Structural, biochemical and immunochemical characterization of an acidic phospholipase A2 from Lachesis acrochorda (Viperidae: Crotalinae) venom.

Viperids of the genus Lachesis, also known as bushmasters, are capable of injecting great amounts of venom that cause severe envenomation incidents. Since phospholipases type A2 are mainly involved in edema and myonecrosis within the snakebite sites, in this work, the isolation, amino acid sequence and biochemical characterization of the first phospholipase type A2 from the venom of Lachesis acrochorda, named Lacro_PLA2, is described. Lacro_PLA2 is an acidic aspartic 49 calcium-dependent phospholipase A2 with 93% similarity to the L. stenophrys phospholipase. Lacro_PLA2 has a molecular mass of 13,969.7 Da and an experimental isoelectric point around 5.3. A combination of N-terminal Edman degradation and MS/MS spectrometry analyses revealed that Lacro_PLA2 contains 122 residues including 14 cysteines that form 7 disulfide bridges. A predicted 3D model shows a high resemblance to other viperid phospholipases. Nevertheless, immunochemical and phospholipase neutralization tests revealed a notorious level of immunorecognition of the isolated protein by two polyclonal antibodies from viperids from different genus, which suggest that Lacro_PLA2 resembles more to bothropic phospholipases. Lacro_PLA2 also showed significantly high edema activity when was injected into mice; so, it could be an alternative antigen in the development of antibodies against toxins of this group of viperids, seeking to improve commercial polyclonal antivenoms.

Unveiling the Venom Composition of the Colombian Coral Snakes Micrurus helleri, M. medemi, and M. sangilensis.

Little is known of the biochemical composition and functional features of the venoms of poorly known Colombian coral snakes. Here, we provide a preliminary characterization of the venom of two Colombian endemic coral snake species, Micrurus medemi and M. sangilensis, as well as Colombian populations of M. helleri. Electrophoresis and RP-HPLC techniques were used to identify venom components, and assays were conducted to detect enzyme activities, including phospholipase A2, hyaluronidase, and protease activities. The median lethal dose was determined using murine models. Cytotoxic activities in primary cultures from hippocampal neurons and cancer cell lines were evaluated. The venom profiles revealed similarities in electrophoretic separation among proteins under 20 kDa. The differences in chromatographic profiles were significant, mainly between the fractions containing medium-/large-sized and hydrophobic proteins; this was corroborated by a proteomic analysis which showed the expected composition of neurotoxins from the PLA2 (~38%) and 3FTx (~17%) families; however, a considerable quantity of metalloproteinases (~12%) was detected. PLA2 activity and protease activity were higher in M. helleri venom according to qualitative and quantitative assays. M. medemi venom had the highest lethality. All venoms decreased cell viability when tested on tumoral cell cultures, and M. helleri venom had the highest activity in neuronal primary culture. These preliminary studies shed light on the venoms of understudied coral snakes and broaden the range of sources that could be used for subsequent investigations of components with applications to specific diseases. Our findings also have implications for the clinical manifestations of snake envenoming and improvements in its medical management.

Nitazoxanide Inhibits the Bifunctional Enzyme GlG6PD::6PGL of Giardia lamblia: Biochemical and In Silico Characterization of a New Druggable Target.

Giardiasis, which is caused by Giardia lamblia infection, is a relevant cause of morbidity and mortality worldwide. Because no vaccines are currently available to treat giardiasis, chemotherapeutic drugs are the main options for controlling infection. Evidence has shown that the nitro drug nitazoxanide (NTZ) is a commonly prescribed treatment for giardiasis; however, the mechanisms underlying NTZ's antigiardial activity are not well-understood. Herein, we identified the glucose-6-phosphate::6-phosphogluconate dehydrogenase (GlG6PD::6PGL) fused enzyme as a nitazoxanide target, as NTZ behaves as a GlG6PD::6PGL catalytic inhibitor. Furthermore, fluorescence assays suggest alterations in the stability of GlG6PD::6PGL protein, whereas the results indicate a loss of catalytic activity due to conformational and folding changes. Molecular docking and dynamic simulation studies suggest a model of NTZ binding on the active site of the G6PD domain and near the structural NADP+ binding site. The findings of this study provide a novel mechanistic basis and strategy for the antigiardial activity of the NTZ drug.

