Protection of the heart from ischemia by a new oligomeric derivative of prostaglandin E1

A lipophilic oligomeric ester was synthesized from prostaglandin E1. The compound was found to protect a Langendorff perfused rat heart from ischemic insult. After 15 minutes cessation of perfusion, the flow was restarted, and the recovery of contraction was measured. In control experiments, the recovery was 16.1 +/- 7.3%. In the case of pre-ischemic addition of the compound (10 micrograms/ml at 10 minutes before ischemic insult), the recovery was 31 +/- 13.2% (n = 7; the difference was not significant). In post-ischemic addition (10 micrograms/ml), the recovery was 75.9 +/- 9.0% (n = 7; p less than 0.01). The compound was also effective in protecting rat heart myocytes against a 60 minutes anoxia/15 minutes reoxygenation injury as judged by the loss of "rod-shaped" intact myocytes. At a 10 micrograms/ml concentration, the compound protected against the loss of rod-shaped myocytes by 30% in pre-anoxia addition and 35% in post-anoxia addition. The levels of significance in these experiments were p less than 0.001. Possible mechanisms of action of this compound are discussed.     

Calcium ionophoretic activity of chemically synthesized oligomeric derivatives of prostaglandin B1

Chemically synthesized dimers, trimers and tetramers of 15-dehydroprostaglandin B1 and 16,16'-dimethyl-15-dehydroprostaglandin B1 facilitate the release of Ca2+ from isolated rat liver mitochondria. The parent monomeric prostaglandins had no significant activity. The rate of release was stimulated by exogenous K+ or Na+, suggesting an antiport exchange of monovalent cations for intra-mitochondrial Ca2+. The activity depended upon the presence of ruthenium red, which prevented recycling of Ca2+; comparison of the activity with A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone indicated that the prostaglandin B1 oligomers were functioning as ionophores and the release of Ca2+ was not caused by an uncoupling of oxidative phosphorylation. The oligomers caused a major decrease in the membrane potential but only when the mitochondria were preloaded with exogenous Ca2+, and even then, the Ca2+ efflux was completed before the membrane potential decreased to less than 90 mV. The oligomeric molecules were able to form supramolecular aggregates in the presence of Ca2+ as detected by light scattering. They extracted Ca2+ into an organic phase, and translocated Ca2+ from one aqueous domain to another across an organic barrier; K+ and Na+ modulated these processes. The prostaglandin B1 derivatives also translocated Rb+ from one aqueous phase to another across an organic barrier when Ca2+ was translocated.     

Calcium ionophore activity of a prostaglandin B1 derivative (PGBx).

A water soluble derivative of PGB₁, designated PGB_X, has been found to stimulate the release of Ca²⁺ from fragmented sarcoplasmic reticulum and heart mitochondria; its activity is almost two orders of magnitude greater than other prostaglandins. PGB_X demonstrates ionophoretic activity in its ability to transfer Ca²⁺ from an aqueous to an organic phase.     

Studies on PGBx, a polymeric derivative of prostaglandin B1: I. Synthesis and purification of PGBx.

PGBx, a new polymeric derivative of PGB1, previously was shown to (a) restore oxidative phosphorylation to degraded isolated rat liver mitochondria in vitro and (b) to reverse the effects of cardiogenic ischemia in monkeys and cerebral ischemia in rabbits. This report describes in detail the synthesis and purification of PGBx via PGB1, starting with azelaic acid. Details of the in vitro mitochondrial assay are also reported. Purified PGBx exhibiting maximal reactivation of mitochondrial phosphorylation has a mean molecular weight of 2350. Yield of PGBx based on azelaic acid is 4% and based on PGB1 is 25%.     

Effect of oligomeric derivatives of prostaglandin B1 on oxidative phosphorylation and their Ca2+ ionophoretic activity

Dimers, trimers and tetramers of 15-dehydro-PGB1 and of 16,16'-dimethyl-15-dehydro-PGB1 have been synthesized and their effect on mitochondrial function evaluated. The trimers and tetramers, and to a lesser extent the dimers, of both series, protected isolated mitochondria from the loss of phosphorylating capacity during in vitro incubation. The monomers were inactive. The trimers and tetramers inhibited between 40 and 50% the F1F0-ATPase of submitochondrial particles. All of the oligomers, but not the monomers, had Ca2+ ionophoretic activity with isolated mitochondria. These activities are qualitatively similar to that reported for the oligomeric mixture of 15-dehydro-PGB1, termed PGBX.     

