Effects of antioxidants on cyclooxygenase and lipoxygenase activities in intact human platelets: Comparison with indomethacin and etya

Five antioxidative agents (BW755C, 1-naphtol, NDGA, propylgallate and quercetin) were compared with indomethacin and ETYA for their effects on (¹⁴C) arachidonic acid metabolism by cyclooxygenase (CO) and lipoxygenase (LPO) enzymes in intact human platelets. All tested compounds inhibited CO activity in a concentration-dependent manner. LPO activity was suppressed by NDGA, propylgallate, quercetin and ETYA but strongly enhanced by BW755C, 1-napthol and indomethacin. Whereas NDGA and ETYA showed almost equipotent inhibitory effects towards both fatty acid oxygenases, propylgallate and quercetin were found to be respectively 6.5 and 4 times better inhibitors of LPO than of CO activities.These data indicate that antioxidants affect arachidonic acid metabolism in intact human platelets in different ways: BW755C and 1-naphtol exerted the same activity as indomethacin, a selective CO blocker, whereas NDGA, propylgallate and quercetin behaved as ETYA, a dual CO-LPO inhibitor. Considering their inhibition selectivity, propylgallate and quercetin may serve as prototypes for more specific blockers of LPO activity.     

Alterations in uterine and placental prostaglandin F and E with gestational age in the rat

Experiments were designed to determine the chronological alterations in placental and uterine prostaglandin F and E (PGF and PGE) during pregnancy in the rat. Pregnant rats (sperm in the vagina = day 0) were sacrified at days 15, 18,19, 20, 21 and delivery (day 21 ) and placental and uterine tissues assayed (RIA) for PGF and PGE immediately (“ ”) or after 1 hour incubation (“ ”). Uterine content of PGF and PGE (ng PG/mg DNA) was increased significantly by day 19 and further increases were seen through delivery. Incubation of uterine tissue resulted in enhanced net production of PGF and PGE (p <.05) per mg DNA (as judged by tissue content and release into the incubation medium) by day 18 of pregnancy vs. day 15. Net production peaked around the time of delivery thus paralleling the alterations in tissue content .By contrast, no differences with gestational age were found in placental content of PGF and PGE , the concentrations throughout late gestation remaining in the range of uterine PGs at day 15. However, production of PGs per mg placental DNA increased markedly during incubation with significant enhancement detected by day 19 vs. 15, achieving levels even greater than the uterus .The and findings for the uterus are consistent with the hypothesis that increases in uterine PGs levels at the end of pregnancy may play an important role in parturition. The experiences with placental tissue suggest that the potential for PG production per placental cell may also increase in late gestation and thereby contribute to the augmented intrauterine availability of PGs at that time.     

Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

Cryosurgery of the monkey (macaque) prostate: I. Humoral immunologic responsiveness following cryostimulation

The effect of in situ freezing of the monkey (macaque) prostate on the development of antibodies reactive with allogeneic and autologous extract preparations of the cranial and caudal lobe of prostatic tissue and their tissue specificity were evaluated by the method of tanned cell hemagglutination. A representative percentage of the animals receiving cryosurgery developed antibodies to prostatic tissue components; however the intensity of this response was considerably modest when contrasted to that obtained following similar treatment of the rabbit prostate (coagulating gland) and generally did not appear, as in the latter, to increase to any significant degree following multiple freezing. The possible relationship of this modest humoral response to the “cryosensitivity” of the target organ and of the animals evaluated in the present study, i.e., the concentration of glandular secretions (autoantigens), physiologic state (elaboration of androgen) and immunocompetence, are considered.     

Elution of cell-bound anti-prostatic epithelial antibodies after multiple cryotherapy of carcinoma of the prostate

Antibodies reactive with the cytoplasmic membrane or intercellular areas of autologous human and monkey prostatic secretory epithelial cells have been demonstrated by immunofluorescence in an eluate prepared from prostatic tissue obtained from a patient with metastatic adenocarcinoma of the prostate (CaP) after multiple cryotherapy of his primary prostatic tumour. While limited to the study of one patient, elution of anti-prostatic antibodies offers further documentation to previous studies pointing toward the participation of immunologic processes in CaP.     