A subfraction obtained from the venom of the tarantula Poecilotheria regalis contains inhibitor cystine knot peptides and induces relaxation of rat aorta by inhibiting L-type voltage-gated calcium channels.

Venoms from tarantulas contain low molecular weight vasodilatory compounds whose biological action is conceived as part of the envenomation strategy due to its propagative effects. However, some properties of venom-induced vasodilation do not match those described by such compounds, suggesting that other toxins may cooperate with these ones to produce the observed biological effect. Owing to the distribution and function of voltage-gated ion channels in blood vessels, disulfide-rich peptides isolated from venoms of tarantulas could be conceived into potential vasodilatory compounds. However, only two peptides isolated from spider venoms have been investigated so far. This study describes for the first time a subfraction containing inhibitor cystine knot peptides, PrFr-I, obtained from the venom of the tarantula Poecilotheria regalis. This subfraction induced sustained vasodilation in rat aortic rings independent of vascular endothelium and endothelial ion channels. Furthermore, PrFr-I decreased calcium-induced contraction of rat aortic segments and reduced extracellular calcium influx to chromaffin cells by the blockade of L-type voltage-gated calcium channels. This mechanism was unrelated to the activation of potassium channels from vascular smooth muscle, since vasodilation was not affected in the presence of TEA, and PrFr-I did not modify the conductance of the voltage-gated potassium channel Kv10.1. This work proposes a new envenomating function of peptides from venoms of tarantulas, and establishes a new mechanism for venom-induced vasodilation.

Biochemical and Kinetic Characterization of the Glucose-6-Phosphate Dehydrogenase from Helicobacter pylori Strain 29CaP.

Helicobacter pylori (H. pylori) has been proposed as the foremost risk factor for the development of gastric cancer. We found that H. pylori express the enzyme glucose-6-phosphate dehydrogenase (HpG6PD), which participates in glucose metabolism via the pentose phosphate pathway. Thus, we hypothesized that if the biochemical and physicochemical characteristics of HpG6PD contrast with the host G6PD (human G6PD, HsG6PD), HpG6PD becomes a potential target for novel drugs against H. pylori. In this work, we characterized the biochemical properties of the HpG6PD from the H.pylori strain 29CaP and expressed the active recombinant protein, to analyze its steady-state kinetics, thermostability, and biophysical aspects. In addition, we analyzed the HpG6PD in silico structural properties to compare them with those of the HsG6PD. The optimal pH for enzyme activity was 7.5, with a T1/2 of 46.6 °C, at an optimum stability temperature of 37 °C. The apparent Km values calculated for G6P and NADP+ were 75.0 and 12.8 µM, respectively. G6P does not protect HpG6PD from trypsin digestion, but NADP+ does protect the enzyme from trypsin and guanidine hydrochloride (Gdn-HCl). The biochemical characterization of HpG6PD contributes to knowledge regarding H. pylori metabolism and opens up the possibility of using this enzyme as a potential target for specific and efficient treatment against this pathogen; structural alignment indicates that the three-dimensional (3D) homodimer model of the G6PD protein from H. pylori is different from the 3D G6PD of Homo sapiens.

Chemical structure of three basic Asp-49 phospholipases A2 isolated from Crotalus molossus nigrescens venom with cytotoxic activity against cancer cells.

Snake venoms are complex mixtures of molecules with several biological activities. Among these molecules, the enzymes with phospholipase A2 activity have been extensively studied in the venoms from snakes because of their importance in the envenomation process and symptoms. The Mexican rattlesnake Crotalus molossus nigrescens is widely distributed in the Mexican plateau. Unlike other crotalids, its venom components have been poorly studied. Here, we characterized the phospholipase activity of one fraction isolated from the venom of this snake and we determined the cytotoxic and neurotoxic effects on brain tumor cells and neuronal primary cultures, respectively. After reverse phase chromatography, we obtained a fraction which was analyzed by mass spectrometry showing higher activity than that from a PLA2 from bee venom used as control. This fraction was enriched with three basic Asp49 phospholipases with molecular masses of 12.5, 13.9 and 14.2 kDa. Their complete amino acid sequences were determined, and their predicted tertiary structures were generated using the model building softwares I-tasser and Chimera. Viability assays revealed that the fraction showed cytotoxic activity against brain tumor cells (C6, RG2 and Daoy) with IC50 values ranging between 10 and 100 ng/ml, whereas an IC50 > 100 ng/ml was exerted in rat primary astrocytes. These findings might be relevant in oncological medicine due to their potential as anticancer agents and low neurotoxic effects compared to conventional drugs.

