RESUMO
Azole-resistant Aspergillus fumigatus (ARAf) fungi have been found inconsistently in the environment in Denmark since 2010. During 2018-2020, nationwide surveillance of clinical A. fumigatus fungi reported environmental TR34/L98H or TR46/Y121F/T289A resistance mutations in 3.6% of isolates, prompting environmental sampling for ARAf and azole fungicides and investigation for selection of ARAf in field and microcosmos experiments. ARAf was ubiquitous (20% of 366 samples; 16% TR34/L98H- and 4% TR46/Y121F/T289A-related mechanisms), constituting 4.2% of 4,538 A. fumigatus isolates. The highest proportions were in flower- and compost-related samples but were not correlated with azole-fungicide application concentrations. Genotyping showed clustering of tandem repeat-related ARAf and overlaps with clinical isolates in Denmark. A. fumigatus fungi grew poorly in the field experiment with no postapplication change in ARAf proportions. However, in microcosmos experiments, a sustained complete (tebuconazole) or partial (prothioconazole) inhibition against wild-type A. fumigatus but not ARAf indicated that, under some conditions, azole fungicides may favor growth of ARAf in soil.
Assuntos
Antifúngicos , Aspergillus fumigatus , Azóis , Farmacorresistência Fúngica , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , Azóis/farmacologia , Dinamarca/epidemiologia , Antifúngicos/farmacologia , Humanos , Aspergilose/epidemiologia , Aspergilose/microbiologia , Aspergilose/tratamento farmacológico , Testes de Sensibilidade Microbiana , Mutação , Fungicidas Industriais/farmacologia , GenótipoRESUMO
Cercospora leaf spot (CLS) is a globally important disease of sugar beet (Beta vulgaris) caused by the fungus Cercospora beticola. Long-distance movement of C. beticola has been indirectly evidenced in recent population genetic studies, suggesting potential dispersal via seed. Commercial sugar beet "seed" consists of the reproductive fruit (true seed surrounded by maternal pericarp tissue) coated in artificial pellet material. In this study, we confirmed the presence of viable C. beticola in sugar beet fruit for 10 of 37 tested seed lots. All isolates harbored the G143A mutation associated with quinone outside inhibitor resistance, and 32 of 38 isolates had reduced demethylation inhibitor sensitivity (EC50 > 1 µg/ml). Planting of commercial sugar beet seed demonstrated the ability of seedborne inoculum to initiate CLS in sugar beet. C. beticola DNA was detected in DNA isolated from xylem sap, suggesting the vascular system is used to systemically colonize the host. We established nuclear ribosomal internal transcribed spacer region amplicon sequencing using the MinION platform to detect fungi in sugar beet fruit. Fungal sequences from 19 different genera were identified from 11 different sugar beet seed lots, but Fusarium, Alternaria, and Cercospora were consistently the three most dominant taxa, comprising an average of 93% relative read abundance over 11 seed lots. We also present evidence that C. beticola resides in the pericarp of sugar beet fruit rather than the true seed. The presence of seedborne inoculum should be considered when implementing integrated disease management strategies for CLS of sugar beet in the future.
Assuntos
Beta vulgaris , Cercospora , Beta vulgaris/microbiologia , Frutas , Doenças das Plantas/microbiologia , Açúcares , VerdurasRESUMO
The snow flea Ceratophysella sigillata, a winter-active species of springtail, produces unique polychlorinated octahydroisocoumarins to repel predators. The structure of the major compound, sigillinâ A, was elucidated through isolation, spectroscopic analysis, and X-ray crystallography. Sigillinâ A showed high repellent activity in a bioassay with predatory ants. A promising approach for the total synthesis of members of this new class of natural compounds was also developed.
