Presence of benzodiazepine binding sites (receptors) and amplification thereof by imprinting inTetrahymena

Summary LiveTetrahymena cells bound³H-diazepam specifically, as demonstrated by autoradiographic evidence of displacement of about 25% of labeled diazepam in the presence of a 1000-fold amount of cold diazepam. The³H-diazepam bound to membrane preparations isolated from untreated (control) cells was not displaced by cold diazepam, whereas cells involved in primary interaction (imprinting) with diazepam showed amplification and specificity of diazepam binding in both in vivo (cell suspension) and in vitro (pellicle) systems, as well as displacement of bound label in the presence of 1000-fold cold diazepam. It appears that diazepam induced imprinting and, consequently, also the formation of specific receptors inTetrahymena.     

Presence in and effects of pineal indoleamines at very low level of phylogeny

The unicellular organism Tetrahymena contains serotonin and is able to take up the hormone from its mileiu. The serotonin content of the cell changes as a function of the presence of foreign exogenous hormones. This indicates a possible role of serotonin as a chemical mediator. Exogenous serotonin stimulates the RNA synthesis of Tetrahymena, and it was the only one among the hormones studied which kept the RNA level durably high. Serotonin stimulates phagocytosis and growth of Tetrahymena, and its precursors also stimulate growth. Serotonin can imprint Tetrahymena, and as a consequence of this the effect of the hormone increases in the case of further encounters. Treatment with serotonin-related molecules soon after imprinting can reduce the effect of imprinting. Melatonin can contract the pigment cells of Planaria; however, its precursors serotonin and tryptamine can do this more intensely. Both melatonin and serotonin can influenc the regeneration of Planaria, with effects which differ when different phenomena are studied. Evolutionary theories are discussed.     

Comparison of dihydrodiazepam enantiomers: Metabolism,serum binding and brain receptor binding

Summary Dihydrodiazepam is a diazepam prodrug, as shown by its in vitro metabolism by rat and mouse liver and brain microsomal fractions, and its displacing activity on brain diazepam binding. The mechanism of bioactivation is discussed. Stereoselectivity of metabolism and of binding to specific benzodiazepine binding sites in brain synpatosomes and serum albumin were studied.Acknowledgment. The authors are grateful to Dr. J. Wolford for the synthesis of³H-diazepam.     

3H-Diazepam binding sites in Roman high- and low-avoidance rats

The binding of 3H-diazepam to membrane benzodiazepine receptors was examined in 2 psychogenetically selected lines of rats, which differ according to the selection criterion in avoidance behaviour (RHA/Verh greater than RLA/Verh) and, in addition, in emotionality (RHA/Verh less than RLA/Verh). RHA/Verh rats tended to show higher specific diazepam binding in all CNS-subregions when compared with RLA/Verh animals. Significant differences were found in the cortex, striatum, hippocampus, thalamic region and pons-medulla. These results reinforce the contention that a system involving benzodiazepine receptors may play a role in emotional behaviour.     

Role of proline in the imprinting developed by dipeptides — in Tetrahymena Possible role in hormone evolution

The effects of proline and serine dipeptides containing phenylalanine, alanine and leucine on the behaviour of receptors of Tetrahymena were investigated. Only proline-containing dipeptides were able to develop positive imprinting, and the activity depended on which other amino acid was present in the dipeptide. In contrast to the positive imprinting effect of the dipeptides Pro-Phe and Pro-Ala, the dipeptide Pro-Pro and Pro-Leu caused negative imprinting. Serine dipeptides produced negative imprinting in all cases. The possible importance of proline in the evolution of hormone specificity is discussed.     

3H-Diazepam binding sites in roman high- and low-avoidance rats

Summary The binding of³H-diazepam to membrane benzodiazepine receptors was examined in 2 psychogenetically selected lines of rats, which differ according to the selection criterion in avoidance behaviour (RHA/Verh>RLA/Verh) and, in addition, in emotionality (RHA/Verh相似文献   

Role of serum components in the binding and phagocytosis of oxidatively damaged erythrocytes by autologous mouse macrophages

To investigate the role of autologous serum components in the recognition of damaged cells by macrophages, we examined the binding and phagocytosis of damage oxidatively damaged red blood cells with Cu2+ and ascorbate (oxRBCs) by autologous resident mouse peritoneal macrophages. The binding of oxRBCs by macrophages was independent of the presence of serum. However, phagocytosis by macrophages increased with serum concentration, and macrophages showed little ingestion of oxRBCs in a serum-free medium. Macrophages neither bound nor appreciably ingested native RBCs (before oxidation) in either the absence or presence of autologous serum. Mouse macrophages ingested significantly more native as well as oxRBCs in the presence of heat-inactivated fetal calf serum than in the presence of heat-inactivated mouse serum. Pretreated oxRBCs with normal serum were rarely ingested by macrophages in a serum-free medium. Phagocytosis of oxRBCs was significantly inhibited by depletion of IgG or calcium from serum, by heat inactivation of complement, or by antiserum against mouse C3. These results demonstrate that serum components such as IgG, C3, and calcium are involved in phagocytosis of oxRBCs by autologous macrophages.     