Novel inhibitors of human glucose-6-phosphate dehydrogenase (HsG6PD) affect the activity and stability of the protein.

BACKGROUND: The pentose phosphate pathway (PPP) has received significant attention because of the role of NADPH and R-5-P in the maintenance of cancer cells, which are necessary for the synthesis of fatty acids and contribute to uncontrollable proliferation. The HsG6PD enzyme is the rate-limiting step in the oxidative branch of the PPP, leading to an increase in the expression levels in tumor cells; therefore, the protein has been proposed as a target for the development of new molecules for use in cancer. METHODS: Through in vitro studies, we assayed the effects of 55 chemical compounds against recombinant HsG6PD. Here, we present the kinetic characterization of four new HsG6PD inhibitors as well as their functional and structural effects on the protein. Furthermore, molecular docking was performed to determine the interaction of the best hits with HsG6PD. RESULTS: Four compounds, JMM-2, CCM-4, CNZ-3, and CNZ-7, were capable of reducing HsG6PD activity and showed noncompetitive and uncompetitive inhibition. Moreover, experiments using circular dichroism and fluorescence spectroscopy showed that the molecules affect the structure (secondary and tertiary) of the protein as well as its thermal stability. Computational docking analysis revealed that the interaction of the compounds with the protein does not occur at the active site. CONCLUSIONS: We identified two new compounds (CNZ-3 and JMM-2) capable of inhibiting HsG6PD that, compared to other previously known HsG6PD inhibitors, showed different mechanisms of inhibition. GENERAL SIGNIFICANCE: Screening of new inhibitors for HsG6PD with a future pharmacological approach for the study and treatment of cancer.

Glucose-6-Phosphatase and α-Glucosidase Inhibitors from Smilax moranensis Roots Identified by Affinity-Directed Fractionation.

The ethanol/water-soluble extracts of the roots of Smilax moranensis M. Martens & Galeotti, Smilacaceae, which have been appreciated since pre-Hispanic times and traditionally used to treat type 2 diabetes in Mexico, were fractionated by the application of the affinity-directed method to identify glucose-6-phosphatase and α-glucosidase inhibitors. Mass spectrometry was used to identify the inhibitor as free ligands after released from the enzymatic complex by denaturing acidic conditions. The affinity study led to the identification of chlorogenic acid as a glucose-6-phosphatase inhibitor, which is the most abundant metabolite present in the analyzed crude drug. In addition, the affinity studies led to the identification of a flavonolignan glycoside as an α-glucosidase inhibitor. In silico analysis with α-glucosidase MAL12 from the yeast Saccharomyces cerevisiae allowed to determine the best conformational rearrangement for the α-glucosidase inhibitors. Graphical abstract. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s43450-020-00116-3.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom.

BACKGROUND: Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. METHODS: The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. RESULTS: P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. CONCLUSION: These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom

Background Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom

Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods: The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results: P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion: These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.(AU)

Cnidarian Neurotoxic Peptides Affecting Central Nervous System Targets.

Natural products from animal venoms have been used widely in the discovery of novel molecules with particular biological activities that enable their use as potential drug candidates. The phylum Cnidaria (jellyfish, sea anemones, corals zoanthids, hydrozoans, etc.) is the most ancient venomous phylum on earth. Its venoms are composed of a complex mixture of peptidic compounds with neurotoxic and cytolitic properties that have shown activity on mammalian systems despite the fact that they are naturally targeted against fish and invertebrate preys, mainly crustaceans. For this reason, cnidarian venoms are an interesting and vast source of molecules with a remarkable activity on central nervous system, targeting mainly voltage-gated ion channels, ASIC channels, and TRPV1 receptors. In this brief review, we list the amino acid sequences of most cnidarian neurotoxic peptides reported to date. Additionally, we propose the inclusion of a new type of voltage-gated sea anemone sodium channel toxins based on the most recent reports.