Cryosurgery of the rabbit prostate. Comparison of the immune response of immature and mature bucks

The intensity and specificity of the immune response developed following each of three independent in situ freezing insults of the coagulating gland (anterior prostate) of immature male rabbits have been compared to that obtained following similar cryostimulation of the coagulating gland of mature bucks by the methods of tanned cell haemagglutination and gel diffusion precipitation. Results of this study offer confirmation to the previously demonstrated secondary or anamnestic immune response observed following multiple freezing of the rabbit coagulating gland and indicate further that the size and physiological status of the coagulating gland, i.e., the concentration of coagulating gland secretory autoantigens, are among the important variables in determining the cryosensitivity of a given animal. This cryosensitivity may be of particular significance in the prospective treatment of patients with prostatic cancer by cryoimmunotherapy. That is, the development of an immune response in such patients may reasonably be related to the concentration and subsequent release of prostatic tumourspecific or tumour-associated antigens.     

Biphasic effect of cryosurgery of the prostate on lymphocyte proliferation

In a preliminary study, cryosurgery of the prostate in patients with prostatic cancer has been observed to possess a biphasic effect on the proliferative responsiveness of peripheral blood lymphocytes (PBL) to the plant mitogen, phytohaemagglutinin. Phytohaemagglutinin-stimulated PBL cultured in autologous serum obtained up to 1 week postoperatively showed reduction from their preoperative levels. In contrast, cultures in homologous serum showed increased responsiveness following cryosurgery. Although depression of lymphocyte proliferation in autologous serum was transitory, gradually returning to approximate or exceeding levels of lymphocytic reactivity observed preoperatively, such depression may facilitate reduced host surveillance to potential infectious agents and to metastatic tumour cells, leading to an alteration of tumour-host homeostasis. Further studies will be required to delineate the possible clinical significance of the present observations.     

14 C-acetate incorporation and nucleic acid synthesis in human lymphocytes stimulated by PHA and tuberculin in vitro

Studies on the timing of incorporation of labeled acetate in relationship to other cellular events in phytohemagglutinin (PHA)-treated lymphocytes have suggested that acetylation of nuclear histones may constitute an important regulatory mechanism for gene activation. In the present investigation, it was shown that PHA stimulation of lymphocytes from a tuberculin-positive patient caused an early increased incorporation of ¹⁴C-acetate prior to RNA and DNA synthesis. Lymphocytes from the same patient, however, repeatedly showed no increased incorporation of ¹⁴C-acetate following exposure to the sensitizing antigen, tuberculin (PPD), even though RNA and DNA synthesis were markedly stimulated. These results suggest that regulatory mechanisms of DNA template activity other than acetylation may be operative in sensitized lymphocytes responding to specific antigen. One possible explanation for the differences in ¹⁴C-acetate incorporation is that the increased uptake of acetate exhibited by PHA-treated cells is an effect related to nonspecific membrane changes caused by the PHA. If this is the case, then template regulation in PHA and antigen-stimulated lymphocytes may be achieved via similar but yet to be defined mechanisms.     

Microfluorometric detection of asymmetry in the centromeric region of mouse chromosomes

A lateral asymmetry in the centromeric region of mouse chromosomes is revealed in studies involving the BUdR quenching of 33258 Hoechst fluorescence. This cytologically detected asymmetry may reflect the unequal distribution of thymidine between the two chains of mouse satellite DNA.     

Effect of a potent gonadotropin releasing hormone antagonist on pulsatile testosterone and gonadotropin secretion in the male nonhuman primate

A potent gonadotropin releasing hormone (GnRH) antagonist [Ac-delta 3Pro1, pFDPhe2, DTrp3,6]-GnRH was given to adult male monkeys to determine the acute effect on pulsatile testosterone and gonadotropin secretion. Blood was drawn at 30 min intervals over 54 h without anesthesia using a mobile vest and tether assembly to support an indwelling catheter. After a 6 h control period, 0.1, 1.0, 2.0, 4.0 mg GnRH antagonist/kg bw in 1 ml corn oil sc, was given to intact adult male monkeys. The highest dose of GnRH antagonist decreased circulating testosterone within 6 h and for approximately 24-36 h duration. These data demonstrate that this GnRH antagonist can reduce serum testosterone both acutely and for intervals greater than 24 h and that the effective dose in intact animals is several-fold (up to 20 times) greater than in castrate animals.     

Modulation of surface proteins by calcium in Chinese hamster lung cells

Investigations on the role of calcium in regulation of cell morphology of Chinese hamster lung cells (V79) revealed that cells grown with additional calcium (5 mM) in the growth medium (Ham's F12) adhere more tightly to the substratum than those grown in F12 alone. Additional calcium in the medium did not cause any changes in the structural membrane proteins or glycoproteins. Radioiodination of the surface membrane proteins of cells grown with or without additional calcium showed distinct differences in the labeling profile. The most striking change observed in cells grown with additional calcium was a very heavily labeled protein band at 70 K molecular weight. Two bands at approx. 100 K and 42 K were also heavily labeled. In contrast, the amount of radioactivity of a protein band at 52 K decreased in the cells grown in additional calcium. In general, cells grown with additional CaCl² were better iodinated than those grown in growth medium alone. The results demonstrate that calcium modulates surface proteins of V79 cells and this modulation may account for the changes observed in the cell morphology.     

