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Liposomes, modified into bioadhesive systems by the covalent anchoring of hyaluronic acid (HA) to their surface, were investigated for their abilities to act as site-adherent and sustained-release carriers of drugs for the topical therapy of wounds and burns. Epidermal growth factor (EGF) served as the test drug for growth factors and monolayers of the A431 cell line served as models of the in vivo designated targets. The hyaluronic acid was radiolabeled to carry 3H, through a deacetylation-reacetylation procedure, yielding specific activities in the range of 1 x 10(6) dpm/mg. The HA was bound to the surface of the liposomes, using carbodiimide to crosslink its carboxyl residues to amine residues at the liposomal surface. The latter was provided through the inclusion of phosphatidylethanolamine in the liposome formulation. Varying the initial HA/lipid ratio over the range 1-500 mg HA/mmol lipid, it was found that the increase in bound HA was linear, providing up to 80 mg HA/mmol lipid. The HA-modified, but not the nonmodified, liposomes were found to bind with high affinity (delta G degree in the range of -7 to -9 Kcal/mol) to monolayers of the A431 cell line. The HA-modified liposomes were found to encapsulate EGF at high yields (% encapsulation, > 87) and to act as sustained-release depots, the rate constant for the release of the encapsulated EGF in the range of 1.4 x 10(-3) h-1. Taking all of these findings together, it is concluded that these liposomes are bioadhesive sustained-release carriers of drugs, as desired, meriting further cellular and in vivo studies.

Cellular distribution of HIV-1 Nef protein was studied by expressing the protein in mammalian cells. Cell extracts were fractionated by low- and high-speed centrifugation and by nonionic detergents. Two Nef-related proteins were expressed in COS cells, Nef-27kD and Nef-25kD. Nef-27kD, an N-myristoylated form of Nef, was found in the cytosol and in association with a particulate fraction of the cytoplasm. Treatment of the particulate cytoplasmic fraction with nonionic detergents, using three different protocols designed to isolate the cytoskeleton matrix, indicated that part of Nef was sensitive and part was resistant to detergent solubilization. These two cellular fractions represent membrane- and cytoskeleton-associated Nef. Nef-25kD, initiated from an in-frame AUG codon, was not modified with myristic acid at the amino terminus. Consequently, this protein was present in a soluble form in the cytosol. Furthermore, a mutant of Nef-27kD, in which the myristoylation signal is deleted, appears as a cytoplasmic soluble protein. To determine domains in Nef that are responsible for its subcellular distribution, successive internal deletions of 14-20 amino acids were introduced at the N-terminal portion of the protein. Five mutants were evaluated with respect to their cellular localization. One mutant (pSVLA-5), from which amino acids 73-88 were deleted, did not copurify with the detergent-insoluble fraction. The protein was, however, present in the particulate cytoplasmic fraction, presumably in association with membranes. Taken together, these results suggest that N-myristoylation of Nef affects its association with both membranes and cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

A cellulase, cellobiohydrolase I (CBH I) from Trichoderma reesei was chemically modified by covalent attachment of pentaammine ruthenium (III) without loss in hydrolytic activity. Data suggest that such a modification endowed CBH I with oxidoreductase activity. The modified enzyme was able to carry out hydrogen peroxide-dependent oxidation of veratryl alcohol, a substrate for lignin peroxidase, at a rate of 0.148 mumol substrate oxidized min-1 mumol-1 enzyme. The effects of pH, temperature, and substrate concentration on the oxidation reaction were examined. The optimal temperature was determined to be 45 degrees C, and the optimal pH was 4.3. The Km and Vmax for veratryl alcohol were determined to be 3.519 mM and 52.27 microM min-1, respectively. Tartrate at concentrations as low as 0.10 mM was found to inhibit the reaction.

It has been reported previously that intravenous administration of normal human immunoglobulins (IVIg) to human patients can suppress the clinical signs of certain autoimmune diseases. However, the mechanism(s) by which normal Ig interferes with the various disorders and the scheduling of treatment have been poorly delineated. To study these questions, we examined IVIg treatment of two experimentally induced T cell autoimmune diseases in rats: experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA). We now report that IVIg treatment (0.4 g/kg) inhibited the active induction of both EAE and AA, and that this treatment did not affect the acquisition of resistance to reinduction of EAE. The importance of the site of administration and schedule of treatment were studied in the AA model. Ig was effective when given intravenously, but not when administrated subcutaneously or intraperitoneally. IVIg treatment was effective when given daily from immunization to outbreak of disease; but it was also effective when given once at the time of immunization or once 2 wk after induction of AA, just at the clinical outbreak of disease. Administration of IVIg between immunization and outbreak of AA was less effective. Prevention of disease by IVIg occurred despite the presence of T cell reactivity to the specific antigens in the disease. In fact, IVIg administrated to naive rats activated T cell reactivity to some self-antigens. Nevertheless, IVIg treatment led to decreased production of the inflammatory cytokine TNF alpha. Thus, IVIg treatment may exert its therapeutic power not by inhibiting T cell recognition of self-antigens, but by inhibiting the biological consequences of T cell recognition.

