Micellar effects and analytical applications of nitro substitution in 4-Nitro-N-alkyl-1,8-naphthalimide by cysteine derivatives.

The aromatic nucleophilic substitution reactions of the nitro group of 4-Nitro-N-alkyl-1,8-naphthalimides by thiolate anions produce fluorescent derivatives and their rates are strongly accelerated by micelles of hexadecyltrimethylammonium chloride even at low pH. Acceleration factors of this reactions can reach million-fold. As the products are oxidant-insensible, this reaction allows the determination of SH- containing compounds such as cysteine, glutathione or proteins even in oxidative conditions. Limits of detection are as low as 5 × 10-7 M, ten times lower than the limit for the classic 5,5'-dithiobis-(2-nitrobenzoic) acid method. Moreover, this reaction can be developed at pHs between 6.5 and 7.5 thereby diminishing the rate of spontaneous oxidation of the thiols. In addition, we demonstrated that 4-Nitro-N-alkyl-1,8-naphthalimides can be used to evidence SH groups in peptides, proteins and living cells.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology.

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Where do we aspire to publish? A position paper on scientific communication in biochemistry and molecular biology

The scientific publication landscape is changing quickly, with an enormous increase in options and models. Articles can be published in a complex variety of journals that differ in their presentation format (online-only or in-print), editorial organizations that maintain them (commercial and/or society-based), editorial handling (academic or professional editors), editorial board composition (academic or professional), payment options to cover editorial costs (open access or pay-to-read), indexation, visibility, branding, and other aspects. Additionally, online submissions of non-revised versions of manuscripts prior to seeking publication in a peer-reviewed journal (a practice known as pre-printing) are a growing trend in biological sciences. In this changing landscape, researchers in biochemistry and molecular biology must re-think their priorities in terms of scientific output dissemination. The evaluation processes and institutional funding for scientific publications should also be revised accordingly. This article presents the results of discussions within the Department of Biochemistry, University of São Paulo, on this subject.

Surface activity of the triflate ion at the air/water interface and properties of N,N,N-trimethyl-N-dodecylammonium triflate aqueous solutions.

The surface activity of salts added to water is orders of magnitude lower than that of surfactants. Sodium trifluoromethanesulfonate (NaTf) produced a change in surface tension with concentration, Δγ/Δc, of -13.2 mN·L/m·mol. This value is ca. 4-fold larger than those of simple salts and that of methanesulfonate. This unexpected surface effect suggested that positively charged micelles containing Tf could exhibit interesting properties. Dodecyltrimethylammonium triflate (DTATf) had a higher Kraft temperature (37 °C) and a lower cmc (5 × 10(-3) M) and degree of dissociation (0.11) than the chloride and bromide salts of DTA. Above the Kraft temperature, at a characteristic temperature t(1), the addition of NaTf above 0.05 M to a DTATf solution induced phase separation. By increasing the temperature of the two-phase system to above t(1), a homogeneous, transparent solution was obtained at a characteristic temperature t(2). These results, together with well-known triflate properties, led us to suggest that the Tf ion pairs with DTA and that the -CF(3) group may be dehydrated in the interfacial region, resulting in new and interesting self-aggregated structures.

Mechanism of 1,4,5,8-naphthalene tetracarboxylic acid dianhydride hydrolysis and formation in aqueous solution.

The study of highly conjugated, carbonyl-containing molecules such as 1,4,5,8-naphthalene tetracarboxylic dianhydride, III, is of interest since reactivity differences and transmission of electronic effects through the conjugated framework can be evidenced. The kinetics of hydrolysis of III in aqueous solution were determined from 5 M acid to pH 10. In basic solution hydrolysis of III yields, sequentially, 1,4,5,8-naphthalene diacid monoanhydride, II, and 1,4,5,8-naphthalene tetracarboxylic acid, I. The second order rate constant for alkaline hydrolysis is 200 fold higher for the first ring opening. The water-catalyzed hydrolysis of III yields a pH-dependent mixture of ionic forms of I and II. The rate constant for water-catalyzed hydrolysis of III is 25 fold higher than that for II. In concentrated acid the rates for reaching equilibrium (I, II and III) increase and III is the major product. The pK(a)s of I (3.24, 5.13 and 6.25) and II (3.05, 5.90) were determined by potentiometric, fluorescence and UV spectroscopy titrations and by quantitative fit of the kinetic and equilibrium data. The apparent, pH-dependent, equilibrium constants, K(EqII), for anhydride formation between I and II were obtained from the UV spectra. The quantitative fit of kinetic and equilibrium data are consistent with the assumption that anhydride formation only proceeds with the fully protonated species for both I and II and permitted the estimation of the equilibrium constants for anhydride formation, K(EqII). The value of K(EqII) (I <==> II) between pH 1 and 6 was ca. 5. Geometry optimization calculations in the gas phase of the reactions of III in alkaline, neutral and acid conditions, at the DFT level of theory, gave electronic distributions that were qualitatively consistent with the experimental results.

pH at the micellar interface: synthesis of pH probes derived from salicylic acid, acid-base dissociation in sodium dodecyl sulfate micelles, and Poisson-Boltzmann simulation.

