RESUMEN

Brown spider envenomation results in dermonecrosis, characterized by an intense inflammatory reaction. The principal toxins of brown spider venoms are phospholipase-D isoforms, which interact with different cellular membrane components, degrade phospholipids, and generate bioactive mediators leading to harmful effects. The Loxosceles intermedia phospholipase D, LiRecDT1, possesses a loop that modulates the accessibility to the active site and plays a crucial role in substrate. In vitro and in silico analyses were performed to determine aspects of this enzyme's substrate preference. Sphingomyelin d18:1/6:0 was the preferred substrate of LiRecDT1 compared to other Sphingomyelins. Lysophosphatidylcholine 16:0/0:0 was preferred among other lysophosphatidylcholines, but much less than Sphingomyelin d18:1/6:0. In contrast, phosphatidylcholine d18:1/16:0 was not cleaved. Thus, the number of carbon atoms in the substrate plays a vital role in determining the optimal activity of this phospholipase-D. The presence of an amide group at C2 plays a key role in recognition and activity. In silico analyses indicated that a subsite containing the aromatic residues Y228 and W230 appears essential for choline recognition by cation-π interactions. These findings may help to explain why different cells, with different phospholipid fatty acid compositions exhibit distinct susceptibilities to brown spider venoms.

Asunto(s)

Fosfolipasa D , Venenos de Araña , Arañas , Animales , Esfingomielinas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Fosfolipasa D/metabolismo , Venenos de Araña/química , Fosfolípidos/metabolismo , Lisofosfatidilcolinas , Arañas/metabolismo

RESUMEN

PAX8 is a master transcription factor that is essential during embryogenesis and promotes neoplastic growth. It is expressed by the secretory cells lining the female reproductive tract, and its deletion during development results in atresia of reproductive tract organs. Nearly all ovarian carcinomas express PAX8, and its knockdown results in apoptosis of ovarian cancer cells. To explore the role of PAX8 in these tissues, we purified the PAX8 protein complex from nonmalignant fallopian tube cells and high-grade serous ovarian carcinoma cell lines. We found that PAX8 was a member of a large chromatin remodeling complex and preferentially interacted with SOX17, another developmental transcription factor. Depleting either PAX8 or SOX17 from cancer cells altered the expression of factors involved in angiogenesis and functionally disrupted tubule and capillary formation in cell culture and mouse models. PAX8 and SOX17 in ovarian cancer cells promoted the secretion of angiogenic factors by suppressing the expression of SERPINE1, which encodes a proteinase inhibitor with antiangiogenic effects. The findings reveal a non-cell-autonomous function of these transcription factors in regulating angiogenesis in ovarian cancer.

Asunto(s)

Neoplasias Ováricas , Factor de Transcripción PAX8 , Factores de Transcripción SOXF , Factores de Transcripción , Animales , Trompas Uterinas/metabolismo , Trompas Uterinas/patología , Femenino , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Ratones , Clasificación del Tumor , Neoplasias Ováricas/metabolismo , Factor de Transcripción PAX8/genética , Factor de Transcripción PAX8/metabolismo , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

AIMS: The present study aimed to develop and evaluate a new dairy by-product nutritional supplement with Buriti fruit to improve malnutrition in mice and elderly woman. BACKGROUND: Malnutrition is a prevalent problem in the elderly; therefore, oral dietary supplementation is an important strategy to reduce this health problem incidence. OBJECTIVE: The present study evaluated the effects of a low-cost food supplement, made from byproducts of the dairy and fruit industry in the Brazilian Cerrado (Buriti), on the nutritional status and on the recovery of the metabolic profile of malnourished animals and elderly women. METHODS: In the pre-clinical phase, Swiss mice were divided into six groups and subjected to malnutrition and renutrition. The clinical phase was carried out with 25 elderly women residing in a long-term institution, aged ≥ 65 years and with malnutrition or risk of malnutrition. RESULTS: The main results showed improvements in anthropometric parameters and an increase in serum albumin levels, in addition to lipid profile improvement in the preclinical phase and an increase in the red blood cells and hemoglobin in the clinical phase. CONCLUSION: The supplement based on Buriti was able to reverse malnutrition promoting improvements in anthropometric and biochemical parameters.

