Biomedical subjects

M M Santoro

Publications and source records attributed to M M Santoro.

At least 19 recordsLinked to original sources

PubMed2006

Leftward shift in the voltage-dependence for Ca2+ currents activation induced by a new toxin from Phoneutria reidyi (Aranae, Ctenidae) venom.

Various neurotoxins have been described from the venom of the Brazilian spider Phoneutria nigriventer, but little is known about the venoms of the other species of this genus. In the present work, we describe the purification and some structural and pharmacological features of a new toxin (PRTx3-7) from Phoneutria reidyi that causes flaccid paralysis in mice. The observed molecular mass (4627.26 Da) was in accordance with the calculated mass for the amidated form of the amino acid sequence (4627.08 Da). The presence of an alpha-amidated C-terminus was confirmed by MS/MS analysis of the C-terminal peptide, isolated after enzymatic digestion of the native protein with Glu-C endoproteinase. The purified protein was injected (intracerebro-ventricular) into mice at dose levels of 5 microg/mouse causing immediate agitation and clockwise gyration, followed by the gradual development of general flaccid paralysis. PRTx3-7 at 1 microM inhibited by 20% the KCl-induced increase on [Ca2+]i in rat brain synaptosomes. The HEK cells permanently expressing L, N, P/Q and R HVA Ca2+ channels were also used to better characterize the pharmacological features of PRTx3-7. To our surprise, PRTx3-7 shifted the voltage-dependence for activation towards hyperpolarized membrane potentials for L (-4 mV), P/Q (-8 mV) and R (-5 mV) type Ca2+ currents. In addition, the new toxin also affected the steady state of inactivation of L-, N- and P/Q-type Ca2+ currents.

Amino Acid Sequence↗

PubMed2006

The Star STING server: a multiplatform environment for protein structure analysis.

Star STING is the latest version of the STING suite of programs and corresponding database. We report on five important aspects of this package that have acquired some new characteristics, designed to add key advantages to the whole suite: 1) availability for most popular platforms and browsers, 2) introduction of the STING_DB quality assessment, 3) improvement in algorithms for calculation of three STING parameters, 4) introduction of five new STING modules, and 5) expansion of the existing modules. Star STING is freely accessible at: http://sms.cbi.cnptia.embrapa.br/SMS/, http://trantor.bioc.columbia.edu/SMS, http://www.es.embnet.org/SMS/, http://gibk26.bse.kyutech.ac.jp/SMS/ and http://www.ar.embnet.org/SMS.

Algorithms↗

PubMed2005

Comparison of the partial proteomes of the venoms of Brazilian spiders of the genus Phoneutria.

The proteomes of the venoms of the Brazilian wandering "armed" spiders Phoneutria nigriventer, Phoneutria reidyi, and Phoneutria keyserlingi, were compared using two-dimensional gel electrophoresis. The venom components were also fractionated using a combination of preparative reverse phase HPLC on Vydac C4, analytical RP-HPLC on Vydac C8 and C18 and cation exchange FPLC on Resource S at pH 6.1 and 4.7, or anion exchange HPLC on Synchropak AX-300 at pH 8.6. The amino acid sequences of the native and S-pyridyl-ethylated proteins and peptides derived from them by enzymatic digestion and chemical cleavages were determined using a Shimadzu PPSQ-21(A) automated protein sequencer, and by MS/MS collision induced dissociations. To date nearly 400 peptides and proteins (1.2-27 kDa) have been isolated in a pure state and, of these, more than 100 have had their complete or partial amino acid sequences determined. These sequences demonstrate, as might be expected, that the venoms of P. reidyi and P. keyserlingi (Family: Ctenidae) both contain a similar range of isoforms of the neurotoxins as those previously isolated from P. nigriventer which are active on neuronal ion (Ca(2+), Na(+) and K(+)) channels and NMDA-type glutamate receptors. In addition two new families of small (3-4 kDa) toxins, some larger protein (>10 kDa) components, and two serine proteinases of the venom of P. nigriventer are described. These enzymes may be responsible for some of the post-translational modification observed in some of the venom components.