Liposomes, modified by covalently-anchoring collagen to their surface, were investigated for their abilities to be bioadhesive and to act as sustained-release drug carriers. These bioadhesive liposomes have the potential to induce significant improvements in topical and regional therapies. The major findings for uni-(ULV) and multilamellar (MLV) bioadhesive liposomes are: (a) Both ULV and MLV release small molecular weight drugs over prolonged periods. For example, rate constants of (6 +/- 0.5) x 10(-3) and (2.6 +2- 0.8) x 10(-3) h-1, were obtained for the release of vinblastine and fluconazole, respectively, from collagen-ULV. (b) For a given drug, that rate constant can be shifted (up or down) by the choice of liposome type and collagen-surface density and the latter, if high enough, lead to the formation of an additional liposome-associated drug reservoir. (c) Using monolayers of the A431 cell line to model the in vivo targets, the bioadhesive (but not the regular) liposomes were found to bind with high affinity to the monolayers. For example, equilibrium dissociation constants of 6.3(+/- 3) microM and 2.7(+/- 0.5) microM were determined for bioadhesive MLV and ULV, respectively, with corresponding saturation occupancies of 3.7(+/- 1) and 4.0(+/- 0.2) pmoles liposomal collagen/monolayer of 10(5) cells. (d) Following the retention of bioadhesive MLV at A431 monolayers for 24 h, it was found that: at 4 degrees C, 24 h did not suffice to reach equilibrium, but at 37 degrees C equilibrium binding was obtained within 3-5 h and there was quantitative liposome retention (per viable monolayer) thereafter. It is concluded that these liposomes are bioadhesive sustained-release carriers, as desired, meriting further cellular and in vivo studies.

The phenomena of the high affinity of porphyrins to the human serum proteins, albumin, high-density lipoproteins (HDL) and low-density lipoproteins (LDL) is well established. Yet, evaluation of the activities of these proteins as endogenous porphyrin carriers, especially with respect to receptor-mediated porphyrin uptake into tumor cells, the merits of which are still in dispute, requires more quantitative protein-porphyrin binding data. As a continuation of previous studies on this issue, the binding of several porphyrin systems to each of the three proteins, employing previously developed spectral methodologies, was studied. The specific systems reported here are hematoporphyrin ester (HPE), which is a novel hematoporphyrin derivative (HPD)-like system, two porphyrin trimers (denoted O1 and O2) and a porphyrin dimer (denoted O3) isolated from HPE. Human serum albumin (HSA) was found to have a single high-affinity site for the monomeric components of HPE, with an equilibrium binding constant of 3.6 x 10(6). The equilibrium parameters determined for the binding of the three HPE-isolated oligomers to each of the serum proteins are: (1) Binding constants (Kb') of 2.3 x 10(6), 6.9 x 10(4) and 1.5 x 10(4) and number of sites per protein molecule (n) of 3, 1 and 5, for the binding of O1, O2 and O3, respectively, to HSA. (2) Kb' values of 15.5 x 10(3), 15.3 x 10(3) and 6.6 x 10(3) and n values of 1, 2 and 2, for the binding of O1, O2 and O3, respectively, to HDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Pentaammineurthenium(III) was covalently attached to cellobiohydrolase I (CBH I, EC 3.2.1.91), the major component of Trichoderma reesei cellulase, resulting in 0.7 mol ruthenium/mol CBH I and an electrode potential of +95 mV. Fractionation of modified CBH I by chromatofocusing resulted in the separation of fractions with a 1.4- to 3.2-fold increase in specific activity toward p-nitrophenylcellobioside, depending on the assay conditions, over that of native enzyme. The extent of the hydrolysis of insoluble cellulosic substrates (Avicel and newsprint) to glucose by modified CBH I was also greater than that observed by the native enzyme.

