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The extensive use of antibiotics in livestock farming poses increased concerns for human health as residues of these substances are present in edible tissues. The aim of this study was the determination of the levels of four groups of antibiotics (sulfonamides-SAs, tetracyclines-TCs, streptomycines-STr and quinolones-QNLs) in meat samples (muscles, livers and kidneys from beef, chicken and pork) and the estimation of the dietary exposure to antibiotics from meat consumption and the potential hazard for human health. Fifty-four samples of raw meat were randomly collected in 2018 from the Cretan market, Greece and analyzed both with an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS). According to the results derived from the ELISA method, only 2% of the meat samples were free from antibiotics, 2% were detected with 4 antibiotics and the great majority of the samples (87%) were detected with 2 to 3 antibiotics. SAs presented the highest detection frequencies for all samples whereas TCs were not detected in any bovine sample. The highest median concentration was detected for STr in bovine muscles (182.10 µg/kg) followed by QNLs (93.36 µg/kg) in pork kidneys whereas the chicken samples had higher burdens of QNLs compared to the other meat samples. LC-MS analysis showed that oxytetracycline (OTC) was the most common antibiotic in all samples. The highest median concentration of all antibiotics was detected for doxycycline (DOX) (181.73 µg/kg in pork kidney) followed by OTC in bovine liver (74.46 µg/kg). Risk characterization was applied for each of the two methods; The hazard quotients (HQ) did not exceed 0.059 for the ELISA method and 0.113 for the LC-MS method for any group of antibiotics, whereas the total hazard indexes (HI) were 0.078 and 0.021, respectively. The results showed the presence of different groups of antibiotics in meat from the Cretan market and that the health risk to antibiotics is low. A risk assessment analysis conducted for meat consumption and corrected for the aggregated exposure revealed no risk for the consumers.
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BACKGROUND: Parabens (PBs) and triclosan (TCS) are generally used as antimicrobials mostly in personal care products. Their wide prevalence in daily products raised an acute need for the biomonitoring of these contaminants and the investigation of possible health impacts. MATERIAL AND METHODS: In this study we aimed to quantitatively determine PBs and TCS levels in urine and amniotic fluid samples using a liquid chromatography - mass spectrometry system (LC-MS). Ninety nine (99) pregnant women took part in this research. The samples were collected during the amniocentesis in the early second trimester of their pregnancy. Women of all ages, education, household income and profession were selected. The exposure and the burden of pregnant women and their infants were also evaluated. RESULTS: The most prevalent compound in urine, among the analyzed, was TCS with 74.7 % positive samples while in amniotic fluid methyl paraben (MePB) with 21.2 % positive samples. MePB was detected at higher concentrations in urine (mean: 378.5 ng/mL) followed by TCS (mean: 55.3 ng/mL), ethyl paraben (EtPB) (mean: 23.2 ng/mL) and butyl paraben (BuPB) (mean: 2.3 ng/mL) while benzyl paraben (BePB) was not detected in any urine sample. Concentrations in amniotic fluid samples were much lower. In particular, the mean concentrations were 6.6 ng/mL for MePB, 9.2 ng/mL for EtPB, 0.4 ng/mL for BuPB, 0.6 ng/mL for BePB and 1.8 ng/mL for TCS. The detected levels of all analytes in urine were correlated with those in amniotic fluid but no statistically significant results arose (p >n0.05). Negative associations were observed between amniotic fluid levels of MePB and maternal age (p = 0.05) while both urinary and amniotic levels of TCS were correlated with maternal BMI (p = 0.04). Somatometric characteristics of the infants showed no statistical significant associations with the detected levels of PBs and TCS. CONCLUSION: This study indicated a strong/possible association between exposure of pregnant women to TCS and higher/lower maternal body weight gain during pregnancy. The same trend was observed between amniotic fluid MePB levels and maternal age. However, no statistically significant associations were observed between neonatal somatometric characteristics or health status and PBs and TCS levels.