Asunto(s)

Arecaceae , Desnutrición , Anciano , Animales , Brasil/epidemiología , Suplementos Dietéticos , Femenino , Frutas , Humanos , Desnutrición/diagnóstico , Desnutrición/prevención & control , Ratones , Estado Nutricional

RESUMEN

Critical developmental "master transcription factors" (MTFs) can be subverted during tumorigenesis to control oncogenic transcriptional programs. Current approaches to identifying MTFs rely on ChIP-seq data, which is unavailable for many cancers. We developed the CaCTS (Cancer Core Transcription factor Specificity) algorithm to prioritize candidate MTFs using pan-cancer RNA sequencing data. CaCTS identified candidate MTFs across 34 tumor types and 140 subtypes including predictions for cancer types/subtypes for which MTFs are unknown, including e.g. PAX8, SOX17, and MECOM as candidates in ovarian cancer (OvCa). In OvCa cells, consistent with known MTF properties, these factors are required for viability, lie proximal to superenhancers, co-occupy regulatory elements globally, co-bind loci encoding OvCa biomarkers, and are sensitive to pharmacologic inhibition of transcription. Our predictions of MTFs, especially for tumor types with limited understanding of transcriptional drivers, pave the way to therapeutic targeting of MTFs in a broad spectrum of cancers.

RESUMEN

Hyaluronidases are low expressed toxins of brown spider venoms, but, as highly active molecules, they present an important role as spreading factors. By degrading extracellular matrix components, these enzymes favor the diffusion of toxins in the affected tissue and at systemic level. Here, a novel isoform of hyaluronidase of Loxosceles intermedia Mello-Leitão (1934) venom was cloned, expressed in a baculovirus-insect cell expression system and fully active purified. This recombinant enzyme, named LiHyal2 (Loxosceles intermedia Hyaluronidase isoform 2), shares high identity with hyaluronidases of other spiders and scorpions. The catalytic and sugar binding amino acid residues are conserved in LiHyal2, human, and honeybee venom hyaluronidases and the molecular model of LiHyal2 shares major similarities with their crystal structures, including the active site. LiHyal2 was expressed as a 45 kDa protein and degraded hyaluronic acid (HA) and chondroitin sulphate as demonstrated by HA zymography and agarose gel electrophoresis. Lectin blot analysis revealed that LiHyal2 is post-translationally modified by the addition of high mannose N-linked carbohydrates. In vivo experiments showed that LiHyal2 potentialize dermonecrosis and edema induced by a recombinant phospholipase-D (PLD) of L. intermedia venom, as well as enhance the increase in capillary permeability triggered by this PLD, indicating that these toxins act synergistically during envenomation. Altogether, these results introduce a novel approach to express spider recombinant toxins, contribute to the elucidation of brown spider venom mechanisms and add to the development of a more specific treatment of envenomation victims.

Asunto(s)

Hialuronoglucosaminidasa , Fosfolipasa D , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Dominio Catalítico , Humanos , Hialuronoglucosaminidasa/genética , Hialuronoglucosaminidasa/metabolismo , Insectos/metabolismo , Hidrolasas Diéster Fosfóricas

RESUMEN

BACKGROUND: Malnutrition induced by dietary restriction produces several metabolic changes that affect body weight, the digestive system, and annex organs, including the liver. Malnutrition generates an inflammatory state and increases oxidative stress. The liver is one of the body vital organs, becoming necessary to analyze the impact of food supplementation on the repair of possible changes that may occur in this organ due to malnutrition. AIMS: To evaluate the effects of a low-cost supplementation derived from Buriti and dairy byproducts on liver recovery in malnourished mice, focusing on the expression of oxidative stressrelated genes, as well as biochemical and histological parameters. METHODS: Swiss mice were divided into six groups and submitted to two treatment phases: food restriction, for malnutrition onset; and renutrition, with mice being fed with different diets. RESULTS: Our results indicate that dietary supplementation was successful in recovering liver damage caused by malnutrition in animal models. The new supplement has been shown to recover liver damage with similar or superior results compared to the commercial reference supplement on the market. CONCLUSION: Our work presents a new composition of low cost food supplement based on buriti and dairy by-products, proven to be effective in the malnutrition treatment of malnutrition. The improvements were proven through the recovery of body weight, reduction of inflammation and oxidative stress.