Composition and biological activities of the aqueous extracts of three scleractinian corals from the Mexican Caribbean: Pseudodiploria strigosa, Porites astreoides and Siderastrea siderea

Background: Scleractinian corals (stony corals) are the most abundant reef-forming cnidarians found in coral reefs throughout the world. Despite their abundance and ecological importance, information about the diversity of their toxins and their biological activities is very scarce. In this study, the chemical composition and the biological activities of the aqueous extracts of Pseudodiploria strigosa, Porites astreoides and Siderastrea siderea, three scleractinian corals from the Mexican Caribbean, have been assessed for the first time. Methods: Toxicity of the extracts was assessed in crickets; the presence of cytolysins was detected by the hemolysis assay; the vasoconstrictor activity was determined by the isolated rat aortic ring assay; the nociceptive activity was evaluated by the formalin test. The presence of phospholipases A2 (PLA2), serine proteases, and hyaluronidases was determined by enzymatic methods. Low-molecular-weight fractions were obtained by gel filtration chromatography and ultrafiltration. Results: Extracts from the three species were toxic to crickets, induced hemolysis in human and rat erythrocytes, produced vasoconstriction on isolated rat aortic rings, and presented phospholipase A2 and serine-protease activity. Despite the fact that these corals are not considered to be harmless to humans, the extracts generated significant nociceptive responses. The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of the low-molecular-weight fractions revealed the presence of peptides within a mass range of 3000 to 6000 Da. These fractions were toxic to crickets and two of them induced a transitory vasoconstrictor effect on isolated rat aortic rings. Conclusion: This study suggests that scleractinian corals produce low-molecular-weight peptides that are lethal to crickets and induce vasoconstriction.(AU)

Composition and biological activities of the aqueous extracts of three scleractinian corals from the Mexican Caribbean: Pseudodiploria strigosa, Porites astreoides and Siderastrea siderea

Scleractinian corals (stony corals) are the most abundant reef-forming cnidarians found in coral reefs throughout the world. Despite their abundance and ecological importance, information about the diversity of their toxins and their biological activities is very scarce. In this study, the chemical composition and the biological activities of the aqueous extracts of Pseudodiploria strigosa, Porites astreoides and Siderastrea siderea, three scleractinian corals from the Mexican Caribbean, have been assessed for the first time. Methods: Toxicity of the extracts was assessed in crickets; the presence of cytolysins was detected by the hemolysis assay; the vasoconstrictor activity was determined by the isolated rat aortic ring assay; the nociceptive activity was evaluated by the formalin test. The presence of phospholipases A2 (PLA2), serine proteases, and hyaluronidases was determined by enzymatic methods. Low-molecular-weight fractions were obtained by gel filtration chromatography and ultrafiltration. Results: Extracts from the three species were toxic to crickets, induced hemolysis in human and rat erythrocytes, produced vasoconstriction on isolated rat aortic rings, and presented phospholipase A2 and serine-protease activity. Despite the fact that these corals are not considered to be harmless to humans, the extracts generated significant nociceptive responses. The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of the low-molecular-weight fractions revealed the presence of peptides within a mass range of 3000 to 6000 Da. These fractions were toxic to crickets and two of them induced a transitory vasoconstrictor effect on isolated rat aortic rings. Conclusion: This study suggests that scleractinian corals produce low-molecular-weight peptides that are lethal to crickets and induce vasoconstriction.

