Influence of the geographic location and house characteristics on the concentration of microplastics in indoor dust.

Microplastics (MPs) are known for their ubiquity, having been detected in virtually any environmental compartment. However, indoor MPs concentrations are poorly studied despite being closely related to human exposure. The present study aims to evaluate the presence of MPs in settled atmospheric dust in 60 houses distributed in 12 districts of the metropolitan city of Lima, Peru, and investigate the influence of their geographical location and house characteristics. MPs concentration ranged from 0.01 to 33.9 MPs per mg of dust. Fibers and blue were the most frequent shape and color (98 % and 69 %, respectively). Also, 82 % of the particles were between 500 µm - 5 mm in size. A higher concentration of MPs was identified in the center-south of the city. The houses located on the highest floors (levels 4 to 13 to ground) displayed higher concentrations. MPs were primarily composed of polyester (PET), polypropylene (PP), and ethylene-vinyl acetate (EVA), among others. The polymers identified suggest that MPs derived from the fragmentation of components frequently found in houses, such as synthetic clothing, food storage containers, toys, carpets, floors, and curtains. The incorporation of MPs from the outside into dwellings is not ruled out. Future studies should evaluate the influence of consumption habits and housing characteristics on the abundance of MPs.

Common Beans as a Source of Amino Acids and Cofactors for Collagen Biosynthesis.

Common beans (Phaseolus vulgaris L.) are widely consumed in diets all over the world and have a significant impact on human health. Proteins, vitamins, minerals, phytochemicals, and other micro- and macronutrients are abundant in these legumes. On the other hand, collagens, the most important constituent of extracellular matrices, account for approximately 25-30 percent of the overall total protein composition within the human body. Hence, the presence of amino acids and other dietary components, including glycine, proline, and lysine, which are constituents of the primary structure of the protein, is required for collagen formation. In this particular context, protein quality is associated with the availability of macronutrients such as the essential amino acid lysine, which can be acquired from meals containing beans. Lysine plays a critical role in the process of post-translational modifications facilitated with enzymes lysyl hydroxylase and lysyl oxidase, which are directly involved in the synthesis and maturation of collagens. Furthermore, collagen biogenesis is influenced by the cellular redox state, which includes important minerals and bioactive chemicals such as iron, copper, and certain quinone cofactors. This study provides a novel perspective on the significant macro- and micronutrients present in Phaseolus vulgaris L., as well as explores the potential application of amino acids and cofactors derived from this legume in the production of collagens and bioavailability. The utilization of macro- and micronutrients obtained from Phaseolus vulgaris L. as a protein source, minerals, and natural bioactive compounds could optimize the capacity to promote the development and durability of collagen macromolecules within the human body.

Pharmaceutical applications of cyclotides.

Cyclotides are cyclic peptides, present in several plant families, that show diverse biological properties. Structurally, cyclotides share a distinctive head-to-tail circular knotted topology of three disulfide bonds. This framework provides cyclotides with extraordinary resistance to thermal and chemical denaturation. There is increasing interest in the therapeutic potential of cyclotides, which combine several promising pharmaceutical properties, including binding affinity, target selectivity, and low toxicity towards healthy mammalian cells. Recently, cyclotides have been reported to be orally bioavailable and have proved to be amenable to modifications. Here, we provide an overview of the structure, properties, and pharmaceutical applications of cyclotides.

Computational Studies of Snake Venom Toxins.

Most snake venom toxins are proteins, and participate to envenomation through a diverse array of bioactivities, such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic or neurotoxic effects. The venom of a single snake species contains hundreds of toxins, and the venoms of the 725 species of venomous snakes represent a large pool of potentially bioactive proteins. Despite considerable discovery efforts, most of the snake venom toxins are still uncharacterized. Modern bioinformatics tools have been recently developed to mine snake venoms, helping focus experimental research on the most potentially interesting toxins. Some computational techniques predict toxin molecular targets, and the binding mode to these targets. This review gives an overview of current knowledge on the ~2200 sequences, and more than 400 three-dimensional structures of snake toxins deposited in public repositories, as well as of molecular modeling studies of the interaction between these toxins and their molecular targets. We also describe how modern bioinformatics have been used to study the snake venom protein phospholipase A2, the small basic myotoxin Crotamine, and the three-finger peptide Mambalgin.

Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake.

