Transient Gene Expression by SV40 Promoter Characterizes Sequential Differentiation of Embryohal Carcinoma F9 Cells into Primitive and Visceral Endoderm

Transient expression of the chloramphenicol acetyl-transferase (CAT) gene under the control of simian virus 40 (SV40), Moloney murine leukemia virus, human T cell leukemia virus, and cytomegalovirus promoters was stimulated by the differentiation of F9 stem cells into primitive endoderm, but repressed again by further differentiation into visceral endoderm. Deletion mutants of the SV40 enhancer showed that a similar set of motifs is critical for CAT expression at all stages of F9 differentiation, but differentiation dependency was observed even in their absence. The stability of transient gene expression under the control of the SV40 promoter was markedly dependent on F9 differentiation. Appreciable expression was detected even in undifferentiated F9 cells immediately after gene transfection, was maximal at 12 h and declined rapidly thereafter. On the other hand, expression in primitive endoderm increased until 72 h. The decline was accelerated again in visceral endoderm. This shift was somewhat specific to the virus promoter since CAT expression in undifferentiated F9 cells under the control of the elongation factor 1α promoter was more stable than for virus promoters tested. Thus, the change in stability of expression is important for differentiation-dependent virus promoter activity.     

Interleukin-5 induces tumor suppression by peritoneal exudate cells in mice

The antitumor activity of peritoneal exudate cells (PEC) induced by murine interleukin-5 (mIL-5) was examined using Meth-A sarcoma cells transplanted into the peritoneal cavity of mice. Although in vitro treatment of Meth-A sarcoma cells with mIL-5 did not result in inhibition of their growth, treatment of mice intraperitoneally with mIL-5 (1 g/day) from day –5 to +5 (tumor cells were inoculated on day 0) led to a significant increase in survival or even rejection of tumor cells. This antitumor effect depended on the dose of mIL-5. Interestingly, there was identical therapeutic activity when the protocol of days –10 to –1 was used as opposed to –5 to +5. In addition, post-treatment with mIL-5 from day +1 to +10 was ineffective. This suggests that the therapeutic activity of IL-5 is largely prophylactic. Under the former condition, the number of PEC was found to increase over 50-fold when compared to levels in control mice. Moreover, the antitumor effect of mIL-5 was completely abolished by subcutaneous injection of anti-mIL-5 monoclonal antibodies. The treatment of mice injected intraperitoneally with human IL-2 also resulted in an increase in survival. Winn assay experiments using PEC recovered from mIL-5-treated mice (1g/day, from day –10 to –1) revealed that these PEC could mediate antitumor activity against Meth-A sarcoma cells. Furthermore, when the cured mice were re-injected with Meth-A sarcoma cells or syngeneic MOPC 104E cells, they could reject Meth-A sarcoma cells but not MOPC 104E cells, indicating that immune memory had been generated. These results suggest that IL-5 augumented the PEC tumoricidal activity but we have no indication that the tumoricidal activity was mediated through a mIL-5-dependent mechanism.     

Differential regulation of IgA production by TGF-beta and IL-5: TGF-beta induces surface IgA-positive cells bearing IL-5 receptor, whereas IL-5 promotes their survival and maturation into IgA-secreting cells.

Transforming growth factor beta (TGF-beta) and IL-5 have been shown to augment IgA production by LPS-stimulated murine B cells. We investigated the effect of TGF-beta on the expression of surface Ig-isotype and IL-5 receptor on LPS-stimulated B cells. TGF-beta increased the proportion of both surface IgA-positive (sIgA+) B cells and sIgG2b+ B cells and enhanced IgA and IgG2b production by LPS-stimulated B cells. TGF-beta synergized with IL-5 only for IgA production of the seven Ig-isotypes and in combination with IL-5 caused a significant increase in the proportion of sIgA+ B cells up to 17.4%. In contrast, IL-5 decreased the proportion of sIgG2b+ B cells and sIgG3+ B cells and inhibited the production of IgG2b and IgG3 by LPS-stimulated B cells. About 50% of sIgA+ cells induced by TGF-beta expressed IL-5 receptor. They secreted peak levels of IgA and seemed to maintain long viability in the presence of IL-5; whereas TGF-beta had the opposite effects on sIgA+ B cells and down-regulated the IL-5 receptor expression. These results indicate that TGF-beta increases the number of sIgA(+)- and IL-5 receptor-positive B cells which respond to IL-5 giving rise to IgA-secreting cells and also support the notions that TGF-beta preferentially induces switching to sIgA+ B cells and IL-5 induces the maturation of postswitch sIgA+ B cells into IgA-secreting cells in a stepwise fashion.     

