RESUMO
Marinas are central hubs of global maritime leisure and transport, yet their operations can deteriorate the environmental quality of sediments. In response, this study investigated the metal contamination history associated with antifouling paint uses in a sediment core collected from Bracuhy marina (Southeast Brazil). Analysis target major and trace elements (Cu, Zn, Pb, Cd and Sn), rare earth elements (REEs), and Pb isotopes. The modification in Pb isotopic ratios and REEs pattern unequivocally revealed sediment provenance disruption following the marina construction. Metal distribution in the sediment core demonstrates that concentrations of Cu and Zn increased by up to 15 and 5 times, respectively, compared to the local background. This severe Cu and Zn contamination coincides with the onset of marina operations and can be attributed to the use of antifouling paints.
Assuntos
Cobre , Monitoramento Ambiental , Sedimentos Geológicos , Pintura , Poluentes Químicos da Água , Sedimentos Geológicos/química , Pintura/análise , Poluentes Químicos da Água/análise , Cobre/análise , Brasil , NaviosRESUMO
The pendulous crop is characterized by excessive distension of the crop musculature, compromising the bird's productivity and welfare. The etiology is still unknown, but it is believed that factors related to the birds' handling might be related to its incidence. The study was conducted in 2 environmental chambers. One was maintained at a comfortable temperature, while the other was set at a much lower temperature. In each chamber, animals were divided into 16 experimental pens (8 received mash feed and the others received pelletized feed) with a density of 12 birds/m2 (an expected stocking density of 32-36 kg/m2 after 42 d). The effects of rearing temperatures were evaluated in terms of broiler performance, specifically weight gain (kg), feed intake (kg), weekly feed intake (kg/wk), and feed conversion (kgfeed/kggrowth). The occurrences of pendulous crop were quantified every 2 d after the 14th day of rearing. Birds grown in thermal comfort and fed a pelletized ration were most susceptible (12%) to pendulous crop, followed by birds fed pelletized feed and reared in cold conditions (6.8%), and birds given mashed feed and reared at either temperature (about 3%). We concluded that feeding pelleted feed combined with warmer rearing temperatures may have caused some alteration of the gastrointestinal system of birds, which caused pendulous crop to be more prevalent.
Assuntos
Ração Animal/análise , Galinhas/anormalidades , Papo das Aves/crescimento & desenvolvimento , Temperatura , Animais , Galinhas/crescimento & desenvolvimento , Dieta/veterinária , MasculinoRESUMO
Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.
Assuntos
Animais , Bovinos , Humanos , /fisiologia , Células Endoteliais/virologia , Hepacivirus/imunologia , Receptores de LDL/fisiologia , Proteínas do Envelope Viral/fisiologia , /imunologia , Linhagem Celular , Escherichia coli , Células Endoteliais/imunologia , Citometria de Fluxo , Proteínas de Membrana , Pichia , Proteínas Recombinantes , Receptores de LDL/imunologiaRESUMO
Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.
Assuntos
Células Endoteliais/virologia , Hepacivirus/imunologia , Receptores de LDL/fisiologia , Tetraspanina 28/fisiologia , Proteínas do Envelope Viral/fisiologia , Animais , Bovinos , Linhagem Celular , Células Endoteliais/imunologia , Escherichia coli , Citometria de Fluxo , Humanos , Proteínas de Membrana , Pichia , Receptores de LDL/imunologia , Proteínas Recombinantes , Tetraspanina 28/imunologiaRESUMO
The hepatitis C virus (HCV) is an enveloped virus that is about 50-70 nm in diameter, has positive-strand RNA, and belongs to the genus Hepacivirus and the family Flaviridae. The detection and quantification of the core antigen, HCV nucleocapsid protein, has been successful in many trials and is considered a marker of viral replication since it presents a sequence of highly conserved amino acids, giving it high sensitivity and specificity. The E2 protein is an envelope glycoprotein of HCV with 11 glycosylation sites; most of these are well-conserved, making it a target antigen. The aim of this study is to develop high-sensitivity, low-cost diagnostic methods for HCV, which could be used for serological screening. The genomic regions encoding the core (part 136 aa) and E2 proteins of HCV were expressed in Escherichia coli Rosetta strain, cloned in expression vector pET-42a, and induced with 0.4 m mol L(-1) IPTG, producing recombinant proteins that were fused to glutathione S-transferase (GST) protein, which was then purified by affinity chromatography. The immunoreactivity was assessed by Western blot, Slot Blot, and developed and improved diagnostic methods (capture, indirect, and immunoblotting enzyme-linked immunosorbent assay (ELISA)). After applying the results to the formulas for determining the quality parameters, obtained for immunoblotting method 100% sensitivity and specificity and for ELISA 100% sensitivity and 87.5% specificity. The methods developed were more sensitive and specific using the mixture of the recombinant proteins fused to GST (core+E2).
Assuntos
Glutationa Transferase/metabolismo , Hepacivirus/isolamento & purificação , Proteínas do Core Viral/metabolismo , Western Blotting , Ensaio de Imunoadsorção Enzimática , Hepacivirus/metabolismoRESUMO
Xylella fastidiosa is responsible for a wide range of economically important plant diseases. We report here the crystal structure and kinetic data of Xylellain, the first cysteine protease characterized from the genome of the pathogenic X. fastidiosa strain 9a5c. Xylellain has a papain-family fold, and part of the N-terminal sequence blocks the enzyme active site, thereby mediating protein activity. One novel feature identified in the structure is the presence of a ribonucleotide bound outside the active site. We show that this ribonucleotide plays an important regulatory role in Xylellain enzyme kinetics, possibly functioning as a physiological mediator.