Detection of soluble immune complexes by their binding to Fc receptors on mastocytoma cells

Adenosine kinase activity in in vitro human peripheral blood monocyte and human pulmonary alveolar macrophage cultures undergoes significant increases, 3- to 10-fold, in both total and specific activity during 14 days culture. Increased activity in monocyte cultures was not detected during the first 3 days of culture. Adenosine kinase activity in both mononuclear phagocyte cell cultures had a pH optimum at 6.0 and activity was dependent on the concentration of ATP and magnesium; 5 mM ATP and 2.5 mM MgCl were optimal. Increased concentrations of ATP or magnesium were inhibitory. Both dATP and GTP served as phosphate donors in the absence of ATP; in contrast, pyrimidine triphosphates were poor donors. Enzyme activity was inhibited by 1 μM p-chloromercuribenzoate and substrate inhibition by excess adenosine was observed in 2-week pulmonary alveolar macrophage cultures but not in freshly isolated cells. The role of increased adenosine kinase activity in in vitro monocyte-macrophage differentiation is considered.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

The effect of beta-endorphin on natural cytotoxicity and antibody dependent cellular cytotoxicity

The ability of the central nervous system to modulate immune responsiveness has received increasing attention. A potential mechanism that would allow the central nervous system to alter the immune system is the release of neuroendocrine and neurotransmitter polypeptides into the peripheral circulation with subsequent modulation of immunocyte function. In this report, we demonstrate that the neuropeptide, beta-[D-ALA2]-endorphin augments natural cytotoxicity but does not effect antibody-dependent cellular cytotoxicity. The observations are discussed in relation to the mechanisms for natural cytotoxicity and antibody dependent cellular cytotoxicity.     

Nucleoside-specific suppression in MRL/MP +/+ mice

The induction of nucleoside-specific nonresponsiveness was further studied in the autoimmune strain MRL/MP +/+ (MRL/n). Experiments were undertaken to determine (i) whether nucleoside-conjugated spleen cells are able to induce specific nonresponsiveness to T-dependent nucleoside antigens in MRL/n mice, and (ii) whether periodic treatment with nucleoside-conjugated spleen cells would retard the development of spontaneous anti-DNA antibodies and associated indicators of autoimmunity. The results show that nonresponsiveness to nucleoside antigens is inducable in male, but not in female, MRL/n mice. Nonresponsiveness in male MRL/n was transferable and mediated by T cells. Treatment of male MRL/n mice with nucleoside-conjugated spleen cells (NSC) appeared to attenuate the progress of autoimmune symptoms in experimental animals. These results are discussed in the context of recent studies exploring the etiology of autoantibody production and the loss of self-tolerance in murine models of autoimmunity.     

An extended series of fluorescent sulfhydryl reagents useful in energy transfer measurements

Synthesis of four new fluorescent sulfhydryl reagents is described. All are isomers of the previously synthesized N-(iodoacetylaminoethyl)-1-naphthylamine-5-sulfonic acid (1,5-I-AEDANS) and its 1,8-isomer (1,8-I-AEDANS). Three of these new probes (1,4-Br-AEDANS, 2,8-Br-AEDANS, and 2,6-I-AEDANS) carry a single sulfonic acid residue and the fourth (3-(2,7)-Br-AEDANS) carries two sulfonic acid residues. The excitation and emission spectrum of each of these probes is distinct when covalently attached to bovine serum albumin. In addition, they all show a single fluorescent lifetime in the range of 8.0 to 20.8 nsec. This extended range of fluorescent isomers can thus be useful for selecting approprlate energy donors in fluorescence energy transfer experiments.     

Characterization of an immune suppressor from transformed human trophoblastic JEG-3 cells

Cultured human choriocarcinoma JEG-3 cells secrete an immunosuppressor that inhibits lymphocyte proliferation stimulated by either an antigen or a mitogen. In this study, the immunosuppressive factor was characterized by three methods: ion-exchange and exclusion chromatography, partition in organic solvents, and thin-layer chromatography on silicic acid. This JEG-3 cell factor appeared to be a protein complex of about 150,000–200,000 Da that contained an immunologically active polar lipid. The structural and functional characteristics of JEG-3 cell immunosuppressor are similar if not identical to those of SIF, a suppressor lymphokine derived from T cells. These secretions from transformed trophoblastic cells may correspond to normal placental products or represent a function of malignant cells.     

