The effects of a polypeptide neurotoxin from Anemonia sulcata on nerve conduction in crayfish giant axons and on frog myelinated fibers have been analyzed. The main features of toxin action are the following: (i) the toxin acts at very low doses and its action is apparently irreversible. (il) The toxin selectively affects the closing (inactivation) of the Na+ channel by slowing it down considerably; it does not alter the opening mechanism of the Na+ channel or the steady-state potassium conductance. (iii) The tetrodotoxin-receptor association is unaffected by previous treatment of the axonal membrane with the sea anemone toxin. (iv) Conversely, the sea anemone toxin can only associate with the membrane when the Na+ channel is o en for Na+; it does not bind when the channel is previously blocked by tetrodotoxin. (v) Besides its effect on the action potential, the sea anemone toxin displays a veratridinetype depolarizing action at low Ca2+ concentration which can be suppressed by tetrodotoxin. The sea anemone toxin greatly stimulates the release of {Y43H]aminobutyric acid from neurotransmitter-loaded rat brain synaptosomes. The apparent dissociation constant of the neurotoxin-receptor complex in this system is 20 nM. The sea anemone toxin effect is antagonized by tetrodotoxin.Neurotoxins are essential tools for the analysis of molecular aspects of nerve conduction and transmission. Toxic molecules already available for study of molecular aspects of conduction include: (i) tetrodotoxin and saxitoxin, which are highly specific for blocking the Na+ channel in most axons (1, 2); (ii) veratridine and batrachotoxin, which depolarize nerve membrane by a selective increase in the resting sodium permeability (2-5); and (iii) scorpion neurotoxin, a miniprotein which affects reversibly the closing of the Na+ channel and the opening of the K+ channel (6-8).A series of neurotoxins was recently isolated in the pure form froln the sea anemone Anemonia sulcata (9-11). The toxins all are small polypeptides. MATERIALS AND METHODS Purification of sea anemone toxins (Anemonia sulcata) was carried out according to Beress et al. (9,10). ATX11 is the most abundant of the three neurotoxic polypeptides (9, 10, 12).Giant axons used in this work were those of the crayfish Astacus leptodactylus and of a cephalopod, the cuttlefish Sepia offlicnalis (axon diameter 200-400 Mum). Giant axons from crustacea were isolated from circumesophageal nerve connectives, those of Sepia from stellar nerves (13). Resting and action potential recordings and voltage clamp experiments have been previously described (8). When the nerve is bathed in a solution containing 0.1 nM ATX11 some of the thin axons begin to fire spontaneously (Fig. IA). More axons are affected at a concentration of 1 nM of ATX11.The giant axon having the maximum diameter (about 100 Mm), which has been used for microelectrode and voltage clamp analysis, is sensitive to ATX11 at concentrations higher than 0.1 AM (Fig. iC). Toxin action on this axon provokes a marked plateau phase of...
The binding of 125I-Tyr4 bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinity, single-site class of receptors in 3 cell lines (KD of 0.75 x 10(-9) and 10(-9) M, Bmax of 0.75 x 10(-13) and 9.7 x 10(-13) M/mg protein in MDA-MB231 and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analogues (natural or synthetic) inhibited the binding of 125I-Tyr4 bombesin in the following order of potency: gastrin-releasing peptide (GRP, EC50 = 1.7 x 10(-10) M) greater than BIM 26159 greater than bombesin, Tyr4 bombesin greater than BIM 26147 greater than litorin greater than neuromedin C. In contrast, 125I-Tyr4 bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex 125I-Tyr4 bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role.