Assuntos
Produtos Biológicos/química , Isocumarinas/química , Animais , Estrutura Molecular , SifonápterosRESUMO
BACKGROUND: Septoria tritici blotch caused by Zymoseptoria tritici continues to be one of the most economically destructive diseases of winter wheat in north-western Europe. Control is heavily reliant on the application of fungicides, in particular those belonging to the azole group. Here we describe the sensitivity of European Z. tritici populations to the novel azole mefentrifluconazole and the analysis of associated mechanisms of resistance. RESULTS: A wide range of sensitivity to mefentrifluconazole was observed amongst the Z. tritici collections examined, with strong cross-resistances also observed between mefentrifluconazole, difenoconazole and tebuconazole. Overall, the Irish population displayed the lowest sensitivity to all azoles tested. Further detailed analysis of the Irish population in 2021 demonstrated differences in sensitivity occurred between sampling sites, with these differences associated with the frequencies of key resistance mechanisms (CYP51 alterations and MFS1 promoter inserts linked to overexpression). Under glasshouse conditions reductions in the efficacy of mefentrifluconazole were observed towards those strains exhibiting the lowest in vitro sensitivities. CONCLUSIONS: This study demonstrates that a large range of sensitivity to mefentrifluconazole exists in European Z. tritici populations. Those strains exhibiting the lowest sensitivity to the azoles tested had the most complex CYP51 haplotypes in combination with the 519 bp insert, associated with enhanced activity of MFS1. The future use of mefentrifluconazole should take these findings into consideration to minimise the selection of these strains. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Ascomicetos , Fluconazol/análogos & derivados , Fungicidas Industriais , Ascomicetos/genética , Fungicidas Industriais/farmacologia , Azóis , Doenças das PlantasRESUMO
BACKGROUND: We investigated the spinopelvic parameters of lumbar lordosis (LL), pelvic incidence (PI), pelvic tilt (PT) and sacral slope (SS) in patients with fragility fractures of the pelvis (FFPs). We hypothesized that these parameters differ from asymptomatic patients. METHODS: All patients treated for FFPs in a center of maximal care of the German Spine Society (DWG) between 2017 and 2021 were included. The inclusion criteria were age ≥ 60 years and the availability of a standing lateral radiograph of the spine and pelvis in which the spine from T12 to S1 and both femoral heads were visible. The baseline characteristics and study parameters were calculated and compared with databases of asymptomatic patients. The two-sample t-Test was performed with p < 0.05. RESULTS: The study population (n = 57) consisted of 49 female patients. The mean age was 81.0 years. The mean LL was 47.9°, the mean PT was 29.4°, the mean SS was 34.2° and the mean PI was 64.4°. The mean value of LLI was 0.7. LL, LLI and SS were significantly reduced, and PI and PT were significantly increased compared to asymptomatic patients. CONCLUSIONS: The spinopelvic parameters in patients with FFPs differ significantly from asymptomatic patients. In patients with FFPs, LL, LLI and SS are significantly reduced, and PI and PT are significantly increased. The sagittal spinopelvic balance is abnormal in patients with FFPs.
RESUMO
BACKGROUND: Fenpicoxamid is a recently developed fungicide belonging to the quinone inside inhibitor (QiI) group. This is the first fungicide within this group to be active against the Zymoseptoria tritici, which causes Septoria tritici blotch on wheat. The occurrence of pre-existing resistance mechanisms was monitored, using sensitivity assays and Illumina sequencing, in Z. tritici populations sampled in multiple European countries before the introduction of fenpicoxamid. RESULTS: Although differences in sensitivity to all three fungicides tested (fenpicoxamid, fentin chloride and pyraclostrobin) existed between the isolate collections, no alterations associated with QiI resistance were detected. Among the isolates, a range in sensitivity to fenpicoxamid was observed (ratio between most sensitive/least sensitive = 53.1), with differences between the most extreme isolates when tested in planta following limited fenpicoxamid treatment. Sensitivity assays