Chlorotoxin (CTX), a disulfide-rich peptide from the scorpion Leiurus quinquestriatus, has several promising biopharmaceutical properties, including preferential affinity for certain cancer cells, high serum stability, and cell penetration. These properties underpin its potential for use as a drug design scaffold, especially for the treatment of cancer; indeed, several analogs of CTX have reached clinical trials. Here, we focus on its ability to internalize into cells-a trait associated with a privileged subclass of peptides called cell-penetrating peptides-and whether it can be improved through conservative substitutions. Mutants of CTX were made using solid-phase peptide synthesis and internalization into human cervical carcinoma (HeLa) cells was monitored by fluorescence and confocal microscopy. CTX_M1 (ie, [K15R/K23R]CTX) and CTX_M2 (ie, [K15R/K23R/Y29W]CTX) mutants showed at least a twofold improvement in uptake compared to CTX. We further showed that these mutants internalize into HeLa cells largely via an energy-dependent mechanism. Importantly, the mutants have high stability, remaining intact in serum for over 24 h; thus, retaining the characteristic stability of their parent peptide. Overall, we have shown that simple conservative substitutions can enhance the cellular uptake of CTX, suggesting that such type of mutations might be useful for improving uptake of other peptide toxins.

Evaluation of plant growth promoting activity and heavy metal tolerance of psychrotrophic bacteria associated with maca (Lepidium meyenii Walp.) rhizosphere.

The high Andean plateau of Peru is known to suffer harsh environmental conditions. Acidic soils containing high amount of heavy metals due to mining activities and withstanding very low temperatures affect agricultural activities by diminishing crop quality and yield. In this context, plant growth promoting rhizobacteria (PGPR) adapted to low temperatures and tolerant to heavy metals can be considered as an environment-friendly biological alternative for andean crop management. The aim of this work was to select and characterize psychrotrophic PGPR isolated from the rhizosphere of maca (Lepidium meyenii Walp.) a traditional andean food crop. A total of 44 psychrotrophic strains isolated from 3 areas located in the Bombon plateu of Junin-Peru were tested for their PGPR characteristics like indole acetic acid (IAA) production, phosphate solubilization and for their ability to improve seed germination. In addition, their capacity to grow in the presence of heavy metals like cadmium (Cd), lead (Pb), cobalt (Co) and mercury (Hg) was tested. Of the total number of strains tested, 12 were positive for IAA production at 22 °C, 8 at 12 °C and 16 at 6 °C. Phosphate solubilization activities were higher at 12 °C and 6 °C than at 22 °C. Red clover plant assays showed that 16 strains were capable to improve seed germination at 22 °C and 4 at 12 °C. Moreover, 11 strains showed tolerance to Cd and Pb at varying concentrations. This study highlight the importance of obtaining PGPRs to be used in high andean plateu crops that are exposed to low temperatures and presence of heavy metals on soil.

Chlorotoxin: Structure, activity, and potential uses in cancer therapy.

Chlorotoxin is a disulfide-rich stable peptide from the venom of the Israeli scorpion Leiurus quinquestriatus, which has potential therapeutic applications in the treatment of cancer. Its ability to preferentially bind to tumor cells has been harnessed to develop an imaging agent to help visualize tumors during surgical resection. In addition, chlorotoxin has attracted interest as a vehicle to deliver anti-cancer drugs specifically to cancer cells. Given its interesting structural and biological properties, chlorotoxin also has the potential to be used in a variety of other biotechnology and biomedical applications. Here, we review the structure, activity and potential applications of chlorotoxin as a drug design scaffold.

The role of disulfide bonds in structure and activity of chlorotoxin.

BACKGROUND: Chlorotoxin is a small scorpion peptide that inhibits glioma cell migration. We investigated the importance of a major component of chlorotoxin's chemical structure - four disulfide bonds - to its tertiary structure and biological function. RESULTS: Five disulfide bond analogs of chlorotoxin were synthesized, with l-α-aminobutyric acid residues replacing each or all of the disulfide bonds. Chemical oxidation and circular dichroism experiments revealed that Cys III-VII and Cys V-VIII were essential for native structure formation. Cys I-IV and Cys II-VI were important for stability of enzymatic proteolysis but not for the inhibition of human umbilical vein endothelial cell migration. CONCLUSION: The disulfide bonds of chlorotoxin are important for its structure and stability and have a minor role in its activity against cell migration.

Racemic and quasi-racemic X-ray structures of cyclic disulfide-rich peptide drug scaffolds.