U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride-sensitive Naf/H' exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Naf uptake experiments, "Cl-influx and efflux experiments.Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi = 6.3 provided that Na+ ions were present in the assay medium. Li' or K' could not substitute for Na'. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate-stimulated uptake pathway for Na' which was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate. The bicarbonate-dependent 22Naf uptake component was reduced by depleting cells of their internal C1-and increased by removal of external CI-. 36C1-efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36C1-at a cell pHi of 6.3. Finally a 36C1-uptake pathway was documented. It was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate = 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na' and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na' dependences were defined. No evidence for a Na+-independent Cl-/HCO; exchange system could be found. The Na' -dependent Cl-/HCO; exchange system was relatively insensitive to (ary1oxy)alkanoic acids which are potent inhibitors of bicarbonate-induced swelling of astroglia and of the Li(Na)CO;/Cl-exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.= 10 1M) and bicarbonate It is now well established that eukaryotic cells are able to regulate their intracellular pH (pH,) after an intracellular acidosis or a cellular alkalosis. In invertebrates the major mechanism that allows cells to normalize their pHi after an acid load is a Na+-dependent Cl-/HCO; exchange system [l-31. Its properties have been defined in detail in snail neurons [4], in barnacle muscles [5] and in squid giant axons [6]. In mammalian cells pHi recovery from an intracellular acidosis is achieved mostly by the amiloride-sensitive Na'/H + exchange system [7,8]. Yet bicarbonate-transport mechanisms are also present in the plasma membrane of vertebrate cells and have been postulated to play a role in pHi regulation. Three such systems have been described: (a) an electrogenic Na'/HCO; cotransport that is independent of C1-in renal proximal tubular cells of the salamander glands [I61 and (c) a presumably electroneutral Na+-dependent Cl-/HCO; exchange system in CC139 fibroblasts [17] and human carcinoma cells [18]. The properties of the latter system have not yet been analyzed in detail. In a previous study we showed that HL60 human leukemic cells use both an amiloride-sen...
Two of the tree toxic compounds used in this work, veratridine and the sea anemone toxin, provoke neurotransmitter release from synaptosomes; the third one, tetrodotoxin, prevents the action of both veratridine and the sea anemone toxin. The half-maximum effects of veratridine and sea anemone toxin actions on synaptosomes are K0.5 = 10 and 0.02 micronM, respectively. Although veratridine and the sea anemone toxin similarly provoke neurotransmitter release, they act on different receptor structures in the membrane. Tetrodotoxin antagonizes the effects of both veratridine and the sea anemone toxin. The half-maximum inhibitory concentration of tetrodotoxin is K0.5 = 4 nM for veratridine and 7.9 nM for ATXII. It is very similar to the dissociation constant measured from direct binding experiments with the radioactive toxin. The analysis of this antagonistic action offers an easy in vitro assay for tetrodotoxin interaction with its receptor.
We have tested the effect of several bile acids on the proliferation and differentiation of the HL60 human promyelocytic leukemia cell line in vitro. Deoxycholate, chenodeoxycholate and lithocholic acid caused dose-dependent inhibition of cell proliferation and induction of differentiation along the monocyte/macrophage pathway as determined by morphology, NBT test, non-specific esterase, and staining by monoclonal antibodies against specific cell-surface antigens. Optimal effects were obtained at 100, 75, and 60 microM of the 3 bile acids respectively. Cell-cycle flow-cytometric analysis showed that a substantial fraction of HL60 cells accumulated at the G0/G1 transition. Protein-kinase-C inhibitors such as sphinganine and H-7 inhibited the differentiation-inducing effect of bile acids, suggesting a possible role for PKC in this regulation. When bile acids were combined with non-effective concentrations of all-trans retinoic acid, enhancement of the monocytic differentiation of THP-1 human leukemia cells was observed. Our findings demonstrate induction of tumor-cell differentiation by bile acids, compounds that present minimal undesirable effects in humans.
