Inhibition of the in vitro formation of dense cells and of irreversibly sickled cells by charybdotoxin, a specific inhibitor of calcium-activated potassium efflux

Charybdotoxin, a specific inhibitor of the calcium-activated potassium channel, was found to inhibit the in vitro formation of irreversibly dehydrated cells and of irreversibly sickled cells, which occur as a result of repeated cycles of sickling and unsickling of sickle red blood cells. The degree of formation of dense cells was measured by Percoll-renografin density gradient centrifugation. 50% inhibition of the formation was achieved at a concentration of 30 nM of charybdotoxin. The approximate half-life of this compound in the circulation of the guinea pig was determined to be 4 h. Charybdotoxin did not inhibit the sickling of sickle cells under deoxygenation. The effects of charybdotoxin in preventing the irreversible changes of sickle cell membranes may be related to the inhibition of calcium-activated potassium efflux in sickle red blood cells.     

6,9-deepoxy-6,9,-(phenylimino)-delta 6,8-prostaglandin I1, (U-60,257), a new inhibitor of leukotriene C and D synthesis: in vitro studies

Addition of the calcium inophore, A 23187, and cysteine to isolated mononuclear cells from rat peritoneal washings causes a marked increase in the formation of thromboxane B2 (TxB2) along with the formation of leukotrienes C and D (LT's). The formation of LT's in this system was inhibited by 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1, U-60,257, or its methyl ester, U-56,467, (ID50 4.6 and 0.31 microM, respectively). There was no inhibition of TxB2 formation. By contrast, two structurally-related compounds, PGI2 and its stable analog, 6-beta-PGI1, did not affect the formation of either LT's or TxB2. The inhibition of LT formation by U-60,257 was rapidly reversed after removal of this compound from the cells. U-60,257 did not inhibit the cyclooxygenase of human polymorphonuclear leukocytes. Nor did it inhibit formation of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) in human platelets. On the other hand, U-60,257 inhibited glutathione S-transferase activity of rat basophil leukemia cells (ID50, 37 microM), suggesting that this compound may inhibit the last step in LTC biosynthesis. In addition to inhibiting LT synthesis, U-60,257 also appears to be a competitive inhibitor of the action of LT on the guinea pig ileum, although this inhibition requires a higher drug concentration than those ordinarily encountered during assay for LT's in U-60,257-treated incubations.     

Viscosity and gelation studies in deer hunting hemoglobins.

Sickling, viscosity and gelling properties of the red cells and the hemoglobins of three Virginia white-tailed deer homozygous for types II and III (the sickling types) and V (the nonsickling type), respectively, have been analyzed. The sickling of erythrocytes of deer with type II or III is inhibited by urea and cyanate at concentrations which are comparable to those used in in vitro studies of red cells from patients with sickle cell anemia. No differences were observed between the viscosities of the three deer hemoglobin types at temperatures of 12 degrees C or above. High concentrations of deer hemoglobin types II and III gelled at 1 degree C and at pH values of 7.4-7.7; the minimum gelling concentration of type II was 33.5 g% and of type III was 38 g%. Gel formation was not observed at pH values between 6.7-7.1. Hemoglobin type V did not gel and prevented the formation of gels of type II and III in mixtures at pH 7.6-7.7.     

Imidazo[1,2-a]pyridine derivatives as inhibitors of TNF-alpha expression in T cells

Novel hexahydroimidazo[1,2-a]pyridines prepared by the addition of ethyl (1-benzylimidazolidin-2-ylidene)acetate (2) to the fungal metabolite podoscyphic acid (1a) and esters of 1a have been evaluated for their ability to inhibit the inducible TNF-alpha promoter activity in T cells. The methyl ester 3b is the most potent, inhibiting the TNF-alpha driven reporter gene expression in Jurkat T cells with an IC(50)-value of 2.0 microg/ml (3.6 microM). In addition, compound 3b inhibited the inducible TNF-alpha production in the myelomonocytic U937 cells with an IC(50)-value of 4.6 microM.     

