Intra-specific phenotypic and genotypic variation in toxic cyanobacterial Microcystis strains.

AIMS: We determined if the intra-specific genetic diversity of Microcystis aeruginosa correlates with phenotypic characteristics. METHODS AND RESULTS: Microcystis aeruginosa isolates from various Japanese waters were characterized using genetic analyses based on the 16S-23S rDNA internal transcribed spacer (ITS) region and DNA-independent RNA polymerase (rpoC1) gene sequences. In addition, morphological and biochemical properties, and the toxicity of M. aeruginosa strains were determined. We found a correlation in phylogenetic clusters of the ITS region and rpoC1 gene sequences. Using a polyphasic approach, genotypic and phenotypic variations in M. aeruginosa showed that the three genetic lineage groups are comprised of a particular phenotype or subgroup of closely related phenotypes. However, some strains had high phenotypic and genotypic diversity compared to the three lineage groups and did not show distinct lineages; therefore, these strains were designated as the 'complex group'. CONCLUSIONS: The 'complex group' consisted of genetically and phenotypically incoherent and high diverse populations in M. aeruginosa, although some genotypes or lineages displayed consistent phenotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: The polyphasic approach combining phenotypic and genetic characterization was effective for comprehending distinct lineages and discriminating the potential complexity of M. aeruginosa populations at the intra-species level.

Multiple metastases of mammary carcinoma cell lines isolated from feral mouse.

Two established cell lines (JYG-A and B) isolated from mammary carcinoma tissues of M. m. musculus Sub-Jyg (a chinese wild mouse) showed multiple metastasis in the lung (100%), liver (40-60%), kidney (40-80%), lymph node (20-60%) and other organs of the nude mice inoculated with these cells subcutaneously or intravenously. In addition, 100% brain metastasis or infiltration was observed only when inoculated with JYG-B cells intravenously.

Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga.

Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially purified by filtration and hydrophobic column chromatography, and was revealed to be sensitive to trypsin-digestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the first study demonstrating antitumor (antimetastatic) effects of M. crassissimum.

Polyclonal and monoclonal antibodies monospecific to MMTV LTR orf protein produced in E. coli.

Monoclonal and polyclonal antibodies specific to an open reading frame of the mouse mammary tumor virus long terminal repeat were generated using an open reading frame-beta-galactosidase fusion protein produced in E. coli. Both antibodies reacted with the open reading frame-beta-galactosidase fusion protein but not with beta-galactosidase alone using an immunoblotting technique. It is concluded that these antibodies were specific for the protein encoded by the open reading frame of the mouse mammary tumor virus long terminal repeat.

Radiation-induced lymphomas in MSM, (BALB/cHeA x MSM) F1 and (BALB/cHeA x STS/A) F1 hybrid mice.

M.MOL-MSM (MSM) mice derived from Mus musculus molossinus progenitors showed extreme resistance to the induction of lymphomas following whole-body X-irradiation with four doses of 1.7 Gy. (BALB/cHeA x MSM) F1 mice between a high lymphoma strain, BALB/cHeA and the MSM showed a high incidence of radiation-induced lymphomas which was quite similar to that in BALB/cHeA mice, but the latent period was prolonged in the hybrids. Susceptibility in incidence was dominant over resistance in these crosses. Incidences of (BALB/cHeA x MSM)F1 hybrids irradiated with four doses of 2.5 Gy X-rays were 77% in females and 88% in males. F1 hybrids between BALB/cHeA and another resistant strain STS/A, (BALB/cHeA x STS/A) F1, also showed a high level of susceptibility, that is, lymphoma incidence was 64% in females and 63% in males. The mean latent period in the (BALB/cHeA x STS/A) F1 hybrids was similar to that in (BALB/cHeA x MSM) F1 hybrids. As all cases of tumors developed in F1 hybrids are informative concerning the detection of the loss of heterozygosity in the loci depending on the combination of two parental strains, the radiation-induced lymphomas obtained from (BALB/cHeA x MSM) F1 and (BALB/cHeA x STS/A) F1 hybrids could be useful for fine analysis of the genetic alterations involved in lymphomagenesis.

Screening a hybridoma producing a specific monoclonal antibody to HLA-A24+Bw4 antigen by cytotoxicity inhibition assay.

A hybridoma secreting a monoclonal antibody (Tsa-1, IgG3) reacting specifically to HLA-A24+Bw4 was screened by cytotoxicity inhibition assay and micrototoxicity test. The R value of the antibody was 0.843.

DNA-DNA reassociation among a bloom-forming cyanobacterial genus, Microcystis.

DNA base composition and DNA-DNA hybridization among the cyanobacterial genus Microcystis were determined using nine axenic Microcystis strains, including the three 'morphological' species of Microcystis aeruginosa, Microcystis viridis and Microcystis wesenbergii. These Microcystis species showed a similar DNA base composition (42.1-42.8 mol% G + C) and demonstrated more than 70% DNA relatedness, confirming their synonymy based on bacterial criteria.

