The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin     

Parallel bioassay of 27 bombesin-like peptides on 9 smooth muscle preparations. Structure-activity relationships and bombesin receptor subtypes

Thirteen natural bombesin-like peptides and 14 synthetic analogues were submitted to parallel bioassay on 9 smooth muscle preparations in order to determine their relative potency, in comparison to bombesin and litorin. The natural peptides of the bombesin subfamily showed a uniformly high or moderate potency on all preparations. However, synthetic bombesins of shorter chain length (hepta- and octapeptides) manifested a good potency only on the rat uterus preparation. Among the peptides of the litorin and phyllolitorin subfamilies, only litorin and ranatensin presented a full spectrum of potency, equalling or even surpassing that of bombesin. All other natural and synthetic members of the two subfamilies showed a sharply dissociated spectrum of potency on the different smooth muscle preparations. The only exception was the rat urinary bladder and, in part, the chicken intestine, on which the peptides displayed a uniformly high potency, comparable to, or even greater than that of bombesin. The present results help to explain structure/activity relationships and suggest the probable existence, in the periphery, of multiple bombesin receptor subtypes.     

IgE receptor on human lymphocytes. IV. Further analysis of its structure and of the role of N-linked carbohydrates

Previous studies have shown that the low affinity receptor for IgE (Fc epsilon R II) on human B lymphocytes was comprised of three components with apparent Mr of 45, 65 to 95, and 37 kDa. The present results indicate that the 37-kDa component is a soluble degradation fragment of the 45-kDa component and they also suggest that the 65- to 95-kDa component is probably made of aggregates of the above components that are formed after solubilization of the cells. The 45-kDa component appears to be a glycoprotein containing several sialic acid residues, O-linked carbohydrates and one N-linked carbohydrate chain that is of the complex type. Partial digestion of the purified 65- to 95-, 45-, and 37-kDa molecules with alpha-chymotrypsin or protease V8 generates several fragments with identical mobility on SDS-PAGE. The 37 kDa is not N-glycosylated but like the IgE-binding factors present in the culture supernatant of Fc epsilon R-bearing cells, it contains sialic acid and O-linked carbohydrates. On incubation with protease inhibitors, the Mr of IgE-binding factors (BF) is shifted from 25-27 to 37 kDa, indicating that IgE-BF are derived from the proteolytic cleavage of the 37-kDa molecule, previously identified as a membrane component of Fc epsilon R. On incubation with N-glycosylation inhibitors, the production of IgE-BF is significantly increased indicating that N-glycosylation inhibits the degradation of Fc epsilon R into IgE-BF. Inasmuch as the effect of glycosylation inhibitors is not prevented by monensin, it is concluded that all the IgE-BF are derived from surface Fc epsilon R and not from their intracellular precursors.     

Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice

We examined whether fibroblasts from subcutaneous, colon or lung tissues of nude mice influence the invasive potential of highly metastatic human colon carcinoma KM12SM cells. Primary cultures of nude mouse fibroblasts from skin, lung and colon were established. Invasive and metastatic KM12SM cells were cultured alone or with fibroblasts. Growth and invasive properties of the KM12SM cells were evaluated as well as their production of gelatinase activity. KM12SM cells were able to grow on monolayers of all three fibroblast cultures but did not invade through skin fibroblasts. The conditioned media of KM12SM cells cocultured with skin, colon or lung fibroblasts were examined for the presence of type IV collagenase (gelatinase). KM12SM growing on plastic and on colon or lung fibroblasts produced significant levels of latent and active forms of 64 kDa type IV collagenase, whereas KM12SM cells cocultivated with nude mouse skin fibroblasts did not. In contrast, human squamous cell carcinoma A431 cells produced significant levels of collagenase type IV when cocultured with nude mouse skin fibroblasts, a tissue they invaded and completely penetrated. Incubation of KM12SM cells in serum-free medium containing recombinant human interferon-beta (fibroblast interferon) was associated with significant reduction in gelatinase activity. Since the production of type IV collagenase by human colon cancer cells is specifically inhibited by mouse skin fibroblasts but not by colon or lung fibroblasts the data suggest that organ-specific fibroblasts can influence the invasive and metastatic properties of KM12SM cells.     

Electronic structure and molecular design of simplified polyimide systems

The electronic structures of newly designed polyimide systems (ethenetetracarboxylic 1,2:1,2-dianhydride-diaminoethyne (PI-A) and ethenetetracarboxylic 1,1:2,2-dianhydride-diaminoethyne(PI-B)) are studied in detail with respect to their optimized geometries on the basis of the one-dimensional tight-binding self-consistent field crystal-orbital method. The computational results have revealed that PI-B shows intriguing properties such as a very small band gap and a wide bandwidth near the frontier level, compared with PI-A and other polyimides. Since PI-B would be a promising candidate for a new electric conducting material, a reaction diagram for this polymer is also proposed.Also affiliated to Central Research Laboratories, Matsushita Electric Industrial Co., Moriguchi 570, Japan.     