Assuntos
Proteínas de Bactérias/química , Cisteína Proteases/química , Modelos Moleculares , Xylella/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/agonistas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Cinética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/agonistas , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação Puntual , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Difosfato de Uridina/química , Difosfato de Uridina/metabolismoRESUMO
Introduction: Pheochromocytomas are rare neuroendocrine tumors, producing catecholamines, which usually affect the adrenal medulla region of the adrenal gland. These tumors may clinically manifest in several ways, presenting themselves in most patients with persistent hypertension or paroxysmal. Ten percent of cases are considered malignant, confirmed by the presence of metastases and approximately 24% of cases are associated with inherited syndromes. Diagnostic confirmation of these syndromes implies preparatory workup, treatment and stringent follow-up, preferably with a multidisciplinary team. Objective: This study is a survey of recent studies to clarify issues related to clinical, diagnosis, genetic and treatment aspects of these patients. Conclusion: It is widely accepted that a significant percentage of patients with sporadic pheochromocytoma may have germline mutations leading to more widespread disease development and/or malignancy, and that surgical treatment in these cases must be complemented by careful clinical surveillance for early diagnosis of recurrences. This study prioritized the importance of conducting a proper pretreatment workup in cases of pheochromocytoma, which provides the additional information required for a rational course of treatment for patients.
Assuntos
Humanos , Feocromocitoma/diagnóstico , Feocromocitoma/epidemiologia , Feocromocitoma/terapia , Tumores NeuroendócrinosRESUMO
The hepatitis C virus (HCV) core protein possesses amino acid sequences highly conserved among HCV isolates and has proved useful for various diagnostic tests. To date, no information has been published regarding the development of an immunochromatographic test for HCV core antigen detection. Therefore, the aim of this research was to develop a rapid, easily performed, highly sensitive and specific test for detection of the HCV core antigen, based on the immunochromatographic strip. The genomic region encoding the core protein (amino acids 1-136) of the hepatitis C virus was expressed in Escherichia coli as a recombinant fusion protein with glutathione S-transferase (GST) cloned into the prokaryotic expression vector pET42a and was confirmed by immunological detection with HCV positive serum. Positive reactions were detected weakly at a 1:15 dilution of the serum and more strongly in 1:10, 1:5, 1:2 and 1:1 dilutions, by the immunochromatographic test. In addition, the test was capable of detecting 0.25-12.0 microg of the recombinant protein. This immunochromatographic technique opens new perspectives for the diagnosis of hepatitis C during the early seroconversion phase and for a rapid core antigen detection.
Assuntos
Hepacivirus/isolamento & purificação , Hepatite C/diagnóstico , Proteínas do Core Viral/análise , Animais , Cromatografia de Afinidade/métodos , Humanos , Imunoensaio/métodos , CamundongosRESUMO
O câncer de esôfago cervical é uma doença rara. O melhor tratamento é o cirúrgico, porém com muitas complicações. Portanto, sugerimos, demonstrando dois casos operados, um acesso minimamente invasivo (laparoscópico e/ou toracoscópico) com faringolaringectomia total e autonomização do tubo gástrico previamente, com o intuito de diminuir a incidência de fistula.
The cervical esophagus cancer is a rare disease. The best treatment is the surgical one. However, it can present many complications. Therefore, we suggest, demostrating two operated cases, a minimally invasive access (laparoscopic and/or thorascopic) with total pharyngolaryngectomy and autonomization of the gastric tube previously in order to decrase the incidence of leak.
Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Carcinoma/cirurgia , Esofagectomia/métodos , Faringectomia/métodos , Laringectomia/métodos , Neoplasias Esofágicas/cirurgia , Laparoscopia , Procedimentos Cirúrgicos Minimamente Invasivos , ToracoscopiaRESUMO
Practical classes on protein expression and purification were given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering." The heterologous expression of the green fluorescent protein (GFP)* of Aequorea victoria is an interesting system for didactic purposes because it can be viewed easily during experiments. The students were provided with basic information about the molecular features and applications of the GFP in molecular biology, the available heterologous expression systems, and the theoretical and experimental details of GFP expression in Escherichia coli and its purification. E. coli BL21-competent cells were transformed with the pET28a expression vector containing the GFP gene fused to a histidine (His) tag. During the induction of a transformed clone by isopropylthiogalactoside, a time course for GFP expression was analyzed by SDS-PAGE, and the expression was also visualized by the increasing green fluorescence of the bacterial culture. After cellular disruption, protein purification was illustrated by affinity chromatography of the His-tagged protein in a nickel column. Eluted fractions containing imidazole in increasing concentrations were analyzed visually and also by SDS-PAGE, demonstrating the role of imidazole in protein recovery by competition with nonspecific proteins and the His-tagged protein. The results obtained and the experimental factors involved in protein expression, solubilization, and folding were discussed following the laboratory experiments. These practical classes allowed several current approaches to molecular biology to be demonstrated rapidly and helped underscore some of the topics taught during the course.