Correlation of the cell phenotype of cultured cell lines with their adhesion to components of the extracellular matrix

The differential adhesion of cultured mammalian clonal cell lines to components of the extracellular matrix was examined by kinetic adhesion and long-term growth assays. Uniform artificial matrices were prepared by air drying collagen Type I solution (C) onto a microtiter well and then air drying a solution containing a single glycosaminoglycan (GAG): hyaluronic acid (HA), chondroitin sulfate-4 (CHS-4), or chondroitin sulfate-6 (CHS-6). The adhesion of [3H]thymidine-prelabeled cells suspended in fibronectin (FN) depleted medium was measured at 2 and 6 hr. Neuroblastoma (N18, Lan 1) and melanoma (B16, G361, S91) cell lines exhibited a significantly greater percentage of cells adhering to one or more C-GAG matrices compared with C matrices. Maximal adhesion at 2 hr was to C-HA. In contrast at 2 hr, two glial, two epithelial, and one fibroblastic cell line showed unchanged or significantly decreased binding to C-GAG compared with C matrices. Further experiments using a neuroblastoma (N18) and a glioma (C6) cell line indicated that the adhesion patterns were not altered either by the method of dissociation from the tissue culture dish, preincubation with exogenous GAG, or the addition of exogenous fibronectin. Assays of N18 and C6 adhesion to matrices made from a non-GAG polyanionic compound, polygalacturonic acid (PGA), did not yield the same adhesion patterns as C-HA matrices. Long-term growth studies of a neuroblastoma (N18) melanoma (S91), and glioma (C6) cell line on nonuniform matrices deliberately prepared with GAG-rich and GAG-poor regions complemented the observations from the kinetic adhesion assays. N18 and S91 cells did not grow on areas which did not contain GAG by toluidine blue staining. However, the C6 cells did not grow on areas which did strongly stain for GAG. A quantitative analysis of the long term growth of N18 and C6 cells substantiated these observations. All these data indicate that the cellular phenotype may be correlated with matrix adhesion. Neuroblastomas and melanomas have a greater affinity for GAG-containing matrices while glial, epithelial, and fibroblastic cells appear to have a greater or equal affinity for collagen matrices.     

Influence of cortisone on immunologic tolerance in vivo: Cortisone prolongs the duration of unresponsiveness but fails to affect its induction

The influence of cortisone administration on either the induction or the duration of immunologic tolerance was examined in vivo. Tolerance induced by isologous IgG coupled to fluorescein was chosen because the hapten-bearing cell can be directly visualized and the hapten-specific immune response to either a TD antigen or a TI₂ antigen can be tested. It was found that cortisone facilitates the maintenance of tolerance, but fails to affect its induction to either class of antigen. Fluorescein-IgG-bearing cells are cortisone resistant. They are seen for a longer period of time in animals treated with cortisone and tolerogen than in animals treated with tolerogen, and fluorescent cells are either T or B cells. We propose that cortisone facilitates the maintenance of tolerance by maintaining a receptor blockade in vivo. This finding might have clinical implications for the treatment of autoimmunity.     

Failure of beta-endorphin antiserum, naloxone, and naltrexone to alter physiologic growth hormone and insulin secretion.

The role of endogenous opiate-like peptides in physiologic regulation of growth hormone (GH) and insulin (IRI) secretion was assessed by passive immunization with β-endorphin antiserum and by administration of the opiate antagonists naloxone and naltrexone. Six-hour secretory profiles were obtained from 5 groups of freely-moving chronically cannulated male rats following the i.v. administration of (I) β-endorphin antiserum, (II) normal rabbit serum, (III) naloxone (1 mg/kg), (IV) naltrexone (1 mg/kg), and (V) normal saline. The typical ultradian rhythm of GH secretion was evident in all groups with most peak GH values >400 ng/ml. No disruption in amplitude of periodicity of the GH rhythm was observed and there was no significant difference in mean 6-hr plasma GH levels. Plasma IRI levels fluctuated minimally over the 6-hr sampling period. There was no significant difference in mean 6-hr IRI levels between groups I and II, or between groups III, IV and V. These data do not support the view that endogenous opiate-like peptides play a physiologically important role in maintaining basal GH and IRI secretion.