Cloning of a truncated Babesia equi gene encoding an 82-kilodalton protein and its potential use in an enzyme-linked immunosorbent assay

To isolate Babesia equi genes encoding immunodominant proteins, a cDNA expression library prepared from B. equi mRNA was immunoscreened with B. equi-infected horse serum. Eighteen positive cDNA clones were obtained, and the clone that showed the strongest immunoreactivity, designated Be82, was further characterized. The Be82 gene consisted of 1,953 bp and contained a partial open reading frame lacking the 5'-terminal sequence. As shown by Western blot analyses, immune sera from mice intraperitoneally injected with the Be82 gene product recognized the 82- and 52-kDa proteins of B. equi but not those of Babesia caballi. The glutathione S-transferase fusion protein expressed in Escherichia coli that was purified and used as the antigen in the enzyme-linked immunosorbent assay reacted specifically with B. equi-infected horse sera. These results suggest that the Be82 gene product is a potential diagnostic antigen candidate in the detection of B. equi infection in horses that will be useful both in the performance of epidemiological studies and in the granting of quarantine passes.     

A new pseudo-peptide of Arg-Gly-Asp (RGD) with inhibitory effect on tumor metastasis and enzymatic degradation of extracellular matrix

A series of pseudo-peptide analogs of the Arg-Gly-Asp (RGD) sequence of fibronectin have been synthe-sized, and their anti-metastatic effects in mice and inhibitory effects on tumor cell invasion in vitro have been examined. The partially modified retro pseudo-peptide of RGD, R_rev-COCH₂CO-D (FC-63), was more effective in inhibiting tumor metastasis than the original RGDS peptide. Replacement of the malonyl moiety of FC-63 with a carboxyethylene linkage (R_rev-COCH₂CH₂-D, FC-303 ) achieved more potent inhibition of lung metastasis of melanoma cells than FC-63. Among the analogs, FC-336, a p-xylylendiamine derivative having two FC-303 moieties, showed the most potent inhibitory effect on experimental lung metastasis produced by i.v. co-injection with B16-BL6 melanoma or colon 26 M3.1 cells in a dose-dependent manner. Multiple administrations of FC-336 after tumor inoculation also showed efficient therapeutic potency against spontaneous lung metastasis of B16-BL6 melanoma in mice. Furthermore, FC-336 effectively inhibited the invasion, migration and adhesion of tumor cells in vitro, but its inhibitory effects were not more than those of RGDS peptide. Zymography analysis revealed that FC-336 inhibited the degradation of gelatin substrate by matrix metalloproteinases (MMPs) produced by tumor cells, while the RGDS peptide did not affect the enzymatic degradation. These findings indicate that the pseudo-peptides of the RGD sequence, possessing the inhibitory property of the degradation by MMPs differently from original RGD-containing peptides, may be advantageous and useful in preventing tumor metastasis. © Rapid Science 1998     

Evaluation of the test method “activated sludge, respiration inhibition test” proposed by the OECD

The test method of "activated sludge, respiration inhibition test" proposed by the OECD was critically carried out and compared with other test methods. Investigation of test conditions showed that the moderate deviation from the test conditions defined by the OECD Test Guidelines did not have much effect on the result, and some modifications were proposed to improve the method. This method had a poor detection limit compared with the LC50 test with Oryzias latipes and EC50 of the growth inhibition test with Tetrahymena pyriformis. The susceptivity of the method was particularly poor for the chemicals which were highly toxic in the other two tests.     

Breast parenchymal activity on scintimammography: Comparison between bone-seeking agents and99mTc-sestamibi

The aim of this study was to evaluate breast parenchymal activity on scintimammography with bone-seeking agents and 99mTc-MIBI. Scintimammography was performed with bone-seeking agents in 61 patients and with 99mTc-MIBI in 33 patients. Activity in the breast parenchyma contralateral to the suspected lesion was visually assessed by two independent observers. Increased breast parenchymal activity was shown in 19 of 61 patients examined with bone-seeking agents, while it was demonstrated in only two of 33 patients examined with 99mTc-MIBI. Breast parenchymal activity of bone-seeking agents was higher in patients aged 50 years or younger than in those older than 50. Increased parenchymal activity of bone-seeking agents may disturb visualization of primary breast cancer especially in relatively young patients. Low parenchymal activity is suggested to be a favorable characteristic of 99mTc-MIBI as a scintimammographic agent.     

Bone Metastases from Soft Tissue Sarcomas

The incidence, distribution, time of appearance, and radiologic findings of bone metastases from soft tissue sarcomas, exclusive of lymphomas, were evaluated in 320 patients with soft tissue sarcomas. Thirty patients (9.4%) had evidence of 58 bone metastases. Five of 30 patients presented with metastases, and 25 of 30 patients developed metastases up to 66 months after presentation with a mean time interval of 21.3 months. The incidence of skeletal metastases differed among histologic subtypes of sarcomas; alveolar soft part sarcoma (5 of 8), dedifferentiated liposarcoma (2 of 4), angiosarcoma (2 of 4), and rhabdomyosarcoma (5 of 16) tended to show a higher incidence of bone metastases. The sarcomas metastasized to the regional bones close to the primary tumor in 16 (53%) of 30 patients and to the axial bones in 18 (60%). On conventional radiographs, the osseous metastases demonstrated predominantly osteolytic changes, and evidence of pathological fracture was observed in 31% of 58 metastases.     

Regulation of phosphatidylcholine biosynthesis by mGluR1alpha expressed in human embryonic kidney 293 cells--A 31P-NMR study

A recent report has demonstrated that inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release plays a crucial role in neurite growth. Here, using 31P-NMR, we examine whether activation of the metabotropic glutamate receptor 1 (mGluR1), which induces the production of IP3, could modulate phospholipid metabolism in human embryonic kidney 293 cells. mGluR1alpha- but not ionotropic glutamate receptor 1-expressing cells stimulated with glutamate exhibited a drastic reduction in the phosphorylcholine level, with corresponding increases in the level of phosphatidylcholine, a major phospholipid component. Quantitative analysis of cell growth revealed that mGluR1alpha-expressing cells cultured with 100microM glutamate were statistically significantly longer than the nontransfected cells. The effect was no longer observed following coincubation with a metabotropic glutamate receptor antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine. These results suggest that mGluR1alpha activation triggers phosphatidylcholine biosynthesis and may contribute to neurite extension.     

Stresgenin B, an inhibitor of heat-induced heat shock protein gene expression, produced by Streptomyces sp. AS-9

Stresgenin B was isolated as an inhibitor of heat-induced heat shock protein (HSP) gene expression from a culture broth of Streptomyces sp. AS-9 by silica gel chromatography and HPLC. The molecular formula of the novel compound was determined as C11H13NO5 by high resolution FAB-MS analysis, and the structure was determined by UV, 1H NMR, 13C NMR, HMQC, HMBC, and NOESY spectra. Stresgenin B inhibited heat-induced luciferase reporter-gene expression directed by the human hsp70B promoter in Chinese hamster ovary (CHO) cells at concentrations lower than the concentrations for inhibition of dexamethasone-induced luciferase reporter-gene expression directed by the mouse mammary tumor virus (MMTV)-LTR promoter. The inhibition of heat-induced reporter gene expression was evident even when cells were exposed to stresgenin B only during heat stress treatment. Moreover, the compound inhibited heat-induced syntheses of hsp72/73, hsp90, and hsp110 and thereby suppressed the induction of thermotolerance. Stresgenin B showed moderate cytotoxic activities against several neoplastic cell lines and also showed antibacterial activities against Micrococcus luteus, Bacillus subtilis and Staphylococcus aureus strains.