The study of the H+ concentration at the micellar interface is a convenient system for modeling the distribution of H+ at interfaces. We have synthesized salicylic acid derivatives to analyze the proton dissociation of both the carboxylic and phenol groups of the probes, determining spectrophotometrically the apparent pK(a)'s (pK(ap)) in sodium dodecyl sulfate, SDS, micelles with and without added salt. The synthesized probes were 2-hydroxy-5-(2-trimethylammoniumacetyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumacetyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumhexanoyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumundecanoyl)benzoate; 2-hydroxy-5-acetylbenzoic acid; and 2-hydroxy-5-dodecanoylbenzoic acid. Upon incorporation into SDS micelles the pK(ap)'s of both carboxylic and phenol groups increased by ca. 3 pH units and NaCl addition caused a decrease in the probe-incorporated pK(ap). The experimental results were fitted with a cell model Poisson-Boltzmann (P-B) equation taking in consideration the effect of salt on the aggregation number of SDS and using the distance of the dissociating group as a parameter. The conformations of the probes were analyzed theoretically using two dielectric constants, e.g., 2 and 78. Both the P-B analysis and conformation calculations can be interpreted by assuming that the acid groups dissociate very close to, or at, the interface. Our results are consistent with the assumption that the intrinsic pK(a)'s of both carboxylic and phenol groups of the salicylic acid probes used here can be taken as those in water. Using this assumption the micellar and salt effects on the pK(ap)'s of the (trialkylammonium)benzoate probes were described accurately using a cell model P-B analysis.

beta 2-glycoprotein I (apolipoprotein H) modulates uptake and endocytosis associated chemiluminescence in rat Kupffer cells.

beta 2-Glycoprotein I (beta 2 GPI) is known to influence macrophage uptake of particles with phosphatidylserine containing surfaces, as apoptotic thymocytes and unilamellar vesicles in vitro. Nevertheless, effects upon macrophage activation induced by this interaction are still unknown. beta 2 GPI influence upon the reactive species production by Kupffer cells was evaluated in order to investigate whether beta 2 GPI modulates the macrophage response to negatively charged surfaces. Chemiluminescence of isolated non-parenchymal rat liver cells was measured after phagocytosis of opsonized zymosan or phorbolymristate acetate (PMA) stimulation, in the presence and absence of large unilamellar vesicles (LUVs) containing 25 mol% phosphatidylserine (PS) or 50 mol% cardiolipin (CL) and complementary molar ratio of phosphatidylcholine (PC). beta 2 GPI decreased by 50% the chemiluminescence response induced by opsonized zymosan, with a 66% reduction of the initial light emission rate. PMA stimulated Kupffer cell chemiluminescence was insensitive to human or rat beta 2 GPI. Albumin (500 micrograms/ml) showed no effect upon chemiluminescence. beta 2 GPI increased PS/PC LUV uptake and degradation by Kupffer cells in a concentration-dependent manner, without leakage of the internal contents of the LUVs, as shown by fluorescence intensity enhancement. LUVs opsonized with antiphospholipid antibodies (aPL) from syphilitic patients increased light emission by Kupffer cells. Addition of beta 2 GPI to the assay reduced chemiluminescence due to opsonization with purified IgG antibodies from systemic lupus erythematosus (SLE or syphilis (Sy) patient sera. A marked net increase in chemiluminescence is observed in the presence of Sy aPL antibodies, whereas a decrease was found when SLE aPL were added to the assay, in the presence or absence of beta 2 GPI. At a concentration of 125 micrograms/ml, beta 2 GPI significantly reduced Kupffer cell Candida albicans phagocytosis index and killing score by 50 and 10%, respectively. The present data strongly suggest that particle uptake in the presence of beta 2 GPI is coupled to an inhibition of reactive species production by liver macrophages during the respiratory burst, supporting the role of beta 2 GPI as a mediator of senescent cell removal.

Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli.

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent. Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.

Effect of liposomes on the rate of alkaline hydrolysis of indomethacin and acemetacin.