The structure-activity relationships among three noncyclic diimide ionophores, designed to be Li+ carriers, were studied in lipid bilayer membranes. These ionophores (ETH1644, ETH1810, and ETH1811) vary in their N-imide substituents, going from two isobutyls to one isobutyl and one cyclohexyl to two cyclohexyls, respectively. ETH1811 was found to form two types of complexes with 1:1 and 1:2 ion-carrier stoichiometries, the former type dominant over most of the ionophore (0.1-10 microM) and salt (0.01-1.0 M) concentration ranges studied. In contrast, ETH1644 and ETH1810 were previously found to form a single type of complex with the 1:2 stoichiometry. The alkali cations selectivity sequence induced by ETH1811 is Li+ (1) greater than Na+ (0.08) greater than K+ (0.02) greater than Cs+ (0.008). These ETH1811-induced ionic selectivites as well as its relative potency in ion transport were found to be inferior to those of ETH1644 and ETH1810 (the latter being the best in this series). The conductance-voltage relationships reported here, for all three ionophores transporting alkali cations, were found to fit with a transport mechanism in which the diffusion of the ion-ionophore complex across the membrane is the single rate-limiting step, with the following exceptions: The addition of the dissociation of the ion-carrier complex as a second rate limiting step, for the Li(+)-ETH1810 and the Li(+)-ETH1811 systems. For ETH1810 the kinetics of the dissociation step is a minor, whereas for ETH1811 case it is a significant, addition (the ratio of the diffusion to dissociation rate constants being 0.08 and 0.2, respectively). The implications of the effects of the continuous structural change of these ionophores on their performance as ion carriers and on the design and synthesis of improved Li(+)-selective ionophores are discussed.

The effects of 17 beta-estradiol versus tamoxifen on the growth and metabolism of MCF7 human breast cancer cells, in culture and in tumors implanted in nude mice, were studied by 31P and 13C nuclear magnetic resonance spectroscopy and by proton magnetic resonance imaging. In culture, the content of the phosphate metabolites including nucleoside triphosphates (NTP), phosphomonoesters, phosphodiesters and inorganic phosphate (Pi) were not affected by tamoxifen treatment. However, in the presence of estrogen the rate of glucose consumption and lactate production via glycolysis (270 and 280 fmol/cell.h, respectively) were twice that of tamoxifen treated cells. Estrogen rescue of tamoxifen treated cells indicated that glycolysis induction occurs at the early stages of the hormonal response. The in vivo studies included recording of proton images that provided an accurate measure of tumor size and distribution of tumor cells, necrotic regions and stromal tissue. Tamoxifen caused enhanced necrosis extending from the center of the tumor during the first two days of treatment (12 h to 6 days). This was followed by growth of reparative tissue along with tumor regression. Tamoxifen also modified the content of the phosphate metabolites, increasing markedly (P less than 0.0002) the ratio of NTP to Pi from 0.41 before treatment to 1.75 9-19 days after treatment. This change was attributed to the enhanced growth of repair tissue. The results provide new information regarding the response of human breast cancer to hormonal treatment and suggest a mechanism for the induction of tumor regression by tamoxifen.

Magnetic resonance 1H-imaging and 31P-localized spectroscopy were utilized to monitor, noninvasively, MCF7 human breast cancer tumors implanted in immunodeficient mice. The tumors were followed during estrogen dependent growth and tamoxifen induced remission. Early after tamoxifen administration enhanced necrosis developed, extending to most of the tumor volume. This was followed by growth of repair tissue along with tumor regression. The short-term tamoxifen treatment also modified the content of the phosphate metabolites, increasing the nucleoside triphosphate to inorganic phosphate ratio from 0.41 +/- 0.15 (n = 14) before treatment to 1.10 +/- 0.70 (n = 8, P less than 0.025) and to 1.75 +/- 0.66 (n = 9, P less than 0.0002) 4-7 days and 9-19 days, respectively, after treatment. This change was attributed to the growth of reparative tissue. The results provide new information regarding the response and the mechanism of action of tamoxifen.

The neutral, noncyclic Li(+)-selective ionophore ETH1810, which is a di-imide, differs structurally from previous similar ionophores by removal of the intramolecular symmetry of the N-imide substituents. Properties of this ionophore, as a potential carrier of lithium, were probed through studies of ionophore-induced changes in electrical properties of lipid bilayer membranes. ETH1810 was found capable of transporting lithium and other monovalent cations, across lipid bilayer membranes, forming 2:1 ionophore:ion membrane-permeating species. It was found to be 10-fold more potent than ETH1644, which was the previous best ionophore of this type. The selectivity sequence among alkali cations was found to be: Li+ (1) greater than Na+ (0.009) greater than K+ (0.004) greater than Cs+ (0.0035). Among the physiological alkali cations, it constitutes a 40 (vs. Na+) to 160% (vs. K+) improvement over ETH1644. ETH1810 was also found to be capable of acting as a carrier of biogenic amines and related molecules, with the following selectivity sequence:tryptamine (20) greater than phenylethylamine (7.8) greater than tyramine (4.3) greater than serotonin (2.5) greater than Li+ (1) greater than NH+4 (0.013) greater than dopamine (0.012). It was found that protons, at physiological concentrations, do not interfere with the lithium transport mediated by ETH1810. The relationship between the improvements in ionic selectivity and potency vs. the differences in structural features is discussed.

