The polymerase chain reaction for Mycoplasma pulmonis

In vitro DNA amplification by polymerase chain reaction was examined to detect Mycoplasma pulmonis. A pair of synthetic oligonucleotide primers was constructed, and used to amplify a unique sequence of M. pulmonis DNA. Amplified products were detected by agarose gel electrophoresis and verified by blot hybridization with a synthetic oligonucleotide probe. This system detected cellular DNA of M. pulmonis but not M. arthritidis or M. neurolyticum, and thus appears to be useful for M. pulmonis diagnosis.     

Induction of tumor growth inhibitory factor (TGIF) in human mononuclear cells by OK-432, a streptococcal preparation

Summary A tumor growth inhibitory factor (TGIF) was induced in the culture supernatant from mixed culture of human peripheral blood mononuclear cells (PBMC) and a streptococcal preparation, OK-432, in vitro. The activity generated in the supernatant increased in a time-dependent fashion and first appeared 6 h after the initiation of culture, reaching its maximum around 48 h. The TGIF was cytostatic against seven of ten human tumor targets, but not against three murine tumor targets. Tumor cell growth was inhibited by a transient contact, i.e., 1 h, with TGIF. The TGIF was produced by lymphocytes but not by monocytes, because the activity was usually enhanced by elimination of plastic-adherent cells from the original PBMC fraction. The TGIF was relatively stable against heating at 56° C for 30 min, but the activity was totally destroyed after heating at 70° C for 5 min. The molecular weight of TGIF was estimated to be about 43×10³ daltons by gel filtration. No interferon (IFN) activity was detected in the TGIF-positive fractions obtained by gel filtration, and the TGIF-positive fractions did not inhibit the growth of tumor necrosis factor (TNF)-sensitive mouse L929 cells. The TGIF activity was not significantly affected in neutralizing tests using specific antibodies against human IFN and TNF. The OK-432 was administered i.p. for management of cancer patients with malignant ascites. Ascites-derived mononuclear cells (ASMC) were obtained before and 3 to 5 days after OK-432 injection. The ASMC obtained after the injection produced TGIF in vitro in the absence of OK-432; the preinjection ASMC showed no such production. A positive correlation was found between TGIF-producing activity by ASMC and the effect of OK-432 injection on ascites volume. These results indicate that TGIF is induced in mononuclear cells by OK-432 not only in vitro but also in vivo and plays an important role in inhibition of tumor growth in cancer patients.     

Characteristics of specific binding of epidermal growth factor (EGF) on human tumor cell lines

Using seventeen human tumor cell lines derived from a variety of tissues, specific binding sites for epidermal growth factor (EGF), a mouse submandibular gland-derived growth factor, has been characterized. A significant amount of membrane-bound EGF receptors, although considerably varied, was demonstrated in all the tumor cell lines studied. Epidermoid carcinoma appeared to have more EGF receptors than adenocarcinoma. One small cell carcinoma of the lung, one choriocarcinoma of the stomach and three bone tumors also possessed EGF receptors comparable to those of epidermoid carcinoma, while one adenoacanthoma of the stomach had less EGF receptors comparable to adenocarcinoma. Among a variety of phorbol esters tested, tetradecanoyl phorbol acetate, a potent tumor promotor, was shown to be the most effective compound in inhibiting 125I-labeled EGF binding to its receptors. Our results indicate that human tumor cells contain varying amounts of membrane-bound receptors for EGF and that phorbol esters interact with these EGF receptor sites. However, the relationship between EGF receptor sites on tumor cells and cellular proliferation and/or differentiation awaits further study.     

Accumulation of Phenylpropanoids in Cotyledons of Morning Glory (Pharbitis nil) Seedlings during the Induction of Flowering by Poor Nutrition

Flowering of Pharbitis nil strain Violet is induced in continuouslight under poor nutritional conditions. High-performance liquidchromatography of extracts of the cotyledons revealed that twocompounds in addition to chlorogenic acid accumulate under suchconditions. The compounds were identified as pinoresinol glucosideand p-coumaroylquinic acid. The endogenous levels of these phenylpropanoidswere correlated with the flowering response when nutrition waspoor. However, activation of phenylpropanoid biosynthesis seemednot to be essential for the induction of flowering. (Received May 17, 1993; Accepted July 26, 1993)     

Monoclonal antibodies recognizing amino-terminal and carboxy-terminal regions of human aldolase A: probes to detect conformational changes of the enzyme.

Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible clinical application of multicytokine-producing cytotoxic mononuclear cell (MCCM) therapy

When peripheral blood mononuclear cells (PBMC) were incubated with a streptococcal preparation, OK-432, for 24 h, PBMC acquired cytolytic activity against cultured and fresh human tumor cells. Such PBMC were called OK-432-activated mononuclear cells (OK-MC). OK-MC produce several kinds of cytokines such as interferon (IFN), IFN, and tumor growth inhibitory factor (TGIF) bothin vitro andin vivo. OK-MC-produced cytokines also inhibited the growth of cultured and fresh human tumor cells. The growth inhibition was examined by human tumor clonogenic assay using a double-layer agar technique. The results indicate that two pathways of anti-tumor activity are induced in OK-MC, i.e., cell-mediated and cytokine-mediated.     

Chirality of the acyl group of β-hydroxy-β-methylglutarylhydroxyabscisic acid

The β-carbon of the acyl group of β-hydroxy-β-methylglutarylhydroxyabscisic acid was shown to possess R-configuration by HPLC analysis of the reduced product.