Effect of insulin on the glucose uptake of protozoa.

Insulin stimulates the glucose uptake of Tetrahymena pyriformis. This shows the presence of insulin receptors in Tetrahymena, consequently receptors may be present in a level of phylogenesis, where the natural contact between the given hormone and the cell is unnecessary and impossible.     

Two phorbol ester receptor affinities in partially transformed human urothelial cells and decrease of receptor binding in desensitized cells

The presence of specific binding sites for phorbol esters was studied in a transformed but non-tumorigenic human urothelial cell line HCV-29 by assay of specific binding of³H-phorbol-12,13-dibutyrate (³H-PDBu) to intact living cells.³H-PDBu bound specifically to HCV-29 cells in a saturable and competitive manner. Scatchard plot analysis of specific binding yielded a curved plot consistent with two binding sites with K_d of 11 nM and 102 nM, respectively. At saturation the corresponding PDBu binding capacities (B_max) were 8.8 pmol/10⁶ cells (5.2×10⁶ molecules bound per cell) and 2.8 pmol/10⁶ cells (1.7×10⁶ molecules bound per cell).³H-PDBu binding was displaced by biologically active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and mezerein,but not by tumor promoters such as L-tryptophan, anthranilic acid and sodium saccharin. In cells desensitized by pretreatment with 1 g/ml (2M) TPA or PDBu for 24 h the level of binding was reduced to 28% of the level in non-exposed cells. The ability of desensitized cells to bind³H-PDBu was gradually restored within 5–6 days. At the same time the cells became sensitive to the morphological alteration induced by PDBu. This suggests that desensitization of HCV-29 cells is due to a decreased receptor-ligand binding capacity probably associated with down regulation of the phorbol ester receptors.     

Presence of specific binding sites for [3H]sarcophytol-A in cultured human skin fibroblasts

The presence of specific binding sites for [3H]sarcophytol-A in human skin fibroblasts was examined using biochemical and morphological methods. The displacement studies clearly revealed that high (KD = 31.0 nM) and low (KD = 6.05 microM) affinity sites were present in the intact cells. Moreover, autoradiographic studies using light microscopy revealed that the specific binding sites may exist in both the cytoplasm and the nuclei.     

Effects of several preoperative medications on fat cell lipolysis, and activity of adipose tissue cyclic AMP phosphodiesterase.

Effects were examined of atropine, diazepam, pethidene, promethazine, scopolamine, omnopon and papaverine on basal and noradrenaline-stimulated lipolysis in rat isolated fat cells and on rat adipose tissue cyclic AMP phosphodiesterase activity. Papaverine at high concentration (1 mM) inhibited both basal and hormone-stimulated lipolysis, whereas diazepam enhanced basal lipolysis. At a 'clinical dose', omnopon increased both basal and noradrenaline-stimulated lipolysis. Adipose tissue cAMP phosphodiesterase activity was strongly inhibited by 1 mM diazepam, papaverine, promethazine and omnopon (280 microgram ml-1). Lack of enhancement of lipolysis by the established cAMP phosphodiesterase antagonist papaverine, is compatible with simultaneous inhibition also of adipose adenyl cyclase. Diazepam-stimulated lipolysis is compatible with its phosphodiesterase inhibitory activity. It is proposed that papaverine-containing omnopon may offer some survival advantages during surgical stress by facilitating a caloric supply.     

