A novel opioid peptide, leumorphin, acts as an agonist at the kappa opiate receptor

The primary structure of the common precursor of porcine beta-neo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a third leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leu-enkephalin in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, like those of beta-neo-endorphin and dynorphin, was antagonized less effectively by naloxone than that of leu-enkephalin, but more effectively by Mr2266, an antagonist relatively specific for the kappa type opiate receptor. The inhibitory action of leumorphin or beta-neo-endorphin on the contraction of the guinea pig ileum muscle strip was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as beta-neo-endorphin and dynorphin inhibits the contraction of the rabbit vas deferens which is known to have only the kappa type opiate receptor. This action was also effectively antagonized by Mr2266. It is concluded that leumorphin has potent opioid activity and acts at the kappa receptor, like other opioid peptides derived from preproenkephalin B.     

Somatostatin mydriasis in mice

Administration of somatostatin intracerebroventricularly to mice produced a dose-dependent mydriasis, whereas intravenous injections were ineffective. Naloxone could prevent or abolish this effect. It is suggested that somatostatin either directly excites opiate receptors or activates endopioid pathways involved in the regulation of pupillary size.     

Electroejaculation of the coyote

Two electroejaculators were used to collect semen from 40 adult male coyotes. The most effective apparatus used a two-ring rectal probe and an AC voltage of 18 (Vrms) at 1000 Hz. With this ejaculator, 11 of 15 coyotes produced a satisfactory semen sample, which averaged 0.9 ml in volume and 70 million spermatozoa per ml.     

The occurrence of polysialogangliosides including ganglio-N-tetraose series in adult bovine nasal cartilage

We extracted glycolipids from adult bovine nasal cartilage and purified some glycolipids by DEAE-Sephadex A-25 and Iatrobeads column chromatography. Cartilage contained 20 nmol of lipid bound sialic acid per gram wet tissue. The relative content of mono, di, tri, and tetrasialo gangliosides were 14%, 40%, 28% and 18%, respectively, as sialic acid content. We characterized some by examining carbohydrate composition, methylation analysis, sialidase treatment and mild acid hydrolysis. The ganglio-N-tetraose series, including GDla, GDlb, GTla, GTlb and GQlb, was identified as one of the major ganglioside groups of this cartilage.     

Thymus-independent immunogenicity in vitro of the divalent antigen DNP-polyethylene oxide

Chemically simple and physically well-defined dinitrophenyl derivatives of polyethylene oxide (DNP-PEO) can be prepared in a wide range of forms and sizes. These materials were used to investigate the molecular basis of immunogenicity and the binding of the antigens to membrane-bound receptors. Both di- and multivalent DNP-PEO activate normal murine B lymphocytes to yield primary anti-DNP antibody response in vitro. The immunogenicity is dependent on the carrier chain length but independent of T cells. Responses comparable to those induced by DNP-conjugated polymerized flagellin are induced by divalent linear materials of medium molecular weights of about 60,000. A highly multivalent material is moderately immunogenic, but at much lower antigen doses than divalent materials. The carrier PEO does not affect B-cell responses to DNP-PEO or T-cell response to succinyl concanavalin A. Moreover, it shows no polyclonal mitogenicity at concentrations as high as 1 mg/ml. Studies of antigen binding to cell surface DNP receptors show that the strongly immunogenic materials of medium molecular weights have an appreciable tendency to bind bivalently and thus potentially to crosslink receptors. The binding of smaller, less immunogenic antigen appears predominantly monovalent.     

Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.     

Modulation of endogenous prostaglandins by thymosin-α1 in lymphocytes

The effects of thymosin-α₁ on the stimulation of specific release of prostaglandin E₂ (PGE₂) from splenic lymphocytes and thymocytes were studied. Experiments were also performed to study in parallel the absolute levels of thymosin-α₁ in the blood and the induction of serum FTS activity and of azathioprine sensitivity of spleen cells from adult thymectomized (ATx) mice. A significant difference in the release of PGE₂ between normal splenocytes and splenocytes from ATx mice was observed. Thymosin-α₁ at certain concentrations was able to stimulate PGE₂ release from lymphocytes of ATx mice while inhibiting release in lymphocytes of normal mice. Also, thymocytes were stimulated to release PGE₂ after incubation with α₁ in a manner similar to that seen in spleen cells of ATx mice. Approximately the same concentration of α₁ was found to also correct the low azathioprine sensitivity of splenocytes from ATx mice. Determinations of FTS-like activity in the blood and the pharmacokinetics of α₁ after administration of this synthetic molecule show a clear dissociation. A maximum peak of α₁ activity was obtained after 1 hr, while maximal FTS-like activity was observed after 24 hr. The inhibition of the induction by α₁ of FTS-like activity and of Thy 1.2 antigen by indomethacin suggests that the action of α₁ requires prostaglandin biosynthesis.     