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Phthalates are used in industry as plasticizers or additives in everyday products and they have been considered as endocrine disrupting chemicals. Maternal exposure during pregnancy has been associated with neonatal exposure, preterm birth and impacts in the reproductive and respiratory systems. The aim of this study is to determine six phthalate metabolites (mono isobutyl phthalate, miBP, mono n-butyl phthalate, mnBP, mono benzyl phthalate, mBzP, mono ethylhexyl phthalate, mEHP, mono 2-ethyl-5-hydroxyhexyl phthalate, mEHHP, mono 2-ethyl-5-oxohexyl-phthalate, mEOHP) in amniotic fluid and urine from 100 pregnant women. Participants answered questionnaires for the use of plastics and cosmetics, dietary habits, health effects, pregnancy problems, health and infant development. Positive amniotic fluid samples ranged from 1% to 21% and urine from 27% to 54%. The median levels for amniotic fluid were 2.3 µg/L - 10.7 µg/L and for urine 4.9 µg/L - 46.7 µg/L. The major results include significant correlations between urinary phthalates indicating their common sources of exposure, the frequent use of deodorant was significantly associated with higher urinary miBP (p = 0.050) and mnBP (p = 0.028) and a weak inverse association was found for the use of make-up products with mBzP (p = 0.053). The frequent use of plastic food containers was significantly associated with urinary mEHP (p = 0.026), and a positive trend was noticed for mEHP in amniotic fluid (p = 0.093). An association although weak was found between urinary mEHP and lower birth length (rs = 0.396, p = 0.062). No other associations were found for infant health problems or development. The daily intake of the total phthalates was calculated 5.4 µg/kg body weight/day which corresponds to hazard index 0.10 and exposure follows the declining trend that has been observed the last decades.
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Pesticides are widely used in agriculture and their proven high toxicity makes the need of monitoring their presence in food imperative. A multi residue method is applied in apples samples (81) collected from the Greek market for the monitoring of 40 analytes. Pesticides levels were evaluated by gas and liquid chromatography-mass spectrometry using a QuEChERs protocol. Risk for consumers was assessed by a newly developed methodology, employing the source related Hazard Quotient (HQs) and the adversity specific Hazard Index (HIA). The 84% of the apple's samples were positive for at least one pesticide, 21% for one pesticide, 55.6% for two to four pesticide residues and 7.4% for more than 4 pesticide residues. The most frequently detected compound was carbendazim (45.7%) followed by chlorpyrifos (44.4.0%). The mean detected concentration levels varied from 0.169 ppm (fluopyram) to 0.005 ppm (triazophos). 19 of the 40 investigated pesticides were not detected in any apple sample. For all individual pesticides, the source related Hazard Quotient (HQs) was <1 indicating no risk. HIA, resulting from the sum of all HQs was found HIA < 1 in nine out of ten toxicity groups, except to the neurotoxicity group, which presented HIA 2.258, indicating moderate risk.
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Contaminación de Alimentos/análisis , Fungicidas Industriales/análisis , Insecticidas/análisis , Malus/química , Residuos de Plaguicidas/análisis , Cromatografía Liquida/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Grecia , Medición de Riesgo , Espectrometría de Masas en Tándem/métodosRESUMEN
Phthalates, bisphenols A and S (BPA, BPS) are used as plasticizers and many of them are documented or suspected of being endocrine disruptors. Several studies indicate that exposure during pregnancy may affect the newborn's health and development. The aim of this cross-sectional study is the biomonitoring of seven phthalate metabolites, BPA and BPS in hair from 100 pregnant women in Crete. The most frequently detected compounds were monoethylhexyl phthalate (mEHP) (68%), mono isobutyl phthalate (miBP) (40%), BPA (37%), BPS (34%) and mono-n-butyl phthalate (mnBP) (28%). Phthalate metabolites were detected at medians from 19.5 to 44.4 pg/mg, BPA at 69.9 pg/mg and BPS at 3.5 pg/mg. Significant positive correlations between phthalate metabolites were found which indicated their common sources of exposure. The frequent use of plastics for food storage was strongly associated with mEHP (p = .013) and a weaker association was found for miBP (p = .063). The frequent use of cosmetics during or before pregnancy was associated with levels of phthalate metabolites in hair. More specifically, the use of hair spray before pregnancy was significantly correlated with monobenzyl phthalate (mBzP) (p = .041) and a trend was found for miBP (p = .066). The use of makeup products during pregnancy was strongly associated with miBP (p = .015) and the use of deodorant during pregnancy was inversely associated with mEHP (p = .021). Strong associations came up between mEHP and lower birth weight (Spearman correlation coefficient, r = -0.302, p = .021) and exposure to BPS was associated with increased body mass index of the participants (p = .036). Although data in literature on biomonitoring of the compounds in hair are limited, the findings of this study are promising and in agreement with existing data in hair or urine.