Role of phospholipid transfer protein in rabbit lung development

A 36-kDa phospholipid transfer protein (PLT-P_R), which preferentially transfers phosphatidyl choline (PC) compared to phosphatidyl inositol (PI), was purified 827-fold from rabbit lung homogenate. Incorporation of cholesterol in unilamellar vesicles reduced the PC transfer activity of PLTP_R. Dipalmitoyl phosphatidyl choline uptake by alveolar type II cells was increased in the presence of the protein, and further enhanced in the presence of surfactant liposomes. However, a decrease in uptake was noted with cholesterol in host membranes. Incorporation of PI into host membranes had a low stimulatory effect on the process. All these effects were more pronounced in adult type II cells compared to premature, term and 3-day-old pups. Received 12 September 2001; accepted 11 October 2001     

Chromatosomes are not produced from Tetrahymena chromatin by micrococcal nuclease digestion

The subnucleosomal organization of Tetrahymena chromatin, which has an unusual H1 histone, was investigated by NaCl extraction and micrococcal nuclease digestion of nuclei. It was found that Tetrahymena histone H1 is extracted with 0.35 M NaCl, whereas bovine thyroid H1 is not. Micrococcal nuclease digestion of Tetrahymena nuclei did not yield chromatosomes as a stable intermediate, whereas digestion of bovine thyroid nuclei did.     

EGF receptor induction and insulin-EGF overlap in Tetrahymena

Offspring generations of Tetrahymena pretreated (imprinted) with insulin showed a greater binding capacity for the hormone than offspring of untreated ones. The epidermal growth factor (EGF) imprinted for insulin to a greater degree than insulin itself, and vice versa: insulin imprinted for EGF more efficiently than EGF itself. These phenomena can be explained by the overlap of insulin and EGF on one another's receptors in Tetrahymena.     

Presence of digoxin detectable by radioimmunoassay inTetrahymena

Summary Digoxin was demonstrated inTetrahymena pyriformis by radioimmunoassay, at a concentration of 4.3 ng/100,000 cells. Pretreatment of the cells with digoxin or ouabain did not significantly alter the digoxin concentration of the progeny generations.     

Identification of cytotoxic cells responsible for lysis of target cells pretreated with concanavalin A in spleen populations of pregnant mice with suppressive activity]

During an alloimmunization, killer cells which lyse target cells only in the presence of a lectin are generated. That these cells, as well as suppressive cells, share immunocytological properties with specific killer cells, leads to the hypothesis that these cells may be concerned with the mechanism of immunosuppression. Two experimental results presented in this paper are consistent with this hypothesis: 1) Spleens from H-2k mice pregnant by H-2d males which bear a high suppressive activity also contain a relatively large number of killer cells having the ability to lyse Concanavalin A treated target cells and 2) supernatants of suppressive systems generated through an MLC block the cytolysis of specific target cells by the bound killer cells.     

Effects of several preoperative medications on fat cell lipolysis,and activity of adipose tissue cyclic AMP phosphodiesterase

Summary Effects were examined of atropine, diazepam, pethidene, promethazine, scopolamine, omnopon and papaverine on basal and noradrenaline-stimulated lipolysis in rat isolated fat cells and on rat adipose tissue cyclic AMP phosphodiesterase activity. Papaverine at high concentration (1 mM) inhibited both basal and hormone-stimulated lipolysis, whereas diazepam enhanced basal lipolysis. At a clinical dose, omnopon increased both basal and noradrenaline-stimulated lipolysis. Adipose tissue cAMP phosphodiesterase activity was strongly inhibited by 1 mM diazepam, papaverine, promethazine and omnopon (280 g ml^–1). Lack of enhancement of lipolysis by the established cAMP phosphodiesterase antagonist papaverine, is compartible with simultaneous inhibition also of adipose adenyl cyclase. Diazepam-stimulated lipolysis is compatible with its phosphodiesterase inhibitory activity. It is proposed that papaverine-containing omnopon may offer some survival advantages during surgical stress by facilitating a caloric supply.The authours are grateful to Dr D. C. Williams for his continued support and encouragement.     

Estimation of nonspecific lectin-mediated staining of glutaraldehyde-fixed cells

Summary Lectin-mediated stainings are widely used for the visualization of carbohydrate-carrying cellular components using the electron microscope. The use of glutarladehyde-fixed cells for these stainings introduces the possibility of low nonspecific lectin-trapping by the glutaraldehyde which coats the cells. This trapping was estimated by means of peroxidase-binding to human leukocytes,Tetrahymena pyriformis andEscherichia coli cells and was shown to be prevented by rinsing the glutaraldehyde-fixed cells in an amino acid solution before exposure to the lectin.     

Rat liver S-adenosyl-L-homocysteine hydrolase purification by affinity column chromatography (author's transl)]

S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) has been purified 240-fold from rat liver by affinity column chromatography on aminohexyl sepharose bound 6-mercaptopurine 9 D-riboside. The purified enzyme was homogeneous by gel electrophoresis.