Binding of ovarian steroids to erythrocytes in patients with sickle cell disease; effects on cell sickling and osmotic fragility

Ovarian steroids appear to influence the manifestations of sickle cell disease (SCD); oestrogens can adversely affect erythrocyte function, whereas progestogens may inhibit sickling and decrease the osmotic fragility of erythrocytes. The aims of the present studies were: (i) to characterise the binding of oestradiol and progesterone to erythrocytes from women with HbSS, HbSC and HbAA genotypes; (ii) to investigate whether steroids modulate susceptibility to sickling or osmotic fragility of HbSS and HbAA erythrocytes. Erythrocytes were incubated for 1h with [3H]-steroids at 4 and 37 degrees C. Binding of both oestradiol and progesterone was independent of temperature and steroid concentration, but was decreased by sequential "washing" of erythrocytes in fresh incubation buffer. Binding capacity was 80 +/- 6% greater for oestradiol (versus progesterone) in all three genotypes, and binding of both steroids was decreased by > or = 70% in HbSS erythrocytes compared to HbSC or HbAA erythrocytes. Pre-incubation of erythrocytes with 35 microM oestradiol or 30 microM progesterone had no significant effect on susceptibility of HbSS and HbAA erythrocytes to sickling, or on osmotic fragility. We conclude that both oestradiol and progesterone bind in a low affinity, non-saturable manner to erythrocytes with decreased binding in cells from women with HbSS. However, steroid binding does not affect susceptibility to sickling or osmotic fragility irrespective of haemoglobin genotype.     

The mechanism of in vitro formation of irreversibly sickled cells and modes of action of its inhibitors

When red blood cells from sickle-cell patients were exposed to repeated cycles of deoxygenation and reoxygenation (one cycle was 5 min), dehydration of the cells was observed after several cycles of the sickling-desickling process. These dehydrated cells still maintained a biconcave form after 1 h of such cycling, but they started to take the form of irreversibly sickled cells after several hours. If red cells were simply kept deoxygenated for 16 h, neither dehydrated cells nor irreversibly sickled cells were formed. The formation of dehydrated cells was inhibited either by elimination of Ca2+ from the medium, or by the increase of K+ concentration in the medium. Under conditions in which dehydrated cells were not formed, i.e., deoxygenation incubation (either in the absence or presence of Ca2+) or the deoxygenation-reoxygenation cycling in the absence of Ca2+, 15-25% of cellular K+ leaked out during 4 h of incubation. When dehydrated cells were formed in deoxygenation-reoxygenation cycling in the presence of Ca2+, 40-50% of K+ was lost in 4 h. Two different types of inhibitor were found. The first type includes inhibitors of the Ca2+-activated K+ efflux, such as quinine, quinidine or tetraethylammonium chloride. These compounds suppressed both the K+ efflux and the formation of dehydrated cells. The second type includes calmodulin-interacting drugs. For example, chlorpromazine (20 microM) inhibited the formation of dehydrated cells almost completely, even though it did not inhibit the K+ efflux remarkably. Several other calmodulin-binding drugs were found to inhibit the formation of dehydrated cells similarly, and the potency of these drugs to inhibit the formation seems to be related to the binding affinity of these drugs to calmodulin.     

Mouse alpha chains inhibit polymerization of hemoglobin induced by human beta S or beta S Antilles chains

A murine model of sickle cell disease was tested by studying the polymerization of hybrid hemoglobin tetramers between alpha mouse and human beta S or beta S Antilles chains were prepared from Hb S Antilles, which was a new sickling hemoglobin inducing a sickle cell syndrome more severe than Hb S. The hybrid molecules did not polymerize in solution, indicating that the mouse alpha chains inhibited fiber formation. Consequently, a mouse model for sickle cell disease requires the transfer and expression of both alpha and beta S or beta S Antilles genes.     

Effects of suramin, an anti-human immunodeficiency virus reverse transcriptase agent, on protein kinase C. Differential activation and inhibition of protein kinase C isozymes.