The anti-inflammatory, analgesic, and antipyretic drugs indomethacin (INDO) and acemetacin (ACE), extensively used for the treatment of diseases of degenerative or inflammatory character, exhibit marked gastric irritant action, have low water solubility at neutral pH, and decompose in alkali. Alternative formulations are being investigated to obtain products with lower toxicity and higher stability. Here we examine the effect of liposome charge on the rate of alkaline decomposition of INDO and ACE using micelles as reference. Binding of ACE and INDO to zwitterionic hexadecylphosphocholine (HDPC) micelles and phosphatidylcholine (PC) liposomes was analyzed using a two-phase separation model to quantify the effect of these aggregates on the rate of alkaline degradation. The substrate association constants to HDPC micelles were 1335 and 2192 M(-1) for INDO and ACE, respectively, whereas the corresponding values for PC vesicles were 612 and 3050 M(-1). The difference was attributed to the additional hydrophobicity of ACE. The inhibitory effect of HDPC micelles and PC vesicles was quantified by calculating the ratio between the rate constants in water (k(w)) and in the aggregate (k(m)). The values of the k(w)/k(m) ratios for INDO and ACE in HDPC micelles were, respectively, 80 and 42, and in PC liposomes these ratios were 21 and 3.7, respectively. Positively charged micelles of hexadecyltrimethylammonium chloride (CTAC) and vesicles containing varying proportions of dioctadecyldimethylammonium chloride (DODAC) and PC increase the rate of INDO and ACE alkaline decomposition. Vesicle effects were very sensitive to the DODAC/PC ratio, with rates increasing with the proportion of DODAC. The data were analyzed quantitatively using a pseudophase model with explicit consideration of ion exchange. The calculated second-order rate constants in micelles and vesicles were lower than that in water. The charge density in the liposome necessary to increase the entrapment efficiency and decrease drug decomposition can be modulated, by judicious choice of pH and ionic strength. These manipulations can lead to more stable formulation with increased efficiency in drug entrapment and controlled effects on drug stability.

Mapping cancer, cardiovascular and malaria research in Brazil.

This paper presents performance indicators for the Brazilian cancer, cardiovascular and malaria research areas from 1981 to 1995. The data show an increasing number of papers since 1981 and author numbers indicate a continuous growth of the scientific community and suggest an expected impact of scientific activity on biomedical education. The data also characterize cardiovascular research as a well-established area and cancer research as a faster growing consolidating field. The 1989-1994 share of Brazilian articles among world publications shows a growing trend for the cancer (1.61) and cardiovascular (1.59) areas, and a decrease for the malaria area (0. 89). The burden of the three diseases on society is contrasted by the small number of consolidated Brazilian research groups, and a questionable balance of thematic activity, especially with regard to malaria. Brazilian periodicals play an important role in increasing the international visibility of science produced in the country. Cancer and cardiovascular research is strongly concentrated in the Southeastern and in Southern regions of Brazil, especially in São Paulo (at least one address from São Paulo in 64.5% of the 962 cancer articles and in 66.9% of the 2250 cardiovascular articles, the second state being Rio de Janeiro with at least one address in 14.1 and 11% of those articles, respectively). Malaria research (468 articles) is more evenly distributed across the country, following the pattern of the endemic distribution of the disease. Surveying these national indicator trends can be useful to establish policies in the decision process about health sciences, medical education and public health.

Mapping cancer, cardiovascular and malaria research in Brazil

This paper presents performance indicators for the Brazilian cancer, cardiovascular and malaria research areas from 1981 to 1995. The data show an increasing number of papers since 1981 and author numbers indicate a continuous growth of the scientific community and suggest an expected impact of scientific activity on biomedical education. The data also characterize cardiovascular research as a well-established area and cancer research as a faster growing consolidating field. The 1989-1994 share of Brazilian articles among world publications shows a growing trend for the cancer (1.61) and cardiovascular (1.59) areas, and a decrease for the malaria area (0.89). The burden of the three diseases on society is contrasted by the small number of consolidated Brazilian research groups, and a questionable balance of thematic activity, especially with regard to malaria. Brazilian periodicals play an important role in increasing the international visibility of science produced in the country. Cancer and cardiovascular research is strongly concentrated in the Southeastern and in Southern regions of Brazil, especially in São Paulo (at least one address from São Paulo in 64.5 percent of the 962 cancer articles and in 66.9 percent of the 2250 cardiovascular articles, the second state being Rio de Janeiro with at least one address in 14.1 and 11 percent of those articles, respectively). Malaria research (468 articles) is more evenly distributed across the country, following the pattern of the endemic distribution of the disease. Surveying these national indicator trends can be useful to establish policies in the decision process about health sciences, medical education and public health

Fusion of vesicles with the air-water interface: the influence of polar head group, salt concentration, and vesicle size.

