The development of new oral vaccines using porous silica.

Ordered mesoporous silica (OMS) was proved to be an efficient oral adjuvant capable to deliver a wide in size variety of different antigens, promoting efficient immunogenicity. This material can be used in single or polivalent vaccines, which have been developed by a group of Brazilian scientists. The experiments performed with the model protein Bovine Serum Albumin (BSA) gave the first promissing results, that were also achieved by testing the virus like particle surface antigen of hepatitis B (HBsAg) and diphtheria anatoxin (dANA). Nanostructured OMS, SBA-15 type, with bi-dimensional hexagonal porous symmetry was used to encapsulate the antigens either in the mesoporous (pore diameter ∼ 10 nm) or macroporous (pore diameter > 50 nm) regions. This silica vehicle proved to be capable to create an inflammatory response, did not exhibit toxicity, being effective to induce immunity in high and low responder mice towards antibody production. The silica particles are in the range of micrometer size, leaving no trace in mice organs due to its easy expulsion by faeces. The methods of physics, usually employed to characterize the structure, composition and morphology of materials are of fundamental importance to develop proper oral vaccines in order to state the ideal antigen load to avoid clustering and to determine the rate of antigen release in different media mimicking body fluids.

The development of new oral vaccines using porous silica

Ordered mesoporous silica was proved to be an efficient oral adjuvant capable to deliver a wide in size variety of different antigens, promoting efficient immunogenicity. This material can be used in single or polivalent vaccines, which have been developed by a group of Brazilian scientists. The experiments performed with the model protein Bovine Serum Albumin (BSA) gave the first promissing results, that were also achieved by testing the virus like particle surface antigen of hepatitis B (HBsAg) and diphtheria anatoxin (dANA). Nanostructured ordered mesoporous silica, SBA-15 type, with bi-dimensional hexagonal porous symmetry was used to encapsulate the antigens either in the mesoporous (pore diameter ~10 nm) or macroporous ( pore diameter > 50 nm) regions. This silica vehicle proved to be capable to create an inflammatory response, did not exhibit toxicity, being effective to induce immunity in high and low responder mice towards antibody production. The silica particles are in the range of micrometer size, leaving no trace in mice organs due to its easy expulsion by faeces. The methods of Physics, usually employed to characterize the structure, composition and morphology of materials are of fundamental importance to develop proper oral vaccines in order to state the ideal antigen load to avoid clustering and to determine the rate of antigen release in different media mimicking body fluids.

Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions.

Hepatitis B virus causes acute and chronic infections in millions of people worldwide and, since 1982, a vaccine with 95% effectiveness has been available for immunization. The main component of the recombinant hepatitis B vaccine is the surface antigen protein (HBsAg). In this work, the effect of pH, ionic strength and temperature on the native state of the HBsAg antigen were studied by a combination of biophysical methods that included small angle X-ray scattering, synchrotron radiation circular dichroism, fluorescence and surface plasmon resonance spectroscopies, as well as in vivo and in vitro potency assays. The native conformation, morphology, radius of gyration, and antigenic properties of the HBsAg antigen demonstrate high stability to pH treatment, especially in the pH range employed in all stages of HBsAg vaccine production and storage. The HBsAg protein presents thermal melting point close to 56 °C, reaching a more unfolded state after crossing this point, but it only experiences loss of vaccine potency and antigenic properties at 100 °C. Interestingly, a 6-month storage period does not affect vaccine stability, and the results are similar when the protein is kept under refrigerated conditions or at room temperature (20 °C). At frozen temperatures, large aggregates (>200 nm) are formed and possibly cause loss of HBsAg content, but that does not affect the in vivo assay. Furthermore, HBsAg has a well-ordered secondary structure content that is not affected when the protein is formulated with silica SBA-15, targeting the oral delivery of the vaccine. The combined results from all the characterization techniques employed in this study showed the high stability of the antigen at different storage temperature and extreme values of pH. These findings are important for considering the delivery of HBsAg to the immune system via an oral vaccine.

Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions

Hepatitis B virus causes acute and chronic infections in millions of people worldwide and, since 1982, a vaccine with 95% effectiveness has been available for immunization. The main component of the recombinant hepatitis B vaccine is the surface antigen protein (HBsAg). In this work, the effect of pH, ionic strength and temperature on the native state of the HBsAg antigen were studied by a combination of biophysical methods that included small angle X-ray scattering, synchrotron radiation circular dichroism, fluorescence and surface plasmon resonance spectroscopies, as well as in vivo and in vitro potency assays. The native conformation, morphology, radius of gyration, and antigenic properties of the HBsAg antigen demonstrate high stability to pH treatment, especially in the pH range employed in all stages of HBsAg vaccine production and storage. The HBsAg protein presents thermal melting point close to 56°C, reaching a more unfolded state after crossing this point, but it only experiences loss of vaccine potency and antigenic properties at 100°C. Interestingly, a 6-month storage period does not affect vaccine stability, and the results are similar when the protein is kept under refrigerated conditions or at room temperature (20°C). At frozen temperatures, large aggregates (>200nm) are formed and possibly cause loss of HBsAg content, but that does not affect the in vivo assay. Furthermore, HBsAg has a well-ordered secondary structure content that is not affected when the protein is formulated with silica SBA-15, targeting the oral delivery of the vaccine. The combined results from all the characterization techniques employed in this study showed the high stability of the antigen at different storage temperature and extreme values of pH. These findings are important for considering the delivery of HBsAg to the immune system via an oral vaccine.

Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions

Hepatitis B virus causes acute and chronic infections in millions of people worldwide and, since 1982, a vaccine with 95% effectiveness has been available for immunization. The main component of the recombinant hepatitis B vaccine is the surface antigen protein (HBsAg). In this work, the effect of pH, ionic strength and temperature on the native state of the HBsAg antigen were studied by a combination of biophysical methods that included small angle X-ray scattering, synchrotron radiation circular dichroism, fluorescence and surface plasmon resonance spectroscopies, as well as in vivo and in vitro potency assays. The native conformation, morphology, radius of gyration, and antigenic properties of the HBsAg antigen demonstrate high stability to pH treatment, especially in the pH range employed in all stages of HBsAg vaccine production and storage. The HBsAg protein presents thermal melting point close to 56°C, reaching a more unfolded state after crossing this point, but it only experiences loss of vaccine potency and antigenic properties at 100°C. Interestingly, a 6-month storage period does not affect vaccine stability, and the results are similar when the protein is kept under refrigerated conditions or at room temperature (20°C). At frozen temperatures, large aggregates (>200nm) are formed and possibly cause loss of HBsAg content, but that does not affect the in vivo assay. Furthermore, HBsAg has a well-ordered secondary structure content that is not affected when the protein is formulated with silica SBA-15, targeting the oral delivery of the vaccine. The combined results from all the characterization techniques employed in this study showed the high stability of the antigen at different storage temperature and extreme values of pH. These findings are important for considering the delivery of HBsAg to the immune system via an oral vaccine.

Gas-phase dissociation study of erythrinian alkaloids by electrospray ionization mass spectrometry and computational methods.

Alkaloids from plants of the genus Erythrina display important biological activities, including anxiolytic action. Characterization of these alkaloids by mass spectrometry (MS) has contributed to the construction of a spectral library, has improved understanding of their structures and has supported the proposal of fragmentation mechanisms in light of density functional calculations. In this study, we have used low-resolution and high-resolution MSn analyses to investigate the fragmentation patterns of erythrinian alkaloids; we have employed the B3LYP/6-31+G(d,p) model to obtain their reactive sites. To suggest the fragmentation mechanism of these alkaloids, we have studied their protonation sites by density functional calculation, and we have obtained their molecular electrostatic potential map and their gas-phase basicity values. These analyses have indicated the most basic sites on the basis of the proton affinities of the nitrogen and oxygen atoms. The protonated molecules were generated by two major fragmentations, namely, neutral loss of CH3 OH followed by elimination of H2 O. High-resolution analysis confirmed elimination of NH3 by comparison with the losses of H2 and •CH3 . NH3 was eliminated from compounds that did not bear a substituent on ring C. The benzylic carbocation initiated the dissociation mechanism, and the first reaction involved charge transfer from a lone pair of electrons in the oxygen atoms. The second reaction consisted of ring contraction with loss of a CO molecule. The presence of hydroxy and epoxy groups could change the intensity or the occurrence of the fragmentation pathways. Given that erythrinian alkaloids are applied in therapeutics and are promising leads for the development of new drugs, the present results could aid identification of several analogues of these alkaloids in biological samples and advance pharmacokinetic studies of new plant derivatives based on MSn and MS/MS analyses. Copyright © 2017 John Wiley & Sons, Ltd.

