Selective killing of smooth muscle cells in culture by the ricin A-chain conjugated with monoclonal antibodies to a cell surface antigen via a dextran bridge

Summary Monoclonal antibodies to a surface antigen of the modulated smooth muscle cells originally isolated from the rat aorta media were conjugated with ricin A-chain via an oxidized dextran bridge. The interaction of cultured cells with the conjugates obtained and with control substances was monitored following incorporation of¹⁴C-leucine radioactivity. It was found that¹⁴C-leucine incorporation was suppressed by 80–90% at a conjugate concentration of 10^–6–10^–7 M. Antigen-negative cells (line IAR; rat hepatocytes) were insensitive to the conjugate at any concentration used. Control use of purified ricin A-chain, native or oxidized dextran, specific and nonspecific IgG did not affect normal¹⁴C-leucine incorporation. The data obtained may be useful for designing targeted drug transport systems and for selective screening of modulated smooth cells in vascular pathology models in vivo.     

Effect of sodium octanoate on leucine incorporation into protein of rat liver slices and of Yoshida ascites hepatoma cells

Summary 7.38×10^–4 M octanoate does not significantly modify leucine incorporation into protein of rat liver slices, while in hepatoma cells a 19% inhibition has been noted.3.69×10^–3 M octanoate reduces leucine incorporation to about the same extent (71–76%) in both liver slices and hepatoma cells.     

Selective antiproliferative effects of thymidine

A substance with antiproliferative bioactivity in an aqueous extract ofCordyline terminalis was purified and identified by mass spectrometry to be the natural nucleoside, thymidine. 10^–5M Thymidine inhibited EL4 cell replication and decreased cell viability after 12–24 h. The effect was highly specific for this nucleoside. Treated cell cultures showed a significant increase in S phase cells and a corresponding decrease in G₁ phase cells. Nitrobenzylthioinosine (which prevented facilitated entry of thymidine) protected cells from the antiproliferative action of thymidine. A human breast cancer cell line (MCF7) was also growth-inhibited by 10^–5M thymidine but a murine lymphoma cell line (K36) was not. Thus, submillimolar thymidine has effects on cell proliferation which are selective both with respect to specificity for the compound and for different tumour cell lines.     

Inhibition and sex specific induction of spawning by serotonergic ligands in the zebra musselDreissena polymorpha (Pallas)

Serotonin (5-hydroxytryptamine, 5-HT) stimulates spawning in the zebra mussel (Dressena polymorpha), a macrofouling European bivalve that has recently invaded North America. To develop methods of controlling zebra mussel spawning, two vertebrate serotonin antagonists, methiothepin and metergoline, known to bind with high affinity to snail 5-HT receptors, were tested for their ability to block 5-HT-induced spawning in zebra mussels. Methiothepin inhibited 5-HT-induced spawning at concentrations as low as 10^–6 M. Metergoline (10^–4 M) inhibited 5-HT-induced spawning; however, at lower concentrations (10^–8 to 10^–5 M), metergoline by itself significantly induced spawning in male, but not female zebra mussels. Metergoline (10^–5 M)-induced male spawning was inhibited by 10^–5 M methiothepin. Thus, methiothepin is the most effective inhibitor and metergoline the most powerful inducer of spawning yet tested in zebra mussels.     

Somatostatin and 3-oxy-methyl-D-glucose (3-OMG) uptake in isolated chicken intestinal epithelial cells

Summary The direct effect of somatostatin on the absorption of 3-oxymethylglucose in epithelial cells isolated from the small intestine of chicken was studied. The presence of somatostatin in the incubation medium at concentrations of 3.5×10^–8 M and 7×10^–8 M produced significant dose-dependent increases in the accumulation of sugar in the enterocytes. This effect might be due to an increase in the cell membrane permeability caused by hormone action.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Thymidine: inhibitor of differentiation in the young chick blastoderm in culture

Summary Differentiation in the young chick blastoderm is affected by thymidine at concentrations higher than 8.2×10^–4 M. Blastoderms at the hypoblastic island stage cultured continuously in the presence of thymidine form an atypical primitive streak which is not capable of inducing the embryonic axis. However, blastoderms with a mature streak escape the effect of thymidine and develop normally.A visiting scientist supported by a grant from the European Molecular Biology Organization (EMBO).     