Amino Acid Sequence↗

PubMed2005

Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to beta-trypsin.

Serine-proteases are involved in vital processes in virtually all species. They are important targets for researchers studying the relationships between protein structure and activity, for the rational design of new pharmaceuticals. Trypsin was used as a model to assess a possible differential contribution of hydration water to the binding of two synthetic inhibitors. Thermodynamic parameters for the association of bovine beta-trypsin (homogeneous material, observed 23,294.4 +/- 0.2 Da, theoretical 23,292.5 Da) with the inhibitors benzamidine and berenil at pH 8.0, 25 degrees C and with 25 mM CaCl2, were determined using isothermal titration calorimetry and the osmotic stress method. The association constant for berenil was about 12 times higher compared to the one for benzamidine (binding constants are K = 596,599 +/- 25,057 and 49,513 +/- 2,732 M(-1), respectively; the number of binding sites is the same for both ligands, N = 0.99 +/- 0.05). Apparently the driving force responsible for this large difference of affinity is not due to hydrophobic interactions because the variation in heat capacity (DeltaCp), a characteristic signature of these interactions, was similar in both systems tested (-464.7 +/- 23.9 and -477.1 +/- 86.8 J K(-1) mol(-1) for berenil and benzamidine, respectively). The results also indicated that the enzyme has a net gain of about 21 water molecules regardless of the inhibitor tested. It was shown that the difference in affinity could be due to a larger number of interactions between berenil and the enzyme based on computational modeling. The data support the view that pharmaceuticals derived from benzamidine that enable hydrogen bond formation outside the catalytic binding pocket of beta-trypsin may result in more effective inhibitors.

Animals↗

PubMed2005

Lutzomyia longipalpis peritrophic matrix: formation, structure, and chemical composition.

Sandflies are vectors of several pathogens, constituting serious health problems. Lutzomyia longipalpis (Lutz & Neiva, 1912) is the main vector of Leishmania chagasi, agent of visceral leishmaniasis. They synthesize a thick bag-like structure that surrounds the bloodmeal, named peritrophic matrix (PM). One of the major roles of PM in blood-fed insects includes protection against ingested pathogens by providing a defensive barrier to their development. We used traditional and modern morphological methods as well as biochemical and immunolabeling tools to define details of the PM structure of the Lu. longipalpis sandfly, including composition, synthesis, and degradation. The kinetics of PM formation and degradation was found to be related to the ingestion and time of digestion of the bloodmeal. The midgut changes its size and morphology after the blood ingestion and during the course of digestion. A striking morphological modification takes place in the midgut epithelium after the stretching caused by the bloodmeal, revealing a population of cells that was not observed in the unfed midgut. The transmission and scanning electron microscopies were used to reveal several morphological aspects of PM formation. The PM looks thicker and well formed 24 h after the bloodmeal. Presence of chitin in the PM was demonstrated by immunolabeling with an alpha-chitin monoclonal antibody. SDS-polyacrylamide gel electrophoresis showed at least five protein bands with molecular masses of 38.7-135 kDa, induced by the protein-free diet. Mouse polyclonal antiserum was produced against PMs induced by protein-free meal and used in Western blotting, which revealed at least three associated proteins.

Animals↗

PubMed2005

Purification and molecular characterization of antibacterial compounds produced by Lactobacillus murinus strain L1.

