers indicated that the asexual populations consisted only of female individuals, whereas sexual populations are composed of both males and females. In culture, female gametes of most strains from asexual populations were able to fuse with male gametes; however, they had little to no detectable sexual pheromones, significantly larger cell sizes, and more rapid parthenogenetic development compared to female/male gametes from sexual populations. Investigations of sporophytic stages in the field indicated that alternation of gametophytic and parthenosporophytic stages occur in an asexual population. These results indicate that the S. lomentaria asexual populations are female populations that lack sexual reproduction and reproduce parthenogenetically. It is likely that females in the asexual populations have reduced a sexual trait (pheromone production) and have acquired asexual traits (larger gamete sizes and rapid parthenogenetic development).     

The endogenous reactive oxygen species promote NF-kappaB activation by targeting on activation of NF-kappaB-inducing kinase in oral squamous carcinoma cells

Reactive oxygen species (ROS) could stimulate or inhibit NF-κB pathways. However, most results have been obtained on the basis of the exogenous ROS and the molecular target of ROS in NF-κB signalling pathways has remained unclear. Here, the oral squamous carcinoma (OSC) cells, with a mild difference in the endogenous ROS level, were used to investigate how slight fluctuation of the endogenous ROS regulates NF-κB activation. This study demonstrates that NF-κB-inducing kinase (NIK) is a critical target of the endogenous ROS in NF-κB pathways. The results indicate that ROS may function as a physiological signalling modulator on NF-κB signalling cascades through its ability to facilitate the activity of NIK and subsequent NF-κB transactivation. In addition, the data are useful to explain why the altered intracellular microenvironment related to redox state may influence biological behaviours of cancer cells.     

A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides

Enzymes capable of hydrolyzing the beta-glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids has been found in microorganisms and invertebrates and designated endoglycoceramidase (EC 3.2.1.123) or ceramide glycanase. Here we report the molecular cloning, characterization, and homology modeling of a novel endoglycoceramidase that hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. The novel enzyme was purified from a culture supernatant of Rhodococcus equi, and the gene encoding 488 deduced amino acids was cloned using peptide sequences of the purified enzyme. Eight residues essential for the catalytic reaction in microbial and animal endoglycoceramidases were all conserved in the deduced amino acid sequence of the novel enzyme. Homology modeling of the enzyme using endocellulase E1 as a template revealed that the enzyme displays a (beta/alpha)8 barrel structure in which Glu234 at the end of beta-strand 4 and Glu341 at the end of beta-strand 7 could function as an acid/base catalyst and a nucleophile, respectively. Site-directed mutagenesis of these glutamates resulted in a complete loss of the activity without a change in their CD spectra. The recombinant enzyme hydrolyzed the beta-galactosidic linkage between oligosaccharides and ceramides of 6-gala series glycosphingolipids that were completely resistant to hydrolysis by the enzymes reported so far. In contrast, the novel enzyme did not hydrolyze ganglio-, globo-, or lactoseries glycosphingolipids. The enzyme is therefore systematically named "oligogalactosyl-N-acylsphingosine 1,1'-beta-galactohydrolase" or tentatively designated "endogalactosylceramidase."     

Therapeutic and life-prolonging effect of intrapleural injection with a streptococcal preparation,OK-432, and IL2-cultured effusion lymphocytes to breast cancer patients with malignant pleural effusion