Membrane negative curvature induced by a hybrid peptide from pediocin PA-1 and plantaricin 149 as revealed by atomistic molecular dynamics simulations.

Antimicrobial peptides (AMPs) are cationic peptides that kill bacteria with a broad spectrum of action, low toxicity to mammalian cells and exceptionally low rates of bacterial resistance. These features have led to considerable efforts in developing AMPs as an alternative antibacterial therapy. In vitro studies have shown that AMPs interfere with membrane bilayer integrity via several possible mechanisms, which are not entirely understood. We have performed the synthesis, membrane lysis measurements, and biophysical characterization of a novel hybrid peptide. These measurements show that PA-Pln149 does not form nanopores, but instead promotes membrane rupture. It causes fast rupture of the bacterial model membrane (POPG-rich) at concentrations 100-fold lower than that required for the disruption of mammalian model membranes (POPC-rich). Atomistic molecular dynamics (MD) simulations were performed for single and multiple copies of PA-Pln149 in the presence of mixed and pure POPC/POPG bilayers to investigate the concentration-dependent membrane disruption by the hybrid peptide. These simulations reproduced the experimental trend and provided a potential mechanism of action for PA-Pln149. It shows that the PA-Pln149 does not form nanopores, but instead promotes membrane destabilization through peptide aggregation and induction of membrane negative curvature with the collapse of the lamellar arrangement. The sequence of events depicted for PA-Pln149 may offer insights into the mechanism of action of AMPs previously shown to induce negative deformation of membrane curvature and often associated with peptide translocation via non-bilayer intermediate structures.

A rail system for circular synthetic aperture sonar imaging and acoustic target strength measurements: design/operation/preliminary results.

A 22 m diameter circular rail, outfitted with a mobile sonar tower trolley, was designed, fabricated, instrumented with underwater acoustic transducers, and assembled on a 1.5 m thick sand layer at the bottom of a large freshwater pool to carry out sonar design and target scattering response studies. The mobile sonar tower translates along the rail via a drive motor controlled by customized LabVIEW software. The rail system is modular and assembly consists of separately deploying eight circular arc sections, measuring a nominal center radius of 11 m and 8.64 m arc length each, and having divers connect them together in the underwater environment. The system enables full scale measurements on targets of interest with 0.1° angular resolution over a complete 360° aperture, without disrupting target setup, and affording a level of control over target environment conditions and noise sources unachievable in standard field measurements. In recent use, the mobile cart carrying an instrumented sonar tower was translated along the rail in 720 equal position increments and acoustic backscatter data were acquired at each position. In addition, this system can accommodate both broadband monostatic and bistatic scattering measurements on targets of interest, allowing capture of target signature phenomena under diverse configurations to address current scientific and technical issues encountered in mine countermeasure and unexploded ordnance applications. In the work discussed here, the circular rail apparatus is used for acoustic backscatter testing, but this system also has the capacity to facilitate the acquisition of magnetic and optical sensor data from targets of interest. A brief description of the system design and operation will be presented along with preliminary processed results for data acquired from acoustic measurements conducted at the Naval Surface Warfare Center, Panama City Division Test Pond Facility. [Work Supported by the U.S. Office of Naval Research and The Strategic Environmental Research and Development Program.].

Chaperone-mediated native folding of a ß-scorpion toxin in the periplasm of Escherichia coli.

BACKGROUND: Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria. METHODS: The ß-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined. RESULTS: Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold. CONCLUSIONS: The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method. GENERAL SIGNIFICANCE: We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.

Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa.