AIMS: The aim of this work was to purify and characterize antibacterial compounds produced by Lactobacillus murinus strain L1. METHODS AND RESULTS: Antagonistic activity was observed in a deferred agar-spot assay against spoilage and pathogenic bacteria, but not against lactobacilli. The inhibitory activity occurred between pH 3.0 and 5.0, and was heat stable. The active compounds were purified by gel filtration chromatography and two peaks of antibacterial activity were observed using Bacillus cereus ATCC 11778 and Shigella sonnei ATCC 11060 as indicator strains. Two active low molecular weight compounds were responsible for this phenomenon and UV spectroscopy, gas chromatography and mass spectrometry were used to characterize them. One of them is lactic acid, while the other is a mono-substituted aromatic ring apparently constituted by group residues of m/z 192 linked in tandem to phenylalanine. CONCLUSIONS: Lactobacillus murinus produces at least two low molecular weight compounds active against B. cereus and Sh. sonnei. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first purification of a new broad-spectrum antibacterial compound from Lact. murinus which inhibits various pathogenic and food spoilage bacteria without acting on other lactobacilli. Using it as a biotechnological control agent of bacterial spoilage may be a promising possibility for the food industry.

Animals↗

PubMed2003

Characterization of -trypsin at acid pH by differential scanning calorimetry.

Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel -sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of -trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM -alanine and 20.0 mM CaCl2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (deltaHcal/deltaHvH) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, deltaHcal/deltaHvH ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, -trypsin unfolded with Tm = 54 C and deltaH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (deltaCp) was estimated to be 2.50 0.07 kcal mol-1 K-1. The stability of -trypsin calculated at 298 K was deltaG D = 5.7 kcal/mol at pH 3.00 and deltaG D = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.

Animals↗

PubMed2003

Reduced production of hyperalgesic substances by mononuclear cells from aged rats incubated with carrageenan: role of interleukin 2 and prostaglandins.

OBJECTIVE AND DESIGN: To compare the production of hyperalgesic substances by cells from aged (A; 24-month) and juvenile (J; 2-month) rats. MATERIAL AND METHODS: 4 x 10(5) purified mononuclear cells from J and A were 2 h-stimulated (test) or not (control) by 250 microg lambda-carrageenan/well. Supernatants (0.1 ml) were intraplantarly (ipl) injected in rat paws and development of mechanical hyperalgesia, in grams, evaluated. Rat interleukin 2 (IL 2) and prostaglandin E2 (PGE2) were also assessed for hyperalgesia development. RESULTS: Test supernatants from A compared with J induced significantly less hyperalgesia (-56 +/- 8.1 and -88.4 +/- 4.6 g, respectively, p < 0.05, ANOVA t test). Local injection of a specific, but not a control, antiserum against IL 2 significantly blocked both pure IL 2- and stimulated supernatants-derived hyperalgesia. In contrast to PGE-like materials, IL 2 content in supernatants was compatible with hyperalgesia development. CONCLUSIONS: Hyperalgesia induced by test supernatants was significantly less intense when derived from aged animals. IL 2 may have accounted for such hyperalgesia.

Aging↗

PubMed2003

The denaturation of alpha, beta and psi bovine trypsin at pH 3.0: evidence of intermediates.

The conformational stability and the folding process of alpha, beta and Psi bovine trypsin at pH 3.0 followed by circular dichroism (CD) and size exclusion in HPLC have been analyzed as a function of urea concentration. The thermodynamic stability for a and b are deltaG = 15.91 +/- 0.28 kcal/mol, deltaG = 15.54 +/- 2.39 kcal/mol. respectively, and y trypsin is deltaG = 16.10 +/- 2.51 kcal/mol. The transition curves for alpha, beta and Psi forms suggest a molten globule state.

Animals↗

PubMed2002

Enzymes with gelatinolytic activity can be found in Tityus bahiensis and Tityus serrulatus venoms.

Enzymes with gelatinolytic activity were detected in Tityus bahiensis and Tityus serrulatus venom. Their activity was optimal at pH 8.0 in SDS-PAGE-gelatin. They were inhibited by PMSF but not by iodoacetamide, pepstatin or phenantrolin in the assay conditions used. This suggests that these enzymes are serine proteases. The presence of metal ions did not affect the proteolytic activity of these enzymes. Several possible functions may be envisaged for these enzymes: in tissue permeabilization, pancreatitis and toxin processing.