Asunto(s)

Arecaceae/metabolismo , Productos Lácteos/análisis , Hepatopatías/dietoterapia , Hígado/lesiones , Desnutrición/complicaciones , Animales , Arecaceae/química , Peso Corporal , Suplementos Dietéticos/análisis , Frutas/química , Frutas/metabolismo , Humanos , Hepatopatías/etiología , Hepatopatías/metabolismo , Hepatopatías/fisiopatología , Masculino , Ratones , Estrés Oxidativo

RESUMEN

Paired Box 8 (PAX8) is a lineage-specific transcription factor that has essential roles during embryogenesis and tumorigenesis. The importance of PAX8 in the development of the reproductive system is highlighted by abnormalities observed upon the loss or mutation of this PAX family member. In cancer, PAX8 expression is deregulated in a key set of neoplasms, including those arising from the Müllerian ducts. The roles of PAX8 in oncogenesis are diverse and include epigenetic remodeling, stimulation of proliferation, inhibition of apoptosis, and regulation of angiogenesis. PAX8 can interact with different protein partners during cancer progression and may exhibit significant function-altering alternative splicing. Moreover, expression of PAX8 in cancer can also serve as a biomarker for diagnostic and prognostic purposes. In this review, we focus on the roles of PAX8 in cancers of the reproductive system. Understanding the diverse mechanisms of action of PAX8 in development and oncogenesis may identify new vulnerabilities in malignancies that currently lack effective therapies.

Asunto(s)

Neoplasias de los Genitales Femeninos/genética , Neoplasias de los Genitales Masculinos/genética , Factor de Transcripción PAX8/fisiología , Animales , Biomarcadores de Tumor/genética , Carcinogénesis/genética , Femenino , Neoplasias de los Genitales Femeninos/diagnóstico , Neoplasias de los Genitales Femeninos/patología , Neoplasias de los Genitales Femeninos/terapia , Neoplasias de los Genitales Masculinos/diagnóstico , Neoplasias de los Genitales Masculinos/patología , Neoplasias de los Genitales Masculinos/terapia , Humanos , Masculino , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , Pronóstico

RESUMEN

Sirtuins (SIRTs) are a protein family with high preservation degree among evolutionary scale. SIRTs are histone deacetylases regulatory enzymes of genetic material deeply involved in numerous physiological tasks including metabolism, brain function and aging. Mammals sirtuins comprise seven enzymatic components (SIRT1-SIRT7). The highest studied sirtuin is SIRT1, which plays an essential position in the prevention and evolution of neuro-disorders. Resveratrol (3,5,4-trihydroxystylbene) (RSV) is a polyphenol, which belongs to a family compounds identified as stilbenes, predominantly concentrated in grapes and red wine. RSV is the must studied Sirtuin activator and is used as food supplementary compound. Resveratrol exhibits strong antioxidant activity, reducing free radicals, diminishing quinone-reductase-2 activity and exerting positive regulation of several endogenous enzymes. Resveratrol is also able to inhibit pro-inflammatory factors, reducing the stimulation of the nuclear factor kB (NF-kB) and the release of endogenous cytokines. Resveratrol treatment can modulate multiple signaling pathway effectors related to programmed cell death, cell survival, and synaptic plasticity. In this context, the present review looks over news and the role of Sirtuins activation and resveratrol effects on modulating target genes, cognition and neurodegenerative disorders.

RESUMEN

Brown spider envenomation results in dermonecrosis with gravitational spreading characterized by a marked inflammatory reaction and with lower prevalence of systemic manifestations such as renal failure and hematological disturbances. Several toxins make up the venom of these species, and they are mainly peptides and proteins ranging from 5-40 kDa. The venoms have three major families of toxins: phospholipases-D, astacin-like metalloproteases, and the inhibitor cystine knot (ICK) peptides. Serine proteases, serpins, hyaluronidases, venom allergens, and a translationally controlled tumor protein (TCTP) are also present. Toxins hold essential biological properties that enable interactions with a range of distinct molecular targets. Therefore, the application of toxins as research tools and clinical products motivates repurposing their uses of interest. This review aims to discuss possibilities for brown spider venom toxins as putative models for designing molecules likely for therapeutics based on the status quo of brown spider venoms. Herein, we explore new possibilities for the venom components in the context of their biochemical and biological features, likewise their cellular targets, three-dimensional structures, and mechanisms of action.