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Monitoreo Biológico , Estudios Transversales , Exposición a Riesgos Ambientales , Contaminantes Ambientales , Femenino , Grecia , Humanos , Recién Nacido , Ácidos Ftálicos , EmbarazoRESUMEN
The aim of this study was the monitoring of the levels of parabens (PBs) and triclosan (TCS) in head hair samples of women collected during the first months of their pregnancy. Personal details concerning somatometric and demographic characteristics, dietary habits, use of personal care products and the medical and obstetrical history of the pregnant women as well as infants' somatometric characteristics and health condition were recorded through relevant questionnaires. Ninety five hair samples were collected, extracted by solid-liquid extraction and analysed using a liquid chromatography-mass spectrometry system (LC-MS). Analysis revealed high percentage of positive samples for all tested compounds (90-100% except from BePB (15.8%)). The mean concentration levels were 4501.2â¯pg/mg (17.6-27,437.0â¯pg/mg) for MePB; 510.1â¯pg/mg (11.0-4224.5â¯pg/mg) for EtPB; 22.9â¯pg/mg (2.1-66.6â¯pg/mg) for BePB; 237.1â¯pg/mg (1.8-2513.7â¯pg/mg) for BuPB and 245.0â¯pg/mg (8.8-8070.2â¯pg/mg) for TCS. Statistical analysis of both analytical results and questionnaires' data showed that the frequent use of personal care and hygiene products, such as makeup, hairspray and sunscreens, is correlated with higher levels of PBs in hair of the pregnant women. Additionally, positive correlation was observed between the BePB levels in hair and the infants' height. Finally, no other correlation was observed between endocrine disruptors' levels in maternal hair and infants' somatometric characteristics or health condition. Our study is the first one that determined PBs and TCS levels in hair samples, simultaneously. At the same time, correlation of the detected levels with the use of personal care products was accomplished, leading to significant association of BePB levels in hair of pregnant women with infants' height.
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Disruptores Endocrinos , Contaminantes Ambientales/análisis , Análisis de Cabello , Exposición Materna/estadística & datos numéricos , Femenino , Cabello/química , Humanos , Parabenos , Embarazo , TriclosánRESUMEN
Human exposure to pesticides can be estimated through different approaches. The approach adopted in this study is based on internal dose measures. Studies published during 2001 and 2017 were collected from PubMed and Scopus databases, filtered and organized. The intake of parent compounds is estimated based on the urinary excretion of different OP metabolites applying a mathematical model previously used for similar purposes. Once defined an Estimated Daily Intake (EDI), risk assessment is performed through comparison with specific guideline values and hazard index (HI) is calculated to assess cumulative health risk. The EDI was expressed as malathion, diazinon, parathion, phorate and dimethoate equivalents. Differences in exposure between pregnant women, general population, children and farmers are highlighted and exposures are presented by country and sampling year. Higher exposure to OPs was calculated for farmers, followed by children whereas pregnant women were less exposed. Median HQ values for children ranged between 0.016 and 0.618, for pregnant women 0.005-0.151, for general population 0.008-0.206 and for farmers 0.009-0.979. Combined exposure to dimethoate and phorate was the worst-case scenario. The annual distribution of the urinary DAPs showed that exposure to OPs since 1998 tends to be stable for both children and adults.
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Compuestos Organofosforados/análisis , Compuestos Organofosforados/metabolismo , Plaguicidas/análisis , Plaguicidas/metabolismo , Animales , Exposición a Riesgos Ambientales/análisis , Monitoreo del Ambiente , Contaminación de Alimentos/análisis , HumanosRESUMEN
Bisphenol A (BPA), triclosan (TCS) and perfluorooctanoic acid (PFOA) are endocrine disruptors linked with negative health effects such as developmental, reproductive and cardiovascular toxicity. The aim of this study was to determine simultaneously the concentration of BPA, TCS and PFOA in hair from children and adults and examine possible associations between biomonitoring data and age, gender, dietary habits and body mass index. Methanolic extraction was applied and the compounds were determined by liquid chromatography-mass spectrometry. Low levels of exposure to PFOA were detected for children and adults at concentrations below limit of quantification. The mean concentration of BPA in children and adults was 20.6 and 16.6 pg mg-1 , while for TCS 275.2 and 687.0 pg mg-1 , respectively. Children were highly exposed to BPA relative to adults (P = .011) although adults had greater exposure to TCS (P = .003). Hair from girls had a greater burden of BPA (P = .06) compared to boys. Moreover, higher TCS levels were depicted for females in both examined groups (children P = .200 and adults P = .213) compared to males, but no statistical differences were observed. Significant differences were also observed between age groups (P = .0007) for TCS. No correlations were found between BPA or TCS levels and body mass index or dietary habits for both children and adults. Children have a greater exposure to BPA compared to adults, whereas exposure of adults to TCS seems to be higher than that in children and elderly people. Exposure to BPA occurs mainly via ingestion whereas exposure to TCS mainly via dermal absorption.