Animals↗

PubMed2002

Receptor tyrosine kinases as target for anti-cancer therapy.

Receptor tyrosine kinases (RTKs) are cell surface transmembrane proteins responsible for intracellular signal transduction. They are expressed in several cell types and, after activation by growth factor binding, trigger a series of intracellular pathways, leading to a wide variety of cell responses (e.g., differentiation, proliferation, migration and invasion, angiogenesis, survival). Over-expression and/or structural alteration of RTKs family members are often associated to human cancers and tumor cells are known to use RTK transduction pathways to achieve tumor growth, angiogenesis and metastasis. Therefore, RTKs represent pivotal target in approaches of cancer therapy. A number of small molecules acting as RTK inhibitors have been synthesized by pharmaceutical companies and are under clinical trials, are being analyzed in animal models or have been successfully marketed. Ligand-dependent downregulation of RTKs is a critical step for modulating their activity and the adaptor Cbl has been indicated as the key protein involved in negative regulation of RTKs, such as EGF and HGF receptors. These data suggest novel potential pharmacological targets for the treatment of human malignancies associated to oncogenic activation of RTKs.

Animals↗

PubMed2001

Structural stabilization displayed in the soluble phase of cocrystallization of flavianic acid with trypsin in acid media.

Coprecipitation and cocrystallization of proteins with synthetic dyes are known to involve reversible denaturation processes which can offer specificity towards a target protein. Although the knowledge of conformational equilibrium and how to control it are central into the basic molecular dynamics of protein precipitation, the exact molecular mechanism of the precipitation remains unknown. Aiming at understanding the events that take place before the coprecipitation step of generic dye-protein systems, we investigated the binding of flavianic acid to bovine trypsin, using approaches of visible and second-derivative ultraviolet spectroscopy, viscosimetry, densimetry and circular dichroism. The results suggest a restricted transconformation of the macromolecule linked to dye binding at a stoichiometry of 1:1. An increase on the protein secondary structures occurred together with an electro-constriction effect on trypsin, a borderline event to the coprecipitation process, suggesting a stabilized structure for trypsin as a ligand-induced molten globule-like state.

Acids↗

PubMed2001

Hyperalgesia and edema responses induced by rat peripheral blood mononuclear cells incubated with carrageenin.

The aim of this study was to verify the role played by mononuclear cells in an acute (nonimmune) inflammatory reaction. Mononuclear cells purified from rat peripheral blood were incubated for 1, 2, or 24 h with 100 or 250 microg/ml carrageenin (Cg). The resultant donor supernatant was injected into recipient rats to test its ability to induce hyperalgesia (reduction in threshold for paw pressure) and edema (increase in paw volume). Mononuclear cell supernatants (MnS) induced a significant time- and dose-dependent hyperalgesia and edema in rat paws, which reached a maximal effect at 3 h, lasted for 6 h, and returned to basal levels at 24 h of injection. Prostaglandins and cytokines (interleukin 1, 2, 6, 8, and tumor necrosis factor alpha) accounted for the hyperalgesia induced by MnS, as it was reduced (40 to 90%) by synthesis inhibitors such as indomethacin, dexamethasone, rolipram, and cyclosporin added to the cultures at a microgram dose-range. Edema was dependent on serotonin release in rat paws. These results indicate that mononuclear cells may be important contributors to acute inflammatory reactions, especially under those conditions where pain is an important component.

Animals↗

PubMed2001

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline.

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 microM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37 degrees C was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 microM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 microM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 +/- 0.8 microM and k cat = 48.4 +/- 1.0 min(-1). The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 +/- 10, 1,098 +/- 91, 38.6 +/- 5.2 and 37,340 +/- 5,400 microM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 +/- 92.8 and 310,500 +/- 38,600 microM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.