Differential assay of salivary and pancreatic alpha-amylase in serum and urine, with use of monoclonal antibody to human salivary amylase immobilized on bacterial cell wall.

We previously reported (Clin Chim Acta 1986;159:89) that bacterial cell wall chemically coated with a monoclonal antibody specific to human salivary (S) amylase (EC 3.2.1.1) could be successfully used to separate S and pancreatic (P) amylase in solution. We have now applied this method to serum and urine samples and found that the activities of S and P amylases so measured correlated well with those measured by the isoamylase inhibitor method. The present method is simple and reliable for routine clinical tests.

Inhibition of cytotoxicity for screening a monoclonal antibody to HLA antigen. Preparation of a highly specific monoclonal antibody to HLA-A2 antigen.

Cytotoxicity inhibition assay was established for the screening of a monoclonal antibody to HLA antigen. The assay involved the inhibition of typing cells with hybridoma culture supernatant and with F(ab')2 fragment of sheep anti-mouse IgG. Using the assay and the conventional microcytotoxicity test, an anti-HLA-A2 monoclonal antibody was screened.

[A new monoclonal antibody (SH-9) recognizes the low molecular mass of ovarian cancer antigen CA125].

The SHIN-3 cell line producing CA125 was established from an ovarian cancer patient. Using the SHIN-3 cell line, we found that the low-molecular-mass antigen (about 50 KDa) might be the main antigenic determinant in CA125-immunoreactive species. A new monoclonal antibody to this low-molecular-mass was raised to examine a new cancer associated antigen by a hybridoma technique. Using enzyme linked immuno sorbent assay, ten clones were selected from among 398 clones. Two clones were IgG1 and eight were IgM. By immunostaining (ABC assay), a new antibody (named SH-9) reacted with normal pulmonary bronchus and uterine cervical glands. No positivity, however, was observed in endometriosis (adenomyosis). In tumorous lesions of ovary, SH-9 antibody reacted specifically with mucinous cystadenoma-benign, borderline or malignant. However, no positivity was found in serous cystadenocarcinoma. In any other carcinomas, only lung cancer (adenocarcinoma, squamous cell carcinoma) showed a clear positivity. Immuno blotting analysis showed that SH-9 antibody recognized a low molecular mass. Therefore, SH-9 is seen to be an extremely unique antibody when compared with OC125 biochemically and histochemically.

Monoclonal antibodies against CA125-bearing antigenic molecule fragments; reactivity with mucinous ovarian tumours and lung cancers.

We first established a cell strain, SHIN-3, from human ovarian serous cystadenocarcinoma, and performed antigen analysis for CA125 which appeared to be massively secreted by the SHIN-3 cells. Protein digestion analysis revealed that a low molecular weight peptide of 49 kDa showed antigen activity. In the present study, we describe a mouse monoclonal antibody against this low molecular peptide, presumed to be part of the CA125-bearing antigenic molecule. Purified antigen prepared from culture supernatant was adsorbed to nitrocellulose membranes and injected intrasplenically in mice. Of the obtained 398 clones, 10 clones were selected by screening in an ELISA test. Of the 10, two were further selected, i.e. SH-9. This hybridoma produces IgG1 monoclonal antibodies. Immunoblotting analysis revealed that SH-9 recognizes the low molecular peptide used as the immunogen. Immunohistological examination with the SH-9 MAb revealed that the antigen reacted with the bronchial epithelium, cervical glands of the uterus and other various normal tissues. Of tumorous tissues, the antibody mainly reacted with ovarian tumours, but positive reactions were also observed with pulmonary adenocarcinomas or squamous cell carcinomas. Surprisingly the positive rate was high in mucinous tumour of the ovary, while no positive reaction was observed in serous tumours. Dot-blot assay using SH-9 revealed that 17/19 (90%) sera of lung cancer patients were positive for the titre suggesting that SH-9 may be useful to set up a serum test for lung cancers.

Mouse Mammary Tumor Virus Proviral Integration in the DD/Tbr Mice.

The patterns of mouse mammary tumor virus (MMTV) integration in the DNA of spontaneous-mammary tumors, salivary glands and livers of DD/Tbr mice were examined using MMTV env, int -1c and int-2c probes. The MMTV env probe revealed 1 to 7 new proviral insertions in all mammary tumors. MMTV integration into int-1 was observed in 10 of 18 mammary tumors, whereas that into int-2 was seen in only 2 of 18 tumors. Of the 13 salivary glands examined, only 3 showed new MMTV proviral integrations, but rearrangement in int-1 or int-2 loci by MMTV was not observed. Immuno-collidal gold electron microscopy revealed the presence of MMTV particles both in mammary tumors and in salivary glands, but no tumors were found to be developed in salivary glands. Taken together these results suggest that salivary glands support MMTV replication, but the virions thus produced may not lead to salivary gland tumorigenesis. It is suggested that the salivary gland is the source of horizontally transmitted MMTV in DD/Tbr mice.