Yuji Irie

The Rice Culture Filtrate of Bacillus cereus Isolated from Emetic-Type Food Poisoning Causes Mitochondrial Swelling in a HEp-2 Cell

Rice culture filtrates of Bacillus cereus SA‐50, an emetic‐type strain, produced a toxin which caused cytoplasmic vacuole formation in HEp‐2 and HeLa cells. Electron microscopic observation revealed that the apparent vacuoles in HEp‐2 cells seen under a light microscope were actually swollen mitochondria. The oxygen consumption of HEp‐2 cells was accelerated by the addition of the rice culture filtrate as was measured with a polarographic oxymeter; a respiratory control ratio was 1.0 for control cells, while 1.4 for ones with the filtrates. The culture filtrates showed a similar effect on the isolated mouse liver mitochondria; respiratory control ratios for the mitochondria with and without the filtrates were 3.6 and 1.0, respectively. The affecting manner of the culture filtrates on the oxygen consumption of mitochondria was similar to that of 2,4‐dinitrophenol, suggesting that the culture filtrate contains a toxin acting as an uncoupler of oxidative phosphorylation in mitochondria. It is likely that the culture filtrates containing the emetic toxin of B. cereus causes mitochondrial swelling with a close relationship to the uncoupling of the oxidative phosphorylation of mitochondria.

Integration and expression of murine retrovirus-related sequences in schistosomes

Antibodies against the retrovirus envelope glycoprotein (gp70) of mouse xenotropic retrovirus, BALB virus 2 (Bv2) reacted with the adult worms of Schistosoma japonicum and S. mansoni. This reaction was completely inhibited after adsorption of the antibodies with virions of retrovirus. The reactive schistosome antigen was located in the subtegumental layer of the adult male fluke and in the vitelline gland of the adult female of S. japonicum and S. mansoni. Proteins extracted from both parasites were examined by immunoblot analysis. Anti-Bv2 gp70 antiserum reacted with those proteins from both schistosomes and the band patterns were different among sexes and species. Southern hybridization of the DNA extracted from adults of S. japonicum and S. mansoni demonstrated the presence of sequences homologous to the env gene of mouse ecotropic and xenotropic retroviruses. DNA sequences homologous to the gag and pol regions of the ecotropic murine leukaemia virus were also detected in the DNAs of schistosomes.

Existence of host-related DNA sequences in the schistosome genome

DNA sequences homologous to the mouse intracisternal A particle and endogenous type C retrovirus were detected in the DNAs of Schistosoma japonicum adults and S. mansoni eggs. Furthermore, other kinds of repetitive sequences in the host genome such as mouse type 1 Alu sequence (B1), mouse type 2 Alu sequence (B2) and mo-2 sequence, a mouse mini-satellite, were also detected in the DNAs from adults and eggs of S. japonicum and eggs of S. mansoni. Almost all of the sequences described above were absent in the DNAs of S. mansoni adults. The DNA fingerprints of schistosomes, using the mo-2 sequence, were indistinguishable from each other and resembled those of their murine hosts. Moreover, the mo-2 sequence was hypermethylated in the DNAs of schistosomes and its amount was variable in them. These facts indicate that host-related sequences are actually present in schistosomes and that the mo-2 repetitive sequence exists probably in extra-chromosome.

Dynamic changes of DNA sequences in Schistosoma mansoni in the course of development

Deletion and/or amplification of DNA sequences in Schistosoma mansoni were demonstrated by Southern blot analysis. Total cellular DNAs and genomic clones derived from S. mansoni miracidia, adult males and females were used as probes. Endonuclease BamHI-restricted DNAs from miracidia, adult males and females of both S. mansoni and S. japonicum were reacted to each probe. Hybridization with a total cellular DNA from S. mansoni miracidia as a probe showed elimination of signals in S. mansoni adults. On the other hand, blot analysis using a total cellular DNA from S. mansoni adult males as a probe revealed elimination of hybridization signals in S. mansoni miracidia. Hybridization with a clone SmE15 DNA from S. mansoni miracidia as a probe showed no signal in the DNAs from S. mansoni adults, indicating these sequences deleted in adults. Hybridization experiments using the probes SmF25 and SmM51 which are 1.3 and 2.2 kb fragments cloned from S. mansoni adult females and males respectively, demonstrated no signal to DNA from S. mansoni miracidia. Our data suggested the existence of stage-specific DNA sequences in S. mansoni. We propose a model for multiple-step rearrangement of DNA sequences in S. mansoni during the course of development.

Detection of host DNA sequences including the H-2 locus of the major histocompatibility complex in schistosomes

The mouse type 2 Alu (B2) sequence was detected in both DNAs of Schistosoma mansoni and S.japonicum except for the cercarial stage by the polymerase chain reaction (PCR). Using several kinds of mouse STMS (sequence tagged microsatellite site) primer sets, PCR products related to the host were found in the DNAs of S. mansoni as well as of S.japonicum. Products could be detected only in the DNA of S. japonicum using certain STMS primer sets. The fact that no products could be amplified from the DNAs of both parasites when other kinds of STMS primer sets were used suggests unequal incorporation of the host DNA into the schistosomes. Furthermore, the sequence of the N-terminal domain of H-2, the mouse major histocompatibility complex (MHC), was detected in the DNAs from S. mansoni miracidium, male adult and S. japonicum adults, whereas the sequence of the C2 domain of H-2 was found only in the DNAs of S. japonicum adults. This evidence that host DNA sequences, including the class I MHC, exist heterogeneously in the DNAs of schistosomes might provide an important insight for further understanding of host-parasite immune interactions.