Role of high-density lipoprotein in transport of circulating bilirubin in rats

The analbuminemic rat strain established by Nagase et al. (Nagase, S., Shimamune, K., and Shumiya, S. (1979) Science 205, 590-591) exhibits hereditary deficiency in albumin biosynthesis. Serum bilirubin concentration is rather lower in homozygous (aa) rats (0.009 +/- 0.002 mg/dl) as compared with heterozygous (Aa) rats (0.047 +/- 0.009 mg/dl) or wild-type Sprague-Dawley (AA) rats (0.034 +/- 0.006 mg/dl) as evidenced by high pressure liquid chromatography analysis of bilirubin. After intravenous administration of various amounts of [heme-3H]hemoglobin in rats, [3H]bilirubin derived from [3H]heme of hemoglobin in vivo is more efficiently excreted into bile in aa rats than in Aa or AA rats. [3H]Bilirubin is exclusively bound with high-density lipoprotein (HDL) in aa rats, and a significant amount of [3H]bilirubin is shown to bind with HDL in Aa or AA rats in vivo. Scatchard plots revealed that [3H]bilirubin is bound with HDL in three binding modes depending on the molar ratio of [3H]bilirubin to HDL: Kd = 0.8 X 10(-7) M (molar ratio, 0.02-0.06), Kd = 1.6 X 10(-6) M (molar ratio, 0.06-0.41), and Kd = 1.2 X 10(-4) M (molar ratio, 0.79-9.02). Even under extreme conditions of excess hemoglobin administration, the molar ratio remains under 0.041; and thus, expected the Kd value would remain around 0.8 X 10(-7) M. Binding of [3H]bilirubin to rat serum albumin revealed two distinct binding modes depending on the molar ratio of [3H]bilirubin to rat serum albumin: Kd = 3.6 X 10(-7) M (molar ratio, 0.03-0.21), and Kd = 5.0 X 10(-6) M (molar ratio, 0.21-2.46). Under physiological conditions in Aa or AA rats, the former mode would be more reliable than the latter. Thus, HDL could bind with approximately 4.5 times higher affinity than rat serum albumin in Aa or AA rats under physiological conditions in vivo.     

Purification and Properties of 1-AminocycIopropane-l-carboxylate Synthase of Mesocarp of Cucurbita maxima Duch. Fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, which formsAGC from S-adenosylmethionine (SAM), was purified to homogeneityfrom sliced and aged mesocarp tissue of Cucurbita maxima Duch.cv Ebisu fruits, and its enzymatic properties were determined.The specific activity of the purified enzyme was 220 mU/mg proteinat 30°C at 50 µM SAM. Native ACC synthase has a relativemolecular mass of 160 ± 10 kDa and consisted of two subunitsof about 84±3 kDa. S-adenosylhomocysteine (SAH), S-methylmethionine(SMM) and L-methionine did not serve as substrate. The enzymereaction was competitively inhibited by aminoethoxyvinylglycine(AVG) (Ki, 2.5 µM), aminooxyacetic acid (Ki, 40 µM)and SAH (Ki, 30 µM). The reaction was also strongly inhibitedby semicarbazide, and less effectively by homocysteine. Theenzyme was rapidly inactivated by its substrate, SAM in thepresence of pyridoxalphosphate (PLP), but in the absence ofPLP, SAM-induced inactivation was much slower. Inactivationdid not occur by SAH and SMM, SAM analogs without substrateactivity. Pyridoxal phosphate was an essential cofactor to beadded to a reaction mixture for maximum activity, but an enzymepreparation from which pyridoxal phosphate was removed by SephadexG-25 gel filtration exhibited one-eighth activity which wasinhibited by semicarbazide, this indicating that a small amountof pyridoxal phosphate is firmly bound to the enzyme. (Received May 6, 1986; Accepted May 20, 1986)     

Effects of ethanol and phenobarbital administration on the metabolism and toxicity of benzene

Effects of ethanol- and phenobarbital(PB)-treatment on the metabolism of benzene in vitro and in vivo, and on the benzene-induced hemotoxicity, were investigated. Ethanol consumption markedly enhanced in vitro metabolism of both benzene and phenol in rat liver, whereas PB-treatment, which enhanced the metabolism of phenol to some degree (about one-third of ethanol-induced enhancement), did not affect the metabolism of benzene. In a single exposure experiment with rats, ethanol increased benzene metabolism in vivo as evidenced by accelerated disappearance of benzene from the blood as well as by elevated urinary excretion of phenol, whereas PB produced little or no significant influence on the metabolism. In a 3-week exposure experiment, ethanol administration accelerated benzene disappearance from the blood in agreement with the single exposure experiment, but it tended to decrease urinary phenol excretion with repetition of exposure, probably due to concomitant stimulation of subsequent phenol metabolism by ethanol. Again, PB-treatment produced only a negligible effect on the metabolism of benzene. Ethanol consumption aggravated benzene-induced hemopoietic disorder as evidenced by a marked decrease in the peripheral white blood cell number. PB produced a protective effect on the toxicity. It is concluded that ethanol potentiates benzene toxicity by accelerating (1) hydroxylation of benzene, a rate-limiting step of benzene metabolism and (2) transformation of phenol into highly toxic metabolites.     

Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.