Micro identificación taxonómica y nuevos caracteres diagnósticos del erizo marino Astropyga pulvinata (Diadematoida: Diadematidae) / Taxonomic microidentification and new diagnostic characters of the sea urchin Astropyga pulvinata (Diadematoida: Diadematidae)

Resumen Astropyga pulvinata es un diademátido que posee cinco pares de series de puntos iridiscentes presentes en los interambulacros. A partir de la observación 20 individuos in vivo a través de un estereoscopio y el análisis con un Microscopio Electrónico de Barrido se identificaron los pedicelarios, las espículas y las rosetas calcáreas de los pies ambulacrales. Los pedicelarios presentes en A. pulvinata son trivalvados y de tres tipos: claviformes (oficéfalos) presentes en ambulacro; tridentados distribuidos en el peristoma; y trifoliados presentes en toda la testa excepto en los interambulacros. Los pedicelarios claviformes son los únicos que no están unidos por un pedúnculo calcáreo de tipo diadematoide. Se obtuvieron diez tipos de espículas de diversos tejidos blandos: uno del intestino; tres de la membrana anal y seis del peristoma, dos de las cuales no han sido descritas hasta el momento. Uno de estos nuevos tipos de espículas reportados posee forma de basto y otro de media luna. De las observaciones in vivo se encontraron puntos iridiscentes más pequeños presentes en el ambitus constituidos por aproximadamente ocho células.

Abstract Astropyga pulvinata is a sea urchin of the family Diadematidae that have five pairs of series of blue iridescent spots on interambulacral zones, from in vivo observation of 20 individuals under stereo-microscope and Scanning Electron Microscope analysis identification, the pedicellariae, spicules, and calcareous rosettes. The pedicellariae of A. pulvinata have three valves and are of three types: claviform, present on ambulacres; tridentates present on peristome and triphyllous present all over the test except in interambulacral areas. So ophicephalous pedicellariae are not bounded with a calcareous stalk. Ten types of spicules were observed from different soft tissues: one type from the intestine; three from the anal membrane; and six from the peristome, two of them constitute new diagnostic characters. One of them having a bat-shaped form and the other one with a waning crescent shape. In vivo observations showed that the blue iridescent spots were present on interambulacral areas as group of 35 cells bounded, and that other smaller spots were present on the ambitus, composed by eight cells. Rev. Biol. Trop. 63 (Suppl. 2): 49-58. Epub 2015 June 01.

Lipoprotein N-acyl transferase (Lnt1) is dispensable for protein O-mannosylation by Streptomyces coelicolor.

A protein glycosylation system related to that for protein mannosylation in yeast is present in many actinomycetes. This system involves polyprenyl phosphate mannose synthase (Ppm), protein mannosyl transferase (Pmt), and lipoprotein N-acyl transferase (Lnt). In this study, we obtained a series of mutants in the ppm (sco1423), lnt1 (sco1014), and pmt (sco3154) genes of Streptomyces coelicolor, which encode Ppm, Lnt1, and Pmt, to analyze their requirement for glycosylation of the heterologously expressed Apa glycoprotein of Mycobacterium tuberculosis. The results show that both Ppm and Pmt were required for Apa glycosylation, but that Lnt1 was dispensable for both Apa and the bacteriophage φC31 receptor glycosylation. A bacterial two-hybrid assay revealed that contrary to M. tuberculosis, Lnt1 of S. coelicolor does not interact with Ppm. The D2 catalytic domain of M. tuberculosisPpm was sufficient for complementation of an S. coelicolor double mutant lacking Lnt1 and Ppm, both for Apa glycosylation and for glycosylation of φC31 receptor. On the other hand, M. tuberculosisPmt was not active in S. coelicolor, even when correctly localized to the cytoplasmic membrane, showing fundamental differences in the requirements for Pmt activity in these two species.

Purification and characterization of structural and functional properties of two lectins from a marine sponge Spheciospongia vesparia.

The purification, structural and functional characterization of two different lectins (named Svl-1 and Svl-2) has been reported from the marine sponge Spheciospongia vesparia. Purification procedure includes ammonium sulfate precipitation, combined with chromatography including Octyl-Sepharose-(NH4)SO4 hydrophobic column and DEAE-Toyopearl anion-exchange column using a high performance liquid chromatography. The similarities in function, specificity for saccharides, molecular weight, amino acid content and the N-terminal sequence of two lectins suggest that these proteins are isolectins. Amino acid composition and fluorescence analyses reveal that they contain an intrachain disulfide bridge, which might contribute to their high thermal stability. Furthermore, the purified lectins exhibit antibacterial activity against the gram-negative bacteria Pseudomonas aeruginosa and E. coli, indicating that they may be involved in a recognition strategy and may play a role in the defense response function of the sponge. This is the first report on the isolation of lectins from the S. vesparia. The purified lectins represent a potential possible candidate for future application in the recognition or treatment of cancer cells.