Chemical properties of the divalent cation binding site on potassium channels

The actions of divalent cations on voltage-gated ion channels suggest that these cations bind to specific sites and directly influence gating kinetics. We have examined some chemical properties of the external divalent cation binding sites on neuronal potassium channels. Patch clamp techniques were used to measure the electrophysiological properties of these channels and Zn ions were used to probe the divalent cation binding site. The channel activation kinetics were greatly (three- to fourfold) slowed by low (2-5 mM) concentrations of Zn; deactivation kinetics were only slightly affected. These effects of Zn were inhibited by low solution pH in a manner consistent with competition between Zn and H ions for a single site. The apparent inhibitory pK for this site was near 7.2. Treatment of the neurons with specific amino acid reagents implicated amino, but no histidyl or sulfhydryl, residues in divalent cation binding.     

A prostaglandin oligomeric derivative inhibits activities of phospholipase and protease: a possible mechanism of membrane protection during ischemia

A prostaglandin oligomeric derivative was synthesized by alkaline treatment of prostaglandin E1. This compound protected the perfused rat heart from global ischemia. This compound was found to inhibit several lipolytic and proteolytic enzymes in vitro. When phospholipase A2 from Naja naja venom was used as an enzyme and phosphatidylcholine was used as a substrate, 50 per cent inhibition was achieved at 50 microM of the prostaglandin derivative. When trypsin and casein were used as enzyme and substrate, 50 per cent inhibition was obtained at 80 microM. A possible mechanism of beneficial effect of this compound in protecting membranes during ischemia is discussed.     

Divalent metal cation binding properties of human prothymosin alpha.

The divalent cation binding properties of human prothymosin alpha, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin alpha retardation on a weak cation chelating resin charged with various divalent cations, specific binding of Zn2+ ions by prothymosin alpha was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn2+ concentrations, prothymosin alpha could bind up to three zinc ions in the presence of 100 mM NaCl and up to 13 zinc ions in the absence of NaCl. Equilibrium dialysis analysis also revealed that prothymosin alpha could bind Ca2+, although the parameters of Ca2+ binding by prothymosin alpha were less pronounced than those of Zn2+ binding in terms of the number of metal ions bound, the KD values, and the resistance of the bound metal ions to 100 mM NaCl. The effects of Zn2+ and Ca2+ on the interaction of prothymosin alpha with its putative partners, Rev of HIV type 1 and histone H1, were examined. We demonstrated that Rev binds prothymosin alpha, and that prothymosin alpha binding to Rev but not to histone H1 was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin alpha interaction with Rev and histone H1 are distinct and that the observed zinc and calcium-binding properties of prothymosin alpha might be functionally relevant.     

Differential binding properties of B7-H1 and B7-DC to programmed death-1

Programmed death-1 (PD-1) is a negative regulatory receptor expressed on activated T and B cells. Two ligands for PD-1, B7-H1 (PD-L1) and B7-DC (PD-L2), have been identified, but their binding properties have not been characterized yet. In this study, we generated soluble Ig fusion proteins of these molecules and examined the kinetics and relative affinities of the interactions between B7-H1 or B7-DC and PD-1 by flow cytometry and surface plasmon resonance. The interaction of B7-DC/PD-1 exhibited a 2-6-fold higher affinity and had different association/dissociation kinetics compared with the interaction of B7-H1/PD-1. Our results suggest that the differential binding properties of B7-H1 and B7-DC may be responsible for differential contributions of these two PD-1 ligands to immune responses.     

B cell triggering properties of a nontoxic derivative of amphotericin B

The immunomodulating properties of amphotericin B (AMB), an antifungal polyene antibiotic, have been reported in multiple studies. However, many findings on the subject are conflicting, and the precise mechanism of AMB action on the immune system is yet unknown. Because toxicity and limited solubility of AMB are likely to be responsible for these discrepancies, we synthesized a nontoxic derivative of AMB (AMBSH), and we investigated its immune modulating effects on murine B cells. Our results show that AMBSH induces a strong proliferative response under conditions where AMB is weakly efficient or toxic, and that AMBSH supports maturation to Ig secretion. When suboptimal doses of LPS (or BCGF) are present together with AMBSH, a synergistic effect on B cell proliferation occurs. Frequency analyses reveal that, although only a limited number of B cells respond to AMBSH alone, a large population of B cells will respond to subthreshold doses of LPS in the presence of this polyene. Finally, we show that incubation of spleen cells with AMBSH results in an increase in Ia expression. These results are discussed in terms of the membrane disorganizing properties of polyene antibiotics.     

Studies on the covalent binding of an intermediate(s) in prostaglandin biosynthesis to tissue macromolecules.