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Compuestos de Bencidrilo/análisis , Caprilatos/análisis , Disruptores Endocrinos/análisis , Monitoreo del Ambiente/métodos , Fluorocarburos/análisis , Cabello/química , Fenoles/análisis , Triclosán/análisis , Adulto , Factores de Edad , Índice de Masa Corporal , Niño , Conducta Alimentaria , Grecia , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores SexualesRESUMEN
The substance 4-methylimidazole (4-MEI) has raised several concerns regarding its toxicity to humans, although no harmonized classification has yet been decided. The regulatory limits for food products set by various authorities in Europe and the USA differ considerably. The purpose of the present study is to compare two liquid chromatography techniques in order to determine the levels of 4-MEI in food products from the Greek market and roughly estimate the possible exposure and relevant health risk for the consumers. A total of thirty-four samples (soft drinks, beers, balsamic vinegars, energy drinks and sauces) were collected and analyzed. The quality parameters for both analytical methodologies (linearity, accuracy, inter day precision, recovery) are presented. No detectable levels of 4-MEI are found in beers and soft drink samples, other than cola type. On the other hand, 4-MEI was detected in all cola type soft drinks (15.8-477.0 ng/ml), energy drinks (57.1%, 6.6-22.5 ng/ml) and vinegar samples (66.7%, 9.7-3034.7 ng/ml), while only one of the sauce samples was found to have a detectable level of 17.5 ng/ml 4-MEI.
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Bebidas/análisis , Bebidas Gaseosas/análisis , Cromatografía Líquida de Alta Presión/métodos , Contaminación de Alimentos/análisis , Imidazoles/análisis , Ácido Acético/análisis , Bebidas/economía , Bebidas Gaseosas/economía , Contaminación de Alimentos/economía , Grecia , HumanosRESUMEN
Bisphenol A (BPA) is one of the most common industrial chemicals and known to exert endocrine disruption activity. The aim of this study was the quantification of BPA in food stuffs on the Greek market. The applied liquid chromatography-mass spectrometry method was validated for linearity, limit of quantification, accuracy, precision and recovery. About 41.7% of the canned solid phase samples, 25.0% of the canned liquid phase samples and 43.8% of the soft drinks were positive. Mean BPA concentrations (range) were 33.4 ± 4.4 ng/g (4.90 ± 0.64-66.0 ± 8.6 ng/g) in canned solid phase, 2.70 ± 0.08 ng/ml (1.90 ± 0.06-3.50 ± 0.11 ng/ml) in canned liquid phase and 2.30 ± 0.18 ng/ml (0.40 ± 0.03-10.2 ± 0.8 ng/ml) in soft drinks. The results of this study are comparable with those reported in the literature according to which higher concentrations of BPA were detected in the solid fraction of canned food compared to their liquid fraction.
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Compuestos de Bencidrilo/análisis , Bebidas Gaseosas/análisis , Disruptores Endocrinos/análisis , Alimentos en Conserva/análisis , Fenoles/análisis , Cromatografía Liquida/métodos , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Grecia , Espectrometría de Masas/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Phthalates are ubiquitous environmental contaminants which are used in industry as plasticizers and additives in cosmetics. They are classified as Endocrine Disrupting Chemicals (EDCs) which impair the human endocrine system inducing fertility problems, respiratory diseases, childhood obesity and neuropsychological disorders. The aim of this review is to summarize the current state of knowledge on the toxicity that phthalates pose in humans based on human biomonitoring studies conducted over the last decade. Except for conventional biological matrices (such as urine and serum), amniotic fluid, human milk, semen, saliva, sweat, meconium and human hair are also employed for the estimation of exposure and distribution of pollutants in the human body, although data are not enough yet. Children are highly exposed to phthalates relative to adults and in most studies children's daily intake surpasses the maximum reference dose (RfD) set from US Environmental Protection Agency (US EPA). However, the global trend is that human exposure to phthalates is decreasing annually as a result of the strict regulations applied to phthalates.