Fusion of vesicles with the air-water interface and consequent monolayer formation has been studied as a function of temperature. Unilamellar vesicles of DMPC, DPPC, and DODAX (X=Cl(-), Br(-)) were injected into a subphase containing NaCl, and the surface pressure (tension) was recorded on a Langmuir Balance (Tensiometer) using the Wilhelmy plate (Ring) method. For the zwitterionic vesicles, plots of the initial surface pressure increase rate (surface tension decrease rate) as a function of temperature show a peak at the phase transition temperature (T(m)) of the vesicles, whereas for ionic ones they show a sharp rise. At high concentrations of NaCl, ionic DODA(Cl) vesicles seem to behave like zwitterionic ones, and the rate of fusion is higher at the T(m). The influence of size was studied comparing large DODA(Cl) vesicles with small sonicated ones, and no significant changes were found regarding the rate of fusion with the air-water interface.

Determination of Halide Concentrations at the Interface of Zwitterionic Micelles by Chemical Trapping: Influence of the Orientation of the Dipole and the Nature of the Cation.

The interfacial concentrations of Cl(-) and Br(-) in aqueous zwitterionic micelles were determined by chemical trapping by analyzing product yields from spontaneous dediazoniation of micelle-bound 2,6-dimethyl-4-hexadecylbenzenediazonium ion. Interfacial concentrations of Cl(-) and Br(-) in 3-(N-hexadecyl-N, N-dimethylammonio) propane sulfonate, HPS, micelles were higher than in bulk solutions prepared with Li(+), Na(+), Rb(+), Cs(+), tetramethylammonium (TMA(+)), Mg(+2), and Ca(+2) salts. In contrast, the interfacial concentrations of Cl(-) and Br(-) were generally lower than in bulk solution in hexadecylphosphoryl choline, HDPC, micelles for all salts except Mg(+2) and Ca(+2). In both HPS and HDPC micelles the interfacial concentration of Br(-) was higher than that of Cl(-), showing that binding is anion selective. The cation had a large effect on the interfacial concentration of halide ions with HDPC micelles decreasing in the order Ca(2+) > Mg(2+) >> Li(+) > Na(+) > K(+) > Cs(+) > Rb(+) >> TMA(+). These results are the first direct and extensive determination of local halide ion concentration at the surface of zwitterionic micelles, and they demonstrate that chemical trapping methodology will work in membranes at physiologically relevant salt concentrations. Copyright 1999 Academic Press.

Induction of antiphospholipid antibodies by immunization with synthetic viral and bacterial peptides.

We previously induced pathogenic antibodies against anionic phospholipids (PL) in experimental animals by immunization with lipid-free purified human beta2glycoprotein I (beta2GPI). We hypothesized that antiphospholipid antibodies (aPL) are induced by in vivo binding of foreign beta2GPI to self-PL, thus forming an immunogenic complex against which aPL antibodies are produced. If this hypothesis is true, other PL-binding proteins that are products of ubiquitous viral/bacterial agents may also induce aPL. To test this hypothesis, groups of NIH/Swiss mice were immunized with synthetic peptides of viral and bacterial origin that share structural similarity with the putative PL-binding region of beta2GPI. Compared with the control groups, animals immunized with the peptides produced significantly higher levels of aPL and anti-beta2GPI antibodies. These findings demonstrate that some PL-binding viral and bacterial proteins function like beta2GPI in inducing aPL and anti-beta2GPI production, and are consistent with a role for such viral and bacterial proteins in inducing aPL antibody production in humans.

Opposite beta2-glycoprotein I requirement for the binding of infectious and autoimmune antiphospholipid antibodies to cardiolipin liposomes is associated with antibody avidity.

