Carlos Munoz-Garay - Academia.edu (original) (raw)

Papers by Carlos Munoz-Garay

European journal of medicinal chemistry, Dec 1, 2017

Therapeutic investigations of novel indoxyl-based indolines: A drug target validation and structu... more Therapeutic investigations of novel indoxyl-based indolines: A drug target validation and structureactivity relationship of angiotensin-converting enzyme inhibitors with cardiovascular regulation and thrombolytic potential,

Biophysical Journal, Feb 1, 2016

Biochemistry, Dec 12, 2003

During intoxication, the Cry protoxins must change from insoluble crystals into membraneinserted ... more During intoxication, the Cry protoxins must change from insoluble crystals into membraneinserted toxins, which form ionic pores. Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer. In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion. Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed. Cry1Ab has nine Trp residues, seven of them in pore-forming domain I. Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent. Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%). Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in λ max of the emission spectrum, indicating a more hydrophobic environment for some Trp residues. In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane. Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membranewater interface. Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns. These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.

The Journal of Membrane Biology, Jul 1, 2006

Pesticide Biochemistry and Physiology, Oct 1, 2012

ABSTRACT Cry and Cyt toxins produced by Bacillus thuringiensis are insecticidal proteins used wor... more ABSTRACT Cry and Cyt toxins produced by Bacillus thuringiensis are insecticidal proteins used worldwide to control different insect pests, either as spray products or expressed in transgenic Bt-crops. Extensive studies have shown that they are safe to the environment and non-toxic to other organisms. However, still there are concerns regarding to the potential impact of Bt-crops on non-target organisms. One possibility to counteract the action of Cry or Cyt toxins is the use of dominant negative mutants also called anti-toxin that could inhibit the toxicity of their corresponding native toxin. Additionally, the evolution of insect resistance threatens the effectiveness of Bt-crops, since several examples of insects resistant to Bt-crops have been recently documented. The study of the mode of action of Cry and Cyt toxins has shown that oligomerization is an important step to form pores in the target midgut cells to eventually kill the larvae. This knowledge allowed us to develop strategies to produce toxin mutants that act as anti-toxins to counter Cry or Cyt toxins action in specific environments and to construct CryMod toxins that are able to kill insects that developed resistance to native Cry toxins.We will review the constructions of Cry and Cyt anti-toxins. The non-toxic helix α-4 mutants of Cry1Ab are able to oligomerize and to interact with native toxin forming inactive hetero-oligomers, inhibiting toxicity of native Cry1Ab. On the other hand, the N-terminal domain of Cyt1Aa induces oligomerization of native toxin, and inhibits its toxicity against mosquitoes as well as its hemolytic activity.Regarding insect resistance, we will review the construction of Cry1AMod toxins lacking helix α-1 that are able to form oligomers in absence of binding to cadherin receptor. CryMod toxins kill resistant insects to native Bt toxins affected in cadherin. We will review and discuss recent data that indicated that CryMod toxins overcome other mechanisms of resistance.Overall the mutants reviewed here support that oligomerization is an important step in the mechanism of action of Cry and Cyt toxins.

Southwestern Entomologist, Oct 1, 2010

The Cry proteins produced by Bacillus thuringiensis Berliner are effective bioinsecticides agains... more The Cry proteins produced by Bacillus thuringiensis Berliner are effective bioinsecticides against different insect pests. The primary threat to their long-term use is evolution of resistance by pests. Cry toxins are produced as protoxins activated in larval-gut lumen. The toxins bind to receptors that induce oligomerization and insertion into the membrane forming ionic pores that kill the cells and the larvae. The Cry1AMod toxins were created by genetic engineering of Cry1A by the deletion of the N-terminal end. These proteins kill resistant insects, in particular, those that have eliminated or modified one of the toxin receptors. Cry1AMod toxins could have potential to be used to control insect resistance. However, for the future use of these proteins in the field, it is important to demonstrate their mechanism of action is similar to the wild-type Cry1A toxins. Here we summarize results regarding the mode of action of CryMod toxins showing they have a similar mechanism as wild-type Cry toxins.