This study was aimed at investigating the purification, biological activity, and some structural properties of three serine protease inhibitors isoforms, denoted ApTIA, ApTIB, and ApTIC from Acacia plumosa Lowe seeds. They were purified from the saline extract of the seeds, using Superdex-75 gel filtration and Mono-S ion exchange chromatography. They were further investigated by mass spectrometry, spectroscopic measurements, surface plasmon resonance, and inhibition assays with proteases and phytopathogenic fungi. The molecular mass of each isoform was estimated at ca. 20 kDa. Each contained two polypeptide chains linked by a disulfide bridge, with different isoelectric points that are acidic in nature. The N-terminal sequences of both chains indicated that they were Kunitz-type inhibitors. Circular dichroism (CD) analyses suggested the predominance of both disordered and beta-strands on ApTI isoforms secondary structure, as expected for beta-II proteins. In addition, it was observed that the proteins were very stable, even at either extreme pH values or at high temperature, with denaturation midpoints close to 75 degrees C. The isoinhibitors could delay, up to 10 times, the blood coagulation time in vitro and inhibited action of trypsin (Ki 1.8 nM), alpha-chymotrypsin (Ki 10.3 nM) and kallikrein (Ki 0.58 microM). The binding of ApTIA, ApTIB, and ApTIC to trypsin and alpha-chymotrypsin, was investigated by surface plasmon resonance (SPR), this giving dissociation constants of 0.39, 0.56 and 0.56 nM with trypsin and 7.5, 6.9 and 3.5 nM with alpha-chymotrypsin, respectively. The growth profiles of Aspergillus niger, Thielaviopsis paradoxa and Colletotrichum sp. P10 were also inhibited by each isoforms. These three potent inhibitors from A. plumosa may therefore be of great interest as specific inhibitors to regulate proteolytic processes.

Chemical constituents of Lychnophora pohlii and trypanocidal activity of crude plant extracts and of isolated compounds.

Crude extracts of Lychnophora pohlii were tested in vitro against trypomastigote forms of Trypanosoma cruzi, and the dichloromethane and methanol crude extracts from leaves plus inflorescences were found to have trypanocidal activity. The bioassay-guided fractionation of the extracts yielded seven active compounds: the sesquiterpene lactones lychnopholide, centratherin, goyazensolide and 15-desoxygoyazensolide in the dichloromethane extract, and caffeic acid and the flavonoids luteolin and vicenin-2 in the methanol extract. One active caffeoyl quinic acid derivative was isolated from the inactive hydroalcoholic extract of leaves plus inflorescences. Chemically, the plant has sesquiterpene lactone type furanoheliangolides, flavonoids, caffeic acid, a caffeoyl quinic acid derivative, which are characteristic of the Vernonieae.

Organ donation and transplantation in Brazil: university students' awareness and opinions.

This study was aimed at assessing university students' awareness and opinions about aspects of the law no. 10.221/01. This exploratory study was performed among students at the São Paulo Federal University-UNIFESP, from 2001 to July 2002, including 1284 undergraduates in the following courses: nursing, medicine, speech and language therapy, biomedicine, and ophthalmic technology. That data show that 47.7% (333) of undergraduates are aware of the kind of donation established by the new law. Among the adolescents of first and second grades, and courses, there was a difference on the awareness about the kind of donation (P =.000). Most undergraduates (57.6%, 402) agree with consented donation. However, the high percentage--35.1% (245)--that chose the alternative "I don't know" is remarkable. Results show that many undergraduates are unaware of the specifications of the technical files for transplants; only 48.3% (337) of them chose the correct answer, and 79.4% (554) do not believe that the order in the waiting list for transplants is respected. The conclusion is that it is worth emphasizing is that there are different opinions and levels of awareness among students, considering age, religion and religious practice, and course and level at school. In spite of unawareness and lack of belief in the principles of distribution of organs and tissue, most students are willing to be donors (68.2%).

Inhibitory activity of flavonoids from Lychnophora sp. on generation of reactive oxygen species by neutrophils upon stimulation by immune complexes.

Formation of circulating immune complexes (ICs) is essential for clearance of invading agents. In some circumstances ICs might deposit on host tissues, leading to an inflammatory process that involves massive activation of neutrophils (PMNs), release of reactive oxygen species (ROS) and lysosomal enzymes and damage to the host tissue. Extracts of plants from Lychnophora sp. are used in Brazilian folk medicine as antiinflammatory agents. In this study, we evaluated the effect of eight flavonoids isolated from L. granmongolense, L. salicifolia and L. ericoides on the generation of ROS by rabbit PMNs stimulated with two kinds of ICs: particles of serum-opsonized zymosan (OZ) and insoluble ICs (ICIgG). ROS production was measured by chemiluminescence (CL) assay. We observed that 5- and 7- dihydroxylated compounds at 5 micromol/L inhibited almost totally ICIgG- and OZ-triggered luminol-CL and OZ-triggered lucigenin-CL. The degree of inhibitory effect among the other flavonoids was different, depending on the kind of ICs used to trigger ROS generation by PMNs and the number and position of methoxy groups. Moreover, under the conditions assessed, the studied flavonoids were not toxic to the rabbit PMNs. These results suggest that the actions of flavonoids on ROS generation by stimulated PMNs are highly dependent on their structures.