Existence of host DNA sequences in schistosomes—horizontal and vertical transmission

The localization of repetitive DNA sequences in the mouse genome such as mouse type 2 Alu sequence (B2) and mouse retrovirus-related sequences was shown in the body of adult Schistosoma japonicum and Schistosoma mansoni by applying an in situ PCR and hybridization technique. Using the same method, mouse major histocompatibility complex (MHC) class I sequence was also found in schistosomes. Furthermore, mouse MHC class I sequence and type A retroviral sequence were detected in S. japonicum and S. mansoni cercarial DNA by blot hybridization. These findings indicated that horizontal and vertical transmission of host DNA sequences occurred in schistosomes. The incorporation and propagation of host sequences in schistosomes and the roles played by such host sequences form the focus of this brief review.

Degenerative changes in the reproductive organs of female schistosomes during maintenance in vitro.

Degenerative alteration of the reproductive organs of female schistosomes in correlation with the change in egg-laying rate of schistosome pairs in vitro was studied by electron microscopy. The production of normal eggs by adult S. japonicum pairs decreased after 4 days in vitro followed by an increase of abnormal egg laying up to day 8. In S. mansoni, the yield of both normal and abnormal eggs decreased gradually from the start of maintenance in vitro in spite of a much higher pairing rate than in S. japonicum. The vitelline gland of 14-day in vitro-maintained S. japonicum stained with Fast red B, while that of S. mansoni did not. The ovary of both species exhibited regressive features after 14 days of maintenance in vitro. Ultrastructural examination showed that the vitelline cells and oocytes of S. japonicum and S. mansoni had already lost their structural integrity after 2 days in vitro and continued to exhibit signs of structural degeneration throughout the 14-day in vitro maintenance period. The regressive changes in reproductive potential of female S. mansoni maintained in vitro for 4 days could be reversed by surgically implanting the parasites into mouse mesenteric veins.

Horizontal and vertical transfer of mouse endogenous retroviral DNA sequences in schistosomes.

The in situ polymerase chain reaction (PCR) results revealed that mouse type A and type C retroviral sequences were transmitted horizontally from the host to schistosomes. The signals to these retroviral sequences were observed in the nuclei of the mesenchymal and reproductive cells of 8-week Schistosoma japonicum. These signals were also detected in the nuclei of the mesenchymal and reproductive cells and in the cytoplasm of the tegumental tubercles of 24-week S. mansoni. Furthermore, mouse type A retroviral sequence was detected in the DNA extracted from the cercariae of both species. However, mouse type C retroviral sequence and mouse type 2 Alu sequence (B2) were difficult to detect in the cercarial DNA of either species. These findings may indicate that some host sequences are propagated in the schistosome progeny, that is to say, not only horizontal but also vertical transfer of the host gene may occur in schistosomes.

Localization of mouse type 2 Alu sequence in schistosomes.

Localization of the type 2 Alu sequence (B2), a highly repetitive DNA sequence in the mouse genome, was examined by in situ polymerase chain reaction (in situ PCR) in schistosomes. The signals to the B2 sequence were detected in the cytoplasm of the tegumental membrane and in the nuclei of the mesenchymal, testicular, ovarian and vitelline cells of 8-week Schistosoma japonicum. In contrast, it was difficult to detect any signals of this sequence in 8-week S. mansoni, whereas in 24-week male S. mansoni the signals were observed in the cytoplasm of the tegumental tubercles and in the nuclei of the mesenchymal and testicular cells. On the other hand, in 24-week female S. mansoni the signals were found in the nuclei of the mesenchymal, ovarian and vitelline cells but not found in the tegument. On the contrary, no hybridization band of the B2 sequence was detected in the amplified DNA of 3-week schistosomula of either species. These observations proved that the host DNA sequences existed in restricted schistosome cells and were accumulated in the schistosome body during their development.

Comparison of protein composition between schistosomula of Schistosoma japonicum and S. mansoni

SDS-PAGE analysis revealed that the protein composition of mechanically transformed schistosomula (ms) of a Japanese strain of Schistosoma japonicum was essentially similar to that of a Philippine strain of S. japonicum. However, the protein components of S. mansoni ms were remarkably different from those of S. japonicum. Specific proteins of Mr 60-65 and 30 kDa were found in ms of S. japonicum and S. mansoni, respectively. In skin-penetrated schistosomula (ss) of the two species, a common protein of 67 kDa was detected. Using two-dimensional electrophoresis, strain-specific polypeptides (9 in a Japanese and 11 in a Philippine strain) were identified in ms of S. japonicum. Mechanically transformed and skin-penetrated schistosomula of S. japonicum (Japanese) had, respectively, 7 and 10 specific proteins. Four and 1 specific polypeptides were also detected in two-dimensional profiles of ms and ss of S. mansoni, respectively.