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Disruptores Endocrinos/análisis , Exposición a Riesgos Ambientales/análisis , Contaminantes Ambientales/análisis , Ácidos Ftálicos/análisis , Adulto , Disruptores Endocrinos/toxicidad , Monitoreo del Ambiente , Contaminantes Ambientales/toxicidad , Salud Global , Humanos , Ácidos Ftálicos/toxicidadRESUMEN
Ingestion of gastrointestinal foreign bodies represents a challenging clinical scenario. Increased morbidity is the price for the delayed diagnosis of complications and timely treatment. We present a case of 57-year-old female patient which was admitted in the emergency room department complaining of a mid-epigastric pain over the last twenty-four hours. Based on the patient's history, physical examination and elevated serum amylase levels, a false diagnosis of pancreatitis, was initially adopted. However, a CT scan confirmed the presence of a radiopaque foreign body in the pancreatic head and the presence of air bubbles outside the intestinal lumen. The patient was unaware of the ingestion of the foreign body. At laparotomy, after an oblique duodenotomy, a fish bone pinned in the pancreatic head after the penetration of the medial aspect of the second portion of the duodenal wall was identified and successfully removed. The patient had an uneventful postoperative recovery. Wide variation in clinical presentation characterizes the complicated fish bone ingestions. The strategically located site of penetration in the visceral wall is responsible for the often extraordinary gastrointestinal tract injury patterns. Increased level of suspicion is of paramount importance for the timely diagnosis and treatment.
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BACKGROUND: The V3 loop of the glycoprotein gp120 of HIV-1 plays an important role in viral entry into cells by utilizing as coreceptor CCR5 or CXCR4, and is implicated in the phenotypic tropisms of HIV viruses. It has been hypothesized that the interaction between the V3 loop and CCR5 or CXCR4 is mediated by electrostatics. We have performed hierarchical clustering analysis of the spatial distributions of electrostatic potentials and charges of V3 loop structures containing consensus sequences of HIV-1 subtypes. RESULTS: Although the majority of consensus sequences have a net charge of +3, the spatial distribution of their electrostatic potentials and charges may be a discriminating factor for binding and infectivity. This is demonstrated by the formation of several small subclusters, within major clusters, which indicates common origin but distinct spatial details of electrostatic properties. Some of this information may be present, in a coarse manner, in clustering of sequences, but the spatial details are largely lost. We show the effect of ionic strength on clustering of electrostatic potentials, information that is not present in clustering of charges or sequences. We also make correlations between clustering of electrostatic potentials and net charge, coreceptor selectivity, global prevalence, and geographic distribution. Finally, we interpret coreceptor selectivity based on the N6X7T8|S8X9 sequence glycosylation motif, the specific positive charge location according to the 11/24/25 rule, and the overall charge and electrostatic potential distribution. CONCLUSIONS: We propose that in addition to the sequence and the net charge of the V3 loop of each subtype, the spatial distributions of electrostatic potentials and charges may also be important factors for receptor recognition and binding and subsequent viral entry into cells. This implies that the overall electrostatic potential is responsible for long-range recognition of the V3 loop with coreceptors CCR5/CXCR4, whereas the charge distribution contributes to the specific short-range interactions responsible for the formation of the bound complex. We also propose a scheme for coreceptor selectivity based on the sequence glycosylation motif, the 11/24/25 rule, and net charge.
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Chlorosomes, the antenna complexes of green bacteria, are unique antenna systems in which pigments are organized in aggregates. Studies on isolated chlorosomes from Chlorobaculum tepidum based on SDS-PAGE, immunoblotting and molecular biology have revealed that they contain ten chlorosomal proteins, but no comprehensive information is available about the protein composition of the entire organelle. To extend these studies, chlorosomes were isolated from C. tepidum using three related and one independent isolation protocol and characterized by absorption spectroscopy, tricine SDS-PAGE, dynamic light scattering (DLS) and electron microscopy. Tricine SDS-PAGE showed the presence of more than 20 proteins with molecular weights ranging between 6 and 70 kDa. The chlorosomes varied in size. Their hydrodynamic radius (R(h) ) ranged from 51 to 75 nm and electron microscopy indicated that they were on average 140 nm wide and 170 nm long. Furthermore, the mass of 184 whole chlorosome organelles determined by scanning transmission electron microscopy ranged from 27 to 237 MDa being on average 88 (±28) MDa. In contrast their mass-per-area was independent of their size, indicating that there is a strict limit to chlorosome thickness. The average protein composition of the C. tepidum chlorosome organelles was obtained by MS/MS-driven proteomics and for the first time a detailed protein catalogue of the isolated chlorosomal proteome is presented. Based on the proteomics results for chlorosomes isolated by different protocols, four proteins that are involved in the electron or ion transport are proposed to be tightly associated with or incorporated into C. tepidum chlorosomes as well as the ten Csm proteins known to date.