Ontogeny of B-lymphocyte function. XIV. Maturation of the capacity of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin

The maturation of the ability of the B-cell population to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit anti-mouse immunoglobulin (RAMIg) was studied in LAF1, C57BL/6, and C57L mice. As demonstrated by previous workers, the B-cell population from immature mice failed to re-express sIg after treatment with RAMIg. We have shown that the age at which the B-cell population acquires the capacity to re-express sIg is different in different strains and that the order in which the B-cell population of the different strains acquires the capacity to re-express sIg is different from the order in which their B-cell populations acquire the capacity to produce high-affinity antibodies. This suggests that these represent distinct differentiation events in the development of the B-cell population. In all of the strains studied the maturation of the capacity to re-express sIg occurred in two steps. After the first maturation step the B-cell population was able to re-express sIg after treatment with RAMIg for 1 hr but did not re-express sIg after treatment with RAMIg for 24 hr. After the second maturation step the B-cell population could re-express sIg even after 24 hr treatment with RAMIg. It has been suggested by previous workers that the inability of the immature B-cell population to re-express sIg could represent one of the mechanisms responsible for the development of B-cell self-tolerance. It is suggested here that the existence of a period during which cells become tolerant only upon prolonged exposure to antigen could protect the developing B cells from becoming unresponsive to transiently experienced foreign antigens but still permit them to become tolerant to self antigens which are continuously present.     

The suppressive effect of peritoneal exudate macrophages on production of antibody to sheep erythrocytes in vitro

The effects of peritoneal exudate macrophages on antibody response to sheep erythrocytes (SRBC) were investigated in mice. Peritoneal exudate macrophages obtained from mice injected intraperitoneally with proteose peptone or Corynebacterium parvum 4 days earlier had stronger ability to phagocytize and degrade SRBC than normal resident macrophages. These macrophages suppressed antibody formation to SRBC in vitro as well as in vivo. This suppression was overridden by increasing the amount of SRBC and diminished completely by pretreatment of the macrophages with iodoacetate and partly by pretreatment with 2-deoxyglucose, both known to be inhibitors of phagocytosis, but not by addition of indomethacin to the in vitro culture. These results suggest that the suppression of antibody response by peritoneal exudate macrophages was due to the increased activity of these cells as scavenger cells, resulting in a reduced amount of effective antigenic stimulation, and that it was not mediated by a prostaglandin-dependent mechanism. The scavenger function of these macrophages may be due to Ia-negative macrophages.     

Inhibition of human lymphocyte activation by wheat germ agglutinin: a model for saccharide-specific suppressor factors

The suppressive effect of wheat germ agglutinin (WGA) on lectin-stimulated blastogenesis and immunoglobulin production was studied. Addition of WGA at 10 micrograms/ml inhibited phytohemagglutinin (PHA)-, concanavalin-A (Con-A)-, and pokeweed mitogen (PWM)-induced mitogenic responses by 70-80%. PWM-driven immunoglobulin synthesis was suppressed by 45% with WGA. The inhibitory effects of WGA were not due to cell death or to interference with lectin binding at the cell surface. Inhibition was dependent on the presence of WGA in the cell culture during the first 24 hr of mitogen exposure and was observed in cultures of both monocyte-depleted peripheral blood mononuclear cells as well as T-cell-enriched populations. WGA-induced inhibition of blastogenesis was blocked by the addition of N-acetylglucosamine (GluNAc) which prevents WGA binding to the cell surface. WGA was found to mimic the suppressive effect of a soluble immune suppressor supernatant (SISS) derived from Con-A-activated mononuclear cell cultures. PHA responses were inhibited by 80 and 95% with SISS and WGA, respectively. The inhibition by both WGA and SISS was totally reversed with addition of GluNAc. Furthermore, WGA and SISS demonstrated competition for the same cell surface receptor site. WGA may therefore be useful as an in vitro model of a saccharide-specific, biologically relevant, soluble mediator for the investigation of mechanisms of immunologic suppression.