We developed a local AIT using PEL cultured with TCGF combined with preadministration of OK-432. Twenty-six patients of breast cancer with pleural effusion have been treated with this therapy since 1983. PEL expanded and tumor cells collapsed by day 9 in culture with TCGF. Cultured PEL possessed significantly higher cytotoxic activity against autologous tumor cells than PBL cultured in the same condition (p < 0.05), but there was no difference between their cytotoxic activities against K562. The proliferation rate of PEL obtained after intrapleural administration of OK-432 was higher than that obtained before OK-432 (p < 0.01). Moreover, the cytotoxic activities against both autologous tumor and K562 of cultured PEL obtained after OK-432 administration was significantly (p < 0.05) higher than those cultured PEL obtained before.Cultured PEL (1 x 10⁸ - 6 x 10⁹) were transferred into the pleural cavity after the intrapleural administration of OK-432 (1–5 KE). The volume of pleural effusion increased temporarily after the administration of OK-432 but significantly (p < 0.01) decreased after AIT. Tumor cells disappeared cytologically in 22 patients at the last puncture of pleural effusion. Pleural effusion disappeared completely in 19 of 26 patients and decreased by more than 50% in volume in 6 patients. Performance status improved in 22 patients. The response rate for OK-432-combined AIT in the present study was 96%. The survival period of the patients treated by OK-432-combined AIT in this trial was significantly (p < 0.002) prolonged compared to that of the patients receiving chemotherapy alone. The side effects were fever and general malaise after OK-432 administration but no critical toxicity was observed.     

Presentation of synthetic peptide antigen encoded by the MAGE-1 gene by granulocyte/macrophage-colony-stimulating-factor-cultured macrophages from HLA-A1 melanoma patients

The recent identification of the sequences of the peptides derived from a number of human melanomaassociated antigens has presented opportunities for developing a specific-peptide-based vaccine in this form of cancer. Since antigen-presenting cells (APC) play a crucial role in the induction of the T-cell-mediated immune response, we examined whether or not ex vivo cultured APC, bearing the appropriate MHC restricting elements, when pulsed with a relevant melanoma-specific cytotoxic-T-lymphocyte(CTL)-determined peptide, can present the peptide to the CTL. Here we show that a population of cells, derived from the monocyte/macrophage lineage from peripheral blood and grown in granulocyte/macrophage-colony-stimulating factor, exhibit many essential characteristics of professional APC (dendritic-type morphology with a proportion of the population, the B7 molecule, and high levels of MHC class I and class II molecules, CD11b and CD54 molecules) and are capable of efficiently presenting the nonapeptide, EADPTGHSY, encoded by the melanoma antigen MAGE-1 gene, to the MAGE-1-specific CTL clone, 82/30. These results suggest that this type of autologous ex vivo cultured population of professional APC, when pulsed with the relevant-CTL-determined peptide, can serve as a novel type of candidate vaccine for active specific immunization against HLAA1-positive patients with melanoma expressing the MAGE-1 antigen.This work was supported by a PHS grant CA 61398 and in part by the Lavery Cancer Research Fund     

Mechanism of freezing injury to erythrocytes: Effect of initial cell concentration on the post-thaw hemolysis

It has been previously reported that the post-thaw hemolysis of erythrocytes, frozen under various conditions, depends upon the initial cell concentration; increasing the cell concentration decreases the proportion of intact cells after freeze-thawing. In the present study, the effect of cell concentration upon post-thaw hemolysis, examined mainly by the morphological observation of freezing patterns in specimens with or without cryoprotectant glycerol, was most marked in concentrated cell suspensions in which the cells had become shrunken as a result of extracellular freezing. The addition of glycerol lessened the packing effect progressively as the concentration was increased. The results thus obtained may be explained by assuming that cells, deformed in the freezing process, and rigid at low temperatures, might undergo mechanical damage when subjected to compression and abnormal contact.     

Subcutaneous cystic granuloma caused byPhialophora verrucosa

A case of subcutaneous cystic granuloma caused byPhialophora verrucosa was reported. The patient was 21-year-old Japanese female with systemic lupus erythematosus (SLE) who had received prolonged steroid therapy. P. verrucosa has occasionally been isolated from patients as one of etiological agents of chromomycosis. There have been, however, no paper published to report an isolation of this fungus from subcutaneous cyst or abscess of a patient. This case is the first report of subcutaneous cystic granuloma induced clinically byP. verrucosa.