Revista Digital Universitaria

Las evaluaciones estandarizadas en el ámbito educativo tienen una larga historia, que inicia a pr... more Las evaluaciones estandarizadas en el ámbito educativo tienen una larga historia, que inicia a principios de siglo XX. El campo de la Psicología y de la Educación ha impactado enormemente el mundo educativo y, últimamente, ha servido para diseñar políticas públicas y para rendir cuentas a la sociedad. La principal característica de las pruebas estandarizadas es que pueden administrarse a una gran cantidad de personas, cuyas respuestas se califican de manera automática con dispositivos electrónicos. Su gran desventaja radica en que utilizan, principalmente, el formato de selección de respuestas que hace un tanto artificial la evaluación. A pesar de esta limitación, las evaluaciones estandarizadas se utilizan con una gran variedad de propósitos: desde la admisión a instituciones educativas hasta la evaluación de la calidad educativa de un país. A lo largo de su historia, las evaluaciones estandarizadas han sido objeto de críticas, algunas de ellas justas y otras no. Dada su importanci...

Methods in Molecular Biology, 2021

The development of new strategies for achieving stable asymmetric membrane models has turned inte... more The development of new strategies for achieving stable asymmetric membrane models has turned interleaflet lipid asymmetry into a topic of major interest. Cyclodextrin-mediated lipid exchange constitutes a simple and versatile method for preparing asymmetric membrane models without the need for sophisticated equipment. Here we describe a protocol for preparing asymmetric supported lipid bilayers mimicking membrane rafts by cyclodextrin-mediated lipid exchange and the main guidelines for obtaining structural information and quantitative measures of their mechanical properties using Atomic force microscopy and Force spectroscopy; two powerful techniques that allow membrane characterization at the nanoscale.

Biocatalysis and Biotransformation, 2020

Laccase treatment of phenolic compounds found in lignocellulosic hydrolysates is an alternative t... more Laccase treatment of phenolic compounds found in lignocellulosic hydrolysates is an alternative to reduce the growth inhibitory effect of these compounds on fermenting microorganisms. In order to determine the main factors that affect the efficiency of this biocatalytic approach, laccase oxidation of individual and mixtures of phenolic compounds was evaluated. Additionally, the effect of phenolic compounds on Saccharomyces cerevisiae cell physiology and their effect on yeast membrane liposome models were evaluated. Trametes versicolor laccase showed complete oxidation of six phenolic compounds, while Agaricus bisporus laccase showed oxidation percentages ranging between 30-100%. High Performance Liquid Chromatography (HPLC) analyses suggested that compounds such as syringic acid, catechol and gallic acid were not completely polymerised after laccase treatment, which could explain the increase of negative effects observed when they were added to S. cerevisiae cultures. Ferulic acid and vanillin oxidation led to a 6.4 and 6.5-fold increase in ethanol production, respectively, compared with the untreated cultures. In phenolic mixtures, chemical interactions between phenolic compounds led to biotransformation of these compounds since different by-products were observed in HPLC chromatograms. Moreover, after laccase treatment, the negative effects of these mixtures were reduced, resulting in an increase in ethanol production yields. No correlation was observed between growth inhibition data, the effect on liposomes and the detoxification efficiencies. Thus, the efficiency of laccase detoxification was not related to the toxicity of the compounds but could be conditioned by the heterogeneity in the chemical structure and properties of these compounds (functional side groups, hydrophobicity, pKa).

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2021

Sphingolipids-enriched rafts domains are proposed to occur in plasma membranes and to mediate imp... more Sphingolipids-enriched rafts domains are proposed to occur in plasma membranes and to mediate important cellular functions. Notwithstanding, the asymmetric transbilayer distribution of phospholipids that exists in the membrane confers the two leaflets different potentials to form lateral domains as next to no sphingolipids are present in the inner leaflet. How the physical properties of one leaflet can influence the properties of the other and its importance on signal transduction across the membrane are questions still unresolved. In this work, we combined AFM imaging and Force spectroscopy measurements to assess domain formation and to study the nanomechanical properties of asymmetric supported lipid bilayers (SLBs) mimicking membrane rafts. Asymmetric SLBs were formed by incorporating N-palmitoyl-sphingomyelin (16:0SM) into the outer leaflet of preformed 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Cholesterol SLBs through methyl-β-cyclodextrin-mediated lipid exchange. Lipid domains were detected after incorporation of 16:0SM though their phase state varied from gel to liquid ordered (Lo) phase if the procedure was performed at 24 or 37 °C, respectively. When comparing symmetric and asymmetric Lo domains, differences in size and morphology were observed, with asymmetric domains being smaller and more interconnected. Both types of Lo domains showed similar mechanical stability in terms of rupture forces and Young's moduli. Notably, force curves in asymmetric domains presented two rupture events that could be attributed to the sequential rupture of a liquid disordered (Ld) and a Lo phase. Interleaflet coupling in asymmetric Lo domains could also be inferred from those measurements. The experimental approach outlined here would significantly enhance the applicability of membrane models.