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Proteínas Bacterianas/química , Chlorobi/química , Chlorobi/citología , Espectrometría de Masas/métodos , Orgánulos/química , Orgánulos/ultraestructura , Proteómica/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Luz , Microscopía Electrónica/métodos , Datos de Secuencia Molecular , Proteoma/análisisRESUMEN
Due to the neurotoxicity of organophosphate (OP) pesticides and nerve agents synthesized as military or terror agents, their safe destruction and disposal is of considerable current importance. A representative OP, trimethyl phosphate (TMP), was adsorbed onto NaX zeolite, two mesoporous modifications, and a low-silica X zeolite. The nucleophilic chemical reactions of TMP with the zeolites were investigated by solid-state 13C and 31P nuclear magnetic resonance (NMR) and the solvent extracts by 1H, 13C, and 31P NMR. Nucleophilic substitution and subsequent hydrolysis reaction schemes are proposed. All of the zeolites have similar TMP decomposition yields, supporting the hypothesis that slow or incomplete diffusion of TMP in the microporous zeolite regions limits TMP decomposition.
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Restauración y Remediación Ambiental/métodos , Organofosfatos/química , Zeolitas/química , Adsorción , Espectroscopía de Resonancia Magnética , Porosidad , Agua/químicaRESUMEN
The third variable (V3) loop is an important region of glycoprotein 120 (gp120) for many biological processes, as it contains the highly conserved GPGR sequence and it represents the binding site for human immunodeficiency virus 1 (HIV-1) antibodies and for CCR5 and CXCR4 host cell coreceptors. The interaction of the principal neutralizing determinant (PND) V3 with the chemokine receptor CCR5 N-terminal region has been reported to be crucial for HIV-1 infection. The goal of this study is to characterize the solution structures of three HIV-1 gp120 V3 subtype B peptides and their interaction with a nonsulfated N-terminal CCR5 peptide. NMR titration experiments revealed that the CCR5Nt-PND V3 interaction is dependent on the number of the positively charged V3 residues, which is in agreement with the observation that increase in positive charge in the V3 sequence correlates with the augmentation of the interaction. As expected for free peptides in solution, the peptides representing the PND V3 region of gp120 exhibit conformational flexibility, but they also exhibit a large number of NOEs which allowed convergence to a dominant conformation. The PND V3 peptides retain the U-turn conformation observed in the crystal structures of gp120 complexes independently of CCR5 presence. The interaction of different regions of the CCR5Nt peptide is gradually increasing proportionally to the positive charge increase in the V3 peptides. The data demonstrate that the PND V3 and CCR5Nt peptide sequences have propensities for interaction even in the absence of sulfated tyrosines and that their binding and selectivity is determined by simple electrostatic attraction mechanisms.
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Fragmentos de Péptidos/química , Receptores CCR5/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Protones , Electricidad EstáticaRESUMEN
The chlorophyll (Chl)-containing membrane protein complexes from the green alga Scenedesmus obliquus have been isolated from the thylakoid membranes by solubilization with dodecyl-beta-maltoside and fractionation using a sucrose density gradient. The Chl-containing protein fractions were characterized by absorption spectroscopy, tricine SDS PAGE, BN-PAGE, and dynamic light scattering (DLS). BN-PAGE showed the presence of seven protein complexes with molecular weights in the range of 68, 118, 157, 320, 494, 828 and 955 kDa, respectively. Furthermore, light scattering reveals the simultaneous presence of particles of different sizes in the 3-4 nm and 6.0-7.5 nm range, respectively. The smaller size is related to the hydrodynamic radius of the trimer Light Harvesting Complex (LHCII), whereas the larger size is associated with the presence of photosystem I and photosystem II reaction centers. Additionally, functional information regarding protein-protein interactions was deconvoluted using coupling 2-D BN-PAGE, MALDI-TOF MS and a detailed mapping of S. obliquus photosynthetic proteome of the solubilized thylakoid membranes is therefore presented.