The effect of the phytoalexins,lubimin, (−)-maackiain,pinosylvin, and the related compounds dehydroloroglossol and hordatine M on human lymphoblastoid cell lines

Summary We have tested the effect of the phytoalexins lubimin, (–)-maackiain and pinosylvin and the related compounds dehydroloroglossol and hordatine M on the growth of the human lymphoblastoid cell lines Molt and Raji. (–)-maackiain, pinosylvin and dehydroloroglossol showed significant growth inhibitory action on the cells. Suppression of [³H] thymidine and [³H] leucine uptake was tested and noted in pinosylvin and dehydroloroglossol. The phytoalexins and related compounds are widespread in plants and provide a potential source of antineoplastic substances.We would like to acknowledge the assistance of J. Hux in preparing the phytoalexins and related compounds. This work was supported by a grant from National Health and Welfare Canada. Correspondence to Dr. L. Skinnider, Department of Pathology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7M OWO.     

Effect of mercuric acetate on mobilization of N and P during germination and seedling growth ofCicer arietinum

Summary Mercuric acetate, at 5.0×10^–5 M, stimulates the mobilization of total nitrogen and phosphate reserves from cotyledons during seedling growth inCicer arietinum cv H208 whereas it suppresses the same process at 2.5×10^–4 M.Thanks are due to Dr D. Banerji for providing facilities and helpful suggestions.     

Effects of corpora cardiaca extract,furosemide and ion substitution on sodium and chloride flux in perfused Malpighian tubules ofLocusta

Treatment with the co-transport inhibitor, furosemide decreased³⁶Cl^– flux across perfused Malpighian tubules ofLocusta. However, exclusion of³⁶Cl^– from the bathing medium had not effect on²²Na⁺ flux whereas substitution of bathing medium Na⁺ by K⁺ increased³⁶Cl^– flux. Diuretic extract of corpora cardiaca increased²²Na⁺ (by 106%) and³⁶Cl^– (by 335%) fluxes differentially.     

Brain factor fromGalleria mellonella (Lepidoptera) stimulating silk gland activity

Brain extracts from day 1–4 last instar larvae ofGalleria mellonella (Lepidoptera) stimulate RNA synthesis in cultured silk glands from day 3 last instar larvae. When the fibroin-synthesizing posterior parts of silk glands were incubated for 3 h in vitro in the presence of brain extract (0.1 brain equivalent), [³H]-uridine incorporation into RNA was stimulated more than twofold. The stimulating effect of brain extract showed a dose response relationship. It is suggested that the heat-resistant and protease-sensitive brain factor is a peptide.     

Modification of theophylline-induced lipolysis in human fat cells after trypsination

Summary Trypsin-treatment of human fat cells results in the potentiation of the lipolytic response and the cAMP accumulation induced by theophylline (5·10^–4 M) but not of those induced by theophylline (5·10^–3 M). The amount of cAMP formed after exposure to theophylline (5·10^–3 M) plus norepinephrine (5·10^–6 M) remains, however, 2.6fold higher in trypsin-treated human fat cells than in the control ones.Acknowledgments. The authors gratefully acknowledge the help of the surgical staff of the C. H. I. of Poissy. This work was supported by grants from the C. H. I. of Poissy and from the Université René Descartes.     

Retinoic acid enhances the proliferation of smooth muscle cells

Summary Retinoic acid (RA, 10^–5–10^–7 M) is shown to enhance the proliferation of cultured rat aortic smooth muscle cells (SMC). This effect is not connected with a synergistic action of RA together with serum mitogens. Moreover, the expression of L1, a surface antigen specific for modulated SMC entering the cell cycle, is amplified by RA treatment.     

Effects of cycloheximide on protein synthesis in human lung tumors,regenerating rat liver and hepatomas

Summary 10^–4 M cycloheximide (CHM) inhibits leucine incorporation to about the same degree in slices of human lung tumors, rat hepatomas, regenerating livers and normal tissues. At 10^–6 M, CHM has a more pronounced effect on tumor tissue and regenerating liver than on normal tissues. 10^–8 M CHM stimulates protein synthesis in normal rat liver slices.     

Hyposmotic shock-induced discharge in acontia ofCalliactis parasitica is blocked by gadolinium

On acontia ofCalliactis parasitica it was observed that mechanical stimuli applied by a gelatin probe, a method effective in tentacles of Anthozoa, do not induce the discharge of nematocytes. Hyposmotic shock, performed by treatment with NaCl solution 35% hyposmotic with respect to sea water, induces, in the presence of Ca²⁺, the discharge that spreads along the acontial filament, as previously observed following treatment with SCN^–. The hyposmotic shock-induced discharge is blocked by Gd³⁺ at a concentration of 1 M. 10 M Gd³⁺ prevents also the SCN^–-induced discharge. These results suggest the presence of stretch activated cation channels either in nematocytes and/or in supporting cells as well as a possible effect of SCN^– on this class of ion channels.     