Suramin inhibited protein kinase C (PKC) type I-III activity in a concentration-dependent manner. Similar inhibitory effects were observed with M-kinase, the constitutively active catalytic fragment of PKC, and autophosphorylation of PKC types I-III. Kinetic experiments indicated that suramin competitively inhibits activity with respect to ATP (Ki = 17, 27, and 31 microM, respectively) and that it can also inhibit by interaction with the substrate histone III-S. With protamine as the Pi acceptor, suramin inhibition was dependent on lipid, being approximately 4-fold less sensitive to inhibition in the absence of phosphatidylserine and diacylglycerol than in their presence. Suramin at low concentrations (10-40 microM), in the presence of Ca2+ and absence of lipid, was able to stimulate kinase activity (approximately 200-400%) in a type-dependent manner and at higher concentrations inhibited activity with histone III-S as substrate. These results indicate that suramin, a hexa-anionic hydrophobic compound, can act as a negatively charged phospholipid analog in activating PKC in the presence of Ca2+ and absence of lipid and can inhibit Ca2+/phosphatidylserine/diacylglycerol-stimulated kinase activity at higher concentrations by competing with ATP or by interaction with the exogenous substrate. Suramin inhibited cAMP-dependent protein kinase much less potently (IC50 = 656 microM) than PKC. The ability of suramin to inhibit PKC-mediated processes in intact cells was tested using the phorbol ester-stimulated respiratory burst of neutrophils as a model system. The respiratory burst of human neutrophils, when preincubated with suramin and then stimulated with phorbol ester, was inhibited in a concentration-dependent manner, suggesting that suramin may also be able to inhibit PKC-mediated processes in intact cells.     

The anti-sickling drug lawsone (2-OH-1,4-naphthoquinone) protects sickled cells against membrane damage

The ability of an anti-sickling drug lawsone, 2-OH-1,4-naphthoquinone, and two related compounds to inhibit the haematoporphyrin-sensitised photohaemolysis of normal and sickle cell erythrocytes has been investigated. The compounds appear to protect the erythrocyte membranes by reaction with transient oxidative species. Differential effects between normal and sickle cells are shown and these are attributed to the different membrane composition of irreversibly sickled erythrocytes. This report describes a possible basis for the decreased formation of irreversibly sickled cells in the presence of lawsone.     

Halo lipids. 10. Synthesis and cytostatic activity of O-alkylglycerophospho-L-serine analogs

The synthesis of O-alkylglycerophospho-L-serine analogs is described, which represent a new class of cytostatically active agents based on phospholipids. The new compounds were obtained by conversion of O-alkylglycerophosphoric ester analogs by means of phospholipase D and by condensing O-alkylglycerophosphoric acid analogs with protected L-serine followed by the removal of protective groups of the resulting intermediates. The structure of the O-alkylglycerophospho-L-serines was confirmed by fast atom bombardment mass spectrometry. The compounds were found to inhibit the growth of Ehrlich ascites tumor cells in vitro. Half maximum inhibition was observed at concentrations between 7 and 15 microM. For the 1-O-alkyl-2-methoxy glycerophosphoserine only a higher value (30 microM) was found. With most of the substances tested growth was completely inhibited at a concentration of 40 microM.     

Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer

Defective dendritic cell (DC) function caused by abnormal differentiation of these cells is an important mechanism of tumor escape from immune system control. Previously, we have demonstrated that the number and function of DC were dramatically reduced in cancer patients. This effect was closely associated with accumulation of immature cells (ImC) in peripheral blood. In this study, we investigated the nature and functional role of those ImC. Using flow cytometry, electron microscopy, colony formation assays, and cell differentiation in the presence of different cell growth factors, we have determined that the population of ImC is composed of a small percentage (<2%) of hemopoietic progenitor cells, with all other cells being represented by MHC class I-positive myeloid cells. About one-third of ImC were immature macrophages and DC, and the remaining cells were immature myeloid cells at earlier stages of differentiation. These cells were differentiated into mature DC in the presence of 1 microM all-trans-retinoic acid. Removal of ImC from DC fractions completely restored the ability of the DC to stimulate allogeneic T cells. In two different experimental systems ImC inhibited Ag-specific T cell responses. Thus, immature myeloid cells generated in large numbers in cancer patients are able to directly inhibit Ag-specific T cell responses. This may represent a new mechanism of immune suppression in cancer and may suggest a new approach to cancer treatment.     

Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.     

Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes

Dimethyl adipimidate (DMA) reduces K⁺ loss from, and dehydration of, red cells containing haemoglobin S (HbS cells). Three membrane transporters may contribute to these processes: the deoxygenation-induced cation-selective channel (P_sickle), the Ca²⁺-activated K⁺ channel (or Gardos channel) and the K⁺-Cl⁻ cotransporter (KCC). We show that DMA inhibited all three pathways in deoxygenated HbS cells. The Gardos channel could be activated following Ca²⁺ loading. Considerable KCC activity was present in oxygenated HbS cells, showing a selective action of DMA on the transporter in deoxygenated cells. Inhibition of sickling correlated strongly with that of P_sickle and moderately with that of KCC activity. We conclude that DMA does not inhibit the K⁺ pathways directly, but acts mainly by preventing HbS polymerisation and sickling. These findings are relevant to the development of novel chemotherapeutic agents for amelioration of sickle cell disease.     

Inhibitory effect of dicationic diphenylfurans on production of type I collagen by human fibroblasts and activated hepatic stellate cells

Excessive production of extracellular matrix is responsible for clinical manifestations of fibroproliferative disorders and drugs which can inhibit excessive synthesis of type I collagen are needed for the therapy. Several dicationic diphenylfurans were synthesized and were found to bind RNA. Two of these type compounds were able to reduce synthesis of type I collagen by human fibroblasts and human activated hepatic stellate cells (HSCs). Activated HSCs are responsible for collagen production in liver fibrosis. When added at 40 microM compound 588 reduced intracellular level and secretion of procollagen alpha1(I) by 50%, while compound 654 reduced these parameters by more than 80% at 20 microM. 654 also significantly reduced secretion of fibronectin. Toxic effects were observed at 80 microM for 588 and 40 microM for 654. 654 reduced expression of a reporter gene with collagen signal peptide, while expression of the same gene without signal peptide was unaffected. Also, expression of intracellular proteins tubulin and calnexin was unchanged. 654 accumulated inside the cell in the cytoplasm and did not change the steady-state level of collagen mRNAs. Treatment of cells with proteosome inhibitor MG132 did not change the inhibitory effect of 654, suggesting that 654 acts as suppressor of translation of proteins containing a signal peptide. Most secreted proteins of fibroblasts and activated HSCs are components of extracellular matrix. Therefore inhibition of their production, as shown here for procollagen alpha1(I) and fibronectin, may be a useful property of some of diphenylfurans, making these compounds a basis for development of antifibrotic drugs.     

Inhibition of invitro sickling by liposome-mediated transport of amino acids into intact human red blood cells

To investigate the role of phenylalanine and tryptophane as potential antisickling agents in intact human SS-red blood cells a liposomal transport system was employed to transfer phenyl-alanine or tryptophane into intact SS-red blood cells. Aromatic amino acids and short peptides containing phenylalanine have been demonstrated to increase the minimum gelling concentration and solubility of deoxy-hemoglobin S in aqueous solution. However, these compounds do not cross the red blood cell membrane under usual incubation conditions. Incorporation of phenylalanine or tryptophane into intact SS-red blood cells $\text{via}$ liposomal transport system markedly inhibited the $\text{in}\text{vitro}$ sickling of deoxy-hemoglobin S. These findings raise the possibility that a nontoxic liposomal transport system which facilitates incorporation of antisickling agents into intact SS-RBC may have significant therapeutic implications in the treatment of sickle cell disease.     

Inhibition of rat brain prostaglandin D synthase by inorganic selenocompounds

Various inorganic selenocompounds dose-dependently inhibited the rat brain prostaglandin (PG) D synthase, both in the purified enzyme preparation and in the crude brain supernatant. All of the quadrivalent selenium compounds tested had a very limited range of IC50 values in the purified enzyme (11-12 microM) and in the brain supernatant (9-15 microM). A divalent selenium compound was also inhibitory, but a hexavalent selenium compound was ineffective. In contrast, organic selenocompounds such as selenomethionine and selenourea had no effect on the PGD synthase activity. Furthermore, sodium sulfate and sodium sulfite up to 10 mM did not inhibit the activity. The inhibition by selenium required the preincubation of the metal with sulfhydryl compounds such as dithiothreitol (DTT), indicating that the formation of selenotrisulfide or some other adduct(s) is essential for the inhibition. Furthermore, the inhibition was reversed by an excess amount of dithiothreitol, suggesting that the selenotrisulfide derivative of DTT binds to the SH group of the PGD synthase. The kinetic analysis revealed the inhibition by selenite to be noncompetitive with a Ki value of 10.1 microM. On the other hand, glutathione-dependent PGD synthase from rat spleen was much less inhibited, and PGF synthase and PGD2 11-ketoreductase activities were not inhibited by the selenium compound.     