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Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteoma/química , Proteoma/metabolismo , Scenedesmus/metabolismo , Tilacoides/química , Tilacoides/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/metabolismo , Scenedesmus/químicaRESUMEN
The V3-loop of the HIV-1 gp120 alters host cell immune function and modulates infectivity. We investigated biophysical parameters of liposome constructs with embedded lipopeptides from the principle neutralizing domain of the V3-loop and their influence on viral infectivity. Dynamic light scattering measurements showed liposome supramolecular structures with hydrodynamic radius of the order of 900 and 1300nm for plain and V3-lipopeptide liposomes. Electron paramagnetic resonance measurements showed almost identical local microenvironment. The difference in liposome hydrodynamic radius was attributed to the fluctuating ionic environment of the V3-lipopeptide liposomes. In vitro HIV-1 infectivity assays showed that plain liposomes reduced virus production in all cell cultures, probably due to the hydrophobic nature of the aggregates. Liposomes carrying V3-lipopeptides with different cationic potentials restored and even enhanced infectivity (p<0.05). These results highlight the need for elucidation of the involvement of lipid bilayers as dynamic components in supramolecular structures and in HIV-1 fusion mechanisms.
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Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Lipoproteínas/química , Péptidos/química , Fenómenos Biofísicos , Biofisica , Espectroscopía de Resonancia por Spin del Electrón , Proteína p24 del Núcleo del VIH/biosíntesis , Humanos , LiposomasRESUMEN
Infection of CD4+ T cells by macrophage-tropic HIV-1 strains involves interaction of viral gp120 with the host cell chemokine receptor CCR5. The principle neutralizing determinant (PND) of the V3-loop of the HIV-1 gp120 was investigated for its interaction with CCR5 by computational modeling methods at atomic resolution and electrostatic calculations to complement experimental findings. The study focused on the recognition step and examined possible peptide-peptide interactions between various PND-derived peptides from the V3-loop and the N-terminal (Nt) domain of CCR5. These recognition interactions are possible because of the complementary character of the spatial distribution of the predominantly positive electrostatic potentials of the PND-derived peptides and the predominantly negative electrostatic potential of the CCR5Nt domain. The CCR5Nt appears more amenable to interaction with the V3 peptides, than the other CCR5 extracellular domains (ECL), because of its length and the domination of its negative electrostatic potential. On the contrary, ECL2 possesses a predominantly positive electrostatic potential. There are positive patches in Nt and negative patches in ECL2, which, following the non-specific recognition of the V3-loop by CCR5 and with the expected local structural rearrangements to facilitate specific binding, may be contributing to the stabilization of the complex. A sequential two-step specific binding, involving different extracellular domains, is conceivable. Although the electrostatic potentials may play a role in a V3-CCR5 interaction, a more specific model cannot be derived in the absence of a three-dimensional structure of a gp120/CD4/CCR5 complex.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Modelos Moleculares , Péptidos/química , Receptores CCR5/metabolismo , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores CCR5/química , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Infection of target host cells by the human immunodeficiency virus-1 (HIV-1) is a multi-step process involving a series of conformational changes in the viral gp120 and gp41 proteins. Gp120 binding to the main cell receptor, CD4, on the surface of cells expressing this molecule, and interaction with the cell chemokine receptors CCR5 and CXCR4, are among the key events for HIV-1 infection. These steps are crucial for the virus and offer potential therapeutic targets. For this reason, understanding the structure and the physicochemical characteristics of the gp120 in relation to these interactions has drawn much attention. This review article focuses on the biologically important V3 region of the gp120 and summarizes the functional role, the sequence variation and the conformational features of V3 peptides, which are important for co-receptor selectivity, specificity and interaction. Synthetic V3 peptides have been extensively studied by NMR spectroscopy and X-ray crystallography, in solution or in solid state, in their free or bound form, and valuable information was generated with the aim to be exploited in the design of new, effective inhibitors of HIV-1 infection. The features of the potential gp120 interacting sites on the two chemokine co-receptors, CCR5 and CXCR4, are also discussed, and co-receptor blocking molecules under clinical trial are also reported.