using fentin chloride suggest some of the observed differences in fenpicoxamid sensitivity are associated with multi-drug resistance. Detailed monitoring of the wider European population using Illumina-based partial sequencing of the Z. tritici also only detected the presence of G143A, with differences in frequencies of this alteration observed across the region. CONCLUSIONS: This study provides a baseline sensitivity for European Z. tritici populations to fenpicoxamid. Target-site resistance appears to be limited or non-existing in European Z. tritici populations prior to the introduction of fenpicoxamid. Non-target site resistance mechanisms exist, but their impact in the field is predicted to be limited. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Fungicidas Industriais , Ascomicetos , Cloretos , Complexo III da Cadeia de Transporte de Elétrons , Fungicidas Industriais/farmacologia , Lactonas , Doenças das Plantas , Piridinas , Quinonas , RespiraçãoRESUMO
Zymoseptoria tritici causes the disease known as septoria leaf blotch in winter wheat and is a major factor in yield loss worldwide. Farmers are inclined to use fungicides to protect their crops; however, the efficacy of these measures is rapidly decreasing due to the natural mechanisms of mutation emergence in pathogen populations. Increasing fungicide resistance is being recorded worldwide, therefore, screening of the current situation in Lithuania is essential to determine the subsequent steps of crop protection strategies. In this study, in vitro fungicide sensitivity tests, mutation detection, and field experiments were carried out. The mean EC50 values for prothioconazole-desthio and mefentrifluconazole were 0.14 and 0.28 mg/l, respectively. Increased frequency of the mutation S524T, linked to DMIs resistance, was observed. Results revealed that the dominant point mutation in the gene CYP51 was I381V, and the most frequent CYP51 haplotype was D13 (V136C, I381V, Y461H, S524T). The mutation G143A, linked to QoI resistance, was detected in ¾ of the population. Mutations conferring resistance to SDHIs were not detected in single pycnidium isolates. Two-year field experiments likewise showed no decline in field efficacy of SDHI fungicide in Lithuania. Moreover, the baseline sensitivity of the Lithuanian Z. tritici population to QiI fungicide fenpicoxamid was established. The findings of this study provide an update on the current status of fungicide resistance in the Lithuanian Z. tritici population.
RESUMO
Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping.
RESUMO
BACKGROUND: Over the past decade, demethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides have been extensively used to control to septoria tritici blotch, caused by Zymoseptoria tritici on wheat. This has led to the development and selection of alterations in the target-site enzymes (CYP51 and SDH, respectively). RESULTS: Taking advantage of newly and previously developed qPCR assays, the frequency of key alterations associated with DMI (CYP51-S524T) and SDHI (SDHC-T79N/I, C-N86S and C-H152R) resistance was assessed in Z. tritici-infected wheat leaf samples collected from commercial crops (n = 140) across 14 European countries prior to fungicide application in the spring of 2019. This revealed the presence of a West to East gradient in the frequencies of the most common key alterations conferring azole (S524T) and SDHI resistance (T79N and N86S), with the highest frequencies measured in Ireland and Great Britain. These observations were corroborated by sequencing (CYP51 and SDH subunits) and sensitivity phenotyping (prothioconazole-desthio and fluxapyroxad) of Z. tritici isolates collected from a selection of field samples. Additional sampling made at the end of the 2019 season confirmed the continued increase in frequency of the targeted alterations. Investigations on historical leaf DNA samples originating from different European countries revealed that the frequency of all key alterations (except C-T79I) has been gradually increasing over the past decade. CONCLUSION: Whilst these alterations are quickly becoming dominant in Ireland and Great Britain, scope still exists to delay their selection throughout the wider European population, emphasizing the need for the implementation of fungicide antiresistance measures. © 2021 Society of Chemical Industry.