We investigated the covalent binding of intermediates in prostaglandin biosynthesis to tissue macromolecules. Following incubation of [1-14C]arachidonic acid with the microsomal fraction from guinea pig lung, ram or bovine seminal vesicle, human platelets, rabbit kidney, or rat stomach fundus, the amount of covalent binding of arachidonic acid metabolites expressed as percentage of total arachidonic acid metabolized varied from tissue to tissue ranging from 3% in human platelets to 18.2% in ram seminal vesicles. In general, the thromboxane synthesizing tissues had less covalently bound metabolites than the other tissues. The amount of covalently bound metabolites was increased in the guinea pig lung microsomes when the thromboxane synthetase inhibitor, N-0164, was added to the incubation mixture. The covalent binding of arachidonic acid metabolite(s) was greatly reduced by the addition of glutathione to the incubation mixture. In addition to the covalently bound metabolites, water-soluble metabolites derived from arachidonic acid metabolism were also observed. The amount of water-soluble metabolites was small in each tissue except for the rat stomach fundus. In the rat stomach fundus the water-soluble metabolites accounted for over 50% of the total metabolites. Conditions which would tend to increase or decrease the levels of free prostaglandin endoperoxides during the incubation of arachidonic acid with the microsomes gave increased or decreased levels of covalent binding. Our data suggest that the prostaglandin endoperoxides are responsible for the covalent binding observed during prostaglandin biosynthesis. This covalent binding to tissue macromolecules may be of physiological and pathological significance.     

Processing, stability, and receptor binding properties of oligomeric envelope glycoprotein from a primary HIV-1 isolate

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is thought to exist on the virion surface as a trimer of non-covalently associated gp120/gp41 molecules. We expressed trimeric envelope glycoprotein from three primary, macrophage tropic HIV-1 isolates in baby hamster kidney cells and analyzed the furin-mediated cleavage, stability, and receptor binding properties of the oligomers. The envelope glycoprotein was secreted in a soluble form deleted of its transmembrane anchor and the intracytoplasmic domain (gp140). A mixture of trimers, dimers, and monomers of gp140 as well as monomeric gp120 was detected on polyacrylamide gels. Analysis by sucrose gradient centrifugation revealed that trimers and dimers were essentially composed of uncleaved gp140, whereas most of the gp120 was found in the monomeric fraction. To analyze the effect of the cleavage of gp140 to gp120/Delta41 on trimerization, we co-expressed the furin protease along with gp140. Surprisingly, furin expression changed the subcellular localization of the envelope glycoprotein, which became in majority sequestered in the major furin compartment, the trans-Golgi network, as judged by confocal laser microscopy. The envelope glycoprotein secreted from furin-co-expressing cells was almost completely cleaved to gp120 and Deltagp41, but gp120 was found exclusively in the monomeric fraction, with a few residual oligomers being composed of uncleaved gp140. Secreted uncleaved gp140 trimers were purified to homogeneity and analyzed for their capacity to interact with cellular receptors CD4 and CC chemokine receptor 5 (CCR5). Receptor binding was analyzed on CD4- and CCR5-expressing cells as well as on peripheral blood mononuclear cells. Trimers showed greatly reduced binding to CD4 as compared with monomers. Neither monomers nor trimers bound directly to CCR5. In conclusion, our results show that the cleaved form of the envelope glycoprotein does not form stable trimers, suggesting that gp120/gp41 oligomers on the virion surface might be stabilized by a yet to be identified mechanism and that the virion might attach to CD4 via a monomeric form of gp120. These results are relevant to the development of an envelope-based vaccine against AIDS.     

Dose dependence of PGBX, a polymeric derivative of prostaglandin B1, for normalization of hereditary diabetes of the mouse

A polymeric derivative of prostaglandin B1, PGBX, which reactivates phosphorylation in damaged mitochondria, is shown to normalize the high blood glucose, obesity, and excessive appetite of the hereditary diabetic mouse. The degree of normalization is shown to vary with dosage.     

