Synthesis and biological evaluation of pNPY fragments

Peptide fragments of pNPY corresponding to the C-terminal segments (13-36) and (25-36), the N-terminal segments (1-12) and (1-24), the segments (6-14) and (7-20), which contain a putative beta-turn, and the internal segments (13-24) and (20-30) were synthesized using solid phase methodology. These fragments were assayed for NPY receptor binding activity in the rat hypothalamus membrane preparation, enhancement of food intake in the rat following ivt administration and inhibition of electrically stimulated muscle contraction in the rat vas deferens. Only the C-terminal fragment (13-36) retained some of the activities of pNPY, appearing to act as a weak agonist, having an additive effect with pNPY on the inhibition of muscle contraction and prolonging the duration of action of pNPY in the feeding assay. It also had considerable alpha-helical character, as did pNPY. None of the other peptide fragments had any agonist or antagonist activity. These results suggest that the expression of full biological NPY activity requires both the C- and the N-terminal segments as well as a putative amphiphilic alpha-helical segment (14-31).     

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     

Interspecific hybridization between Nicotiana repanda Willd. and N. tabacum L. through the pollen irradiation technique and the egg cell irradiation technique

Summary Two techniques were useful in overcoming hybrid inviability between N. repanda and N. tabacum. These techniques combine gamma-ray irradiation to pollen or to egg cells (in ovules) with in vitro culture of fertilized ovules. When in vitro culture of fertilized ovules from in situ hybridization of N. repanda x N. tabacum was combined without gamma-ray irradiation to pollen or to egg cells (in ovules), all of the resulting seedlings developed chlorosis and died. Furthermore, in the case of in situ hybridization of N. repanda x N. tabacum with gamma-ray irradiated N. tabacum pollen, no viable seeds were obtained. By using both techniques, combining gamma-ray irradiation to N. tabacum pollen or to egg cells in (N. repanda ovules) with in vitro culture of fertilized ovules, we were successful in obtaining flowering hybrid plants. Thus, it appears that it may be possible to overcome hybrid inviability to a certain extent using both the pollen irradiation technique and the egg cell irradiation technique, i.e., gamma-ray irradiation to pollen or to egg cells (in ovules) before pollination and in vitro culture of fertilized ovules.The research reported in this paper is in partial fulfillment of PhD requirements for the senior author     

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.     

A Comparative Study of the Structures of {alpha}-Glucans from Suspension-Cultured Nonglutinous and Glutinous Rice Cells

-Glucans (average mol wt, 1.3 ? 10⁴) extracted with perchloricacid from 8-day-old suspension-cultured nonglutinous (var. Sasanishiki)and glutinous rice (var. Miyakogane) cells were compared. Theresults of hydrolysis by alpha;-, ß- and iso-amylasesand methylation analysis of the -glucans suggested that theirbasic structures are almost the same. These -glucans are highly-branchedpolysaccharides with an average chain length of about 9–10,with exterior and interior chain lengths of about 6–7and 2–3, respectively. ¹Current address: Laboratory of Food Science, Faculty of Education,Hirosaki University, Hirosaki, Aomori 036, Japan. (Received April 27, 1987; Accepted March 2, 1988)     

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.     

Heparanases and tumor metastasis

The successful penetration of endothelial basement membranes is an important process in the formation of hematogenous tumor metastases. Heparan sulfate (HS) proteoglycan is a major constituent of endothelial basement membranes, and we have found that HS-degradative activities of metastatic B16 melanoma sublines correlate with their lung-colonizing potentials. The melanoma HS-degrading enzyme is a unique endo-beta-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites and is detectable in a variety of cultured human malignant melanomas. The treatment of B16 melanoma cells with heparanase inhibitors that have few other biological activities, such as N-acetylated N-desulfated heparin, results in significant reductions in the numbers of experimental lung metastases in syngeneic mice, indicating that heparanase plays an important role in melanoma metastasis. HS-degrading endoglycosidases are not tumor-specific and have been found in several normal tissues and cells. There are at least three types of endo-beta-D-glucuronidases based on their substrate specificities. Melanoma heparanase, an Mr approximately 96,000 enzyme with specificity for beta-D-glucuronosyl-N-acetylglucosaminyl linkages in HS, is different from platelet and mastocytoma endoglucuronidases. Elevated levels of heparanase have been detected in sera from metastatic tumor-bearing animals and malignant melanoma patients, and a correlation exists between serum heparanase activity and extent of metastases. The results suggest that heparanase is potentially a useful marker for tumor metastasis.     

Effects of CCK antagonists on CCK-induced suppression of locomotor activity in mice.

Sulfated cholecystokinin octapeptide (CCK-8) was administered either intraperitoneally or into the cerebral ventricle of fully conscious mice, and locomotor activity was quantified. CCK-8 administered by either route suppressed locomotor activity. Subcutaneously administered selective CCK-A receptor antagonist, L-364,718 (1 mg/kg), reversed the inhibitory effect of centrally as well as peripherally administered CCK-8, but the selective CCK-B receptor antagonist, L-365,260 (1 mg/kg), did not. These results demonstrate that centrally as well as peripherally administered CCK-8 suppresses locomotor activity in mice through an interaction with CCK-A, but not CCK-B, receptors.     

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.