The equilibrium binding of hydroxyethyl vinyl deuteroporphyrin (HVD) and of irreversible porphyrin aggregates to human serum albumin was studied at the molecular level. This protein may function as an endogenous drug carrier for porphyrins in photodynamic therapy of tumours. HVD-protein binding studies revealed two types of binding sites, which are attributed to the two HVD isomers. The binding constant for the high-affinity isomer, 2.1 (+/- 0.3) x 10(8) M-1, is similar to that previously determined for protoporphyrin. At the same time the binding constant for the lower-affinity HVD isomer, 1.8(+/- 0.3) x 10(6) M-1, is similar to that previously determined for haematoporphyrin. Irreversible porphyrin aggregates were purified from the haematoporphyrin derivative and from Photofrin and are defined by spectral and chromatographic data. Gel-exclusion studies indicate that the dominant size of these aggregates is ten porphyrin monomeric units. The protein-binding constant of these aggregates is 1.7(+/- 0.2) x 10(5) M-1, with four binding sites per protein molecule. The distinction between the HVD isomers along the porphyrin-protein affinity sequence gives insight into the relationship of porphyrin structure to porphyrin-albumin binding. On the basis of this study an evaluation of human serum albumin as an endogenous carrier for porphyrins (at various aggregation states) in photodynamic therapy of tumours is presented.

The use of X-ray photoelectron spectroscopy (XPS) for determination of the preferred orientation of proteins on metal surfaces is demonstrated. A myoglobin derivative in which a pentaamineruthenium (III) group is attached to a specific histidine (residue 81) is used for this purpose, with the Ru and Fe acting as a double marker. The relative intensities of the XPS photoelectron spectra for these two markers are shown to be a sensitive measure of orientation of the protein film. On both aluminum and indium-tin-oxide surfaces, XPS indicates that the Ru-Fe axis of the myoglobin derivative is preferentially oriented with the Ru furthest from the substrate.

The binding equilibrium of deuteroporphyrin IX to human serum albumin and to bovine serum albumin was studied, by monitoring protein-induced changes in the porphyrin fluorescence and taking into consideration the self-aggregation of the porphyrin. To have control over the latter, the range of porphyrin concentrations was chosen to maker dimers (non-covalent) the dominant aggregate. Each protein was found to have one high-affinity site for deuteroporphyrin IX monomers, the magnitudes of the equilibrium binding constants (25 degrees C, neutral pH, phosphate-buffered saline) being 4.5 (+/- 1.5) X 10(7) M-1 and 1.7 (+/- 0.2) X 10(6) M-1 for human serum albumin and for bovine serum albumin respectively. Deuteroporphyrin IX dimers were found to bind directly to the protein, each protein binding one dimer, with high affinity. Two models are proposed for the protein-binding of porphyrin monomers and dimers in a porphyrin system having both species: a competitive model, where each protein molecule has only one binding site, which can be occupied by either a monomer or a dimer; a non-competitive model, where each protein molecule has two binding sites, one for monomers and one for dimers. On testing the fit of the data to the models, an argument can be made to favour the non-competitive model, the equilibrium binding constants of the dimers, for the non-competitive model (25 degrees C, neutral pH, phosphate-buffered saline), being: 8.0 (+/- 1.8) X 10(8) M-1 and 1.2 (+/- 0.6) X 10(7) M-1 for human serum albumin and bovine serum albumin respectively.

Canavalin is a tetramer with 6 tyrosines per subunit. In the work presented here, we have classified these tyrosines by their spectrophotometric and fluorometric pH titration and their ability to be quenched I-. Of the 6 residues, 2 were found to be exposed to the solvent. One (pK = 10.2) contributes 28% of the total fluorescence intensity; the second has a pK of 11.50, and a lower quantum yield, contributing only 16% of the total intensity. The remaining 4 residues (pK = 12.5, contributing 54% of fluorescence intensity) are buried; their titration is irreversible, requiring protein denaturation.