Amidohydrolases↗

PubMed2001

A time-dependent, two-step binding mode of the nitro dye flavianic acid to trypsin in acid media.

Synthetic dyes bind to proteins causing selective coprecipitation of the complexes in acid aqueous solution by a process of reversible denaturation that can be used as an alternative method for protein fractionation. The events that occur before precipitation were investigated by equilibrium dialysis using bovine trypsin and flavianic acid as a model able to cause coprecipitation. A two-step mode of interaction was found to be dependent on the incubation periods allowed for binding, with pronounced binding occurring after 42 h of incubation. The first step seems to involve hydration effects and conformational changes induced by binding of the first dye molecule, following rapid denaturation due to the binding of six additional flavianate anions to the macromolecule.

Animals↗

PubMed2000

The Ron oncogenic activity induced by the MEN2B-like substitution overcomes the requirement for the multifunctional docking site.

Oncogenic activation of the Ron tyrosine kinase (Macrophage Stimulating Protein receptor) relies on substitutions of two highly conserved residues in the catalytic domain (D1232V and M1254T), which result in ligand-independent activation of the receptor, in vivo tumorigenesis and metastasis. We show here that the Y/F conversion of the Y1317 residue in the kinase domain impairs tumorigenic and metastatic properties of Ron activated by the MEN2B-like mutation (RonM1254T), but not by other two oncogenic substitutions. Furthermore, RonM1254T lacking the multifunctional docking site retains transforming and metastatic activity. These data reveal that the transforming activity of RonM1254T mutant is dependent on Y1317 phosphorylation, suggesting a shift in intramolecular substrate specificity. Consistently, a shift of RonM1254T kinase substrate specificity was observed by in vitro peptide phosphorylation assays and in vivo receptor auto-phosphorylation. The Y1317 phosphorylation elicits by itself activation of PI-3K/Akt and MAPK signalling pathways. Our data indicate that the accomplishment of the full oncogenic phenotype of RonM1254T requires the phosphorylation both of the canonical C-terminal docking site and of the unique Y1317 residue in the tyrosine kinase domain.

Cell Transformation, Neoplastic↗

PubMed2000

Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits 3H-choline uptake in guinea pig ileum.

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of 3H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0. 03-1 microM) and up to 10 min of treatment, crotoxin decreased 3H-choline uptake by 50-75% compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 microM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on 3H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits 3H-choline uptake with high affinity (EC25 = 10 +/- 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission.

Animals↗

PubMed1999

Thermal stability studies of hyperimmune horse antivenoms.

Ampoules of horse antivenoms raised against Bothrops spp and Crotalus durissus (final product) produced by Fundação Ezequiel Dias (FUNED) were fractionated on the molecular filtration chromatography (SUPEROSE 12) and the expected MW species of F(ab')2 fragments were observed. It has been known that high temperatures promote aggregation and formation of protein precipitates. Phenol is used in preparations of antivenoms as preservative; however, as thus is a hydrophobic substance, it can also induce an increase in protein denaturation. Sorbitol and glycerol were used as osmolyte (natural substance or organic compound capable of stabilizing proteins) and decreased the formation of protein precipitates in solutions of antibodies; as judged by a spectrophotometric assay (280 nm), by nephelometry or when tested by the ELISA. In 1.0 M concentration, the sorbitol presented the best results when compared with glycerol. Circular Dichroism (CD) was used to study the spectra of antibodies in the presence of PBS, glycerol or sorbitol. Up to 1.0 M concentration of the osmolyte, there is no significant perturbation of the antibodies spectra in the amide region, in control samples not incubated (kept at room temperature with no phenol added) or incubated at 62 degrees C in presence of phenol. Nephelometry and gel SDS-PAGE techniques were used in assays that demonstrated the effects of phenol (denaturing) and the osmolytes (stabilizing) in horse antivenoms in high temperatures.

Animals↗