Bactericidal activity againstPseudomonas aeruginosa is acquired by cultured human monocyte-derived macrophages after uptake of myeloperoxidase

Myeloperoxidase (MPO) is an enzyme located within polymorphonuclear neutrophils capable of producting cytotoxic oxidant species that are particularly active against bacteria with polysaccharide capsules.Pseudomonas aeruginosa (10⁶ bacteria per 1 ml) are killed within 1 h in vitro by a MPO/H₂O₂/Cl^– system (48 mU=132 ng of MPO). The question arose as to whether human macrophages would acquire cytotoxic activity when loaded with this enzyme. Monocytes were therefore isolated from human blood and cultured for up to ten days to induce maturation to macrophages. These cells lost endogenous MPO within five days while H₂O₂ production in response to stimulation by phorbol myristate acetate (10^–6M) decreased to 23% within ten days. On the other hand, their capacity to take up exogenous MPO increased fourfold from day three to day ten. Human macrophages cultured from eight days (when both H₂O₂ production and MPO uptake were sufficient) were therefore used to study the effects of MPO uptake on cytocidal activity againstPseudomonas aeruginosa. After a 1 h MPO loading period, macrophages (5×10⁵ cells per ml) were incubated in the presence of bacteria (0.5 to 2×10⁶ bacteria per ml) for 2 h at 37°C. At a bacteria/macrophage ratio of 1, only 34.8±7.0% of bacteria survived (compared to killing by non-loaded macrophages), while 74.4±9.3% survived at a ratio of 4. From these results, we conclude that loading macrophages with exogenous MPO could enhance their microbicidal activity, suggesting a potentially useful therapeutic application.     

Effect of high doses of somatostatin on adenylate cyclase activity in peripheral mononuclear leukocytes from normal subjects and from acute leukemia patients

Summary In normal lymphocytes somatostatin non-competitively inhibited basal (ID₅₀ 5×10^–4 M) and isoproterenol- and forskolin-stimulated adenylate cyclase activity (Ac). In acute leukemia blasts, non-responsive to isoproterenol, forskolin, which activates the catalytic subunit, stimulated and somatostatin inhibited Ac, thus indicating the leukemic enzyme, though defective, retains the inhibitory pathway and catalyst function.     

Phosphoramidon enhances allatostatin-mediated inhibition of juvenile hormone biosynthesis in the corpora allta of the cockroach,Diploptera punctata

Use of the enkephalinase inhibitor phosphoramidon in the in vitro radiochemical assay for juvenile hormone biosynthesis enhanced allatostatin-mediated inhibition of hormone production by corpora allata of the cockroach,Diploptera punctata. Significant increases in inhibition in day 2 virgin female CA by AST 1 (at 10^–7 M) and AST 4 (10^–8–10^–7 M) were observed in the presence of phosphoramidon (10^–5M or greater). No significant increases in inhibition were seen in CA from day 6 mated females with AST 4 (10^–9–10^–7M) and phosphoramidon combined. Phosphoramidon alone had no effect on JH biosynthesis. Analysis of allatostatin content of the CA, as determined by ELISA, revealed that addition of phosphoramidon to the medium increased the endogenous allatostatin conten in CA of virgin and mated females. The similarity in primary structure between allatostatins and enkephalin-like peptides and their similar distribution makes it probable that phosphoramidon acts by preventing breakdown of allatostatins within the CA.     

Evidence of a direct action of triiodothyronine (T3) on the cell membrane of GH3 cells: an electrophysiological approach

Summary Electrophysiological experiments demonstrate that triiodothyronine (T₃) exerts a direct effect on the membrane of a strain of cultured rat pituitary tumor cells, GH₃/B₆. These cells respond to pressure application of T₃ (2–5 nl, concentration 1·10^–10 M) with an increase in the membrane resistance (R_m) and a hyperpolarization. Spontaneously firing cells become silent.     

Effects of retinoic acid on embryonic development of mice in culture

Summary The effects of all-trans-retinoic acid (RA) (tretinoin) on the craniofacial development of mouse embryos were examined using whole embryo culture. In day 8 embryos cultured for 48 h, embryonic growth was inhibited concentration-dependently by all-trans-RA treatment. Most of the treated embryos exhibited hypoplasia of the primary palatal processes and a reduction in the development of the first viscreral arches. In day 10 embryos cultured for 48 h, although embryonic growth was not inhibited at any concentrations of all-trans-RA, median cleft lip (93%), hypoplasia of the primary palatal processes (37%) and limb reduction deformities (48%) occurred commonly. Furthermore, RA treatment greatly reduced the size of the secondary palatal processes. The incorporation of³H-thymidine in the treated maxillary processes was decreased to 65% of the control value at 1.0×10^–7 M alltrans-RA. These findings indicate that all-trans-RA is teratogenic in mouse whole embryo culture.