Structural remodeling of an A + U-rich RNA element by cation or AUF1 binding

Association of AUF1 with A + U-rich elements (AREs) induces rapid cytoplasmic degradation of mRNAs containing these sequences, involving the recruitment or assembly of multisubunit trans-acting complexes on the mRNA. Recently, we reported that Mg(2+)-induced conformational changes in the ARE from tumor necrosis factor alpha mRNA inhibited AUF1 binding and oligomerization activities on this substrate (Wilson, G. M., Sutphen, K., Chuang, K., and Brewer, G. (2001) J. Biol. Chem. 276, 8695-8704). In this study, resonance energy transfer was employed to characterize structural changes in RNA substrates in response to cation- and AUF1-binding events. An RNA substrate containing the tumor necrosis factor alpha ARE displayed a weak conformational transition in the absence of added cations but was cooperatively stabilized by Mg(2+). Additional assays demonstrated a strong preference for small, multivalent cations, suggesting that the folded RNA structure was stabilized by counterion neutralization at discrete regions of high negative charge density. Association of AUF1 with cognate RNA substrates also induced formation of condensed RNA structures, although distinct from the folded structure stabilized by multivalent cations. Taken together, these experiments indicate that association of AUF1 with an ARE may function to remodel local RNA structures, which may be a prerequisite for subsequent recruitment of additional trans-acting factors.     

Inhibition of lipid peroxidation by prostaglandin oligomeric derivatives.

The inhibition of lipid peroxidation by oligomeric derivatives synthesized from prostaglandin E1 (PGE1) and PGB2 was studied using two rat models. In an in vitro model, the brain was exposed to decapitation-ischemia, the cortex was removed and homogenized, and the formation of thiobarbituric acid reactive substances (TBAR) was measured after exposing the homogenate to in vitro reoxygenation either in the presence or absence of oligomers. It was found that these oligomers could inhibit lipid peroxidation, and that their activities were higher than that of superoxide dismutase (SOD). In an in vivo administration model, either the oligomer or the vehicle was injected i.p. 30 min before decapitation. The brain was exposed to decapitation-ischemia, the cortex was homogenized and exposed to 'in vitro' reoxygenation, after which TBAR value was determined. Ester-type compounds had a greater activity than free-acid type compounds in inhibiting lipid peroxidation. A possible mechanism of the protective effect of these oligomers in ischemia/reperfusion injury may be to scavenge oxygen free radicals.     

Studies on the binding of 5-N-methylated quindoline derivative to human telomeric G-quadruplex

Quindoline derivatives as telomeric quadruplex ligands have shown good biological activity for telomerase inhibition. In the present study, we used spectroscopic and calorimetric methods to investigate the interactions between a quindoline derivative (5-methyl-11-(2-morpholinoethylamino)-10-H-indolo-[3,2-b]quinolin-5-ium iodide, compound 1) and human telomeric G-quadruplex. The thermodynamic studies using isothermal titration calorimetry (ITC) indicated that their binding process was temperature-dependent and enthalpy–entropy co-driven. The significant negative heat capacity was obtained experimentally from the temperature dependence of enthalpy changes, which was consistent with that from theoretical calculation, and all suggesting significant hydrophobic contribution to the molecular recognition process. Based on the results from UV–vis, ITC, steady-state and time-resolved fluorescence, their binding mode was determined as two ligand molecules stacking on the quartets on both ends of the quadruplex. These results shed light on rational design and development of quindoline derivatives as G-quadruplex binding ligands.     

Sequence-selective binding of an ellipticine derivative to DNA.

The DNA sequence specificity of an ellipticine derivative bearing an aminoalkyl side chain has been determined by a variety of footprinting methods. The drug exhibits sequence selective binding and discriminates against runs of adenines or thymines. Binding is shown to occur at various sequences with a preference for GC rich regions of DNA. A large enhancement of DNAase I and of hydroxyl radical cleavage in regions rich in A's or T's is observed together with hyperreactivity of adenines towards diethylpyrocarbonate in the presence of drug. This indicates the occurrence of drug-induced changes in critical conformational features of DNA. The total absence of hyperreactivity of guanine residues towards diethylpyrocarbonate appears to be related to the sequence selectivity of drug binding. No alteration of the dimethyl sulphate and methylene blue-induced cleavage of DNA is observed. Irradiation of ellipticine derivative-DNA complexes with UV light followed by alkali treatment leads to selective photocleavage at guanine residues, consistent with the deduced degree of selectivity of the binding reaction.     

Experimental study of the pharmacokinetics of an amphotericin B derivative

Absorption, distribution and excretion of a new water soluble derivative of amphotericin B (NWSDA) were studied after its administration by different routes. After the antibiotic intravenous administration the therapeutic concentrations in blood, organs and urine were shown to remain for prolonged periods. The likely sites of NWSDA deposition were detected with microbiological and radionuclide methods. The most prolonged periods of antibiotic preservation were stated in the renal cortex, spleen and lungs. The ways of NWSDA excretion were studied in operated animals. Only 3.5 per cent of the antibiotic was excreted with urine and bile for 24 hours. The pharmacokinetic parameters of NWSDA after its intravenous administration were estimated. The bioavailability of the antibiotic after its intramuscular and oral administration was found to be low.