Assuntos
Fungicidas Industriais , Ascomicetos , Europa (Continente) , Fungicidas Industriais/farmacologia , Doenças das Plantas , Succinato Desidrogenase/genética , Ácido Succínico , TriazóisRESUMO
The C(H)3 domain of antibodies is characterized by two antiparallel beta-sheets forming a disulfide-linked sandwich-like structure. At acidic pH values and low ionic strength, C(H)3 becomes completely unfolded. The addition of salt transforms the acid-unfolded protein into an alternatively folded state exhibiting a characteristic secondary structure. The transition from native to alternatively folded C(H)3 is a fast reaction. Interestingly, this reaction involves the formation of a defined oligomer consisting of 12-14 subunits. Association is completely reversible and the native dimer is quantitatively reformed at neutral pH. This alternatively folded protein is remarkably stable against thermal and chemical denaturation and the unfolding transitions are highly cooperative. With a t(m) of 80 degrees C, the stability of the alternatively folded state is comparable to that of the native state of C(H)3. The defined oligomeric structure of C(H)3 at pH 2 seems to be a prerequisite for the cooperative unfolding transitions.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Regiões Constantes de Imunoglobulina/química , Regiões Constantes de Imunoglobulina/metabolismo , Ácidos/farmacologia , Animais , Ânions/farmacologia , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Cinética , Luz , Camundongos , Peso Molecular , Concentração Osmolar , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína , Estrutura Quaternária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Subunidades Proteicas , Sais/farmacologia , Espalhamento de Radiação , Solventes , Temperatura , Termodinâmica , UltracentrifugaçãoRESUMO
The simplest naturally occurring model system for studying immunoglobulin folding and assembly is the non-covalent homodimer formed by the C-terminal domains (CH3) of the heavy chains of IgG. Here, we describe the structure of recombinant CH3 dimer as determined by X-ray crystallography and an analysis of the folding pathway of this protein. Under conditions where prolyl isomerization does not contribute to the folding kinetics, formation of the beta-sandwich structure is the rate-limiting step. beta-Sheet formation of CH3 is a slow process, even compared to other antibody domains, while the subsequent association of the folded monomers is fast. After long-time denaturation, the majority of the unfolded CH3 molecules reaches the native state in two serial reactions, involving the re-isomerization of the Pro35-peptide bond to the cis configuration. The species with the wrong isomer accumulate as a monomeric intermediate. Importantly, the isomerization to the correct cis configuration is the prerequisite for dimerization of the CH3 domain. In contrast, in the Fab fragment of the same antibody, prolyl isomerization occurs after dimerization demonstrating that within one protein, comprised of highly homologous domains, both the kinetics of beta-sandwich formation and the stage at which prolyl isomerization occurs during the folding process can be completely different.
Assuntos
Anticorpos Monoclonais/química , Prolina/química , Dobramento de Proteína , Dicroísmo Circular , Dimerização , Fluorometria , Isomerismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peptidilprolil Isomerase , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Triptofano/químicaRESUMO
By operating in a region of liquid-liquid equilibrium, hot acetic acid-water mixtures can be used to simultaneously clean, fractionate, and solvate Kraft black-liquor lignins. Lignin-rich liquid phases of controlled molecular weight with key metals contents reduced to <50 ppm are obtained without a washing step.
Assuntos
Lignina/química , Lignina/isolamento & purificação , Ácido Acético/química , Peso Molecular , Água/químicaRESUMO
Binocular depth inversion represents an illusion of visual perception that is sensitive to various behavioural and psychiatric conditions. It is affected by cannabinoids, reflecting associated changes in perception. The present study investigated the differences in binocular depth inversion of different classes of natural and artificial objects and the effect of synthetic delta9-tetrahydrocannabinol (Dronabinol) on these illusionary perceptions. Using this model, the effects of orally administered Dronabinol on binocular depth inversion were investigated in 17 healthy male volunteers. Pictures from natural and artificial objects were presented stereoscopically and the depth perception of the volunteers was scored in an operationalized way. The timecourse of the effects of Dronabinol on binocular depth inversion was analyzed with regard to the stimulus classes (natural and synthetic objects). Significant differences in binocular depth inversion of the different groups of stimuli were revealed. Objects with a higher degree of everyday familiarity were generally seen as more illusionary than those with a lower degree of everyday familiarity. A strong impairment of binocular depth inversion due to Dronabinol was found in most classes of objects. Analysis of different stimulus classes provides further information on the underlying perceptual processing of binocular depth inversion. An impairment of top-down processing of visual sensory data by Dronabinol is suggested. The anandamidergic system seems to be involved in areas of visual information processing.