Monocytic differentiation of U937 cells induced by retinoic acid is accompanied by a 0.2-pH-unit cell alkalinisation. The effect of retinoic acid on intracellular pH (pHi) develops slowly and it precedes the differentiation of the cells by 24 h. Heterogeneity in cellular pHi values was assessed using flow cytometry. It was higher at the differentiated stage than at the undifferentiated stage. It was reduced under conditions of clamped pHi values. Two membrane mechanisms allow U937 cells to recover from an intracellular acidosis. These are the Na'/ H' exchange system and a Na+-dependent HCO;/Cl-exchange system. The increase in the pHi observed after monocytic differentiation resulted from a twofold increase in the maximum activity of the Na+/H' exchange system with no change in the activity of the bicarbonate-dependent system. The properties of interaction of the Na'/H' exchanger of U937 cells with Na', Li+, amiloride and its derivatives were defined and appeared to be unique to human leukemic cells.Retinoids have profound effects on differentiation and growth of a wide range of both normal and transformed cells [l]. For instance the human myeloid leukemic cell line HL60 acquires granulocyte properties after treatment with retinoic acid [2,3]. In another human leukemic cell line, U937, retinoic acid promotes the appearance of monocytic properties [4 -61. The molecular mechanisms underlying the effect of retinoic acid on hematopoietic cell differentiation are still unknown. In a previous study we showed that retinoic acid produced a cellular alkalinisation of HL60 cells that preceded the appearance of granulocytic properties by several hours. The increase in the intracellular pH (pHi) of HL60 cells was further shown to result from a selective activation of the amiloride-snsitive Na+/Hf exchange system [7]. In this study we analyze the effect of retinoic acid on the pHi of U937 cells in relation to the acquisition of monocytic properties. This study also documents the use of flow cytometry for measuring pHi and assessing its variability in populations of leukemic cells. MATERIALS AND METHODS Materials 3-O-methyl-~-[l-~H]glucose
Specific chemical modifications of Escherichia coli acyl-carrier protein have been carried out. The salient results are:1. The deletion of the N-terminal hexapeptide destroys the information required for molecular stability. A very drastic conformational change occurs which produces a total loss of biological activity. The fundamental structural rble of the N-terminal sequence is compared to that found for proteolytic enzymes such as chymotrypsin or elastase. The possible existence of a salt-bridge between the ol-ammonium of serine-1 and the side-chain of aspartic acid-35 (the neighbour of serine-36 which binds the 4'-phosphopantetheine group) is discussed.2. Tyrosine-71 is not easily modified by tetranitromethane. Nitration decreases the pK of the phenol function from 10.4 to 7.3 a t 20 "C. Nitrated acyl-carrier protein and nitrated palmitylacyl-carrier protein have a decreased thermal stability as compared to their native forms. Activities in the malonyl-CoA-CO, exchange reaction and in fatty-acid biosynthesis are similar for acyl-carrier protein and nitrated acyl-carrier protein. Nitrated palmityl-acyl-carrier protein has an activity in phospholipid biosynthesis of only one third that of its native form.3. The side-chain of methionine-44 scarcely reacts with iodoacetamide in the native acylcarrier protein. Its alkylation is easy, however, after the unfolding of the molecule in 9 M urea. Alkylation of methionine-44 does not appear to significantly influence the conformation or the therma,l stability of the protein. However, modification of methionine-44 diminishes the activity in total fatty-acid biosynthesis by a factor of 3 and changes drastically the relative amounts of p-hydroxyacids and saturated acids. Alkylated palmityl-acyl-carrier protein is also less active than its native form in phospholipid biosynthesis. 4.The t,ransformation of 11 of the 22 carboxylates into amide functions induces practically no change in the structural properties of acyl-carrier protein. Nevertheless the biological activity is completely lost.5. The structural consequences of acylation of the -SH group of 4'-phosphopantetheine by bulky hydrophobic groups, the influence of cumulative chemical modifications, and the thermodynamic aspects of the thermal denaturation of acyl-carrier protein and its derivatives are discussed.The acyl-carrier protein of Escherichia coli is a small protein comprising 77 amino acids (M, 8847)
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