The Bark Lectin of Robinia pseudoacacia: Purification and Partial Characterization

A lectin was purified from the bark of Robinia pseudoacaciaby sequential ion-exchange chromatography on DEAE-Sepharoseand CM-Toyopearl. The purified lectin was estimated to havea molecular weight of 106 kDa and to be a homotetramer of subunitswith a molecular weight of 29 kDa. Antibodies raised againstthe bark lectin cross-reacted with a 29-kDa polypeptide duringWestern blot analysis, showing that the antibodies are specificfor the bark lectin. The antibodies against the lectin fromRobinia bark cross-reacted with polypeptides in extracts ofthe seeds and bark of Sophora japonica, indicating that thelectin from Robinia bark is immunologically related to the lectinsof Sophora. However, the antibodies did not cross-react withproteins from Robinia seeds and leaves. The first twenty aminoacid residues from the N-terminus of the lectin from Robiniabark were determined and compared with those of the Sophoralectins. (Received July 13, 1991; Accepted December 12, 1991)     

Tissue distribution of antihypertensive dipeptide, Val-Tyr, after its single oral administration to spontaneously hypertensive rats.

The distribution of an antihypertensive dipeptide, Val-Tyr (VY), in the tissues of spontaneously hypertensive rats (SHR) was investigated in this study. A single oral administration of VY (10 mg/kg) to 18-week-old SHR resulted in a prolonged reduction of systolic blood pressure (SBP) up to 9 h (SBP0h 198.0+/-3.6 mmHg; SBP9h 154.6+/-3.5 mmHg). As a result of VY determination, a roughly 10-fold higher increment of plasma VY level was observed at 1 h than that at 0 h, whereas thereafter the level declined rapidly. In tissues, VY was widely accumulated in the kidney, lung, heart, mesenteric artery and abdominal aorta with the area under the curve over 9 h of more than 40 pmol h/g tissue; of these a higher VY level was observed in the kidney and lung. In addition, a mean resident time (MRT) for each tissue (>5 h except for liver) revealed that VY preferably accumulated in the tissues rather than in the plasma (MRT 3.8 h). Significant reductions of tissue angiotensin I-converting enzyme activity and angiotensin II level were found in the abdominal aorta as well as in the kidney, suggesting that these organs could be a target site associated with the antihypertensive action of VY.     

Purification and immunological properties of cathepsin B in carpCyprinus carpio

Cathepsin B was purified from the crude extract of carp (Cyprinus carpio) hepato-pancreas by the method involving ammonium sulfate fractionation and five sequential chromatographies monitored the activity with Z-Arg-Arg-MCA as a substrate, and the specific activity increased about 11,400 fold with a 2% recovery. Although the homogeneity of the purified cathepsin B was established on Native-PAGE, it migrated as two bands of 29,000 and 25,000 molecular weights by the single and heavy chains on SDS-PAGE, respectively. The monospecific antibody against the homogeneous cathepsin B was purified by the affinity chromatography on cathepsin B-Sepharose 4B, and did not immunologically react with rat cathepsin B, carp cathepsins H and L but only with carp cathepsin B by immunoelectrophoretic blot analysis. As the result of the tissue and liver distributions of cathepsin B, the remarkable immunological reactivities in the extracts of spleen, kidney and hepato-pancreas in carp and those of pacific cod, yellow fin tuna, skip jack tuna and common mackerel in pisces were detected with the anti-carp hepato-pancreas cathepsin B at molecular weight of nearby 29,000 or 25,000.     

Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis

We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl₂ and MnCl_{_2}.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Levamisole in postoperative adjuvant immunochemotherapy for gastric cancer

Summary The usefulness of LMS in postoperative immunochemotherapy of gastric cancer was investigated. In compliance with the protocol, MMC was given at a dose of 20 mg on the day of gastrectomy, and an additional 10 mg on the next day IV. The patients receiving 600 mg Tegafur daily were then divided into two groups according to whether LMS was also given or not. LMS was administered for 3 days before the operation in a daily dose of 150 mg and for 1 year or more after operation according to a schedule of 3 days' administration followed by an 11-day interval. The 2-year follow-up demonstrated that in stage III patients, the LMS (+) regimen was superior to the LMS (–) regimen, since the former prolonged the relapse-free interval significantly. The survival rate for stage III disease was also significantly higher in the LMS (+) than in the LMS (–) group. There was no significant difference in the incidence of subjective or objective side-effects between two groups. The incidence of agranulocytosis was comparable in the two groups.Gastrointestinal Cancer Research Group, Japan Levamisole Research AssociationChairmen of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationController of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationMembers of the Data Collection and Analysis SubcommitteeThis study was carried out by the Gastrointestinal Cancer Research Group, Japan LMS Research Association (directed by Prof. Kiyoshi Inokuchi, Dept. of Surgery, Kyushu University and Prof. Eiro Tsubura, Dept. of Internal Medicine, Tokushima University). The results were presented in part at the 19th General Meeting of the Japanese Society for Gastroenterological Surgery in February, 1982