The neutral noncyclic, lithium-selective ionophore ETH1644, which is structurally different from previously available ionophores of this type, is a selective carrier of Li+ in lipid bilayer membranes of various lipid composition. The ionophore forms a 2:1 carrier/cation complex, and the rate-limiting step in the overall transport process is the diffusion of the carrier/ion complex across the membrane. The selectivity sequence for lithium vs. other ions normally found in biological systems is: Li+ (1) greater than Na+ (0.017) greater than or equal to K+ (0.017) greater than Cl- (0.001), Ca2+ and Mg2+ are impermeant. At neutral pH protons do not interfere with the Li+-carrying ability of this ionophore. On the basis of structural differences and supported by conductance data, it is argued that the improved selectivity of Li+ over the other alkali cations is due more to a decrease in the affinities of the ionophore for the latter cations that to an increase of its affinity to Li+. This ionophore can also act as a carrier of biogenic amines (catecholes, indoles and derivatives), with the structure of the permeant species and mechanism of permeation similar to that observed with the alkali cations. The selectivity sequence is: tryptamine (18.1) greater than phenylethylamine (11.6) greater than tyramine (2.4) greater than Li+ (1) greater than serotonin (0.34) greater than epinephrine (0.09) greater than dopamine (0.05) greater than norepinephrine (0.02), showing the ionophore to be more selective to Li+ than to any of the neurotransmitters studied.

The binding of hematoporphyrin derivative to membrane was studied at the molecular level, employing fluorimetric techniques and using liposomes (large unilamellar) to model biological membranes. Two specific issues were probed: (a) the effect of increasing the porphyrin-liposome incubation period (37 degrees C, neutral pH, in the dark) from 2 h up to 24 h, with liposomes composed of PC or PC/cholesterol 7:3 (molar ratio); (b) the effects of membrane lipid composition, in terms of mol% cholesterol in PC/cholesterol liposomes, on the porphyrin-membrane binding equilibrium, for a long incubation period (16 h). The data were processed and found to be in good agreement with the following proposed model: With time (t greater than 2 h), the porphyrin fractions into three components, two of them binding to membrane with high and low affinities, respectively, the time effect reaching a plateau at 16 h. Linkage was observed between the slow process and changes in the available pools of each fraction. At sufficiently long incubations, the magnitude of each of the two binding fractions was found to be (roughly) 30%, independent of the membrane lipid composition. On the other hand, the magnitude of the binding constants was found to depend on the lipid composition, that of the high-affinity fraction decreasing from 9000 M-1 for 0% cholesterol, to 3000 M-1 for 40% cholesterol, then increasing back to 5000 M-1 upon further increase in cholesterol (to 50% mol) and that of the low-affinity fraction going from 1000 M-1, to 100 M-1, to 300 M-1 for similar lipid compositions. The origin of the time effect, in terms of porphyrin-specific processes, and the biological relevance of the present findings are discussed.

A simple screening method for determining the fibrinolytic activity of plasma is described. A streptokinase activated system is used, which measures the result of the interplay of all components of the fibrinolytic system with the exception of activators, which are added in excess. Mean lysis time and standard deviation with this method is 6.9 +/- 1.4 min. The sensitivity of the method in detecting minor delays in clot lysis time is demonstrated.

The reaction between a5RuH2O2+ (a is NH3) and Pseudomonas aeruginosa azurin at pH 7, followed by oxidation, yields a5Ru(His-83)3+-azurin(Cu2+) as the major product. Spectroscopic measurements (UV-visible, CD, EPR, and resonance Raman) indicate that the native structure is maintained in the modified protein. The site of ruthenium binding (His-83) was identified by peptide mapping. The a5RuHis/Cu ratio in the modified protein, determined from the EPR spectrum, is 1:1, and the reduction potentials (vs. normal hydrogen electrode, pH 7.0, 25 degrees C) are blue copper (Cu2+/1+), 320 +/- 2 mV; a5Ru(His-83)3+/2+, 50 +/- 10 mV. From measurements of the reduction potentials at several temperatures in the 5-40 degrees C range, delta H degree for intramolecular Ru2+ ----Cu2+ electron transfer was estimated to be -12.4 kcal mol-1 (1 cal = 4.184 J). Analysis of kinetic data in light of the electron transfer exothermicity indicates that the reorganizational enthalpy of the blue copper site can be no larger than 7.1 kcal mol-1.

The thermodynamics of zinc hematoporphyrin (ZnHP) dimerization and ZnHP-membrane binding were studied. The dimerization equilibrium was determined over the temperature range 19-40 degrees C, using fluorometric techniques. The dimerization constant obtained at 37 degrees C (neutral pH in phosphate-buffered saline) is 4.6 (+/- 0.6) X 10(4) M-1. The dimerization was found to decrease with temperature over the range 19-36 degrees C, the data allowing the extraction of the following thermodynamic parameters for the temperature range 19-31 degrees C: delta G0 = -9.3 kcal/mol, delta H0 = -7.4 kcal/mol, delta S0 = -6.4 eu. For temperatures above 36 degrees C the dimerization was found to be temperature independent, giving the following parameters: delta G0 = -6.6 kcal/mol, delta H0 = 0 kcal/mol, delta S0 = 21.2 eu. On the basis of the data the case is made for the existence of two types of ZnHP dimers, differing in the location of the fifth Zn2+ ligand and in the nature of the contribution of the solvent to the dimerization. For the membrane binding, large unilamellar liposomes served to model biological membranes. The binding of ZnHP to the liposomes was found to be similar, quantitatively, to the corresponding metal-free molecule, namely, fitting a case of one type of site and giving a binding constant of 1600 +/- 160 M (neutral pH and 37 degrees C) which is independent of the length of the porphyrin-liposome.

