A potentiometric and spectrofluorimetric approach to unravel inhibitory effects of semi- and thiosemicarbazones on mushroom tyrosinase activity.

The inhibitory effects on mushrooms tyrosinase activity of some semi- and thiosemicarbazones were investigated. While the semicarbazones are inactive, the thiosemicarbazones are, in general, more active than the reference (kojic acid, IC50 = 70 µM), with maximum activity obtained with benzaldehyde thiosemicarbazone (IC50 = 7 µM). These inhibitors probably act by coordination of the copper(II) metal ions in the active site of tyrosinase: effectively, potentiometric studies conducted in water solutions confirm that the most active thiosemicarbazone is a good ligand for copper(II) ions. The tyrosinase CD spectra do not show any significant difference by addition of an inhibitor or an inactive compound. On the contrary, interesting results were obtained by spectrofluorimetric titrations of mushrooms tyrosinase aqueous solutions with some of the investigated compounds, giving helpful information about possible mechanism of action. The thiosemicarbazones here reported are not cytotoxic on human fibroblasts and do not activate cells in a pro-inflammatory way.

Salivary biomarkers for diagnosis of systemic diseases and malignant tumors. A systematic review.

BACKGROUND: Saliva evaluation could be a possible alternative to blood and/or tissue analyses, for researching specific molecules associated to the presence of systemic diseases and malignancies. The present systematic review has been designed in order to answer to the question "are there significant associations between specific salivary biomarkers and diagnosis of systemic diseases or malignancies?". MATERIALS AND METHODS: The Preferred Reporting Item for Systematic Reviews and Meta-analysis (PRISMA) statement was used to guide the review. The combinations of "saliva" and "systemic diseases" or "diagnosis" or "biomarkers" or "cancers" or "carcinoma" or "tumors", were used to search Medline, Scopus and Web of Science databases. Endpoint of research has been set at May 2019. Studies were classified into 3 groups according to the type of disease investigated for diagnosis: 1) malignant tumors; 2) neurologic diseases and 3) inflammatory/metabolic/cardiovascular diseases. Assessment of quality has been assigned according to a series of questions proposed by the National Institute of Health. Level of evidence was assessed using the categories proposed in the Oxford Center for Evidence-Based medicine (CEMB) levels for diagnosis (2011). RESULTS: Seventy-nine studies met the inclusion and exclusion criteria. Fifty-one (64%) investigated malignant tumors, 14 (17.5%) neurologic and 14 (18.5%) inflammatory/cardiovascular/metabolic diseases. Among studies investigating malignant tumors, 12 (23.5%) were scored as "good" and 11 of these reported statistically significant associations between salivary molecules and pathology. Two and 5 studies were found to have a good quality, among those evaluating the association between salivary biomarkers and neurologic and inflammatory/metabolic/cardiovascular diseases, respectively. CONCLUSIONS: The present systematic review confirms the existence of some "good" quality evidence to support the role of peculiar salivary biomarkers for diagnosis of systemic diseases (e.g. lung cancer and EGFR).

Safety of leucodepleted salvaged blood in oncological surgery: an in vitro model.

Intra-operative blood cell salvage (IOCS) is mainly avoided in onco surgery due to the suspicion that it could increase metastasis' risk. We simulated IOCS followed by leucodepletion: HCT116 (human colorectal cancer) cells were inoculated into packed red blood cells units, and their distribution was evaluated, step-by-step, by flow cytometry and immunohistochemistry. Most of HCT116 cells were lost during washing, and almost completely removed after filtration. IOCS plus leucodepletion could be of great advantage for oncological patients, where allogenic blood transfusion could influence tumour progression.

Ligands turning around in the midst of protein conformers: the origin of ligand-protein mating. A NMR view.

Protein-ligand binding is a puzzling process. Many theories have been devised since the pioneering key-and-lock hypothesis based on the idea that both the protein and the ligand have a rigid single conformation. Indeed, molecular motion is the essence of the universe. Consequently, not only proteins are characterized by an extraordinary conformational freedom, but ligands too can fluctuate in a rather vast conformational space. In this scenario, the quest to understand how do they match is fascinating. Recognizing that the inherent dynamics of molecules is the key factor controlling the success of the binding and, subsequently, their chemical/biological function, here we present a view of this process from the NMR stand point. A description of the most relevant NMR parameters that can provide insights, at atomic level, on the mechanisms of protein-ligand binding is provided in the final section.