Asunto(s)

Hidrolasas Diéster Fosfóricas , Venenos de Araña , Analgésicos/farmacología , Animales , Antiinflamatorios/farmacología , Antineoplásicos/farmacología , Humanos , Inmunoterapia , Insecticidas/farmacología , Fármacos Neuroprotectores/farmacología , Péptidos/farmacología , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/farmacología , Proteínas Recombinantes/farmacología , Inhibidores de Serina Proteinasa/farmacología , Venenos de Araña/química , Venenos de Araña/farmacología , Proteína Tumoral Controlada Traslacionalmente 1

RESUMEN

Objetivo: descrever as vias de parto a partir dos indicadores de assistência. Método: estudo quantitativo, epidemiológico descritivo, documental, desenvolvido a partir dos indicadores obstétricos neonatais das parturientes atendidas em uma maternidade. O instrumento de coleta de dados foi por meio de consulta aos dados disponíveis por cópias das planilhas do aplicativo Microsoft Excel® 2010. Os dados foram apresentados em tabelas. Resultados: a taxa de parto normal prevaleceu, enquanto a via cesariana foi acima do preconizado pela Organização Mundial de Saúde. Conclusão: achados não diferem de dados encontrados na literatura e evidenciam a necessidade de transformação do modelo de atenção ao pré-natal e ao parto.(AU)

Asunto(s)

Humanos , Femenino , Embarazo , Niño , Adolescente , Adulto , Cesárea , Parto Humanizado , Parto , Partería , Parto Normal , Enfermería Obstétrica , Estudios Epidemiológicos , Educación en Salud , Salud Materno-Infantil

RESUMEN

Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (4-40 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field.

RESUMEN

Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Proteínas de Artrópodos/antagonistas & inhibidores , Araña Reclusa Parda/enzimología , Diseño de Fármacos , Fosfolipasa D/antagonistas & inhibidores , Venenos de Araña/antagonistas & inhibidores , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Bencimidazoles/farmacología , Araña Reclusa Parda/genética , Araña Reclusa Parda/patogenicidad , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hemólisis/efectos de los fármacos , Humanos , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Necrosis , Fosfolipasa D/química , Fosfolipasa D/genética , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Piperidinas/farmacología , Conejos , Proteínas Recombinantes/genética , Piel/efectos de los fármacos , Piel/patología , Picaduras de Arañas/tratamiento farmacológico , Picaduras de Arañas/enzimología , Venenos de Araña/química , Venenos de Araña/genética , Suramina/farmacología

RESUMEN

Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from Loxosceles intermedia and Corynebacterium pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. J. Cell. Biochem. 118:2053-2063, 2017. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Proteínas de Artrópodos/toxicidad , Proteínas Bacterianas/toxicidad , Permeabilidad Capilar/efectos de los fármacos , Corynebacterium pseudotuberculosis/química , Hidrolasas Diéster Fosfóricas/toxicidad , Arañas/química , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Corynebacterium pseudotuberculosis/enzimología , Corynebacterium pseudotuberculosis/patogenicidad , Eritrocitos/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Hemólisis/efectos de los fármacos , Caballos , Humanos , Ratones , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Oveja Doméstica , Piel/efectos de los fármacos , Piel/patología , Arañas/enzimología , Arañas/patogenicidad

RESUMEN

Abstract Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (440 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field.

RESUMEN

Abstract Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (4-40 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field.(AU)

Asunto(s)

Animales , Venenos de Araña , Arañas , Toxicología , Metaloproteasas , Serina Proteasas