Factor XI is a circulating trace plasma protein composed of two similar or identical chains of about 80 000 daltons which upon activation undergo proteolytic cleavage. Recently, we have shown that trypsin activation leads to an active factor XI (factor XIa) which, on reduction, yields three chains of 46 000, 37 000 and 26 000 daltons. Herein, we re-evaluate the effect of contact activation of factor XI at an activating surface both in normal human plasma and in a mixture of purified factors XI, XII, and high molecular weight kininogen (HMWK). Mixtures were analysed by coagulant activity and by reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis using [125I]factor XI. In the purified system, fully activated factor XI on reduction yielded chains of 46 000, 37 000 and 23 000 daltons. In contrast, factor XI activated by surface contact in plasma yielded on reduction only chains of 46 000 and 37 000 daltons in addition to some uncleaved 80 000 chain. We propose that factor XIa containing only 46 000 and 37 000 chains be designated factor XIa alpha, and that factor XIa containing the third chain of 23 000 daltons be designated factor XI a beta. Sequential elution of contact activated plasma factor XI revealed that factor XIa was attached to the glass surface through the 46 000 dalton chain.

Porphyrins are photodynamic drugs employed in an experimental tumor-treatment modality in which cell membranes are one of the primary drug-action sites. To gain insight into the nature of the interaction of these drugs with those primary sites we have studied the affinity of porphyrins to the lipid moieties of biological membranes, at the molecular level. The association of porphyrins to large unilamellar liposomes, modeling the lipid regions of biological membranes was studied (at equilibrium) for deuteroporphyrin IX and protoporphyrin IX, at neutral pH and 37 degrees C, taking into account porphyrin aggregation. Two thermodynamic approaches were investigated: (i) Simple partition equilibria between the external aqueous phase and the lipid bilayer, for drug monomers and dimers. (ii) Binding equilibria of drug monomers and dimers to the lipid bilayer. Using two types of experimental design and processing the data according to the expectations of both approaches, three different models for the binding (differing in the participation assigned to the dimer) were considered. Our major findings are: (a) The data clearly do not fit with the expectations for simple partition equilibria, nor with binding models assuming direct participation of the dimers. (b) The data fit well with a binding process, in which the membrane binds the porphyrin monomers only, with the dimers participating indirectly through the aqueous dimerization equilibrium. (c) At 37 degrees C and neutral pH, for liposomes composed of phosphatidylcholine/cholesterol at molar ratios of 3:2, we found for both investigated species a binding constant of 2.3 x 10(4) M-1. (d) For each species the binding constant is independent of the initial and final states of drug aggregation in the aqueous phase.

Recently we have described a normal plasma activity that modulates contact activation by inhibiting adsorption of factor XI to activating surfaces. Here we report the first identified case in which a patient has abnormal clotting tests due to an excess of a similar activity. The patient's plasma had a prolonged partial thromboplastin time and low apparent factor XI assay. His plasma prolonged the partial thromboplastin time of normal plasma and partially neutralized normal factor XI activity in vivo and in vitro. Analysis in dilute plasma revealed normal amounts of factor XI activity and antigen. Factor XI adsorption from plasma to activating surfaces was tested by adding a small amount of 125I-labeled purified factor XI to plasma, exposing the mixture to a glass tube or kaolin, and determining the amount of factor XI adsorbed to the surface. Whereas normal plasma and plasmas deficient in factor XII, factor XI, or Fletcher factor yielded about 4% adsorption to glass, factor XI adsorption from patient's plasma was less than 1%, indicating the presence of an adsorption inhibitor. This inhibitor did not affect factor XI activation or the activity of preformed factor XIa. It was not adsorbed by AI(OH)3 and was present in serum and the macroglobulin peak on gel filtration of the plasma through Sephadex G-200. The patient's history does not allow a definitive conclusion as to whether this inhibitor was associated with abnormal bleeding.

