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

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative 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 (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 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.     

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.     

3-Hydroxy-3-methylglutaric acid and triton-induced hyperlipidemia in rats.

3-Hydroxy-3-methylglutaric acid (HMG) significantly decreased cholesterol, triglyceride and phospholipid levels in whole serum, serum beta-lipoproteins and liver of Triton-induced hyperlipidemic rats. Therapeutically 50 mg HMG/kg is equivalent to 200 mg nicotinic acid/kg in lowering all these lipid parameters. HMG may exert its hypolipidemic effect through inhibition of lipoprotein synthesis.     

Influence of nitrogen components of wine on the growth of lactic acid bacteria]

Three different fractions of nitrogen compounds have been extracted from wine: proteins, peptides, amino acids. They have been tested to support the growth of malolactic bacteria in acid medium: only the peptidic fraction supports this growth.     

Les tests microbiologiques pour la détermination des vitamines

Summary The qualities required by a microorganism to work as test object are studied, so as the utilized methods to measure development.The different, watersoluble vitamins from the group B: thiamin, riboflavin, nicotinic acid, adermin (pyridoxin), pantothenic acid, -alanin, biotin, p-aminobenzoic acid, mesoinositol, enable the creation of a useful test with a selected microorganism.The problem of the vitaminic factors of the soil is one of those which can only be studied with help of microbiological tests. The presence of vitamins in the soil is proved. Their origin is discussed. Their action and that of vitamins voluntary added to the soil is studied. It is shown that the qualities of soil and manures have a marked influence on the thiamin rate of the studied plant.A cycle is suggested to show how the action of growth factors enable to understand the natural relations existing between the auxo-heterotrophic and the auxo-autotrophic organisms. The growth factors of the soil participate to the biological balance ruling life in and on the soil. The necessity of an ecological vitaminology is being considered.     

Morphological differentiation of neuroblastoma by 1 methyl cyclohexane carboxylic acid (CCA) and some C1 derivatives]

We describe the induction of neuroblastoma morphological differentiation by 1 methyl cyclohexane carboxylic acid (CCA) and by some C1 derivatives of CCA. This induction proceeds in a medium which allows, in the absence of inducer, a normal growth of neuroblastoma cells and contains 7.5% fetal Calf serum. In order to establish a clear-dose response relationship and to compare the relative potencies of different drugs, the morphological changes were assessed by examining "long neurite-bearing cells".     

L'acide borique,inhibiteur de la copulation gamétique chezAllomyces

Summary Addition of a tiny crystal of boric acid to a drop of buffered water containing male and female gametes ofEuallomyces javanicus completely inhibits copulation in this water mold.Pure gametophytic growth can thus be secured by addition of 1/15,000 boric acid to the starch-yeast extract medium.

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Communication annoncée mais non présentée au VIII^e Congrès international de Botanique, Paris 1954.     

Nicotinic acetylcholine receptors in attention circuitry: the role of layer VI neurons of prefrontal cortex

Cholinergic modulation of prefrontal cortex is essential for attention. In essence, it focuses the mind on relevant, transient stimuli in support of goal-directed behavior. The excitation of prefrontal layer VI neurons through nicotinic acetylcholine receptors optimizes local and top-down control of attention. Layer VI of prefrontal cortex is the origin of a dense feedback projection to the thalamus and is one of only a handful of brain regions that express the α5 nicotinic receptor subunit, encoded by the gene chrna5. This accessory nicotinic receptor subunit alters the properties of high-affinity nicotinic receptors in layer VI pyramidal neurons in both development and adulthood. Studies investigating the consequences of genetic deletion of α5, as well as other disruptions to nicotinic receptors, find attention deficits together with altered cholinergic excitation of layer VI neurons and aberrant neuronal morphology. Nicotinic receptors in prefrontal layer VI neurons play an essential role in focusing attention under challenging circumstances. In this regard, they do not act in isolation, but rather in concert with cholinergic receptors in other parts of prefrontal circuitry. This review urges an intensification of focus on the cellular mechanisms and plasticity of prefrontal attention circuitry. Disruptions in attention are one of the greatest contributing factors to disease burden in psychiatric and neurological disorders, and enhancing attention may require different approaches in the normal and disordered prefrontal cortex.     

Effect of benzimidazole on nicotinamide adenine dinucleotide phosphate phosphomonoesterase activity in wheat leaves.

Nicotinamide adenine dinucleotide phosphate phosphomonoesterase was isolated and partially purified from wheat (Triticum aestivum L. var. Selkirk) leaves. The enzyme had KNADP value of 1.4 X 10(-4) M and a pH optimum of 5.9. In vitro activity of this enzyme was unaffected by precursors of NAD (nicotinamide and nicotinic acid) or cytokinins (kinetin and benzimidazole). However, when detached wheat leaves were treated with solutions of these compounds, the precursors lowered the specific activity while the cytokinins enhanced the activity. It is suggested that spatial separation and compartmentation of the enzyme and its substrate NADP account for the similar effect of benzimidazole on both.     

Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

Summary We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

Selective inhibition of reproduction in aminopterin-treated nematodes

Summary Aminopterin was applied to the free-living nematode Caenorhabditis briggsae and subsequent growth was recorded. Nematode populations, containing all developmental stages and selected juvenile stages, were exposed to the drug in both growth-promoting and non-promoting media. It is suggested that aminopterin creates a specific requirement for thymine in thymine-free medium. In otherwise growth-promoting medium, aminopterin-induced thymine deficiency will lead to progressively unbalanced growth and maturation and hence to sterility even after removal of the drug. The omission of essential amino acids from the medium during thymine starvation prevents larval growth and results in better reproduction and faster proliferation in aminopterin-free medium. The 4 juvenile stages exhibit a different response to thymine starvation created by aminopterin.     

Insulin requirement of human leukemic cell lines

Summary The growth characteristics of human leukemic cell lines in serum supplemented medium and in serum free medium with and without the addition of insulin were investigated. No relation was found between the insulin binding capacity of the cells and their hormone-dependence for growth.This work has been supported by grant CT82.00191.04 of CNR Rome and by Regione Piemonte.     

Zur Umwandlung von β-Pyridylcarbinol in Nicotinsäure im tierischen Organismus

Summary By way of the perfusion technique, it is shown that the liver of the rat readily converts-pyridyl-carbinol to nicotinic acid. The turnover attains a rate of 0.5M/g/min.     

Endogenous copper is cytotoxic to a lymphoma in primary culture which requires thiols for growth

Summary With the use of bathocuproine sulfonate, a copper-specific chelator as an indicator, we have demonstrated that copper ions, present as a natural medium constituent are toxic to the growth of a lymphoma in primary culture and are principally responsible for the growth requirement of mercaptoethanol and other thiols. By chelating trace copper normally present in the medium, bathocuproine sulfonate retarded the oxidation of cysteine to poorly utilized cystine, thus permitting its direct utilization by the cells for growth.     

Endogenous copper is cytotoxic to a lymphoma in primary culture which requires thiols for growth

With the use of bathocuproine sulfonate, a copper-specific chelator as an indicator, we have demonstrated that copper ions, present as a natural medium constituent are toxic to the growth of a lymphoma in primary culture and are principally responsible for the growth requirement of mercaptoethanol and other thiols. By chelating trace copper normally present in the medium, bathocuproine sulfonate retarded the oxidation of cysteine to poorly utilized cystine, thus permitting its direct utilization by the cells for growth.     

3-Hydroxy-3-Methylglutaric acid and triton-induced hyperlipidemia in rats

Summary 3-Hydroxy-3-methylglutaric acid (HMG) significantly decreased cholesterol, triglyceride and phospholipid levels in whole serum, serum -lipoproteins and liver of Triton-induced hyperlipidemic rats. Therapeutically 50 mg HMG/kg is equivalent to 200 mg nicotinic acid/kg in lowering all these lipid parameters. HMG may exert its hypolipidemic effect through inhibition of lipoprotein synthesis.Acknowledgments. We are grateful to Dr.M. Saleemuddin for encouragement, Dr.P. E. Schurr, Upjohn Co. USA for generous gift of Triton W. R 1339 and Lady Tata Memorial Trust (India) for financial assistance to one of us (SYKY).     

β-(6-Bromopyridin-3-yl)alanine. A new plant growth regulator

Summary The heterocyclic amino acid, -(6-bromopyridin-3-yl)alanine [DL-a-amino--(6-bromopyridin-3-yl)propanoic acid], interferes with the morphological development of the cruciferArabidopsis thaliana when incorporated in the growth medium. It appears to overcome the normal apical dominance control mechanism within the seedling, producing multirather than single-stemmed plants.     

Nicotinamide/nicotinic acid mononucleotide adenylyltransferase, new insights into an ancient enzyme

Nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT) has long been known as the master enzyme in NAD biosynthesis in living organisms. A burst of investigations on NMNAT, going beyond enzymology, have paralleled increasing discoveries of key roles played by NAD homeostasis in a number or patho-physiological conditions. The availability of in-depth kinetics and structural enzymology analyses carried out on NMNATs from different organisms offer a powerful tool for uncovering fascinating evolutionary relationships. On the other hand, additional functions featuring NMNAT have emerged from investigations aimed at unraveling the molecular mechanisms responsible for complex biological phenomena such as neurodegeneration. NMNAT appears to be a multifunctional protein that sits both at the core of central metabolism and at a crossroads of multiple cellular processes. The resultant wealth of biochemical data has built a robust framework upon which design of NMNAT activators, inhibitors or enzyme variants of potential medical interest can be based.