The aim of this study was to investigate the interaction of antiphospholipid antibodies (aPL) from two different populations (patients with autoimmune or infectious disorders) with cardiolipin (CL) arranged in a defined bilayer. beta2-Glycoprotein I (beta2GPI), an apolipoprotein that plays a critical role in the aPL binding to phospholipids, was quantified by dot blot in purified IgG-aPL samples, further classified according to apparent avidity to CL. In solid-phase assays, beta2GPI increased, preferentially, the binding of low-avidity autoimmune aPL to CL but inhibited the binding of low-avidity syphilitic aPL. In the absence of beta2GPI, both autoimmune and infectious aPL induced the leakage of the entrapped fluorescent probe, carboxyfluorescein (CF), from small unilamellar vesicles containing CL. aPL-induced probe leakage was protein concentration-dependent and characterized by a lag-phase onset of 100-120 min. beta2GPI increased the leakage rate induced by low-avidity autoimmune aPL only and inhibited the leakage induced by all syphilitic aPL. The following conclusions were provided: (1) in the absence of beta2GPI, autoimmune and infectious aPL bind to CL in a bilayer, inducing liposome leakage; (2) the leakage mechanism induced by aPL is suggested to be intravesicular; (3) beta2GPI requirement for phospholipid binding in both solid and fluid phase is associated to aPL avidity; (4) CL alone or the CL-beta2GPI complex are the most likely epitopes for autoimmune aPL; (5) aPL from syphilis patients can only form the CL-aPL complex, supporting that beta2GPI is not (part of) the target epitope.

Stability and activity modulation of chymotrypsins in AOT reversed micelles by protein-interface interaction: interaction of alpha-chymotrypsin with a negative interface leads to a cooperative breakage of a salt bridge that keeps the catalytic active conformation (Ile16-Asp194).

The stability of alpha-chymotrypsin and delta-chymotrypsin was studied in reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. alpha-Chymotrypsin is inactivated at the interface and at the water pool, while delta-chymotrypsin is inactivated only at the water pool. The mechanism of inactivation at the interface is related to the interaction of N-terminal group alanine 149 (absent in delta-chymotrypsin) with the negative interface. The dependence of enzyme activity on water content of these two enzymes in reversed micelles of AOT is also related with the interface interaction, since delta-chymotrypsin does not have a bell-shaped curve as observed for alpha-chymotrypsin.

Effect of hexadecyltrimethylammonium bromide-based microemulsions on the rate of decomposition of the beta-lactam antibiotic cephaclor.

Microemulsions of hexadecyltrimethylammonium bromide (HTAB)/n-butanol/hexadecane/water catalyze the intramolecular degradation of cephaclor. The rate increase is a sensitive function of the microemulsion volume fraction and salt concentration. The effects of microemulsions, analyzed quantitatively using a pseudophase ion-exchange model, assumed that the extent of ion dissociation from the microemulsions varies with volume fraction. Comparison of micellar and microemulsion effects on the same reaction shows that microemulsions are less effective catalysts. Acceleration decreased significantly by increasing the relative proportion of n-butanol ratio in microemulsions and by addition of n-butanol in HTAB micelles. Comparison of the activation parameters of the reaction in aqueous solution, microemulsions, and micelles suggests that catalysis by both aggregates is driven mainly by entropic contributions.

Structure-activity relationships in the fusion of small unilamellar phosphatidylcholine vesicles induced by a model peptide.

Limited proteolysis of fatty acid-free bovine serum albumin by pepsin yields several well characterized peptides, one of which (P9, M(r) 9,000), induces fusion of small unilamellar vesicles (SUV) of phosphatidylcholine at pH 3.6. Circular dichroism (CD) of P9 solutions confirmed that the peptide undergoes a reversible transition between pH 7 and pH 3.6. The spectral changes observed with CD suggest that in the low pH conformation there is a decrease in the alpha-helical contents and an exposure of hydrophobic residues. CD and differential ultraviolet spectroscopy demonstrated that P9 binds to micelles of hexadecylphosphorylcholine and the binding produces changes in the tertiary structure of the peptide. Reduction and carboxymethylation of the two disulfide bridges of P9 produced loss of the ability to induce fusion of SUV, although the reduced peptide binds to vesicles, induces loss of entrapped marker and produces vesicle disruption. In the active form P9 exposes hydrophobic groups, one amphiphilic alpha-helix and requires the integrity of the disulfide bridge-stabilized tertiary structure.

Anticardiolipin antibodies from syphilis and systemic lupus erythematosus induce leakage in cardiolipin vesicles.

Anticardiolipin antibodies from sera of patients with systemic lupus erythematosus or syphilis induced leakage of entrapped carboxyfluorescein (CF) from cardiolipin (CL)/phosphatidylcholine(PC) vesicles prepared by sonication of equimolar mixtures of CL:PC. The sera dilution used here was 1:7500. IgG (5-20 micrograms/ml) from the same sera, not containing beta 2GPI, also produced a concentration-dependent leak. Vesicle leakage was inhibited by salt and was not detected with vesicles prepared exclusively with phosphatidylcholine. The demonstration of antibody-induced vesicle leakage offers a convenient system to investigate the mechanism of antibody-lipid binding as well as a potential diagnostic tool.