The aggregations of protoporphyrin IX and haematoporphyrin IX in aqueous solutions were studied by fluorimetric techniques. Porphyrin concentrations were limited to 0.001-0.1 microM and 0.01-1 microM for protoporphyrin and haematoporphyrin respectively, where dimerization is the dominant aggregation process. The dimerization equilibrium constants (at 25 degrees C, neutral pH, 50 mM-Tris/HCl buffer) were determined to be 3 X 10(7) M and 4 X 10(5) M for the proto and the haemato derivatives respectively. The fluorescence intensity of a given protoporphyrin solution (within the range indicated above) was markedly decreased by salts in the system, over the salt concentration range 0.1-7 mM at constant ionic strength, in the sequence CaCl2 greater than MgCl2 greater than KCl greater than NaCl. The direction of this effect, fluorescence quenching, suggests that these salts promote an increase in aggregation. The differences in the magnitudes of the effect, among different salt species sharing a common anion, at constant ionic strength, imply that the effect is cation-specific. In contrast, the fluorescence intensity of a given solution of haematoporphyrin (within the range indicated above) was unaffected by these salts, under similar concentrations, nor was it sensitive to the total buffer concentration, or to the type of buffer in the system.

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 X 10(6)M-1 and 5.4 X 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van't Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = -36. 4kJ X mol-1; delta H0 = -46. 0kJ X mol-1 and delta S0 = -32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the ' solvophobic force' theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.

The term "effective activator" of plasminogen is proposed, to denote the resultant of activator-antiactivator interaction, and a method for the determination of the level of these activators is described. By adding axcess plasminogen to the euglobulin fraction of plasma the influence of the level of endogenous plasminogen and of the antiplasmin is eliminated. It is shown that the level of fibrinogen has very little bearing on the results. An effective activator unit is defined as equal to 1 CTA unit of urokinase activity on a fibrinogen-plasminogen substrate.

Two processes of porphyrins in heterogeneous systems containing aqueous and membrane phases have been studied with hematoporphyrin and hematoporphyrin derivative: Dimerization equilibrium in the aqueous phases and porphyrin-membrane binding equilibrium using liposomes as models for biological membranes. The interrelationship of aqueous aggregations and membrane binding was probed and the porphyrin aggregation state in the membrane, at equilibrium, was assessed. Fluorimetric techniques were employed. The dimerization equilibrium constants, at neutral pH and 37 degrees C were found to be 2.8 X 10(5) M-1 and 1.9 X 10(6) M-1 for hematoporphyrin and its derivative, respectively. Over a porphyrin concentration range going from monomer-dominant to dimer-dominant systems, we have found that only monomers are bound to the membrane. The respective monomer-liposome binding constants, found to be independent of the initial monomer/dimer distribution in the aqueous phase, were determined to be 1.6 X 10(3) M-1 and 4.1 X 10(3) M-1 at neutral pH and 37 degrees C for hematoporphyrin and its derivative, respectively. The monomer-liposome interaction was found to perurb the initial monomer/dimer distribution in the aqueous phase, so that the monomers residing at equilibrium in the membrane originate from both monomers and dimers in the aqueous phase.

The appearance of new additional bands, at 580 and 630 nm, in the fluorescence spectra of porphyrins, upon the aging of their aqueous solutions, was investigated for four porphyrin systems: The deutero, the hemato, the hemato-derivative, and the proto. The conditions for quantitative conversion to the new spectra were sought. To that end the concentration levels investigated were adjusted to have each porphyrin system in a monomer-dominant state. The species fluorescing at 580 nm was denoted f580 and its rate of appearance was found to fit first-order kinetics, for each porphyrin, the range of the rate constants being 0.03-0.10 hr-1. Two key issues were probed: (a) The identity of f580, the effort directed towards zincporphyrin. (b) Its origins--a contaminant unmasked, or a species newly formed, during the aging. Testing f580 for zincporphyrin properties, spectral at neutral pH, acid/base de/remetalation, EDTA competition, and chromatography gave in each case positive results, allowing the identification of f580 as zincporphyrin. On the basis of the data obtained and the known sources for the contamination of aqueous solutions by zinc, the case is made for zinc porphyrin formation upon aging. The relevance of the phenomena to the therapeutic activities of porphyrins is discussed.