Trypanocidal activity of Lychnophora staavioides Mart. (Vernonieae, Asteraceae).

In the continuing search for new compounds with trypanocidal activity for use in blood banks to prevent the transmission of Chagas' disease, a trypanocidal extract of Lychnophora staavioides Mart. (Vernonieae, Asteraceae) was fractionated using several chromatographic techniques and afforded the following flavonoids: tectochrysin, pinostrobin, pinobanksin, pinobanksin 3-acetate, pinocembrin, chrysin, galangin 3-methyl ether, quercetin 3-methyl ether, chrysoeriol and vicenin-2. The most active compound was quercetin 3-methyl ether, which showed no blood lysis activity and which represents a promising compound for use against T. cruzi in blood banks.

Genotoxic action of the sesquiterpene lactone centratherin on mammalian cells in vitro and in vivo.

Centratherin is a sesquiterpene lactone known for its antimicrobial, anti-inflammatory, and trypanocidal activities. The aim of this study was to determine the clastogenic and cytotoxic potential of centratherin in human lymphocytes and in mice. Human lymphocytes in culture were submitted to either continuous treatment or treatment during G(2) phase of the cell cycle. After continuous treatment the 0.2 microg/ml concentration induced a significant increase in total of chromosomal aberrations (CA) and sister chromatid exchange compared to control, and it reduced the mitotic index (MI). In the treatment during G(2) phase, centratherin induced a significant increase in the frequency of CA for all concentrations tested (0.1, 0.3, and 0.5 microg/ml). In the in vivo test system all three concentrations tested in mice (3.3, 6.7, and 13.3 mg/kg b.w.) induced a significant increase in CA compared to the negative control. On the basis of these results, centratherin showed clastogenic and cytotoxic activity on in vitro and in vivo mammalian systems.

Acoustic transmission across a roughened fluid-fluid interface.

A set of tank experiments was performed to investigate acoustic transmission across a roughened fluid-fluid interface with the intention to test heuristic Bragg scattering predictions used to explain observations of anomalous transmission in field experiments. In the tank experiments, two immiscible fluids (vegetable oil floating on glycerin) formed the layers. Small polystyrene beads were floated at the interface to simulate roughness. An array of hydrophones placed in the bottom layer (glycerin) was used to measure the acoustic levels transmitted across the interface. This array was also employed as a beamformer to determine the apparent angle and sound speed of the scattered signals. Data were acquired at subcritical grazing angles in the frequency range of 100-200 kHz for three different bead diameters and for various configurations in which the locations of the beads floating on the interface were varied. Results of these measurements demonstrated that a significant amount of acoustic energy can be scattered into the bottom layer by beads floating at the interface. The scattered levels increased with increasing bead diameter. However, discrepancies occurred between observed propagation properties and the Bragg predictions. By comparing the processed tank data to a computer simulation of the same it was determined that these discrepancies are a consequence of near-field reception of the scattering by the bead array and ignoring the directionality of the scattering by the beads. Consequences to observations made in field experiments are discussed.

Assessment of the cytotoxic and clastogenic activities of the sesquiterpene lactone lynchnopholide in mammalian cells in vitro and in vivo.

Lychnopholide (LNP), a sesquiterpene lactone with antitumor, trypanocidal, and antimicrobial activities, was isolated from Vanillosmopsis erythropappa. The present study was carried out to assess the cytotoxic and clastogenic potential of this new agent in human cultured lymphocytes and Swiss bone marrow cells before the agent was used in medicine. The mitotic index, chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and proliferation index were investigated. There was no alteration in the number of CAs and SCEs in the continuous in vitro treatment. However, the highest concentration (0.2 microg/ml) of LNP was cytotoxic. LNP (0.1, 0.2, and 0.4 microg/ml) induced a significant increase in CA frequency at the G2 phase in all treated cultures. Only the highest concentration (26.67 mg/kg) caused a significant increase in the total number of CAs in the in vivo investigation. On the basis of these results, LNP had a clastogenic effect on both test systems and a cytotoxic effect in vitro.