Positive inotropic effects of Tityus cambridgei and T. serrulatus scorpion venoms on skeletal muscle.

Toxins that block voltage-dependent K+ channels and those that modify Na+ channel gating exhibit positive inotropic effect on skeletal muscle. We compared the effect of the venom of Tityus cambridgei (Tc) and Tityus serrulatus (Ts) scorpions on mouse diaphragm force, in vitro. In indirect and direct (using D-tubocurarine 7.3 microM) stimulation, Tc, 10microg/mL, increased the contractile force, an effect prevented by tetrodotoxin (TTX) while Ts, 0.5 microg/mL, potentiated only indirectly stimulated diaphragm, thus indicating its activity is mainly mediated through acetylcholine release from nerve terminal. This effect is prevented by TTX and attenuated by the K+ channel opener cromakalim. In conclusion, our data show that while the positive inotropic effect of both venoms appears associated to the activity of Na+ and K+ channels, only Tc venom acts also directly on skeletal muscle. This finding call for further studies on Tc venom to identify the toxin responsible for its direct inotropic activity as it may have clinical applications.

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.

Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein Molecular, functional, and immunoprotection analysis

The Schistosoma mansoni Sm14 antigen belongs to the fatty acid-binding protein family and is considered a vaccine candidate against at least two parasite worms, Fasciola hepatica and S. mansoni. Here the genomic sequence and the polymorphism of Sm14 have been characterized for the first time. We found that the conserved methionine at position 20 is polymorphic, being exchangeable with threonine (M20T). To evaluate the function of the amino acid residue at this position, we have also constructed the mutant Sm14-A20 besides the two native isoforms (Sm14-M20 and Sm14-T20). The three purified recombinant His6-tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant â-barrel structure as shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other forms (rSm14M20>rSm14-T20>rSm14-A20). All of the Sm14 proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA) without substantial difference in the binding affinity. However, rSm14-M20 exhibited a higher affinity for natural fatty acids than the rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments against DAUDA (rSm14-M20>rSm14-T20> rSm14-A20). The rSm14-M20 or rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce significant protection against S. mansoni cercariae challenge in immunized mice. The level of protection efficacy correlates with the extent of structure stability of the recombinant Sm14 isoforms and mutant.

The cytochrome c fold can be attained from a compact apo state by occupancy of a nascent heme binding site.

NMR techniques and 8-anilino-1-napthalenesulphonate (ANS) binding studies have been used to characterize the apo state of a variant of cytochrome c(552) from Hydrogenobacter thermophilus. In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A). CD studies show that the apo state contains approximately 14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R(h), 16.6 A). The apo state binds 1 mol of ANS/mol of protein, and a linear reduction in fluorescence enhancement is observed on adding aliquots of hemin to a solution of apo C11A/C14A cytochrome c(552) with ANS bound. These results suggest that the bound ANS is located in the heme binding pocket, which would therefore be at least partially formed in the apo state. Consistent with these characteristics, the formation of the holo state of the variant cytochrome c(552) from the apo state on the addition of heme has been demonstrated using NMR techniques. The properties of the apo state of C11A/C14A cytochrome c(552) reported here contrast strongly with those of mitochondrial cytochrome c whose apo state resembles a random coil under similar conditions.

Amyloid fibril formation by a helical cytochrome.

The substitution of alanines for the two cysteines which form thioether linkages to the haem group in cytochrome c(552) from Hydogenobacter thermophilus destabilises the native protein fold. The holo form of this variant slowly converts into a partially folded apo state that over prolonged periods of time aggregates into fibrillar structures. Characterisation of these structures by electron microscopy and thioflavin-T binding assays shows that they are amyloid fibrils. The data demonstrate that when the native state of this cytochrome is destabilised by loss of haem, even this highly alpha-helical protein can form beta-sheet structures of the type most commonly associated with protein deposition diseases.

Initial denaturing conditions influence the slow folding phase of acylphosphatase associated with proline isomerization.