Assuntos
Dronabinol/farmacologia , Alucinógenos/farmacologia , Visão Binocular/efeitos dos fármacos , Adulto , Dronabinol/síntese química , Eletrofisiologia , Alucinógenos/síntese química , Humanos , Ilusões/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Neuropsicologia , Observação , Percepção/efeitos dos fármacosRESUMO
OBJECTIVES: The purpose of this study is to determine the temperature dependence of the flexural strength of a dental porcelain (IPS-Classic Dentin; manufacturer: Ivoclar, Liechtenstein) with temperature between its glass transition (T(g) = 581.7 degrees C) and room temperature. METHODS: The flexural strength was measured in three-point bending tests on an Instron 4204 testing apparatus. The strength values were determined for the temperatures T = 20, 300, 400, 450, 500, 550, and 600 degrees C. RESULTS: In the temperature interval 20 < or = T < or = 400 degrees C the flexural strength decreased slightly from approximately 80 to approximately 73 MPa (mean values), as temperature increased. That is a decay of less than 10%. At higher temperatures the flexural strength increased to a maximum of approximately 98 MPa at 500 degrees C, probably due to the closure of microcracks in the surface on account of the onset of viscous flow. A further increase of the temperature delivered again decreasing strength values. At its glass transition temperature the porcelain's flexural strength was approximately 76 MPa which is only about 5% less than the value at room temperature. SIGNIFICANCE: In order to be able to evaluate the risk of fracture of ceramometallic crowns and bridges due to high temperature gradients and accompanying large transient thermal stresses in the veneer during the fabrication process, flexural strength values at high temperatures must be known. This study was carried out to fill this knowledge gap because to the authors' knowledge there was little published in the literature.
Assuntos
Porcelana Dentária , Silicatos de Alumínio , Análise Diferencial Térmica , Teste de Materiais , Ligas Metalo-Cerâmicas , Maleabilidade , TemperaturaRESUMO
The binding of six different FITC-labelled lectins to mesenchyme, blastemal cells and cartilage was investigated in limb buds of mouse embryos during their development from day 10 to day 13. Concanavalin A, wheat germ agglutinin and phaseolus vulgaris agglutinin labelled mesenchymal cells of earlier stages, day 10 or 11, distinctly more than those of later stages. Chondrogenic blastema, basement membrane and muscle were always strongly stained. The galactosamine-specific ricinus communis agglutinin (RCA) bound preferentially to the blastema, whereas the mesenchyme was only weakly labelled. The galactose-specific peanut agglutinin (PNA), however, stained solely the blastema. In the mesenchyme, no binding was detectable light microscopically with this lectin. In cartilage, RCA- and PNA-staining was found to a lesser extent. With the fucose-specific lectin Lotus A, no staining was detectable. Due to the apparent differences in the binding of PNA in mesenchyme and blastema, peroxidase-labelled PNA was used to study the binding behaviour electron microscopically. It is shown that peroxidase-PNA very strongly labelled the intercellular matrix and the plasma membrane of cells in the late blastemal stages, whereas in young blastema no reaction product was detectable. In contrast to light microscopic findings, some label could be demonstrated also in the mesenchyme. The results show a general reduction of lectin receptors in more developed mesenchyme of later stages and the occurrence of galactose and galactose derivatives during early chondrogenesis in the matrix and at the cell membrane. The significance of these changes for chondrogenesis, however, remains to be elucidated.
Assuntos
Cartilagem/embriologia , Extremidades/embriologia , Lectinas , Animais , Sítios de Ligação , Cartilagem/metabolismo , Extremidades/metabolismo , Histocitoquímica , Camundongos , Microscopia Eletrônica , Aglutinina de AmendoimRESUMO
The appearance of the basement membrane components laminin, heparan sulphate proteoglycan and Type IV collagen was investigated by indirect immunofluorescence during the onset of vascularisation of the neuroepithelium of mouse embryos (Days 10, 11 and 12 of development). All three basement membrane components could initially be seen in the embryonic CNS anlage along with the appearance of capillaries in the neuroepithelial cell layer. From this direct correlation between vascularisation and appearance of basement membrane components in the embryonic anlage one can assume that the basement membrane components laminin, heparan sulphate proteoglycan and collagen Type IV are involved in the initial vascularisation of the CNS anlage of 10 to 12 day old mouse embryos.