Cyclic disulfide-rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X-ray structures of these peptides to assist in drug design applications, but disulfide-rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L- and D-forms of three prototypic cyclic disulfide-rich peptides: SFTI-1 (14-mer with one disulfide bond), cVc1.1 (22-mer with two disulfide bonds), and kB1 (29-mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25 Što 1.9 Å. Additionally, we obtained the quasi-racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25 Šand 2.3 Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides.

Resolution of the direct interaction with and inhibition of the human GLUT1 hexose transporter by resveratrol from its effect on glucose accumulation.

Resveratrol acts as a chemopreventive agent for cancer and as a potential antiobesity and antidiabetic compound, by leading to reduced body fat and improved glucose homeostasis. The exact mechanisms involved in improving hyperglycemic state are not known, but most of the glucose uptake into mammalian cells is facilitated by the GLUT hexose transporters. Resveratrol is structurally similar to isoflavones such as genistein, which inhibit the glucose uptake facilitated by the GLUT1 hexose transporter. Here we examined the direct effects of resveratrol on glucose uptake and accumulation in HL-60 and U-937 leukemic cell lines, which express mainly GLUT1, under conditions that discriminate transport from the intracellular substrate phosphorylation/accumulation. Resveratrol blocks GLUT1-mediated hexose uptake and thereby affects substrate accumulation on these cells. Consequently, we characterized the mechanism involved in inhibition of glucose uptake in human red cells. Resveratrol inhibits glucose exit in human red cells, and the displacement of previously bound cytochalasin B revealed the direct interaction of resveratrol with GLUT1. Resveratrol behaves as a competitive blocker of glucose uptake under zero-trans exit and exchange kinetic assays, but it becomes a mixed noncompetitive blocker when zero-trans entry transport was assayed, suggesting that the binding site for resveratrol lies on the endofacial face of the transporter. We propose that resveratrol interacts directly with the human GLUT1 hexose transporter by binding to an endofacial site and that this interaction inhibits the transport of hexoses across the plasma membrane. This inhibition is distinct from the effect of resveratrol on the intracellular phosphorylation/accumulation of glucose.

Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site.

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.

Hexose transporter GLUT1 harbors several distinct regulatory binding sites for flavones and tyrphostins.

The facilitative hexose transporter GLUT1 activity is blocked by tyrosine kinase inhibitors that include natural products such as flavones and isoflavones and synthetic compounds such as tyrphostins, molecules that are structurally unrelated to the transported substrates [Vera, et al. (2001) Biochemistry, 40, 777-790]. Here we analyzed the interaction of GLUT1 with quercetin (a flavone), genistein (an isoflavone), and tyrphostin A47 and B46 to evaluate if they share one common or have several binding sites on the protein. Kinetic assays showed that genistein, quercetin, and tyrphostin B46 behave as competitive inhibitors of equilibrium exchange and zero-trans uptake transport and noncompetitive inhibitors of net sugar exit out of human red cells, suggesting that they interact with the external surface of the GLUT1 molecule. In contrast, tyrphostin A47 was a competitive inhibitor of equilibrium exchange and zero-trans exit transport and a noncompetitive inhibitor of net sugar entry into red cells, suggesting that it interacts with the cytoplasmic surface of the transporter. Genistein protected GLUT1 against iodide-elicited fluorescence quenching and also decreased the affinity of d-glucose for its external binding site, while quercetin and tyrphostins B46 and A47 promoted fluorescence quenching and did not affect the external d-glucose binding site. These findings are explained by a carrier that presents at least three binding sites for tyrosine kinase inhibitors, in which (i) genistein interacts with the transporter in a conformation that binds glucose on the external surface (outward-facing conformation), in a site which overlaps with the external binding site for d-glucose, (ii) quercetin and tyrphostin B46 interact with the GLUT1 conformation which binds glucose by the internal side of the membrane (inward-facing conformation), but to a site accessible from the external surface of the protein, and (iii) the binding site for tyrphostin A47 is accessible from the inner surface of GLUT1 by binding to the inward-facing conformation of the transporter. These data provide groundwork for a molecular understanding of how the tyrosine kinase inhibitors directly affect glucose transport in animal cells.

Endofacial competitive inhibition of the glucose transporter 1 activity by gossypol.

Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a K(i) value of 20 microM. Likewise, GLUT1 fluorescence was quenched approximately 80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of D-glucose. Gossypol did not alter the affinity of D-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero-trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.