Role of interferons in the control of Lassa virus replication in human dendritic cells and macrophages

Lassa fever is a hemorrhagic fever caused by Lassa virus (LV), which primarily targets human dendritic cells (DC) and macrophages (MP). Massive numbers of viral particles are released with no effect on the viability, activation or maturation of these cells. LV does not inhibit the activation of cells induced by sCD40L or LPS. We report here the consequences of exogenous activation of LV-infected human DC and MP for viral replication. The activation of cells with lipopolysaccharide or exogenous poly(I-C) and the transfection of cells with poly(I-C) strongly inhibited LV replication, at least partly by inducing type I interferon (IFN) synthesis. In contrast, cell stimulation with sCD40L did not induce type I IFN responses or inhibit LV release. Recombinant type I IFNs strongly inhibited LV replication in both cell types, whereas IFNgamma and IFNlambda did not. The modest type I IFN production observed in LV-infected MP, but not in DC, was involved in controlling LV replication in MP. These results provide an explanation for the slower replication of LV in MP than in DC, and suggest that type I IFNs are crucial in the control of LV.     

Synthesis of novel diaryl ethers and their evaluation as antimitotic agents

A series of novel diaryl ethers possessing various functional groups were synthesized and evaluated for antiproliferative activity in human myeloid leukemia HL-60 cells. Among the compounds examined, compounds 10, 17, 20, 24, and 33 showed moderate to potent antiproliferative activity. These derivatives were further examined in terms of their abilities to inhibit tubulin polymerization; however, all of the tested compounds were relatively ineffective. The reference compound E7010 with an IC(50) of 0.34 microM exhibited potent antiproliferative activity and significantly inhibited tubulin polymerization in a dose-dependent manner.     

Effects of sulfhydryl reagents on the anti-sickling activity of some membrane-interacting compounds

Under deoxygenated conditions (PO2 = 0 mmHg), sulfhydryl reagents such as N-ethylmaleimide and iodoacetamide had no effect on sickling, but they did inhibit the anti-sickling activity of chlorpromazine. At the same concentration, these sulfhydryl reagents inhibited the cup formation of chlorpromazine in an oxygenated state (PO2 = 143 mmHg). This supports our previous finding that the anti-sickling effect of membrane-interacting compounds is related to their ability to form a cup-shaped red cell.     

Intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and cell-free solutions.

We have measured the intermolecular interactions of oxygenated sickle hemoglobin molecules in cells and in cell-free solutions, and have compared the results with similar data for liganded normal adult hemoglobin. The experiments involve the measurement of the spin-lattice relaxation time T1 of protons of solvent water molecules, as a function of an externally applied static magnetic field. From such data, one can derive a correlation time tauc, for each sample, which is a measure of the time taken for a hemoglobin molecule to randomize its orientation due to Brownian motion. Thus tauc is a measure of the freedom of rotational motion, on a molecular or microscopic level, of hemoglobin molecules. Intermolecular interactions will reduce this freedom of motion and lengthen tauc. We find that oxygenated sickle hemoglobin molecules have an additional intermolecular interaction not found for normal hemoglobin. This extra interaction is increased by the presence of either inorganic phosphate or diphosphoglycerate, and is greater for sickle hemoglobin within cells than in cell-free solutions. By comparing the present results with published data on the viscosity of oxygenated sickle and normal hemoglobin, we conclude that, at concentrations comparable to intracellular values, oxygenated sickle hemoglobin molecules form aggregates several tetramers in size. The possibility exists that these aggregates are the earliest stage of fiber formation itself, the physical basis of the sickling phenomena.