Assuntos
Sistema Nervoso Central/metabolismo , Colágeno/metabolismo , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Laminina/metabolismo , Animais , Capilares/citologia , Capilares/embriologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Sistema Nervoso Central/irrigação sanguínea , Sistema Nervoso Central/embriologia , Colágeno/fisiologia , Epitélio/irrigação sanguínea , Epitélio/embriologia , Epitélio/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Feminino , Heparitina Sulfato/fisiologia , Imuno-Histoquímica , Laminina/fisiologia , Camundongos , GravidezRESUMO
This work describes a technique which permits study of the postembedding lectin histochemistry for WGA-binding sites at the light and electron microscopical level on the same resin embedded tissue without removing or etching of the resin. Unfixed kidney pieces or kidney pieces fixed in 4% formaldehyde were embedded in the hydrophilic polyhydroxy aromatic resins LR-Gold and LR-White, following dehydration in up to 70% ethanol, 90% ethanol or 100% ethanol. LR-Gold was cryopolymerised at -25 degrees C using the light sensitive initiator benzil, whereas LR-White was heat-cured at +50 degrees C. The localisation of WGA-binding sites at the light microscopical level was investigated using FITC-labelled WGA. The ultrastructural localisation of WGA-binding sites was performed using 15 nm gold-labelled WGA. The best fluorescence staining results were obtained on fixed or unfixed tissue dehydrated in up to 70% ethanol and embedded in LR-Gold. At the ultrastructural level, the best staining results for WGA-binding sites were seen on tissue sections, dehydrated in up to 90% ethanol prior to embedding in LR-Gold.
Assuntos
Córtex Renal/metabolismo , Aglutininas do Germe de Trigo/metabolismo , Resinas Acrílicas , Animais , Sítios de Ligação , Feminino , Fluoresceína-5-Isotiocianato , Fluoresceínas , Histocitoquímica , Córtex Renal/ultraestrutura , Camundongos , Microscopia Eletrônica , TiocianatosRESUMO
A total of 41 pregnant Mexican free-tailed bats (Tadarida brasiliensis) were collected from Carlsbad Caverns, New Mexico, and Vickery Cave, Oklahoma, in May and June 1990 and May 1991 for organochlorine pesticide analyses. Residues of p,p'-DDE were detected by gas chromatography in 40 embryos at levels highly correlated with brain concentrations (r = 0.496) but were not correlated mother's body tissues (r = 0.060). Embryonic levels also did not correlate with either embryonic fat content (r = 0.018) or maternal fat content (r = 0.300), suggesting that placental membranes offer only marginal protection for developing embryos against exposure to lipophilic organochlorine pesticides.
Assuntos
Quirópteros/metabolismo , Hidrocarbonetos Clorados , Inseticidas/farmacocinética , Troca Materno-Fetal , Resíduos de Praguicidas/farmacocinética , Animais , Encéfalo/metabolismo , Quirópteros/embriologia , Feminino , New Mexico , Oklahoma , GravidezRESUMO
The C(H)2 domain, one of the constant domains of the murine monoclonal antibody MAK33 (immunoglobulin subtype K/IgG1) was expressed in Escherichia coli forming insoluble inclusion bodies (IBs) and purified by a three-step process including a denaturation-renaturation step, hydrophobic interaction and gel permeation chromatography. After disrupting the cells, the soluble protein fraction was removed by several centrifugation steps. The isolation of the IBs from the cell fragments was achieved by solubilizing the IBs with 6 M guanidinium hydrochloride (GdmCl) and 0.1 M 1,4-dithioerythrit (DTE) to reduce all disulfide bonds. After refolding the C(H)2 domain, 1.5 M (NH4)2SO4 was added to the protein solution in order to precipitate contaminations. Then the protein was loaded on a Butyl-Sepharose fast flow column and eluted with a linear gradient [1.5-0 M (NH4)2SO4]. As the last purification step a gel permeation chromatography was run on a Superdex 75 prep grade. Finally, the purity of the C(H)2 protein was determined by a silver-stained sodium dodecyl sulfate polyacrylamide gel. We achieved a typical yield of 0.5 mg pure protein per 1 g of wet cells.