A study of the trypanocidal and analgesic properties from Lychnophora granmongolense (Duarte) Semir & Leitão Filho.

Crude extracts from the aerial parts of Lychnophora granmongolense (Asteraceae) were bioassayed for trypanocidal (trypomastigote forms of Trypanosoma cruzi) and analgesic (writhing test) activities. The crude ethyl acetate extract from the leaves plus inflorescences exhibited trypanocidal activity but no analgesic activity in the writhing model of pain. The bioassay-guided fractionation of this extract yielded three trypanocidal compounds: the sesquiterpene lactones centratherin (lychnophorolide A) and goyazensolide and the flavonoid eriodictyol. The flavonoids homoeriodictyol, eriodictyol 7, 3'-dimethyl ether, velutin, chrysoeriol, dihydroisorhamnetin, rhamnazin and the sesquiterpene lactone lychnophorolide B were also isolated from the ethyl acetate extract. Such flavonoids did not show any trypanocidal activity. The isolated amount of lychnophorolide B was not enough to account for the full activity against T. cruzi.

Effect of naturally occurring flavonoids on lipid peroxidation and membrane permeability transition in mitochondria.

The ability of eight structurally related naturally occurring flavonoids in inhibiting lipid peroxidation and mitochondrial membrane permeability transition (MMPT), as well as respiration and protein sulfhydryl oxidation in rat liver mitochondria, was evaluated. The flavonoids tested exhibited the following order of potency to inhibit ADP/ Fe(II)-induced lipid peroxidation, estimated with the thiobarbituric acid assay: 3'-O-methyl-quercetin > quercetin > 3,5,7,3',4'-penta-O-methyl-quercetin > 3,7,3',4'-tetra-O-methyl-quercetin > pinobanksin > 7-O-methyl-pinocembrin > pinocembrin > 3-O-acyl-pinobanksin. MMPT was estimated by the extent of mitochondrial swelling induced by 10 microM CaCl2 plus 1.5 mM inorganic phosphate or 30 microM mefenamic acid. The most potent inhibitors of MMPT were quercetin, 7-O-methyl-pinocembrin, pinocembrin, and 3,5,7,3',4'-penta-O-methyl-quercetin. The first two inhibited in parallel the oxidation of mitochondrial protein sulfhydryl involved in the MMPT mechanism. The most potent inhibitors of mitochondrial respiration were 7-O-methyl-pinocembrin, quercetin, and 3'-O-methyl-quercetin while the most potent uncouplers were pinocembrin and 3-O-acyl-pinobanksin. In contrast 3,7,3',4'-tetra-O-methyl-quercetin and 3,5,7,3',4'-penta-O-methyl-quercetin showed the lowest ability to affect mitochondrial respiration. We conclude that, in general, the flavonoids tested are able to inhibit lipid peroxidation on the mitochondrial membrane and/or MMPT. Multiple methylation of the hydroxyl substitutions, in addition to sustaining good anti-lipoperoxidant activity, reduces the effect of flavonoids on mitochondrial respiration, and therefore, increases the pharmacological potential of these compounds against pathological processes related to oxidative stress.

Anodontites trapesialis: a biological monitor of organochlorine pesticides.

The mussel Anodontites trapesialis (Lam, 1819) was used as an indicator of organochlorine pollutants in the Pardo River, located in the municipality of Ribeirão Preto (21 degrees 07'S and 47 degrees 45'W), State of São Paulo, Brazil. Biological monitoring was performed for one year at the site of a sugar cane grove on the left bank of the river. Forty-three animals were placed in two aluminum enclosures on the river bottom at this site and 4 animals of each enclosure were sacrificed for pesticide analysis at 3-month intervals, each collection corresponding to one season of the year. The animals were found to have been exposed to DDT, lindane, heptachlor, aldrin and dieldrin. Endrin was not detected in any of the analyses.