RESUMEN

Inhibitor cystine knots (ICKs) are a family of structural peptides with a large number of cysteine residues that form intramolecular disulfide bonds, resulting in a knot. These peptides are involved in a variety of biological functions including predation and defense, and are found in various species, such as spiders, scorpions, sea anemones, and plants. The Loxosceles intermedia venom gland transcriptome identified five groups of ICK peptides that represent more than 50 % of toxin-coding transcripts. Here, we describe the molecular cloning of U2-Sicaritoxin-Lit2 (U2-SCRTX-Lit2), bioinformatic characterization, structure prediction, and molecular dynamic analysis. The sequence of U2-SCRTX-Lit2 obtained from the transcriptome is similar to that of µ-Hexatoxin-Mg2, a peptide that inhibits the insect Nav channel. Bioinformatic analysis of sequences classified as ICK family members also showed a conservation of cysteine residues among ICKs from different spiders, with the three dimensional molecular model of U2-SCRTX-Lit2 similar in structure to the hexatoxin from µ-hexatoxin-Mg2a. Molecular docking experiments showed the interaction of U2-SCRTX-Lit2 to its predictable target-the Spodoptera litura voltage-gated sodium channel (SlNaVSC). After 200 ns of molecular dynamic simulation, the final structure of the complex showed stability in agreement with the experimental data. The above analysis corroborates the existence of a peptide toxin with insecticidal activity from a novel ICK family in L. intermedia venom and demonstrates that this peptide targets Nav channels.

Asunto(s)

Miniproteínas Nodales de Cistina/química , Modelos Moleculares , Venenos de Araña/química , Arañas/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína

RESUMEN

Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of symptoms found in victims after accidents is called loxoscelism and is characterized by two clinical conditions: cutaneous loxoscelism and systemic loxocelism. The only specific treatment is serum therapy, in which an antiserum produced with Loxosceles venom is administered to the victims after spider accidents. Our aim was to improve our knowledge, regarding the immunological relationship among toxins from the most epidemiologic important species in Brazil (Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta). Immunoassays using spider venoms and L. intermedia recombinant toxins were performed and their cross-reactivity assessed. The biological conservation of the main Loxosceles toxins (Phospholipases-D, Astacin-like metalloproteases, Hyaluronidase, ICK-insecticide peptide and TCTP-histamine releasing factor) were investigated. An in silico analysis of the putative epitopes was performed and is discussed on the basis of the experimental results. Our data is an immunological investigation in light of biological conservation throughout the Loxosceles genus. The results bring out new insights on brown spider venom toxins for study, diagnosis and treatment of loxoscelism and putative biotechnological applications concerning immune conserved features in the toxins.

Asunto(s)

Antivenenos/inmunología , Venenos de Araña/inmunología , Arañas , Animales , Proteínas de Artrópodos/química , Biología Computacional , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Venenos de Araña/química , Venenos de Araña/enzimología , Proteína Tumoral Controlada Traslacionalmente 1

RESUMEN

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP2) to phosphoinositide-3,4,5-trisphosphate (PIP3), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP2, a membrane lipid.

RESUMEN

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.

Asunto(s)

Fosfatidilinositol 3-Quinasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Compuestos de Boro/química , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Conformación Proteica , Células Sf9 , Transducción de Señal , Spodoptera , Dominios Homologos src

RESUMEN

INTRODUCTION: The levels of cadmium (Cd) and lead (Pb) in foods should be monitored as a function of health risks. OBJECTIVE: To evaluate Cd and Pb levels in oral hospital diets and in an oral food complement (OFC) according to their respective consumption by patients, and to estimate the patient's exposition risk. METHODS: The levels of Cd and Pb were determined by ICP-OES in samples of regular, blend, soft and renal diets and OFC, collected on 6 weekdays. About 14.3% of the diets and OFC served were analyzed. RESULTS AND DISCUSSION: 163 patients participated, with mean weights and ages of 62.7 kg and 56.5 years, respectively, the majority being men (59.5%). The mean Cd content consumed was greater for men fed the regular and blend diets and similar amongst the sexes for the soft diet. The consumption of Cd (max. 21.02 µg/day) was below the provisional tolerable monthly intake (PTMI). The mean Pb ingested (max. 199.49 µg/day) was similar amongst the sexes. The soft diet showed the highest Pb content in September/2010, whereas the other showed no variation according to season. In September/2010 and January/2011, the soft and regular diets associated with the OFC offered 207.50 and 210.50 µg/day of Pb, respectively. CONCLUSIONS: The combination of the diet with the OFC increased the risk of an excessive ingestion of Pb, and the vulnerability of the patients to an excessive exposition to Pb could be greater due to water and medications. It was concluded that whereas the calculated ingestion of Cd conformed to the PTMI, the Pb level and ingestion represented a risk to the health of the patients.

Asunto(s)

Cadmio/análisis , Plomo/análisis , Brasil , Dieta , Análisis de los Alimentos , Servicio de Alimentación en Hospital , Humanos , Medición de Riesgo