The folding kinetics of human common-type acylphosphatase (cAcP) from its urea- and TFE-denatured states have been determined by stopped-flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53-Pro54 peptide bond. However, this slow phase is absent when refolding is initiated by dilution of the highly a-helical denatured state formed in the presence of 40% trifluoroethanol (TFE). NMR studies of a peptide fragment corresponding to residues Gly53-Gly69 of cAcP indicate that only the native-like trans isomer of the Gly-Pro peptide bond is significantly populated in the presence of TFE, whereas both the cis and trans isomers are found in an approximately 1:9 ratio for the peptide bond in aqueous solution. Molecular modeling studies in conjunction with NMR experiments suggest that the trans isomer of the Gly53-Pro54 peptide bond is stabilized in TFE by the formation of a nonnative-like hydrogen bond between the CO group of Gly53 and the NH group of Lys57. These results therefore reveal that a specific nonnative interaction in the denatured state can increase significantly the overall efficiency of refolding.

Structure of two fragments of the third cytoplasmic loop of the rat angiotensin II AT1A receptor. Implications with respect to receptor activation and G-protein selection and coupling.

The structural bases that render the third intracellular loop (i3) of the rat angiotensin II AT1A receptor one of the cytoplasmic domains responsible for G-protein coupling are still unknown. The three-dimensional structures of two overlapping peptides mapping the entire i3 loop and shown to differently interact with purified G-proteins have been obtained by simulated annealing calculations, using NMR-derived constraints collected in 70% water/30% trifluoroethanol solution. While the NH2-terminal half, Ni3, residues 213-231, adopts a stable amphipathic alpha-helix, extending over almost the entire peptide, a more flexible conformation is found for the COOH-terminal half, Ci3, residues 227-242. For this peptide, a cis-trans isomerization around the Lys6-Pro7 peptide bond generates two exchanging isomers adopting similar conformations, with an alpha-helix spanning from Asn9 to Ile15 and a poorly defined NH2 terminus. A quite distinct structural organization is found for the sequence EIQKN, common to Ni3 and Ci3. The data do suggest that the extension and orientation of the amphipathic alpha-helix, present in the proximal part of i3, may be modulated by the distal part of the loop itself through the Pro233 residue. A molecular model where this possibility is considered as a mechanism for G-protein selection and coupling is presented.

Structure of the C-terminal fragment 300-320 of the rat angiotensin II AT1A receptor and its relevance with respect to G-protein coupling.

Angiotensin II AT1A receptor is coupled to G-protein, and the molecular mechanism of signal transduction is still unclear. The solution conformation of a synthetic peptide corresponding to residues 300-320 of the rat AT1A receptor, located in the C-terminal cytoplasmic tail and indicated by mutagenesis work to be critical for the G-protein coupling, has been investigated by circular dichroism (CD), nuclear magnetic resonance (NMR) and restrained molecular dynamics calculations. The CD data indicate that, in acidic water, at concentration below 0.8 mM, the peptide exists in a predominantly coil structure while at higher concentration it can form helical aggregates; addition of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. Using NMR-derived constraints, an ensemble of conformers for the peptide has been determined by restrained molecular dynamics calculations. Analysis of the converged three-dimensional structures indicates that a significant population of them adopts an amphipathic alpha-helical conformation that, depending upon experimental conditions, presents a variable extension in the stretch Leu6-Tyr20. An equilibrium with nonhelical structured conformers is also observed. We suggest that the capability of the peptide to modulate its secondary structure as a function of the medium dielectric constant, as well as its ability to form helical aggregates by means of intermolecular hydrophobic interactions, can play a significant role for G-protein activation.

Spin-labeled extracellular loop from a seven-transmembrane helix receptor: studies in solution and interaction with model membranes.

A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N-terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mass oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin-labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (aN). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of aN vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in aN centered at pH 6.5 was ascribed to the titration of the histidines. Values of calculated rotational correlation times were indicative of a pH-induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction.

Conformational changes upon binding of a receptor loop to lipid structures: possible role in signal transduction.

The mas oncogene codes for a seven transmembrane helix protein. The amino acid sequence 253-266, from the third extracellular loop and beginning of helix 7, was synthesized either blocked or carrying an amino acid spin label at the N-terminus. Peptide binding to bilayers and micelles was monitored by ESR, fluorescence and circular dichroism. Binding induced tighter lipid packing, and caused an increase of peptide secondary structure. While binding to bilayers occurred only when peptide and phospholipid bore opposite charges, in micelles the interaction took place irrespective of charge. The results suggest that changes in lipid packing could modulate conformational changes in receptor loops related to the triggering of signal transduction.