The neutral, noncyclic, imide and ether containing ionophore AS701, has been developed as Li+-selective molecule, to be used potentially as an aid in the Li+-therapy of manic-depressive illness. The present report is a characterization of this molecule in neutral lipid bilayer membranes. This ionophore was found to render the bilayers Li+-selective, acting as a selective carrier of monovalent cations. In addition, this molecule was found to be capable of acting as a selective carrier of monovalent anions. For both types of ions, the rate-limiting step in the process of permeation was found to be the diffusion of the carrier-ion complex through the membrane. The membrane-permeating species were found to be 2 : 1 carrier-ion complexes, carrying either a monovalent cation or a monovalent anion. The selectivity sequence among the ions studied being: Li+(1) greater than ClO4-(0.7) greater than Na+(0.07) greater than K+(0.016) greater than Rb+(0.0095) greater than Cs+(0.0083) greater than Cl-(0.001). Mg2+ and SO42- were found to be impermeant (under present experimental conditions). This sequence shows that the AS701 molecule has low selectivity for ions present in biological media, among those studied (i.e. Na+, K+, Mg2+, Cl- and SO42-). This indicates that these ions will not interfere in the Li+ permeability induced by this carrier in vivo, and that the carrier will not interfere in the normal transport processes of these ions.

A series of structurally-related Li+-selective ionophores were studied in planar lipid bilayer membranes, to assess their potential ability to act as Li+-selective carriers in vivo. The ionophores are acyclic, neutral molecules of similar structure: N,N'-diheptyl-N,N'-diR-5,5-dimethyl-3,7-dioxanonane diamide. The structural differences among them are the N-amide substituents (the R residues) as follows: an aliphatic ether (AS701), tetrahydrofuran (AS706), furan (AS708), an ester (AS702) and an amide (AS704). For each ionophore, the steady-state, single salt, membrane conductances and conductance-voltage behaviors were determined in the presence of LiCl, NaCl and MgCl2. Membrane zero-current potentials were measured for NaCl/LiCl and MgCl2/LiCl mixtures. All five ionophores were found to operate as "equilibrium-domain" carriers of monovalent ions. All select lithium over sodium, but with different magnitudes of selectivity, ranging from PLi/PNa of 13 (for AS701) to PLi/PNa of 2 (for AS708). The ionophores also differ in their ability to mediate Li+ membrane permeation, the order of decreasing potency being: AS701 greater than or equal to AS706 greater than AS702 greater than AS704 greater than or equal to AS708. Of the five molecules studied, the AS701 molecule was found to have the best Li+ over Na+ selectivity and highest potency. These findings indicate that this molecule has the best potential for mediating lithium-selective membrane permeation in vivo, among the group studied.

The potential of albumin (bovine serum) microspheres as delivery systems for haematoporphyrin and/or its zinc derivative was probed at the molecular level. The microspheres, prepared by heat denaturation, were stable to detergents and organic solvents but could be degraded by proteolytic enzymes. Drug loading was either by encapsulation during microsphere formation or by binding to preformed microspheres. The efficiency of encapsulation was found to depend on the initial drug/protein ratio with a saturable pattern. Binding of both drugs, or each alone, indicated the presence of two types of affinities of each drug to the microspheres. The leakage (release) of drug(s) from the microspheres into the aqueous medium over a period of several hours showed biphasic behaviour for each type of preparation, bound and encapsulated, for one or both drugs. A model assuming the microsphere-bound drug is distributed into two pools, differing in their drug-microsphere affinities, with no interference between the two drugs if both were bound, was found to account well for the equilibria data. Binding constants for respective pools were found to be similar for both drugs, of the order of 0.10 and 2 x 10(-4) ml/micrograms protein for the high and low affinity pools, respectively. The low affinity pools had the higher drug population. The two-pool model also fits well with the kinetic data, the pools differing in the rates of release. Rate constants for both drugs were found to be similar, of the order of 0.3 per min and 0.025 per hour for the fast and slow releasing pools, respectively. The population of the slow-releasing pool was higher, the high-affinity pool being the fast-releasing one, the low-affinity pool being the slow-releasing one. Implications for the microsphere-mediated delivery of these drugs in vivo are discussed.

A systematic investigation of the fibrinolytic system during the period after acute myocardial infarction in 42 patients showed a progressive reduction of fibrinolytic activity, with the longest lysis times on days 4 to 5 after the event. There is a concomitant rise in the levels of fibrinogen, plasminogen, and antiplasmin; only the latter parallels the delay in lysis. After 12 to 15 months, normal lysis times and fibrinogen levels were found in 24 patients re-examined. It is concluded that the reduced fibrinolytic activity in these patients was a transient phenomenon after myocardial infarction.

The reaction of [(H2O)(NH3)5RuII]2+ with bleomycin forms at least two stable products following oxidation to the Ru(III) analog. Spectroscopic and electrochemical measurements indicate monodentate binding of [(NH3)5RuIII] to the imidazole and pyrimidine moieties, with coordination to the latter involving the exocyclic amine nitrogen. DNA cleavage studies show the complexes to be ineffective in DNA strand scission. In vitro biological studies reveal these adducts to be cytotoxic.