Biochemical Journal, 2019

Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) str... more Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) strains. Once in circulation, a low concentration of the toxin induces an increase in intracellular calcium that activates calpains — which proteolyse cytoskeleton proteins — and also favours the exposure of phosphatidylserine (PS) in the outer leaflet of erythrocyte membranes. All these events are considered part of eryptosis, as well as the delivery of microvesicles (MVs). Within this context, we studied the delivery of MVs by erythrocytes treated with sublytic concentrations of HlyA and demonstrated that HlyA-treated erythrocytes secrete MVs of diameter ∼200 nm containing HlyA and PS by a mechanism involving an increment of intracellular calcium concentration and purinergic receptor activation. Despite the presence of toxin in their membrane, HlyA-MVs are not hemolytically active and do not induce ATP release in untreated erythrocytes, thus suggesting that the delivery of HlyA-MVs might ...

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2019

Regulation of extracellular ATP of human erythrocytes treated with αhemolysin. Effects of cell vo... more Regulation of extracellular ATP of human erythrocytes treated with αhemolysin. Effects of cell volume, morphology, rheology and hemolysis.

The Journal of Membrane Biology, 2012

Journal of Biological Chemistry, 2007

Journal of Biological Chemistry, 2004

FEBS Letters, 2000

This study provides evidence for a novel mechanism of voltage‐gated Ca2+ channel regulation in ma... more This study provides evidence for a novel mechanism of voltage‐gated Ca2+ channel regulation in mammalian spermatogenic cells by two agents that affect sperm capacitation and the acrosome reaction (AR). Patch‐clamp experiments demonstrated that serum albumin induced an increase in Ca2+ T current density in a concentration‐dependent manner, and significant shifts in the voltage dependence of both steady‐state activation and inactivation of the channels. These actions were not related to the ability of albumin to remove cholesterol from the membrane. In contrast, β‐estradiol significantly inhibited Ca2+ channel activity in a concentration‐dependent and essentially voltage‐independent fashion. In mature sperm this dual regulation may influence capacitation and/or the AR.

Biochimica et Biophysica Acta (BBA) - General Subjects, 2013

Plasmodium berghei Background: Microbial antibiotic resistance is a challenging medical problem n... more Plasmodium berghei Background: Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity. Methods: Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured. Results: A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26−Α29), and K4N Δ(K12-Q18; Ν26−Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25 μM and 5 to 25 μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900 μM for their EC 50 , compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells. Conclusions: These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections. General significance: Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.

FEBS Letters, 1998

Several site directed mutations were introduced into a synthetic Noxiustoxin (NTX) gene. Alanine ... more Several site directed mutations were introduced into a synthetic Noxiustoxin (NTX) gene. Alanine scanning of the nonapeptide at the N‐terminal segment of NTX (threonine 1 (T1) to serine 9 (S9)) was constructed and the recombinant products were obtained in pure form. Additionally, lysine 28 (K28) was changed to arginine (R) or glutamic acid (E), cysteine 29 was changed to alanine, and residues 37–39 (Tyr‐Asn‐Asn) of the carboxyl end were deleted. The recombinant mutants were tested for their ability to displace 125I‐NTX from rat brain synaptosome membranes, as well as for their efficiency in blocking the activity of Kv1.1 K+ channels expressed in Xenopus laevis oocytes. The main results indicate that residues K6, T8 at the amino end, and K28 and the tripeptide YNN at the carboxyl end are involved in specific interactions of NTX with rat brain and/or Kv1.1 K+ channels.