Role of vitamin B12 on methylmalonyl-CoA mutase activity.

Vitamin B(12) is an organometallic compound with important metabolic derivatives that act as cofactors of certain enzymes, which have been grouped into three subfamilies depending on their cofactors. Among them, methylmalonyl-CoA mutase (MCM) has been extensively studied. This enzyme catalyzes the reversible isomerization of L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (AdoCbl) as a cofactor participating in the generation of radicals that allow isomerization of the substrate. The crystal structure of MCM determined in Propionibacterium freudenreichii var. shermanii has helped to elucidate the role of this cofactor AdoCbl in the reaction to specify the mechanism by which radicals are generated from the coenzyme and to clarify the interactions between the enzyme, coenzyme, and substrate. The existence of human methylmalonic acidemia (MMA) due to the presence of mutations in MCM shows the importance of its role in metabolism. The recent crystallization of the human MCM has shown that despite being similar to the bacterial protein, there are significant differences in the structural organization of the two proteins. Recent studies have identified the involvement of an accessory protein called MMAA, which interacts with MCM to prevent MCM's inactivation or acts as a chaperone to promote regeneration of inactivated enzyme. The interdisciplinary studies using this protein as a model in different organisms have helped to elucidate the mechanism of action of this isomerase, the impact of mutations at a functional level and their repercussion in the development and progression of MMA in humans. It is still necessary to study the mechanisms involved in more detail using new methods.

Bioactive peptides from marine organisms: a short overview.

Marine organisms are an immense source of new biologically active compounds. These compounds are unique because the aqueous environment requires a high demand of specific and potent bioactive molecules. Diverse peptides with a wide range of biological activities have been discovered, including antimicrobial, antitumoral, and antiviral activities and toxins amongst others. These proteins have been isolated from different phyla such as Porifera, Cnidaria, Nemertina, Crustacea, Mollusca, Echinodermata and Craniata. Purification techniques used to isolate these peptides include classical chromatographic methods such as gel filtration, ionic exchange and reverse-phase HPLC. Multiple in vivo and in vitro bioassays are coupled to the purification process to search for the biological activity of interest. The growing interest to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer drugs now in clinical trials. This review presents examples of interesting peptides obtained from different marine organisms that have medical relevance. It also presents some of the common methods used to isolate and characterize them.

Glucose exerts a negative effect over a peroxidase from Trichosporon asahii, with carotenoid cleaving activity.

Tobacco aroma compounds were generated via lutein cleavage by the combined action of a yeast and a bacterium identified as Trichosporon asahii and Paenibacillus amylolyticus, respectively. In this study, an inverse relationship between glucose concentration and the generation of three compounds, present in the tobacco aroma profile, was observed in mixed cultures. In order to identify the organism sensitive to the sugar effect, both were grown separately. The presence of glucose suppressed beta-ionone production by T. asahii grown with lutein. However, the biotransformation of the ionone into its reduced derivatives (7,8-dihydro-beta-ionone and 7,8-dihydro-beta-ionol) by P. amylolyticus was not affected by the sugar. This pointed to the cleavage of lutein, a step within the process necessary for the synthesis of beta-ionone, as the target of the glucose effect. In vitro studies with crude extracts and concentrated cell-free medium derived from T. asahii cultures showed that the carotenoid breakdown activity was located extracellularly and only detected in supernatants from yeast cells grown in the absence of the sugar. Rather than an inhibition or a mechanism affecting the enzyme secretion, the glucose effect on lutein degradation comprised another regulatory level. Further experiments showed that the enzyme responsible for lutein breakdown and susceptible to the sugar effect exhibited a high degree of identity to fungal peroxidases, studied as well, for their involvement in carotenoid cleavage.